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Retinal pigment epithelium is a monolayer of cells located beneath photoreceptors of the retina maintaining their functionality. Malfunction of RPE leads to retinal degenerative diseases, such as age-related macular degeneration and Stargardt disease. Ca2+ is a ubiquitous ion that takes part in regulation of vital cellular processes. The knowledge of Ca2+ dynamics is essential for understanding RPE physiology. This is especially important for functionality assessment of cells intended for transplantation and for drug testing. The aim of this thesis was to study spontaneous and mechanically induced Ca2+ activity in human RPE and to assess the effect of cellular maturation and wounding on the [Ca2+]i dynamics. For this, various methods, such as fluorescent Ca2+ imaging, immunofluorescence staining, PCR, and mathematical modeling were applied. In addition, novel methods were developed to analyze large amounts of Ca2+ imaging data. ARPE-19 and human embryonic stem cell-derived RPE cells (hESC-RPE) were used as RPE cell models. In this thesis, it was shown that both ARPE-19 and hESC-RPE exhibit intercellular Ca2+ waves upon mechanical stimulation. With live-cell Ca2+ imaging and mathematical modeling, it was demonstrated that in ARPE-19 cells, the mechanically induced Ca2+ waves propagate intracellularly through gap junctions and extracellularly involving diffusion of a paracrine factor. By applying in-house image analysis tools for the experimental fluorescence time-series, it was found that in hESC-RPE cells, spontaneous [Ca2+]i transients and the ability to propagate intercellular Ca2+ waves upon mechanical stimulation strongly depend on the maturation status of the cells. Finally, it was demonstrated that wounding affects spontaneous Ca2+ activity close to the wound edges, and cells within the healed areas resemble Ca2+ dynamics of immature hESC-RPE. To conclude, this thesis has provided important insights into human RPE Ca2+ dynamics, as well as into the events of single cell mechanical stimulation and large scale monolayer wounding. In addition, it was demonstrated that maturation drastically affects RPE Ca2+ dynamics. This knowledge and the developed image analysis algorithms contribute to understanding RPE physiology and can facilitate establishment of novel tools for assessment of RPE functionality prior to transplantation and in drug testing assays.

The proprotein convertase enzyme FURIN processes immature pro-proteins into functional end- products. FURIN is upregulated in activated immune cells and it regulates T-cell dependent peripheral tolerance and the Th1/Th2 balance. FURIN also promotes the infectivity of pathogens by activating bacterial toxins and by processing viral proteins. Here, we evaluated the role of FURIN in LysM+ myeloid cells in vivo. Mice with a conditional deletion of FURIN in their myeloid cells (LysMCre-fur(fl/fl)) were healthy and showed unchanged proportions of neutrophils and macrophages. Instead, LysMCre-fur(fl/fl) mice had elevated serum IL-1β levels and reduced numbers of splenocytes. An LPS injection resulted in accelerated mortality, elevated serum pro-inflammatory cytokines and upregulated numbers of pro-inflammatory macrophages. A genome-wide gene expression analysis revealed the overexpression of several pro-inflammatory genes in resting FURIN-deficient macrophages. Moreover, FURIN inhibited Nos2 and promoted the expression of Arg1, which implies that FURIN regulates the M1/M2-type macrophage balance. FURIN was required for the normal production of the bioactive TGF-β1 cytokine, but it inhibited the maturation of the inflammation-provoking TACE and Caspase-1 enzymes. In conclusion, FURIN has an anti-inflammatory function in LysM+ myeloid cells in vivo.

ObjectivesTo determine the effects of various monomers on conversion and cytocompatibility of dental composites and to improve these properties without detrimentally affecting mechanical properties, depth of cure and shrinkage.MethodsComposites containing urethane dimethacrylate (UDMA) or bisphenol A glycidyl methacrylate (Bis-GMA) with poly(propylene glycol) dimethacrylate (PPGDMA) or triethylene glycol dimethacrylate (TEGDMA) were characterized using the following techniques: conversion (FTIR at 1 and 4mm depths), depth of cure (BS EN ISO 4049:2009 and FTIR), shrinkage (BS EN ISO 17304:2013 and FTIR), strength and modulus (biaxial flexural test) and water sorption. Cytocompatibility of composites and their liquid phase components was assessed using three assays (resazurin, WST-8 and MTS).ResultsUDMA significantly improved conversion, BFS and depth of cure compared to Bis-GMA, without increasing shrinkage. UDMA was cytotoxic at lower concentrations than Bis-GMA, but extracts of Bis-GMA-containing composites were less cytocompatible than of those containing UDMA. PPGDMA improved conversion and depth of cure compared to TEGDMA, without detrimentally affecting shrinkage. TEGDMA was shown by all assays to be highly toxic. Resazurin, but not WST-8 and MTS, suggested that PPGDMA exhibited improved cytocompatibility compared to TEGDMA.SignificanceThe use of UDMA and PPGDMA results in composites with excellent conversion, depth of cure and mechanical properties, without increasing shrinkage. Composites containing UDMA appear to be slightly more cytocompatible than those containing Bis-GMA. These monomers may therefore improve the material properties of dental restorations, particularly bulk fill materials. The effect of diluent monomer on cytocompatibility requires further investigation.

Hypertrophic cardiomyopathy (HCM) is a genetic cardiac disease, which affects the structure of heart muscle tissue. The clinical symptoms include arrhythmias, progressive heart failure, and even sudden cardiac death but the mutation carrier can also be totally asymptomatic. To date, over 1400 mutations have been linked to HCM, mostly in genes encoding for sarcomeric proteins. However, the pathophysiological mechanisms of the disease are still largely unknown. Two founder mutations for HCM in Finland are located in myosin-binding protein C (MYBPC3-Gln1061X) andα-tropomyosin (TPM1-Asp175Asn) genes. We studied the properties of HCM cardiomyocytes (CMs) derived from patient-specific human induced pluripotent stem cells (hiPSCs) carrying eitherMYBPC3-Gln1061XorTPM1-Asp175Asnmutation. Both types of HCM-CMs displayed pathological phenotype of HCM but, more importantly, we found differences between CMs carrying eitherMYBPC3-Gln1061XorTPM1-Asp175Asngene mutation in their cellular size, Ca2+handling, and electrophysiological properties, as well as their gene expression profiles. These findings suggest that even though the clinical phenotypes of the patients carrying eitherMYBPC3-Gln1061XorTPM1-Asp175Asngene mutation are similar, the genetic background as well as the functional properties on the cellular level might be different, indicating that the pathophysiological mechanisms behind the two mutations would be divergent as well.

Background: Odontogenic tumors such as ameloblastic fibro-odontoma (AFO) are rare conditions in children and are often asymptomatic. AFOs are found by routine clinical and radiological examination or when they cause obvious intra- or extra-oral swelling. Materials and Methods: A case of an AFO in a 7-year-old girl is described, and 107 cases from the literature and this report are analyzed. Results: The total of 108 cases revealed the average age at presentation of AFO to be 6.3 years in boys and 9.6 years in girls. There was a slight male predilection and AFO lesions most often occurred in the posterior mandible. AFO was almost always associated with an unerupted tooth or teeth. Conclusions: While the recurrence rate of AFO was found to be 5.5%, long-term postoperative clinical and radiological follow-up is advised to ensure no future signs of aggressive recurrence.

Epithelial to mesenchymal transition (EMT) of cancer cells involves loss of epithelial polarity and adhesiveness, and gain of invasive and migratory mesenchymal behaviours. EMT occurs in prostate cancer (PCa) but it is unknown whether this is in specific areas of primary tumours. We examined whether any of eleven EMT-related proteins have altered expression or subcellular localisation within the extraprostatic extension component of locally advanced PCa compared with other localisations, and whether similar changes may occur in in vitro organotypic PCa cell cultures and in vivo PCa models. Expression profiles of three proteins (E-cadherin, Snail, and α-smooth muscle actin) were significantly different in extraprostatic extension PCa compared with intra-prostatic tumour, and 18/27 cases had an expression change of at least one of these three proteins. Of the three significantly altered EMT proteins in pT3 samples, one showed similar significantly altered expression patterns in in vitro organotypic culture models, and two in in vivo Pten-/- model samples. These results suggest that changes in EMT protein expression can be observed in the extraprostatic extension component of locally invasive PCa. The biology of some of these changes in protein expression may be studied in certain in vitro and in vivo PCa models.

Summary Trichinellosis is a helminthic infection where different species of T richinella nematodes are the causative agents. Several molecular assays have been designed to aid diagnostics of trichinellosis. These assays are mostly complex and expensive. The genomes of T richinella species contain certain parasite‐specific genes, which can be detected by polymerase chain reaction (PCR) methods. We selected β‐carbonic anhydrase (β‐ CA) gene as a target, because it is present in many parasites genomes but absent in vertebrates. We developed a novel β‐CA gene‐based method for detection of T richinella larvae in biological samples. We first identified a β‐CA protein sequence from T richinella spiralis by bioinformatic tools using β‐CAs from C aenorhabditis elegans and D rosophila melanogaster. Thereafter, 16 sets of designed primers were tested to detect β‐CA genomic sequences from three species of T richinella, including T . spiralis, T richinella pseudospiralis and T richinella nativa. Among all 16 sets of designed primers, the primer set No. 2 efficiently amplified β‐CA genomic sequences from T . spiralis, T . pseudospiralis and T . nativa without any false‐positive amplicons from other parasite samples including T oxoplasma gondii, T oxocara cati and P arascaris equorum. This robust and straightforward method could be useful for meat inspection in slaughterhouses, quality control by food authorities and medical laboratories.

Small nucleolar RNAs (snoRNAs) are dynamically regulated in different tissues and affected in disease. SnoRNAs are processed further to stable smaller RNAs. We sequenced the small RNA transcriptome of prostate cancer (PCa) at different PCa stages and generated a quantified catalogue of 3927 small non-coding RNAs (sncRNAs) detected in normal and malignant prostate tissue. From these, only 1524 are microRNAs. The remaining 2401 sncRNAs represent stable sncRNAs species that originate from snoRNA, tRNA and other sncRNAs. We show that snoRNA-derived RNAs (sdRNAs) display stronger differential expression than microRNAs and are massively upregulated in PCa. SdRNAs account for at least one third of all small RNAs with expression changes in tumor compared to normal adjacent tissue. Multiple sdRNAs can be produced from one snoRNA in a manner related to the conservation of structural snoRNA motifs. Q-PCR analysis in an independent patient cohort (n=106) confirmed the processing patterns of selected snoRNAs (SNORD44, SNORD78, SNORD74 and SNORD81) and the cancer-associated up-regulation of their sdRNAs observed in sequencing data. Importantly, expression of SNORD78 and its sdRNA is significantly higher in a subset of patients that developed metastatic disease demonstrating that snoRNA and sdRNAs may present as novel diagnostic and/or prognostic biomarkers for PCa. This article has supplementary files, which can be found here:http://dx.doi.org/10.18632/oncotarget.4172

Levels of total human kallikrein 2 (hK2), a protein involved the pathology of prostate cancer (PCa), could be used as a biomarker to aid in the diagnosis of this disease. In this study, we report on a porous silicon antibody immunoassay platform for the detection of serum levels of total hK2. The surface of porous silicon has a 3-dimensional macro- and nanoporous structure, which offers a large binding capacity for capturing probe molecules. The tailored pore size of the porous silicon also allows efficient immobilization of antibodies by surface adsorption, and does not require chemical immobilization. Monoclonal hK2 capture antibody (6B7) was dispensed onto P-Si chip using a piezoelectric dispenser. In total 13 × 13 arrays (169 spots) were spotted on the chip with its single spot volume of 300 pL. For an optimization of capture antibody condition, we firstly performed an immunoassay of the P-Si microarray under a titration series of hK2 in pure buffer (PBS) at three different antibody densities (75, 100 and 145 µg/mL). The best performance of the microarray platform was seen at 100 µg/mL of the capture antibody concentration (LOD was 100 fg/mL). The platform then was subsequently evaluated for a titration series of serum-spiked hK2 samples. The developed platform utilizes only 15 µL of serum per test and the total assay time is about 3 h, including immobilization of the capture antibody. The detection limit of the hK2 assay was 100 fg/mL in PBS buffer and 1 pg/mL in serum with a dynamic range of 106 (10(-4) to 10(2) ng/mL).

Prostate cancer is the third most common cause of male cancer death in developed countries, and one of the most comprehensively characterized human cancers. Roughly 60% of prostate cancers harbor gene fusions that juxtapose ETS-family transcription factors with androgen regulated promoters. A second subtype, characterized by SPINK1 overexpression, accounts for 15% of prostate cancers. Here we report the discovery of a new prostate cancer subtype characterized by rearrangements juxtaposing the SMAD inhibitor SKIL with androgen regulated promoters, leading to increased SKIL expression. SKIL fusions were found in 6 of 540 (1.1%) prostate cancers and 1 of 27 (3.7%) cell lines and xenografts. 6 of 7 SKIL-positive cancers were negative for ETS overexpression, suggesting mutual exclusivity with ETS fusions. SKIL knockdown led to growth arrest in PC-3 and LNCaP cell line models of prostate cancer, and its overexpression led to increased invasiveness in RWPE-1 cells. The role of SKIL as a prostate cancer oncogene lends support to recent studies on the role of TGF-β signaling as a rate-limiting step in prostate cancer progression. Our findings highlight SKIL as an oncogene and potential therapeutic target in 1-2% of prostate cancers, amounting to an estimated 10,000 cancer diagnoses per year worldwide. This article has supplementary files, which can be found here:http://dx.doi.org/10.18632/oncotarget.3359

In addition to vaccines, noninfectious virus-like particles (VLPs) that mimic the viral capsid show an attractive possibility of presenting immunogenic epitopes or targeting molecules on their surface. Here, functionalization of norovirus-derived VLPs by simple non-covalent conjugation of various molecules is shown. By using the affinity between a surface-exposed polyhistidine-tag and multivalent tris-nitrilotriacetic acid (trisNTA), fluorescent dye molecules and streptavidin–biotin conjugated to trisNTA are displayed on the VLPs to demonstrate the use of these VLPs as easily modifiable nanocarriers as well as a versatile vaccine platform. The VLPs are able to enter and deliver surface-displayed fluorescent dye into HEK293T cells via a surface-attached cell internalization peptide (VSV-G). The ease of manufacturing, the robust structure of these VLPs, and the straightforward conjugation provide a technology, which can be adapted to various applications in biomedicine.

The IFIH1 gene encodes the pattern recognition receptor MDA5. A common polymorphism in IFIH1 (rs1990760, A946T) confers increased risk for autoimmune disease, including type 1-diabetes (T1D). Coxsackievirus infections are linked to T1D and cause beta-cell damage in vitro. Here we demonstrate that the rs1990760 polymorphism regulates the interferon (IFN) signature expressed by human pancreatic islets following Coxsackievirus infection. A strong IFN signature was associated with high expression of IFNλ1 and IFNλ2, linking rs1990760 to the expression of type III IFNs. In the high-responding genotype, IRF-1 expression correlated with that of type III IFN, suggesting a positive-feedback on type III IFN transcription. In summary, our study uncovers an influence of rs1990760 on the canonical effector function of MDA5 in response to an acute infection of primary human parenchymal cells with a clinically relevant virus linked to human T1D. It also highlights a previously unrecognized connection between the rs1990760 polymorphism and the expression level of type III IFNs.

We have created a transgenic reporter for virus infection and used it to study Nora virus infection in Drosophila melanogaster. The transgenic construct, Munin, expresses the yeast transcription factor Gal4, tethered to a transmembrane anchor via a linker that can be cleaved by a viral protease. In infected cells, liberated Gal4 will then transcribe any gene that is linked to a promoter with a UAS motif, the target for Gal4 transcription. For instance, infected cells will glow red in the offspring of a cross between the Munin stock and flies with a UAS-RFPnls transgene (expressing a red fluorescent protein). In such flies we show that after natural infection, via the faecal-oral route, 5–15% of the midgut cells are infected, but there is little if any infection elsewhere. By contrast, we can detect infection in many other tissues after injection of virus into the body cavity. The same principle could be applied for other viruses and it could also be used to express or suppress any gene of interest in infected cells.

BACKGROUND: It is generally acknowledged that a functional understanding of a biological system can only be obtained by an understanding of the collective of molecular interactions in form of biological networks. Protein networks are one particular network type of special importance, because proteins form the functional base units of every biological cell. On a mesoscopic level of protein networks, modules are of significant importance because these building blocks may be the next elementary functional level above individual proteins allowing to gain insight into fundamental organizational principles of biological cells. RESULTS: In this paper, we provide a comparative analysis of five popular and four novel module detection algorithms. We study these module prediction methods for simulated benchmark networks as well as 10 biological protein interaction networks (PINs). A particular focus of our analysis is placed on the biological meaning of the predicted modules by utilizing the Gene Ontology (GO) database as gold standard for the definition of biological processes. Furthermore, we investigate the robustness of the results by perturbing the PINs simulating in this way our incomplete knowledge of protein networks. CONCLUSIONS: Overall, our study reveals that there is a large heterogeneity among the different module prediction algorithms if one zooms-in the biological level of biological processes in the form of GO terms and all methods are severely affected by a slight perturbation of the networks. However, we also find pathways that are enriched in multiple modules, which could provide important information about the hierarchical organization of the system. publishedVersion

Background Horizontal gene transfer (HGT) is a movement of genetic information occurring outside of normal mating activities. It is especially common between prokaryotic endosymbionts and their protozoan, insect, and nematode hosts. Although beta carbonic anhydrase (β-CA) plays a crucial role in metabolic functions of many living organisms, the origin of β-CA genes in eukaryotic species remains unclear. Methods This study was conducted using phylogenetics, prediction of subcellular localization, and identification of β-CA, transposase, integrase, and resolvase genes on the MGEs of bacteria. We also structurally analyzed β-CAs from protozoans, insects, and nematodes and their putative prokaryotic common ancestors, by homology modelling. Results Our investigations of a number of target genomes revealed that genes coding for transposase, integrase, resolvase, and conjugation complex proteins have been integrated with β-CA gene sequences on mobile genetic elements (MGEs) which have facilitated the mobility of β-CA genes from bacteria to protozoan, insect, and nematode species. The prokaryotic origin of protozoan, insect, and nematode β-CA enzymes is supported by phylogenetic analyses, prediction of subcellular localization, and homology modelling. Conclusion MGEs form a complete set of enzymatic tools, which are relevant to HGT of β-CA gene sequences from prokaryotes to protozoans, insects, and nematodes. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1415-7) contains supplementary material, which is available to authorized users.

The Drosophila mutant tko(25t) exhibits a deficiency ofmitochondrial protein synthesis, leading to a global insufficiency of respiration and oxidative phosphorylation. This entrains an organismal phenotype of developmental delay and sensitivity to seizures induced bymechanical stress. We found that the mutant phenotype is exacerbated in a dose-dependent fashion by high dietary sugar levels. tko(25t) larvae were found to exhibit severe metabolic abnormalities that were further accentuated by high-sugar diet. These include elevated pyruvate and lactate, decreased ATP and NADPH. Dietary pyruvate or lactate supplementation phenocopied the effects of high sugar. Based on tissue-specific rescue, the crucial tissue in which this metabolic crisis initiates is the gut. It is accompanied by down-regulation of the apparatus of cytosolic protein synthesis and secretion at both the RNA and post-translational levels, including a novel regulation of S6 kinase at the protein level., Academy of Finland; Tampere University Hospital Medical Research Fund; Sigrid Juselius Foundation

Human adipose tissue is a good source of mesenchymal (adipose) stem cells which can be used in bone tissue engineering both in studying differentiation and for treating patients. Bone morphogenetic protein 2 (BMP-2) is often used in culture medium to differentiate mesenchymal stem cells in vitro and in vivo, even thiough its efficacy has been questioned. The mechanical properties of the substrate the cells grow on have been found important to the outcome of differentiation. The aim of this study was to investigate the effects of an electrospun BMP-2 releasing polymer scaffold which enables controlled and targeted drug-release on the adipogenic and osteogenic differentiation of human adipose stem cells in vitro. Human adipose stem cells (hASCs) from four different donors were cultured on the electrospun polymer material, loaded with BMP-2. The properties of BMP-2 scaffolds and control scaffolds (CTRL) were compared in basal medium (BM) or osteogenic medium (OM) with or without added BMP-2. The osteogenicity of the materials was evaluated using quantitative alkaline phosphatase activity assay (qALP), as well as Alizarin red staining and gene and protein expression of marker genes using quantitative real time polymerase chain reaction (qPCR) and Western blotting (WB). The proliferation and morphology of hASCs were studied using total DNA quantification and immunofluorescence respectively. The adipogenicity of the material was evaluated using qPCR as well as Oil Red O staining and fluorescence imaging. It was found that hASCs seemed to grow in aggregates on the BMP-2 scaffolds when compared to CTRL scaffolds. The BMP-2 released from scaffolds did not significantly increase the cells' ALP activity and mineralization of ECM. Instead the BMP-2 releasing scaffolds and CTRL scaffolds seemed to bolster adipogenic differentiation based on the shape of the cells and increased intracellular fat as well as the qPCR results. However, no statistically significant differences were observed between cells grown in different conditions when the results from different cell lines were pooled. It seemed however from the morphological data that the cells grown on BMP-2 releasing scaffolds were more similar to adipocytes than osteoblasts. It was determined that BMP-2 releasing scaffold material is more beneficial for adipogenic than osteogenic differentiation. This is probably a result of the material not being stiff enough and being too hydrophobic. It is likely that the amount of loaded BMP-2 is too low, as it was significantly lower than the concentrations regularly used in differentiation protocols.

Periytyvä eturauhassyöpäalttius Suomessa - tunnettuja ja uusia kandidaattigeenejä Noin joka neljäs ihminen sairastuu elämänsä aikana syöpään. Suomessa miesten yleisin syöpäsairaus on eturauhassyöpä, joka todetaan vuosittain noin 5000 miehellä. Suuri osa eturauhassyöpäalttiudesta selittyy perinnöllisillä tekijöillä. Eturauhassyöpä on kuitenkin geneettisesti huomattavan heterogeeninen sairaus. Toistaiseksi tunnetaan vain muutamia varsinaisia alttiusgeenejä, joiden virheet liittyvät korkeaan sairastumisriskiin. Näiden harvinaisten muutosten lisäksi on löydetty useita yleisempiä, matalan riskin geenivirheitä. Suomalaisissa korkean riskin perheissä tunnetut geenivirheet selittävät kuitenkin vain pienen osan eturauhassyöpätapauksista, eikä sairastumisriskissä olevia perheenjäseniä ole tällä hetkellä mahdollista luotettavasti tunnistaa. Tutkimuksen tavoitteena oli saada uutta tietoa eturauhassyövälle altistavista perinnöllisistä tekijöistä suomalaisväestössä. Tutkimuksessa keskityttiin etsimään geenivirheitä erityisesti kromosomialueilta 2q37 ja 17q11.2-q22, joiden on jo aiemmin todettu liittyvän kohonneeseen eturauhassyöpäalttiuteen. Alueiden hienokartoitus perustui DNA:n ja RNA:n syväsekvensointiin. Havaittujen, mahdollisesti haitallisiksi arvioitujen geenivirheiden yhteyttä eturauhassyöpään selvitettiin tutkimalla näiden muutosten esiintyvyyttä laajoissa potilas- ja verrokkiaineistoissa mm. TaqMan-kemiaa ja Sequenom MassARRAY-teknologiaa hyödyntäen. Lisäksi eturauhassyöpäpotilailta etsittiin geenien kopioluvun muutoksia koko perimän alueelta. Kopiolukuanalyysissä käytettiin sirupohjaista vertailevaa genomista hybridisaatiomenetelmää (ns. array-CGH). Kromosomialueelle 2q37 on aiemmissa tutkimuksissa paikannettu kaksi kandidaattigeeniä, HDAC4 ja ANO7, ja alueelle 17q11.2-q22 samoin kaksi kandidaattigeeniä, HOXB13 ja ZNF652. Näistä erityisesti HOXB13-geenin p.G84E-muutoksen on havaittu liittyvän periytyvään eturauhassyöpäalttiuteen. Tässä tutkimuksessa selvitettiin kyseisen p.G84E-muutoksen yleisyyttä Suomessa. Muutos oli osoitettavissa 8.4 %:lla potilaista, jotka kuuluivat korkean riskin eturauhassyöpäperheisiin, sekä 3.6 %:lla satunnaista eturauhassyöpää sairastavista potilaista, kun taas verrokeista vain 1.0 % todettiin muutoksen kantajiksi. HOXB13-geenin p.G84E-muutos on siis toistaiseksi yleisin eturauhassyövälle altistava geenivirhe Suomessa. ZNF652-geenistä tunnistettiin kaksi uutta, haitalliseksi ennustettua muutosta, jotka liittyivät kohonneeseen eturauhassyöpäalttiuteen sekä perheaineistossa että satunnaista eturauhassyöpää sairastavilla potilailla. Lisäksi HDAC4-geenistä löydettiin uusi, harvinainen periytyvään eturauhassyöpäalttiuteen liittyvä muutos ja ANO7-geenistä kahdeksan mahdollisesti haitallista muutosta, joskin näiden geenivirheiden kliinisen merkityksen arvioiminen edellyttää vielä lisäselvityksiä. Tutkimuksessa löydettiin myös uusia mahdollisia eturauhassyövän kandidaattigeenejä, esimerkiksi HOXB3 ja EFCAB13 alueelta 17q21.3. Kopiolukuanalyysissä tunnistettiin neljä kopiolukumuutosta, jotka olivat rikastuneet korkean riskin eturauhassyöpäperheisiin kontrolleihin verrattuna. Näistä kuitenkin vain yhden muutoksen, kromosomialueella 3p11.1 sijaitsevan EPHA3-geenin deleetion voitiin osoittaa liittyvän kohonneeseen eturauhassyöpäalttiuteen. EPHA3-deleetio oli noin kaksi kertaa yleisempi perinnöllistä eturauhassyöpää sairastavilla potilailla (11.6 %) kuin verrokeilla (6.1 %). Väitöskirjatyössä sekä tunnetuista että uusista kandidaattigeeneistä tunnistettiin useita mahdollisesti haitallisia, kohonneeseen eturauhassyöpäalttiuteen liittyviä geenivirheitä. Erityisesti vahvistui HOXB13-geenin keskeinen osuus perinnöllisen eturauhassyövän riskitekijänä suomalaisväestössä. Uusien löydösten kliininen merkitys tulee vielä varmentaa jatkotutkimuksissa. On kuitenkin mahdollista, että väitöskirjatyössä saatua tietoa voidaan tulevaisuudessa hyödyntää esimerkiksi eturauhassyövän seulonnassa, diagnostiikassa ja hoitomuotojen valinnassa. Prostate cancer is the most frequently diagnosed male malignancy in industrialized Western countries. In Finland, approximately 5000 new cases emerge each year, which is equal to more than one-third of all male cancers. Following lung cancer, prostate cancer is the second most common cause of cancer death in Finland (Finnish Cancer Registry). The burden not only to the patients and their families but also to the national health care system is, therefore, significant. While the etiology of prostate cancer is not yet fully understood, a few specific risk factors have been recognized, including advanced age, ethnic origin and positive family history. In addition to genetic predisposition, environmental factors, diet and hormones likely modify the disease risk. A majority of prostate cancer cases are sporadic, but approximately 5-10% of cases can be classified as hereditary cancers, which result from inherited germline variants predisposing their carriers to the disease. In prostate cancer, genetic factors play an essential role and have been estimated to explain as much as 58% of the cancer risk. Unlike other common cancers, such as breast or colorectal cancer, prostate cancer is genetically very heterogeneous, which has made the identification of genetic susceptibility factors extremely challenging. Only a few high-risk candidate genes and variants have been found, and the risk effect of the more common variants is typically low. As a consequence, in many Finnish prostate cancer families, the underlying causative gene defects remain unknown. The aim of this thesis study was to identify novel genetic factors contributing to prostate cancer predisposition in Finland. The search focused especially on two chromosomal regions, 2q37 and 17q11-q22, which have repeatedly shown a strong linkage with increased prostate cancer risk in various populations. These two loci were characterized by sequencing samples representing both familial and unselected prostate cancer patients, as well as unaffected controls. In addition, a genome-wide copy number variation analysis was performed on familial prostate cancer patients to locate genomic alterations associated with increased risk of hereditary prostate cancer. Additional evidence for a role in prostate carcinogenesis was obtained for several previously reported candidate genes, including HOXB13 and ZNF652 at 17q21.3 and HDAC4 and ANO7 at 2q37. In particular, the importance of the HOXB13 variant p.G84E was established in this study. This variant was observed at a frequency of 8.4% among familial prostate cancer patients (vs. 1.0% in controls), making it the most common prostate-cancer-associated risk variant detected in Finland thus far. This variant was also associated with earlier age at disease onset (

Eturauhas- ja rakkosyövän molekyylimekanismit Syöpä on monimutkainen sairaus, jonka aiheuttavat normaaliin soluun kertyvät geneettiset muutokset. Tällaiset geneettiset muutokset häiritsevät normaalien solujen homeostaasia ja johtavat hallitsemattomaan solujen lisääntymiseen. Näiden muutosten tunteminen on tärkeää, jotta voitaisiin kehittää entistä tehokkaampia syövän hoitomuotoja ja diagnostisia menetelmiä tappavan tautimuodon tunnistamiseksi. Virtsarakon syöpä on yleisin virtsateiden maligniteetti. Useimmat virtsarakon syövät syntyvät välimuotoisesta epiteelistä (uroteliaaliset karsinoomat). Alle 5 % virtsarakon syöpätapauksista on levyepiteeliperäisiä. Virtsarakon syöpä on heterogeeninen sairaus, jolle ovat ominaisia erilaiset geneettiset muutokset, jotka johtavat eri polkuja syövän kehittymiseen ja etenemiseen. Monet näistä geneettisistä muutoksista vaikuttavat geenien kopiolukumäärään johtaen onkogeenien monistumaan ja kasvurajoitegeenien häviämään. Tässä tutkimuksessa käytettiin sirupohjaista, vertailevaa genomista hybridisaatiomenetelmää (aCGH) geenikopiolukumäärän analysoimiseksi kliinisissä rakkosyöpänäytteissä sekä solulinjoissa. Yhdeksi uudeksi monistuma-alueeksi tunnistettiin kromosomialue 1p21-22. Työssä kartoitettiin ko. aluetta tarkemmin ja osoitettiin, että ns. minimaalinen monistuma-alue käsitti yhden miljoonan emäsparin DNA-jakson sisältäen yhteensä 11 tunnettua geeniä. Korkein monistuma-aste löytyi SCaBER-levyepiteelisyöpäsolulinjasta. Näistä neljä geeniä, TMED5, DR1, RPL5 ja EVI5, yli-ilmeni SCaBER-solulinjassa, DR1 kaikkein eniten. Julkisten tietokantojen perusteella DR1 yli-ilmenee myös kliinisissä näytteissä, jotka edustavat yleistä virtsarakkosyövän histologista tyyppiä. Eturauhassyöpä on miesten toiseksi yleisin diagnosoitu syöpä maailmassa ja yleisin kehittyneissä maissa. Taudin histologisen ja kliinisen käyttäytymisen monimuotoisuuden taustalla olevia mekanismeja tunnetaan huonosti. Eturauhassyövän molekyylitason mekanismien tunteminen mahdollistaisi uusien taudin aggressiivisuutta kuvastavien biomarkkerien löytämisen. Viime aikoina on yhä enemmän kiinnitetty huomiota mikroRNA:iden (miRNA) merkitykseen syövän kehittymisessä. Useat tutkimukset ovat osoittaneet, että miRNA-ilmentymistasot ovat muuntuneita eturauhassyövässä. Onkin mahdollista, että niitä voitaisiin käyttää ennusteellisina biomarkkereina. Kuten muissa syövissä, on epäselvää, koostuuko eturauhassyöpä eri solupopulaatioista, joilla on erilainen jakaantumiskyky, kuten syövän kantasolumallit ennustavat. Useissa tutkimuksissa on käytetty virtaussytometriaa eturauhasen kantasolujen eristämiseen. Tässä tutkimuksessa tehtiin genominlaajuinen miRNA-ilmentymisen analyysi potilaista peräisin olevista kantasolun kaltaisista soluista (SC), välivaiheen soluista ja sitoutuneista (CB) soluista. Jokainen solualapopulaatio poikkesi toisistaan miRNA-ilmentymisprofiililtaan riippumatta siitä, oliko kyseessä syöpänäyte vai ei. MiR-548c-3p:n havaittiin yli-ilmentyvän noin viisinkertaisesti SC-soluissa verrattuna CB-soluihin. Toiminnallisissa tutkimuksissa miR-548c-3p:n yli-ilmentyminen CB-soluissa vähensi erilaistumista kohti kantasoluilmiasua. MiR-548c-3p oli myös merkittävästi yli-ilmentynyt kastraatioresistentistä eturauhassyövästä (CRPC) eristetyissä soluissa verrattuna eturauhasen hyvänlaatuisesta liikakasvusta (BPH) eristettyihin epiteelisoluihin. Tämä viitaa siihen, että miR-548c-3p voisi olla eturauhassyövän aggressiivisuuden biomarkkeri. Tunnistaaksemme uusia, eri tavoin ilmentyviä miRNA:ita, syväsekvensoimme joukon kliinisiä eturauhassyöpänäytteitä. MiR-1247-5p, miR-1249, miR-1269a, miR1271-5p, miR-1290, miR-1291 ja miR-1299 ilmentyivät eri lailla syövässä ja normaalissa eturauhasessa. Niinpä näitä tutkittiin laajemmassa materiaalissa qRT-PCR-menetelmällä. MiR-1247-5p ilmentyi merkittävästi enemmän CRPC-näytteissä verrattuna ei-maligniin eturauhaseen. Ennusteohjelmien perusteella miR-1247-5p:n yksi kohdegeeni voisi olla MYCBP2 (myc:iä sitova proteiini 2). miR-1247-5p:n yli-ilmentyminen PC-3- ja LNCaP-syöpäsolulinjoissa johti MYCBP2:n ilmentymisen laskuun sekä mRNA- että proteiinitasolla. Lusiferaasireportterikoe vahvisti, että MYCBP2 on miR-1247-5p:n kohde. Useissa uuden sukupolven sekvensointitutkimuksissa on syövissä löydetty uusia ryhmiä ei-koodaavia RNA:ita. Eturauhassyöpäsolulinjoissa miRNA:iden jälkeen toiseksi yleisin ryhmä ei-koodaavia RNA:ita ovat tRNA:ista peräisin olevat fragmentit (tRFs). Niiden ominaisuudet, runsas ilmentyminen ja tarkka sekvenssi viittaavat siihen, että nämä molekyylit eivät ole satunnaisia tuotteita tRNA:n hajoamisesta. tRF:ien tarkka rooli on kuitenkin edelleen epäselvä. Tässä tutkimuksessa tRF:ien ilmentymistä analysoitiin potilaan kudosnäytteistä. Tutkimuksessa tunnistettiin yhteensä 598 erilaista tRF:ää, jotka näyttivät ilmentyvän eri lailla syövässä kuin normaalissa kudoksessa. Suurin osa tunnistetuista tRF:ista on peräisin kypsän sytosolisen tRNA:n 5’-ja 3’-päistä, mutta myös muita fragmentteja löytyi. 5’-päästä peräisin olevat tRF:t olivat eniten yli-ilmentyneitä ja 3’-päästä peräsin olevat ali-ilmentyneitä syövässä. Kolmen tRF:n ilmentymiserot varmennettiin qRT-PCR:llä. Normalisoitu tRNALys:stä ja tRNAPhe:stä peräisin olevien tRF-315:n ja tRF-544:n ilmentyvyyssuhde ennusti hyvin taudin etenemistä. Yhteenvetona tutkimuksessa löydettiin uusi monistuma-alue, jossa saattaa sijaita rakkosyövän kehityksen kannalta tärkeitä onkogeenejä. Lisäksi tunnistettiin useita eturauhassyövässä poikkeavasti ilmentyviä ns. ei-koodaavia RNA:ita. Nämä saattavat olla mekanistisesti tärkeitä eturauhassyövän kehityksen kannalta ja mahdollisia syövän aggressiivisuuden markkereita. Cancer is a complex disease, caused by accumulation of genetic alterations in normal cells. The consequence of these genetic alterations is the disruption of normal cell number homeostasis and uncontrolled cell proliferation. Understanding the molecular mechanisms behind tumorigenesis is essential in order to identify aggressive and possibly lethal form of the disease as well as to plan effective cancer therapeutic strategies. Urinary bladder cancer is the most common malignancy of the urinary tract. Most of the tumors arise from the epithelium lining the inside of the urinary bladder (urothelial carcinomas). Squamous cell carcinomas represent less than 5% of bladder cancer cases. Bladder cancer is a heterogeneous disease, characterized by different genetic alterations, leading to diverse pathways of cancer development and progression. Many of these genetic alterations consist of region-specific gains and losses of DNA copy number. Regions of DNA copy number gain or amplification commonly harbor oncogenes, whereas deleted regions harbor tumor suppressor genes. In this study, array-comparative genomic hybridization (aCGH) was performed in bladder cancer clinical samples and cell line models, revealing a common amplification at chromosomal region 1p21-22. The minimal region of the amplification was mapped to a region of about one Mb in size, containing a total of 11 known genes. The highest amplification was found in SCaBER squamous cell carcinoma cell line. Four genes, TMED5, DR1, RPL5 and EVI5, showed significant overexpression in the SCaBER cell line compared to all the other samples tested. DR1 was found to be the most significantly overexpressed in the SCaBER cell line. According to published clinical sample cohorts, DR1 is overexpressed also in superficial and infiltrating bladder cancers. Prostate cancer (PC) is the second most commonly diagnosed cancer among males worldwide and the most frequently diagnosed malignancy in developed countries. The heterogeneity of histologic and clinical features of PC is well known, but the mechanisms underlying the heterogeneity are not understood. Deeper understanding of the molecular mechanisms of PC tumorigenesis is needed to discover more specific biomarkers of aggressive form of the disease. Recently, there has been increasing attention on the role of microRNAs (miRNAs) in cancer development. Several expression-profiling studies have provided evidence of aberrant expression of miRNAs in prostate cancer and have highlighted the potential use of specific miRNA expression signatures as prognostic or predictive markers. Similarly to other solid tumors, it is at present unclear whether prostate cancer is organized hierarchically into populations of cells with different proliferative potentials, as cancer stem cell (CSC) model suggests. Several studies have used flow cytometry-based approaches to isolate putative prostate stem cells. Here, genome-wide miRNA expression analysis was performed on patient-derived, stem-like cells (SC), transit-amplifying cells and committed basal (CB) cells, enriched from briefly cultured primary prostate epithelial cells. Each cell subpopulation showed a distinct miRNA expression profile, regardless of its pathologic status. MiR-548c-3p was found to be overexpressed approximately fivefold in SCs, compared with CBs. Functional studies of miR-548c-3p overexpression in CBs showed increased dedifferentiation to a more stem-like phenotype. MiR-548c-3p was also found to be significantly upregulated in CRPC-derived epithelial cells, compared with BPH-derived epithelial cells, suggesting that miR-548c-3p is a functional biomarker for PC aggressiveness. In order to identify new, differentially expressed miRNAs, the expression data obtained from recent deep-sequencing experiments on pools of clinical specimens were analyzed. MiR-1247-5p, miR-1249, miR-1269a, miR1271-5p, miR-1290, miR-1291 and miR-1299 showed differential expression in malignant samples, compared to benign ones and were selected for validation by qRT-PCR. Significant up-regulation of miR-1247-5p was found in castration-resistant prostate cancer (CRPC), compared to non-malignant prostate. The expression of miR-1247-5p was subsequently studied in PC cell lines where an up-regulation of miR-1247-5p was observed in the androgen-independent PC-3 model. According to on-line target prediction tools MYCBP2 (myc-binding protein 2) is a high-scoring potential target of miR-1247-5p. Down-regulation of MYCBP2 at both mRNA and protein levels was demonstrated by overexpression of miR-1247-5p in PC-3 and LNCaP models. Next, MYCBP2 was confirmed as a target of miR-1247-5p by using luciferase reporter assay. Several high-throughput sequencing studies in human cancers have recently led to the discovery of additional groups of non-coding RNAs. Next to miRNAs, the most abundant non-coding RNAs in prostate cancer cell lines were found to be fragments derived from tRNAs that are termed tRNA-derived RNA fragments (tRFs). The characteristic and abundant expression of the fragments, as well as their precise sequence, indicate that these molecules are not random products of tRNA degradation. However, the precise role of tRFs is still unclear. In this study, the expression of tRFs was analyzed in normal adjacent prostate and different stages of PC by RNA-sequencing. A total of 598 unique tRFs were identified, many of which appear to be deregulated in cancer samples, when compared to controls. Most of the identified tRFs are derived from the 5’ and 3’ end of mature cytosolic tRNAs, but tRFs produced from pre-tRNA trailers and leaders were also found, as well as tRFs from mitochondrial tRNAs. The 5’-derived tRFs comprised the most abundant class of tRFs and represented the major class among upregulated tRFs, while 3’-derived tRFs types were dominant among downregulated tRFs in PC. The expression of three tRFs (tRF-544, tRF-315 and tRF-562) was validated in PC using qRT-PCR. Interestingly, the normalized expression ratio of tRF-315 and tRF-544, derived from tRNALys and tRNAPhe respectively, emerged as a good indicator of progression-free survival and as a candidate prognostic marker. In conclusion, a novel amplification, which may harbour important oncogenes, was identified in bladder cancer. In addition, several differentially expressed non-coding RNAs were discovered in prostate cancer. They may be important drivers of prostate tumorigenesis and putative biomarkers of aggressive form of the disease.

Verkkokalvon pigmenttiepiteeli (retinal pigment epithelium, RPE) on tiivis yksikerroksinen epiteelisolukerros, joka sijaitsee silmän takaosassa verkkokalvon ja suonikalvon välissä. RPE:llä ja sen alapuolella sijaitsevalla tukirakenteella, Bruchin kalvolla, on merkittävä tehtävä ylläpitää verkkokalvon toimintoja. Peruuttamattomat vauriot RPE:n toiminnassa johtavat asteittaiseen näkökyvyn heikkenemiseen ja lopulta sokeutumiseen verkkokalvon rappeumasairauksissa kuten verkkokalvon ikärappeumassa. Tällä hetkellä verkkokalvon ikärappeuman kuivaan muotoon ei ole olemassa parantavaa hoitokeinoa. Solusiirtoa, jossa vaurioituneet RPE-solut korvataan terveillä toiminnallisilla soluilla, pidetään mahdollisena tulevaisuuden hoitokeinona verkkokalvon rappeumasairauksiin. Ihmisen alkion kantasoluista erilaistetut RPE-solut (hESC-RPE) ovat osoittautuneet lupaavaksi solulähteeksi näihin solusiirtoihin eläinkokeissa ja ovat parhaillaan tutkimuksen kohteena ensimmäisissä kliinisissä hoitokokeissa. RPE-solujen siirtämisen tiiviinä yksisolukerroksena tukirakenteen päällä on todettu auttavan solusiirteen selviytymistä ja sopeutumista vaurioituneelle verkkokalvon alueella. Useita biomateriaali-kasvualustoja on tutkittu mahdollisina tukirakenteina RPE-soluille, mutta nämä aiemmin ehdotetut rakenteet harvoin täyttävät tukirakenteelle asetettuja vaatimuksia. Lisäksi, nämä kasvualustat eivät muistuta koostumukseltaan RPE-solujen luontaista ympäristöä, Bruchin kalvoa, mikä saattaa vaikuttaa solujen tuotantoon laboratoriossa sekä solujen toimintakykyyn solusiirroissa. Useat tutkituista kasvualuista sisältävät myös ihmiselle vieraita aineita, ja kasvualustojen testaukset tehdään pääosin kaupallisilla kuolemattomiksi tehdyillä solulinjoilla kliiniseen käyttöön soveltumattomissa olosuhteissa. Tämän väitöskirjatutkimuksen tavoitteena oli kehittää biomateriaalipohjaisia kasvualustoja hESC-RPE soluille. Tutkimuksessa keskityttiin kehittämään ihmisperäisiä ja synteettisiä kasvualustoja, jotka muistuttavat RPE-solujen luontaista ympäristöä silmässä ja tukevat hESC-RPE solujen kasvua seerumittomissa viljelyolosuhteissa. Tavoitetta lähestyttiin kolmella eri tavalla. Ensiksi tutkittiin useita ihmisen Bruchin kalvon luontaisia proteiinipinnoitteita sekä kaupallisia soluviljelypinnoitteita hESC-RPE solujen kasvatusalustana solujen erilaistuksessa sekä kypsymisessä. Siitä huolimatta, että hESC-RPE solujen alkuvaiheen erilaistuksessa ei löydetty eroja eri proteiinipinnoitteiden välillä, solujen kypsyessä proteiinipinnoitteiden huomattiin vaikuttavan merkittävästi solujen rakenteeseen, toimintaan sekä soluväliaineproteiinien tuotantoon. Seuraavaksi Bruchin kalvon rakennetta ja koostumusta jäljittelevä kasvualusta tehtiin ihmisperäisistä kollageeneista Langmuir-Schaefer (LS) tekniikalla. Valmistuksessa käytettiin vain bioyhteensopivia ainesosia. LS-kalvojen kerroksittainen ja kuitumainen rakenne muistutti Bruchin kalvon RPE:n läheisen kerroksen rakennetta. Lisäksi LS-kalvot tukivat paremmin hESC-RPE solujen kypsymistä ja toiminnallisuutta seerumittomissa viljelyolosuhteissa pelkkään proteiinipinnoitteiseen verrattuna. Viimeiseksi tässä väitöskirjatyössä valmistettiin biohajoava siirtomateriaali hESC-RPE soluille synteettisestä polymeeristä sähkökehruutekniikalla. Valmistettu siirtomateriaali pintakäsiteltiin ja päällystettiin kollageeni-proteiinipinnoituksella hESC-RPE solujen kiinnittymisen ja kypsymisen edistämiseksi. Nämä biohajoavat siirtomateriaalit koostuivat satunnaisesti järjestäytyneistä säikeistä ja läpäisivät pienimolekyylistä yhdistettä. Materiaalien säiemäinen ja huokoinen rakenne muistutti Bruchin kalvon rakennetta. Pintamuokatut ja proteiini pinnoitetut biohajoavat siirtomateriaalit tukivat toiminnallisen hESC-RPE:n muodostumista seerumittomassa viljely-ympäristössä, minkä vuoksi nämä ovat lupaavia tukirakenteita verkkokalvon kudosteknologisiin sovelluksiin. Tämä väitöskirjatyö on tuonut merkittävästi lisätietoa hESC-RPE solujen vuorovaikutuksesta biomateriaali-kasvualustoilla. Tässä työssä esitetään useita lupaavia tekniikoita biomimeettisen ympäristön valmistukseen hESC-RPE solujen in vitro-tuotantoa varten. Lisäksi, tässä väitöskirjassa esiteltyjen töiden tuloksia voidaan hyödyntää biomateriaalirakenteiden suunnittelussa ja soluterapia sovelluksissa verkkokalvon kudosteknologiassa. The retinal pigment epithelium (RPE) is a monolayer of polarized and pigmented cells that resides between the neural retina and the choroid. Together with the underlying Bruch’s membrane, the RPE has a pivotal role in the proper function, homeostasis and survival of the adjacent retinal photoreceptors. Irreversible damage and loss of the RPE is a fundamental factor in the development of degenerative retinal diseases such as age-related macular degeneration (AMD). In AMD, degeneration of the RPE and photoreceptors in the macular area of central vision lead to a gradual loss of visual acuity and eventually blindness. Currently, there is no treatment for the dry form of AMD. However, replacement of the dysfunctional and damaged RPE with a population of healthy cells is considered as a potential therapeutic strategy for AMD and related diseases. Cell transplants of human embryonic stem cell derived RPE (hESC-RPE) cells have shown potential for these cell therapies in animal models and are currently investigated in clinical setting. An approach where cells are delivered to the subretinal space as a sheet on a biomaterial substrate, has shown improved cell survival upon transplantation. Several biomaterial substrates have been investigated as prospective carriers for RPE, but they often fail to fulfill the requirements set for subretinal implantation. Importantly, these substrates do not mimic the composition and structure of Bruch’s membrane, the natural environment of the RPE, which might affect their capacity to differentiate in vitro and subsequent performance in cell transplantation. Moreover, the majority of currently used biomaterial substrates contain animal-derived products. In addition, testing of these substrates is usually carried out with immortalized cells lines under culture conditions not suitable for clinical production. The work presented in this dissertation aimed at finding and developing biomaterial substrates for hESC-RPE cells that bear a resemblance to the native microenvironment of the RPE. A special focus was paid to exploring biomaterial substrates of human or synthetic origin that would support the formation of mature hESC-RPE in serum-free culture conditions. Consequently, three approaches were developed to fabricate a biomimetic environment for hESC-RPE in vitro. To begin with, the role of several human extracellular matrix (ECM) proteins found in the Bruch’s membrane and commercial basement membrane matrices was evaluated in adherent hESC-RPE cell differentiation and maturation cultures under serum-free conditions. Although there were no significant differences between the studied protein coatings in early-stage differentiation, the protein coatings had a major effect on the structure, function and basal lamina production of hESC-RPE cells upon further maturation of the cultures. Thereafter, a biomimetic microenvironment simulating the layered structure of the Bruch’s membrane was fabricated with Langmuir-Schaefer technology from human derived collagens for the production of hESC-RPE cells. Only biocompatible components were involved in the manufacturing process. A thorough characterization of the substrate demonstrated that the fabricated collagen films had a layered structure with oriented fibers resembling the architecture of the two uppermost layers of Bruch’s membrane. Furthermore, the fabricated collagen films were superior in supporting hESC-RPE cell maturation and functionality compared to collagen IV dip-coated controls in serum-free culture conditions. Finally, biodegradable biomaterial substrates were fabricated from synthetic polymer with an electrospinning method. The substrates were surface modified and coated with an additional collagen layer to increase hESC-RPE cell adhesion and maturation. The fabricated substrates consisted of unaligned fibers and were permeable for small molecular weight substance. Thus, these substrates bore a resemblance to the fibrous structure of Bruch’s membrane. Moreover, these biodegradable surface modified biomaterial substrates supported the formation of functional hESC-RPE in serum-free culture medium, therefore demonstrating the potential of biomaterial substrates for subretinal transplantation. In conclusion, this dissertation has increased understanding of hESC-RPE cell interaction and performance on biomaterial substrates. Moreover, the results of this dissertation offer a range of methods to provide a biomimetic environment for the in vitro production of hESC-RPE cells without the use of animal-derived substrates and serum. These results can be exploited in future applications and biomaterial design for the retinal tissue engineering field.

Eturauhassyövän geneettiset alttiustekijät kromosomialueella 11q13.5 Eturauhassyöpä on Suomessa kuten myös useissa muissa länsimaissa miesten yleisin syöpätyyppi ja toiseksi suurin syöpäkuolleisuuden aiheuttaja. Eturauhassyöpä on monitekijäinen sairaus, jonka syntymekanismeja ei vielä täysin tunneta. Riskitekijöinä ovat ikä, sukuhistoria ja etninen tausta. Suuri osa kliinisesti havaittavista eturauhaskasvaimista on hyvänlaatuisia ja hidaskasvuisia, mutta osa kehittyy pahanlaatuisiksi kasvaimiksi, jotka leviävät etäpesäkkeinä ja aiheuttavat syöpäkuolemia. Uusia biologisia merkkiaineita eli biomarkkereita tarvitaan erottamaan eturauhassyöpään sairastuvista miehistä ne, joilla on kohonnut riski sairastua nopeasti etenevään aggressiiviseen syöpätyyppiin, mikä mahdollistaisi tehokkaamman yksilöidyn hoitosuunnitelman tekemisen. Perintötekijät vaikuttavat eturauhassyövän syntyyn. Eturauhassyöpään ei ole yhdistetty korkean riskin perintötekijöitä samaan tapaan kuin esimerkiksi rintasyövässä, ja syövän on havaittu olevan geneettisesti hyvin monimuotoinen, sillä useita eri kromosomialueita on liitetty kohonneeseen syöpäriskiin. Yksi näistä kromosomialueista on 11q13-14, joka on yhdistetty syöpään suomalaisissa eturauhassyöpäsuvuissa ja jonka on havaittu altistavan etenkin aggressiiviselle syöpätyypille. Alueelta ei kuitenkaan ole vielä tunnistettu syöpäkasvaimen kehittymiselle altistavaa perimän muutosta. Alueella sijaitseva EMSY-geeni on yhteydessä rinta- ja munasarjasyöpään ja on tämän vuoksi mielenkiintoinen tutkimuskohde. Väitöskirjatutkimuksen tavoitteena oli tunnistaa alueella 11q13.5 sijaitsevia eturauhassyövälle altistavia muutoksia ja tutkia niiden toimintaa. Aineistona käytettiin potilasnäytteitä ja -tietoja suomalaisilta eturauhassyöpäpotilailta sekä näytteitä mieskontrolleilta. Muutosten yhteys syöpäalttiuteen selvitettiin tapaus-verrokkitutkimuksella. Syöpäriskiä lisäävien muutosten vaikutus geenien ilmenemisen säätelyyn tutkittiin syöpäkasvaimissa, veressä ja lymfoblastisolulinjoissa. Tutkimuksessa havaittiin yhteensä yhdeksän syöpäalttiuteen liittyvää perimän muutosta. EMSY-geenissä havaittiin kolme intronista eturauhassyöpäriskiä nostavaa muutosta, joista yksi, populaatiossa harvinaisesti esiintyvä muutos altisti etenkin aggressiiviselle syövälle suomalaisessa populaatiossa. Muutos havaittiin myös kolmessa eturauhassyöpäsuvussa miehillä, jotka sairastivat aggressiivista eturauhassyöpää. Yhteensä kuusi, geenien välisellä alueella sijaitsevaa, populaatiossa yleisesti esiintyvää muutosta yhdistyi kohonneeseen syöpäkuoleman riskiin. Muutokset olivat matalan riskin muutoksia. Yksittäisten muutosten lisäksi alueella tunnistettiin myös eturauhassyöpäriskiä lisääviä alleeliyhdistelmiä eli haplotyyppejä. Syöpäkuoleman alttiuteen yhdistetyt muutokset sijaitsivat geenien säätelyelementtien alueella, ja niiden havaittiin vaikuttavan lipidien biosynteesissä toimivan DGAT2-geenin ilmentymiseen eturauhassyöpäkasvaimissa ja AP001189.4-geenin ilmentymiseen veressä. AP001189.4 on RNA:ta koodaava geeni, jonka toimintaa ei vielä tiedetä. Näiden tulosten perusteella muutokset säätelevät geenien toimintaa kudosspesifisesti. Tässä tutkimuksessa saadut tulokset viittaavat siihen, että kromosomialueella 11q13.5 sijaitsee pahanlaatuiselle eturauhassyövälle altistavia perimän muutoksia, joista osa vaikuttaa geenien toiminnan säätelyyn eturauhassyöpäkasvaimissa ja veressä. Lisätutkimuksia tarvitaan selvittämään miten tunnistetut kohdegeenit altistavat syöpäkasvaimen kehittymiselle. Lisäksi tutkimusta tarvitaan määrittämään havaittujen muutosten vaikutus syöpäalttiuteen muissa populaatioissa. Tutkimuksessa tunnistettuja pahanlaatuiselle syövälle altistavia muutoksia voidaan mahdollisesti käyttää kehittämään biomarkkeripaneeli, joka tunnistaa kohonneen riskin sairastua pahanlaatuiseen syöpään. Prostate cancer is a significant public health concern worldwide. It is the most common cancer affecting men in Finland and in other Western countries. The increased incidence of prostate cancer can be at least partly explained by the development of prostate-specific antigen testing, which results in a high rate of overdiagnosis and overtreatment. Therefore, improved biomarkers are needed to identify men at high risk of aggressive prostate cancer among the majority of patients for tailored monitoring and treatment of this disease. The main risk factors for prostate cancer are increased age, family history and ethnicity. Inherited genetic factors contribute to the risk of prostate cancer, which has the highest estimated genetic contribution among common cancers. Studies of prostate cancer susceptibility have suggested that prostate cancer is a genetically complex disease with susceptibility factors located in multiple chromosomal regions. The chromosomal region 11q13-14 has been found to contain prostate cancer-predisposing factors in both linkage and association studies. This region has been specifically linked to prostate cancer in Finnish prostate cancer families, and it has been reported to contribute to the aggressive form of this disease. No candidate gene or other causal factor has been identified in the region to date. One interesting gene in this region is EMSY, located at 11q13.5, which has been identified as a candidate gene for breast and ovarian cancers. The aim of the thesis was to study the region 11q13.5 in relation to prostate cancer susceptibility in Finnish men. Samples and patient information from Finnish prostate cancer patients and samples from male controls were obtained for use in this study. Fine mapping of this region using imputation and more precise screening of EMSY by Sanger sequencing resulted in identification of three intronic EMSY single-nucleotide polymorphisms (SNPs) and six intergenic variants that predispose to prostate cancer. A rare EMSY mutation was found to increase the risk, particularly that of aggressive prostate cancer, in Finnish population, and it was detected in men in Finnish prostate cancer families with aggressive disease. Intergenic common variants, which were correlated with each other, were strongly associated with the risk of prostate cancer death. In addition, haplotypes including the identified risk SNPs were found to contribute to disease predisposition. Functionality of the variants was assessed by functional annotation and by examining effects of the variants on gene expression by expression quantitative trait loci (eQTL) analysis. The prostate cancer death-associated risk SNPs coincided with enhancer elements in multiple cell types and were observed to affect the expression of DGAT2 in prostate tumours and of AP001189.4 in whole blood, suggesting tissue-specific gene regulation as a mechanism promoting tumour development. The functional annotations indicated that the EMSY SNPs may affect messenger RNA splicing of EMSY, but this finding warrants experimental confirmation. In conclusion, alterations in the chromosomal region 11q13.5 contribute to prostate cancer susceptibility in Finnish men, increasing the risk particularly of aggressive cancer and life-threatening disease progression. Replication of the associations of these variants in other populations, as well as additional analyses of the identified target genes, are necessary for detailed characterization of their tumour-promoting properties. This thesis has revealed novel information regarding prostate cancer susceptibility that could be used in the development of a biomarker panel specific for detection of an increased risk of aggressive and advanced prostate cancer.

According to international human genome sequencing consortium 2004[43], it was known that only less than 2% of the total human genome code for proteins. This ignited quite a surprise in the scientific community. Since then, a lot of researchers are attracted towards the noncoding part of the genome. There are explosion of researches addressing the role of the 98% of the human untranslated regions of the genome. This shows that the transcription is not only limited to the protein coding regions of the genome rather more than 90% of the genome are likely to be transcribed. [43] This will result in the transcription of tens and thousands of the long noncoding RNAs (lncRNAs) with little or no coding potential. However, the molecular mechanism and function of long noncoding RNAs are still an open research topic. Although the functions of limited lncRNAs are identified, there is still a gap in identifying the function of novel lncRNAs. This project implements different computational methods to predict the function of novel lncRNAs identified from TCGA glioblastoma multiforme samples. The methods used in this functional prediction include both expression and sequence-based analysis approach. In expression-based analysis, the co-expressing genes with lncRNAs are used to predict the possible functional relation. In sequence based analysis, the gene-protein and lncRNA-protein interactions together with miRNA-lncRNA interactions are considered towards the possible functional predictions. The result from the integrated functional prediction on the novel lncRNAs show that TCGA_gbm3-153501 novel lncRNA which is co-expressed together with the THBS1 gene with correlation coefficient of more that 0.5 is predicted to function in cell-cell and cell-to-matrix interactions, platelet aggregation, angiogenesis, and tumorigenesis. [202] MSI1, RBM3 and RBM8A are RNA binding proteins (RBPs) that have binding site on both the first top five differentially expressed lncRNAs which are TCGA_gbm-2-104096501, TCGA_gbm-3-153501, TCGA_gbm-5-63687001 and TCGA_gbm-17-10671251 and IGF2 which is among the top 10 differentially expressed genes. Therefore, these lncRNAs are predicted to have functional role in cell proliferation and maintenance of stem cells in the central nervous system.

Prostate cancer (PCa) is the most common cancer among men in developed countries. Although its genetic background is thoroughly investigated, rather little is known about the role of small non-coding RNAs (sncRNA) in this disease. tRNA-derived fragments (tRFs) represent a new class of sncRNAs, which are present in a broad range of species and have been reported to play a role in several cellular processes. Here, we analyzed the expression of tRFs in fresh frozen patient samples derived from normal adjacent prostate and different stages of PCa by RNA-sequencing. We identified 598 unique tRFs, many of which are deregulated in cancer samples when compared to normal adjacent tissue. Most of the identified tRFs are derived from the 5’- and 3’-ends of mature cytosolic tRNAs, but we also found tRFs produced from other parts of tRNAs, including pre-tRNA trailers and leaders, as well as tRFs from mitochondrial tRNAs. The 5’-derived tRFs comprise the most abundant class of tRFs in general and represent the major class among upregulated tRFs. The 3’-derived tRFs types are dominant among downregulated tRFs in PCa. We validated the expression of three tRFs using qPCR. The ratio of tRFs derived from tRNALysCTT and tRNAPheGAA emerged as a good indicator of progression-free survival and a candidate prognostic marker. This study provides a systematic catalogue of tRFs and their dysregulation in PCa and can serve as the basis for further research on the biomarker potential and functional roles of tRFs in this disease. This article has supplementary files, which can be found here:http://dx.doi.org/10.18632/oncotarget.8293

Introduction Hip arthroplasty is the standard treatment of a painful hip destruction. The use of modern metal-on-metal (MOM) bearing surfaces gained popularity in total hip arthroplasties during the last decade. Recently, worrisome failures due to adverse reaction to metal debris (ARMD), including pseudotumor response, have been widely reported. However, the pathogenesis of this reaction remains poorly understood. The aim of the present study was to investigate the ARMD response by flow cytometry approach. Methods Sixteen patients with a failed Articular Surface Replacement (ASR) hip prosthesis were included in the study. Samples of pseudotumor tissues collected during revision surgery were degraded by enzyme digestion and cells were typed by flow cytometry. Whole blood chromium and cobalt concentrations were analyzed with mass spectrometry before revision surgery. Results Flow cytometry analysis showed that the peri-implant pseudotumor tissue expressed two principal phenotypes, namely macrophage-dominated and T-lymphocyte-dominated response; the average portions being 54% (macrophages) and 25% (T-lymphocytes) in macrophage-dominated inflammation and 20% (macrophages) and 54% (T-lymphocytes) in T-lymphocyte-dominated response. The percentages of B-lymphocytes and granulocytes were lower in both phenotypes. Interestingly, the levels of blood chromium and cobalt were significantly higher in patients with macrophage-dominated response. Conclusions The results suggest that the adverse tissue reactions induced by MOM wear particles contain heterogeneous pathogeneses and that the metal levels are an important factor in the determination of the inflammatory phenotype. The present results support the hypothesis that higher metal levels cause cytotoxicity and tissue injury and macrophages are recruited to clear the necrotic debris. On the other hand, the adverse response developed in association with lower metal levels is T-lymphocyte-dominated and is likely to reflect hypersensitivity reaction. Public Library of Science open access

This paper describes work presented at the Nordic Symposium on Digital Pathology 2015, in Linkoping, Sweden. Prostatic intraepithelial neoplasia (PIN) represents premalignant tissue involving epithelial growth confined in the lumen of prostatic acini. In the attempts to understand oncogenesis in the human prostate, early neoplastic changes can be modeled in the mouse with genetic manipulation of certain tumor suppressor genes or oncogenes. As with many early pathological changes, the PIN lesions in the mouse prostate are macroscopically small, but microscopically spanning areas often larger than single high magnification focus fields in microscopy. This poses a challenge to utilize full potential of the data acquired in histological specimens. We use whole prostates fixed in molecular fixative PAXgene™, embedded in paraffin, sectioned through and stained with H&E. To visualize and analyze the microscopic information spanning whole mouse PIN (mPIN) lesions, we utilize automated whole slide scanning and stacked sections through the tissue. The region of interests is masked, and the masked areas are processed using a cascade of automated image analysis steps. The images are normalized in color space, after which exclusion of secretion areas and feature extraction is performed. Machine learning is utilized to build a model of early PIN lesions for determining the probability for histological changes based on the calculated features. We performed a feature-based analysis to mPIN lesions. First, a quantitative representation of over 100 features was built, including several features representing pathological changes in PIN, especially describing the spatial growth pattern of lesions in the prostate tissue. Furthermore, we built a classification model, which is able to align PIN lesions corresponding to grading by visual inspection to more advanced and mild lesions. The classifier allowed both determining the probability of early histological changes for uncategorized tissue samples and interpretation of the model parameters. Here, we develop quantitative image analysis pipeline to describe morphological changes in histological images. Even subtle changes in mPIN lesion characteristics can be described with feature analysis and machine learning. Constructing and using multidimensional feature data to represent histological changes enables richer analysis and interpretation of early pathological lesions.

Background: Microelectrode array (MEA) is a widely used technique to study for example the functional properties of neuronal networks derived from human embryonic stem cells (hESC-NN). With hESC-NN, we can investigate the earliest developmental stages of neuronal network formation in the human brain.Methods: In this paper, we propose an in silico model of maturating hESC-NNs based on a phenomenological model called INEX. We focus on simulations of the development of bursts in hESC-NNs, which are the main feature of neuronal activation patterns. The model was developed with data from developing hESC-NN recordings on MEAs which showed increase in the neuronal activity during the investigated six measurement time points in the experimental and simulated data.Results: Our simulations suggest that the maturation process of hESC-NN, resulting in the formation of bursts, can be explained by the development of synapses. Moreover, spike and burst rate both decreased at the last measurement time point suggesting a pruning of synapses as the weak ones are removed.Conclusions: To conclude, our model reflects the assumption that the interaction between excitatory and inhibitory neurons during the maturation of a neuronal network and the spontaneous emergence of bursts are due to increased connectivity caused by the forming of new synapses.

Perintötekijöillä on merkittävä rooli rinta- ja munasarjasyöpäalttiudessa. Kaksi tärkeintä rinta- ja munasarjasyöpäalttiusgeeniä ovat BRCA1 ja BRCA2. Myös lukuisia muita alttiusgeenejä tunnetaan. Kuitenkaan valtaosassa korkean rinta- ja/tai munasarjasyöpäriskin perheistä altistavia geenivirheitä ei tiedetä, jonka vuoksi perheiden perinnöllisyysneuvonta on haastavaa. Tutkimuksen tavoitteena oli saada uutta tietoa rinta- ja/tai munasarjasyövälle altistavista perintötekijöistä suomalaisissa korkean riskin rinta- ja/tai munasarjasyöpäriskin perheissä, joissa ei esiinny tunnettuja BRCA1- ja BRCA2-geenien perustajamuutoksia. Tutkimuksessa hyödynnettiin kolmea eri menetelmällistä lähestymistapaa geneettisten alttiustekijöiden tunnistamiseksi: 1) kandidaattigeenien mutaatioanalyysi 2) koko perimän kattava kopiolukumuutosanalyysi sekä 3) koko perimän proteiinia koodaavan alueen (eksomi) sekvensointi. Kandidaattigeenien mutaatioanalyysissä löydettiin BRCA1- ja CHEK2-geenien tunnettujen haitallisten muutosten esiintyvän 13.4 %:lla korkean rinta- ja/tai munasarjasyöpäriskin perheistä. Näistä CHEK2-geenin muutosten osuus oli huomattava ja kliinisesti olennainen. Uusi ja haitalliseksi ennustettu muutos löytyi BRCA2-geenistä. Kopiolukumuutosanalyysissä tunnistettiin potentiaalisia perinnölliseen rinta- ja/tai munasarjasyöpäalttiuteen vaikuttavia kopiolukumuutoksia 3p11.1, 5q15, 8p23.2 ja 19q13.41 alueilla. Näistä muutokset 3p11.1 ja 8p23.2 alueilla sijaitsevat EPHA3 ja CSMD1-geenien ei-koodaavilla alueilla ja muutos 19q13.41 ERVV-2 geenin koodaavalla alueella. Muutos 5q15 alueella sijaitsi geenien välisellä alueella mutta sen havaittiin mahdollisesti vaikuttavan säätelyelementteihin. Eksomisekvensoinnissa keskityttiin muutoksiin, jotka sijaitsivat DNA:n korjausreitin geeneissä. Tutkimuksessa tunnistettiin viisi muutosta ATM, MYC, PLAU, RAD1, ja RRM2B geeneissä, ja muutosten havaittiin rikastuneen rinta- ja/tai munasarjasyöpäperheissä kontrolleihin verrattuna viitaten siihen, että virheet liittyvät mahdollisesti matalasta keskisuuren syöpäriskiin. BRCA1-geenistä tunnistettiin lisäksi harvinainen virhe, joka voi olla kliinisesti tärkeä. Myös RAD50-geenistä tunnistettiin virhe, joka voi mahdollisesti altistaa miesrintasyövälle. Eksomianalyysissä keskityttiin myös potilasjoukkoon, johon kuului hyvin varhaisella iällä rintasyöpään sairastuneita naisia, ja näillä potilailla tunnistettiin virheitä uusissa potentiaalisissa kandidaattigeeneissä, jotka osallistuvat etenkin DNA:n korjaukseen ja replikaatioon, signalointiin, apoptoosiin ja solusykliin. Mielenkiintoisia kandidaattigeenejä ovat esimerkiksi DENND2D, TICRR, BNIPL, EDN3 ja FOCAD. Väitöskirjatyössä työssä tunnistettiin potentiaalisia perinnölliselle rinta- ja/tai munasarjasyövälle altistavia ituradan muutoksia jo tunnetuissa alttiusgeeneissä ja uusissa kandidaattigeeneissä. Uudet löydökset vaativat varmennusta ja tarjoavat erinomaisen lähtökohdan jatkotutkimuksille. Saatua tietoa voidaan hyödyntää suunniteltaessa uusia tehokkaampia strategioita rinta- ja munasarjasyövän diagnostiikkaan, seulontaan, ehkäisyyn, hoitomuotoihin ja perinnöllisyysneuvontaan. Breast cancer is the most frequent cancer and the most common cause of cancer deaths among females worldwide. Ovarian cancer is a highly lethal gynecologic malignancy that is the seventh most common cancer and the eight cause of death from cancer in women worldwide. In Finland, 4694 new breast cancer cases and 471 new ovarian cancer cases were diagnosed in 2012. Both breast and ovarian cancers are heterogeneous groups of diseases that can be divided into several subtypes; each subtype has distinct biological and clinical characteristics and responses to therapies. The major risk factors of breast and ovarian cancers include age and family history, and genetic predisposition accounts for as much as 10% of breast and 15% of ovarian cancers. The two major susceptibility genes for both diseases are BRCA1 and BRCA2, and several other susceptibility genes have been identified. However, in the majority of high-risk breast and/or ovarian cancer (HBOC) families, the genetic predisposition factors remain unidentified, making the genetic counseling of these families challenging. The aim of this study was to obtain new information about the genetic factors that predispose individuals to breast and ovarian cancer in the high-risk Finnish BRCA1/2-negative HBOC families. Three different methodological approaches were utilized to identify genetic predisposition factors in high-risk Finnish BRCA1/2 founder mutation-negative HBOC families: 1) mutational screening of candidate genes by Sanger sequencing, TaqMan genotyping assays, the HRM-method, and MLPA; 2) genome-wide copy number variation analysis using a SNP genotyping array; and 3) exome sequencing by target enrichment of the protein coding region of the genome and next-generation sequencing. A candidate gene approach revealed that previously known pathogenic mutations in BRCA1 and CHEK2 contribute to 13.4% of cancer cases in HBOC families. The proportion of CHEK2 mutations was remarkable and clinically relevant. Additionally, a novel and possibly pathogenic variant was detected in BRCA2. Copy number variation analysis identified several potential copy number variations that likely increase the risk of HBOC susceptibility and explain the fraction of breast and ovarian cancer cases. Chromosomal aberrations at 3p11.1, 5q15, 8p23.2, and 19q13.41 were of special interest. Of these, deletions at 3p11.1 and 8p23.2 affected intronic regions of EPHA3 and CSMD1, respectively, whereas duplication at 19q13.41 disrupted the coding region of the ERVV-2 gene. Moreover, a deletion at 5q15 was located in a non-genic region but was determined to affect regulatory elements. Exome sequencing analysis focused on DNA damage repair (DDR) pathway genes. Five variants in DDR genes (ATM, MYC, PLAU, RAD1, and RRM2B) were enriched in a cohort of HBOC cases compared to controls, suggesting that these variants may be low-to-moderate risk alleles. A rare variant that may have clinical relevance was detected in BRCA1. Additionally, a rare variant in RAD50 gene was suggested to predispose to male breast cancer. Moreover, defects in novel candidate genes targeting other pathways, such as DNA repair and replication, signaling, apoptosis, and the cell cycle, were identified in early-onset breast cancer patients. The interesting candidate genes included, for instance, DENND2D, TICRR, BNIPL, EDN3, and FOCAD. In conclusion, potential germline sequence alterations and copy number variations were detected in known susceptibility genes, as well as in novel candidate genes, and the roles of the variations in HBOC predisposition were indicated. These findings warrant further confirmation and provide an excellent premise for further studies.The obtained information can be utilized in designing more efficient diagnostic, screening, prevention, and therapeutic strategies for breast and ovarian cancer, as well as new tools for genetic counseling.

Background: Germline mutations within DNA-repair genes are implicated in susceptibility to multiple forms of cancer. For prostate cancer (PrCa), rare mutations in BRCA2 and BRCA1 give rise to moderately elevated risk, whereas two of ~100 common, low-penetrance PrCa susceptibility variants identified so far by genome-wide association studies implicate RAD51B and RAD23B. Methods: Genotype data from the iCOGS array were imputed to the 1000 genomes phase 3 reference panel for 21 780 PrCa cases and 21 727 controls from the Prostate Cancer Association Group to Investigate Cancer Associated Alterations in the Genome (PRACTICAL) consortium. We subsequently performed single variant, gene and pathway-level analyses using 81 303 SNPs within 20 Kb of a panel of 179 DNA-repair genes. Results: Single SNP analyses identified only the previously reported association with RAD51B. Gene-level analyses using the SKAT-C test from the SNP-set (Sequence) Kernel Association Test (SKAT) identified a significant association with PrCa for MSH5. Pathway-level analyses suggested a possible role for the translesion synthesis pathway in PrCa risk and Homologous recombination/Fanconi Anaemia pathway for PrCa aggressiveness, even though after adjustment for multiple testing these did not remain significant. Conclusions: MSH5 is a novel candidate gene warranting additional follow-up as a prospective PrCa-risk locus. MSH5 has previously been reported as a pleiotropic susceptibility locus for lung, colorectal and serous ovarian cancers.

Objectives: In a tissue engineering field, co-culture techniques of different kind have been widely researched in recent years to form physiologically relevant cell structures for different tissue reconstructions. The treatment methods of that kind are promising opportunity to be used also in the treatment of corneal blindness, which is still mainly treated with traditional tissue graft from a donor. Tissue grafts cannot still be used to treat one of the most common cause behind the corneal blindness -limbal stem cell deficiency (LSCD), due the lack of host stem cells. Thus, alternative opportunities are needed. Objectives of this research were to find a suitable medium for human adipose stem cells (hASCs), from those that had already been shown to be suitable for human corneal epithelial cells (hCECs), and use it to co-culture hydrogel- embedded hASCs with hCECs. Results will possibly help us to evaluate the functionality of this kind of 3D structure better as a treatment method of the corneal damages and LSCD. Materials and methods: HASCs were first cultured in three different mediums to find a suitable one to use in co-culturing. Following 2D co-culturing occurred within indirect- and direct contact between the immortalized hCECs and hASCs. Research was accomplished by culturing the hASCs inside the hyaluronic acid- hydrogel. Cells' behaviour during these experiments was evaluated with phase-contrast microscope, proliferation- and live/dead assays and immunofluorescent stainings. Results and conclusions: HASCs grew well in tested co-culture medium and appeared to have enhancing effect on the growth and differentiation of the co-cultured immortalized hCECs. When cultured in 3D hydrogel structure, hASCs were able to survive and proliferate within right cell densities. These results indicate that 3D co-cultures with hCECs and hydrogel- embedded hASCs might have potential for future applications in ocular surface reconstruction.

Yhden nukleotidin variantti-genotyypitys (SNV-genotyypitys) on menetelmä, jota voisi soveltaa rutiininomaisesti syöpä-diagnostiikassa. Sitä ei käytetä, koska teknologiat joita menetelmä hyödyntää ovat suhteellisen uusia. Genotyypitys-laitteiden tulee olla luotettavia ja tarkkoja, koska syöpä-DNA:n määrä kudosnäytteissä voi olla hyvin vähäinen. Formaliinilla kiinnitetyt ja parafiiniin valetut näytteet (FFPE-näytteet) ovat esimerkki kudosnäytteistä, joissa on pieni määrä DNA:ta. Toisen sukupolven sekvensointia (NGS) ja reaali-aikaista kvantitatiivista PCR:ä (qPCR) voisi mahdollisesti käyttää SNV-genotyypityksessä, kun kudosnäytteissä on pieniä määriä DNA:ta. Tässä tutkimuksessa genotyypitettiin näytteitä, joissa oli eri osuuksia syöpä-DNA:ta kahdella eri menetelmällä: NGS:lla Illuminan MiSeqillä ja qPCR:lla Fluidigmin BioMark HD:llä. Tulokset osoittavat, että MiSeq kykenee systemaattisesti havaitsemaan yhden nukleotidin variantteja näytteistä, joissa on 10 % syöpä-DNA:ta, joka tarkoittaa 22,5 ng DNA:ta. BioMark HD kykenee havaitsemaan yhden nukleotidin variantteja näytteistä, joissa on 20 % syöpä-DNA:ta, joka tarkoittaa 12,0 ng DNA:ta. Näiden tulosten lisäksi, BioMark HD on luotettavampi, koska se havaitsi kahdeksan kymmenestä variantista, kun MiSeq havaitsi vain neljä.

Although genome-wide association studies have identified over 100 risk loci that explain ~33% of familial risk for prostate cancer (PrCa), their functional effects on risk remain largely unknown. Here we use genotype data from 59,089 men of European and African American ancestries combined with cell-type-specific epigenetic data to build a genomic atlas of single-nucleotide polymorphism (SNP) heritability in PrCa. We find significant differences in heritability between variants in prostate-relevant epigenetic marks defined in normal versus tumour tissue as well as between tissue and cell lines. The majority of SNP heritability lies in regions marked by H3k27 acetylation in prostate adenoc7arcinoma cell line (LNCaP) or by DNaseI hypersensitive sites in cancer cell lines. We find a high degree of similarity between European and African American ancestries suggesting a similar genetic architecture from common variation underlying PrCa risk. Our findings showcase the power of integrating functional annotation with genetic data to understand the genetic basis of PrCa.

Väestön ikääntymisen myötä onnettomuuksien ja sairauksien aiheuttamien luuvaurioiden määrä kasvaa jatkuvasti. Luun kudosteknologia on nuori tieteenala, joka yhdistää biomateriaalitukirakenteen, solut sekä solujen proliferaatiota ja erilaistumista tukevia tekijöitä, ja tarjoaa näin lupaavan menetelmän luuvaurioiden hoitoon ilman perinteisenä hoitomuotona käytettyjen luusiirteiden haittoja. Luun kudosteknologiaa ajatellen rasvan kantasolut ovat suotuisa soluvalinta, sillä ne ovat erilaistumiskyvyltään multipotentteja ja eristettävissä suurella saannolla helposti saatavilla olevasta rasvakudoksesta. Luun kudosteknologiaan perustuvat, rasvan kantasoluja käyttävät hoidot ovatkin jo tuottaneet lupaavia tuloksia useissa yksittäisissä potilastapauksissa. Huolimatta kudosteknologisten menetelmien nopeasta kehityksestä, molekyylitason soluvasteet erilaisiin kemiallisiin sekä biomateriaalin aikaansaamiin ärsykkeisiin tunnetaan kuitenkin vielä huonosti. Tässä väitöskirjatyössä tutkittiin luun morfogeneettinen proteiini-2 (BMP-2) – kasvutekijän sekä bioaktiivisen lasin aiheuttamia luu- ja rasvaerilaistumisvasteita sekä niihin liittyviä solunsisäisiä signalointivaikutuksia ihmisen rasvan kantasoluissa. Kliinisestikin käytettyä BMP-2:a pidetään voimakkaana luuerilaistajana, mutta viimeisimmän tutkimustiedon valossa sen aiheuttamat soluvasteet eivät ole yhteneviä. Tämän vuoksi BMP-2:n vaikutusta luu- ja rasvaerilaistumiseen sekä kanoniseen Smad-signalointireittiin analysoitiin usealta eri luovuttajalta peräisin olevissa rasvan kantasoluissa. Lisäksi tutkittiin kasvatusliuoksen (ihmisseerumi-/naudan seerumipohjainen) sekä BMP-2:n tuottoalkuperän (Escherichia coli/nisäkässolut) vaikutusta kasvutekijän toimintaan. Mitä tulee luun kudosteknologiassa käytettyihin biomateriaaleihin, bioaktiivisia laseja pidetään erityisen hyödyllisinä niiden luuerilaistumista stimuloivain vaikutuksen vuoksi. Ilmiön tarkkaa mekanismia ei kuitenkaan juuri tunneta. Tämän vuoksi S53P4-, 2-06-, 1-06- ja 3-06-laseista valmistettujen ekstraktien luuerilaistavaa vaikutusta tutkittiin ilman suoraa solu-biomateriaali-kontaktia. Lisäksi lasien aiheuttaman varhaisen luuerilaistumisen mekanismeja liittyen solujen kiinnittymiseen ja kiinnittymisen käynnistämään signalointiin tutkittiin kasvattamalla rasvan kantasoluja S53P4- ja 1-06-bioaktiivisista laseista valmistetuilla levyillä. BMP-2-stimuloinnin havaittiin aiheuttavan toiminnallisen kanonisen Smad-signalointivasteen kaikilta luovuttajilta peräisin olevissa rasvan kantasoluissa. Tästä huolimatta eri luovuttajien solujen erilaistumisvasteet erosivat merkittävästi toisistaan: toisten luovuttajien solut erilaistuivat BMP-2:n vaikutuksesta luun ja toisten rasvan suuntaan. BMP-2:n aiheuttama soluvaste oli voimakkain ihmisen seerumia sisältävässä mediumissa sekä nisäkässoluissa tuotetulla kasvutekijällä. Mitä tulee lasiekstrakteihin, lasista vapautuneet ionit sekä perinteisesti käytetyt kemialliset luuerilaistustekijät yhdistävä kasvatusliuos aiheutti poikkeuksellisen nopean ja voimakkaan luuerilaistusvasteen rasvan kantasoluissa verrattuna lasi-ionittomaan luuerilaistusmediumiin. Lasiekstrakteista 2-06 ja 3-06 indusoivat luuerilaistumista voimakkaimmin. Ilman luuerilaistusmediumia lasiekstraktit eivät kuitenkaan aiheuttaneet luuerilaistumista. Viljeltäessä S53P4- ja 1-06-biolasilevyillä ilman kemiallisia luuerilaistustekijöitä rasvan kantasolut ilmensivät luuerilaistumisen varhaisia markkereita. Solut kiinnittyivät lasilevyille epätyypillisellä mekanismilla: fokaaliadheesiokohdat olivat pieniä ja tasaisesti ympäri soluja levittäytyneitä, ja aktiinitukiranka oli epäjärjestynyt, mutta tästäkin huolimatta integriiniβ1:n sekä vinkuliinin tuoton havaittiin lisääntyvän lasien vaikutuksesta. Solut myös muokkasivat allaan olevaa biolasipintaa. Bioaktiivisen lasin aiheuttaman varhaisen luuerilaistumisen havaittiin riippuvan fokaaliadheesiokinaasista (FAK) sekä mitogeeniaktivoituvista proteiinikinaaseista (MAPK) ERK1/2 ja JNK. MAPK p38 oli sen sijaan toiminnaltaan merkityksettömämpi. Yhteenvetona BMP-2:n aiheuttamien soluvasteiden havaittiin olevan voimakkaasti riippuvaisia rasvan kantasolujen luovuttajasta, mutta myös kasvatusmediumin koostumuksesta sekä kasvutekijän tuottoalkuperästä, mikä aiheuttaa haasteita BMP-2:n onnistuneelle kliiniselle käytölle. Kaksijakoisesta erilaistumisvasteesta huolimatta BMP-2:n aiheuttama Smad-signalointi oli yhtenevää kaikissa soluissa, minkä vuoksi myös muiden signalointimekanismien täytyy säädellä BMP-2:n indusoimaa erilaistumisvastetta. Mitä tulee biolasiekstrakteihin, luuerilasitustekijöiden läsnä ollessa lasi-ionien luuerilaistava vaikutus oli poikkeuksellisen voimakas, mikä saattaa tarjota näille mediumeille lukuisia käyttökohteita tehokasta luuerilaistumista edellyttävissä sovelluksissa. Viljeltäessä rasvan kantasoluja biolasilevyillä, solujen huomattiin kiinnittyvän laseille poikkeuksellisella tavalla, ja kiinnittymiseen kytkeytyvän FAK-MAPK–signalointireitin havaittiin olevan merkittävässä asemassa lasien aiheuttaman varhaisen luuerilaistumisen säätelyssä. Nämä havainnot luovat pohjaa bioaktiivisten lasien aiheuttaman solusignalointivasteen tarkemmille analyyseille. Due to the aging population, the incidence of bone defects, caused either by trauma or disease, is constantly increasing. Bone tissue engineering (TE), combining a biomaterial scaffold, cells and cell growth and differentiation stimulating factors, has recently emerged as a promising approach to treat these defects without the drawbacks associated with the traditionally used bone grafts. With respect to bone TE, adipose stem cells (ASCs) are considered a highly favorable cell choice due to their multipotent differentiation capability and high abundance in the easily obtainable adipose tissue. In fact, bone TE treatments utilizing adipose stem cells have already shown promising results in a number of clinical case studies. However, despite the fast progress of the bone TE approaches, the molecular level cellular responses to the various chemical and biomaterial-elicited stimuli are still poorly understood. In this thesis, the osteogenic and adipogenic differentiation responses stimulated by bone morphogenetic protein-2 (BMP-2) and bioactive glass (BaG), as well as the underlying intracellular signaling events in human ASCs (hASCs), were evaluated. Accepted also for clinical use, BMP-2 is thought to be a strong bone formation inducer, but based on recent findings, it seems to elicit highly variable cellular responses. To shed light on this, the effect of BMP-2 on osteogenic and adipogenic differentiation, as well as on the canonical Smad signaling pathway was analyzed in hASCs derived from several donors. Moreover, the role of the culture medium (either human serum (HS) or fetal bovine serum (FBS) supplemented) and the production origin of BMP-2 (Escherichia coli or mammalian cells) in the BMP-2 function was evaluated. With respect to the biomaterial component of the bone TE approach, BaGs have been considered highly advantageous due to their inherent ability to stimulate osteogenic differentiation. However, the exact mechanism of this favorable phenomenon is not well known. Thus, the osteogenesis-stimulating effect of ionic extracts from the BaGs S53P4, 2-06, 1-06 and 3-06, in the absence of the cell-biomaterial contact, was determined. Secondly, the mechanisms of BaG-induced early osteogenic differentiation, with respect to cell attachment and attachment-mediated signaling, were evaluated when hASCs were cultured on S53P4 and 1-06 BaG discs. The stimulation with BMP-2 was observed to elicit functional canonical Smad signaling response in all the hASC donor lines studied. However, with respect to differentiation, hASCs from some donors adopted an osteogenic fate while hASCs from other donors committed towards adipocytes in response to BMP-2. Moreover, the cellular response to BMP-2 was observed to be the strongest in HS condition and with mammalian cells originated BMP-2. With respect to the BaG ionic extracts, the combination of the ions released from the BaGs and the traditionally used osteogenic medium (OM) induced exceptionally fast and extensive osteogenic differentiation of hASCs when compared to the control OM. Of the different BaGs, 2-06 and 3-06 OM extracts seemed to be the strongest stimulators of osteogenic differentiation. As such, however, the BaG extracts could not induce the osteogenic commitment of hASCs. When the hASCs were cultured in contact with the S53P4 and 1-06 discs, the glasses stimulated the early osteogenic differentiation even in the absence of OM. The cell attachment mode on the BaGs was shown to be atypical, with small and dispersed focal adhesion sites and disorganized actin cytoskeleton, but increased production of integrinβ1 and vinculin. The cells also modified the BaG surface underneath them. The BaG-induced early osteogenic differentiation was shown to be highly dependent on focal adhesion kinase (FAK) and mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK), whereas the role of MAPK p38 was less significant. In conclusion, the BMP-2-induced cellular responses were observed to be highly dependent on the hASC donor but also on culture medium composition and BMP-2 production origin, which poses challenges to the clinical utility of this growth factor. Moreover, despite the varying differentiation responses, the BMP-2-induced Smad signaling was similar in all the hASC donor lines, implying that additional signaling mechanisms must be involved in the BMP-2-induced differentiation. In case of the BaG extracts, the exceptionally strong osteogenesis-inducing ability of the OM-based BaG extracts might be highly applicable in various approaches requiring effective osteogenic differentiation. When cultured in direct contact with the BaGs, a unique cell attachment mode of hASCs was observed and, furthermore, the attachment-related FAK-MAPK signaling pathway was shown to play a central role in the BaG-induced early osteogenic response. These observations set the basis for a more detailed evaluation of the cell signaling events on BaGs, an area with currently only very little knowledge.

Eturauhassyöpä on miesten yleisin syöpä länsimaissa. Syövän vaarallisuuden määrää suurelta osin sen kyky tunkeutua ympäröivään kudokseen ja lähettää etäispesäkkeitä. Eturauhassyövän leviämiseen ja metastaasiin johtavat molekulaariset mekanismit tunnetaan kuitenkin yhä suurelta osin heikosti. Nämä mekanismit täytyy tuntea, jotta voidaan löytää uusia apuvälineitä eturauhassyövän diagnosointiin ja tarkemman ennusteen tekemiseen sekä suunnitella parempia terapioita syövän leviämisen pysäyttämiseksi. Tässä työssä tutkittiin pitkälle edenneessä syövässä yli-ilmentyvän androgeenireseptorin säätelemän mikroRNA:n, miR-32:n, vaikutusta peptidaasi-inhibiittori 15:n (PI15) ilmentymiseen eturauhassyövän solulinjoissa PC-3 ja LNCaP. Tämä miRNA kohdegeeni-pari herätti kiinnostuksen, sillä kastraatioresistentit syövät ovat tyypillisesti hyvin aggressiivisia ja peptidaaseilla ja proteaaseilla, joita PI15 inhiboi, on tähän suuri vaikutus. Hypoteesina on, että pitkälle edenneessä syövässä miR-32 vaimentaa PI15:n ilmentymisen, jolloin peptidaasien toiminta on aktiivisempaa ja syövän leviäminen kiihtynyttä. Edellä mainittuja solulinjoja transfektoitiin PI15 geenin sisältävällä plasmidilla saman aikaisesti pre- miR-32:lla ja vaikutuksia PI15:n ilmentymiseen mitattiin qRT-PCR:lla ja western blot-menetelmällä. Lisäksi tehtiin reportterivektori, jossa lusiferaasi-geeni kytkettiin PI15:n 3' säätelyalueeseen, jolloin voidaan mitata miR-32:n säätelyvaikutusta lusiferaasin ilmentymiseen suoraan proteiinitasolla. Myös PI15:n yli-ilmentämisen vaikutuksia tutkittiin kloonaamalla PI15 geenin koodaava alue uuteen plasmidiin, jossa on nisäkässoluissa toimiva selektiogeeni, ja transfektoimalla PC-3 solulinja pysyvästi hyödyntäen antibiootin aikaan saamaa selektiopainetta. Näin saatujen PI15:ttä stabiilisti yli-ilmentävien PC-3 kloonien kasvunopeutta ja solujen elinkelpoisuutta mitattiin verrattuna tyhjällä plasmidilla transfektoituihin klooneihin. PI15:n ja miR-32:n yhtäaikaisen transfektion aiheuttama hajonta osoittautui liian suureksi pitävien johtopäätösten tekemiseksi miR-32:n vaikutuksesta PI15:n ilmentymiseen. Tulevaisuudessa tehtävä lusiferaasi-reportterikoe voisi vastata paremmin tähän kysymykseen. PI15:n ilmentymistä ei tämän työn puitteissa kyetty todentamaan proteiinitasolla. Geenin yli-ilmentäminen kuitenkin johti selvästi alentuneeseen kasvunopeuteen ja solujen elinkelpoisuuteen sekä fenotyypin muuttumiseen suurimmalla ilmentämisen tasolla. Jatkotutkimusta vaaditaan, jotta voidaan osoittaa PI15 proteiinin aikaan saavan nämä muutokset.

During replication of nuclear ribosomal DNA (rDNA), clashes with the transcription apparatus can cause replication fork collapse and genomic instability. To avoid this problem, a replication fork barrier protein is situated downstream of rDNA, there preventing replication in the direction opposite rDNA transcription. A potential candidate for a similar function in mitochondria is the mitochondrial transcription termination factor 1 (MTERF1, also denoted mTERF), which binds to a sequence just downstream of the ribosomal transcription unit. Previous studies have shown that MTERF1 prevents antisense transcription over the ribosomal RNA genes, a process which we here show to be independent of the transcription elongation factor TEFM. Importantly, we now demonstrate that MTERF1 arrests mitochondrial DNA (mtDNA) replication with distinct polarity. The effect is explained by the ability of MTERF1 to act as a directional contrahelicase, blocking mtDNA unwinding by the mitochondrial helicase TWINKLE. This conclusion is also supported by in vivo evidence that MTERF1 stimulates TWINKLE pausing. We conclude that MTERF1 can direct polar replication fork arrest in mammalian mitochondria.

Background and aims: Adipose tissue is a source of multipotent adipose stromal/stem cells (ASCs). ASCs have excellent proliferation and multilineage differentiation capacity, low immunogenicity and promising immunosuppressive capacity. The immunosuppressive properties of ASCs have been previously studied but detailed intracellular mechanisms are still unknown. The aim of this study was to investigate the suppressive potential of ASCs in direct versus indirect co-culture with peripheral blood mononuclear cells (PBMCs) and to identify the signaling pathways (STAT3, STAT1, NF-κB and Smad1/5) that are activated during ASC-mediated immunosuppression. Methods: Four different ASC donors were used to study the immunosuppressive capacity of ASCs. Mixed lymphocyte reactions (MLR) using direct and indirect co-cultures of ASCs and PBMCs were used to study the immunosuppressive capacity of ASCs. Immunosuppression was analyzed using BrdU-ELISA. Activation of intracellular signaling pathways were analyzed using Western Blot analysis from direct and indirect MLR samples. Results: Strong immunosuppression on PBMC proliferation was obtained with three ASC donors. ASCs in direct co-culture with PBMCs had stronger immunosuppressive capacity compared to indirect co-cultures. ASCs cultured with MLR2 combination possessed stronger immunosuppressive capacity compared to MLR1 combination. There was variation in the activation of intracellular signaling pathways between different donors. Two signaling pathways (NF-κB and Smad1/5) were activated only in direct reactions compared to pathways STAT3 and STAT1, which were activated also in indirect reactions. NF-κB phosphorylation was inhibited in ASCs in direct reactions. Activation of Smad1/5 was donor-specific and it was activated with other ASC donor and inhibited with another ASC donor. In direct reactions STAT3 phosphorylation was inhibited and in indirect reactions STAT3 was mainly activated. STAT1 was phosphorylated in PBMCs with three ASCs donors and in two ASC donors in indirect reactions. Conclusion: ASCs have immunosuppressive capacity when co-cultured with PBMCs in both direct and indirect cultures. More studies with more ASC and PBMC donors are needed to obtain more detailed information about intracellular signaling behind the immunosuppression of ASCs.

Background and aims: Cell factories are currently used to produce biochemicals for nutrition, pharma, medical, and energy industries with a high future potential and interest among these and other industries. General products of cell factories are complex biochemicals difficult to derive, and robust ways of controlling the flux of carbon in the metabolic reactions of the cell are required. In this work, we propose a three step synthetic biology workflow to perform pathway optimization in Saccharomyces cerevisiae with tryptophan pathway as an example. First, metabolic modelling is performed on the chosen metabolic pathway to identify suitable genes to regulate. Then, the promoters of the selected genes are replaced by synthetic ones, hence setting the gene expression on a chosen level. The derived S. cerevisiae strains are then screened for the production of the chosen molecule. Thirdly, machine learning is utilized, fitting a regression model on the data derived from the screening measurements, and new strains are produced according to the prediction given by the model. Methods: We identified tryptophan pathway enzymes worth regulating with flux balance analysis (FBA) on yeast stoichiometric metabolic model. The capability of the system to grow and produce tryptophan under anaerobic conditions were investigated in simulated conditions where essential fatty acids were unconstrained. With CRISPR/Cas9 genome editing technology, the promoters of the selected three genes were replaced by synthetic ones in a randomized manner. The promoters were designed to carry 0-8 binding sites for bacterial LexA domain which in turn is a part of a chimeric synthetic transcription factor protein in a randomized manner. We used an S. cerevisiae strain harbouring a VioABE cassette to convert tryptophan to our screening compound, green prodeoxyviolacein. We derived yeast strains with clearly different levels of prodeoxyviolacein, and screened them in an in-house robot facility. We attempted to find the genotypes of the produced strains with capillary electrophoresis with no success, so we fit linear and gam models with simulated genotypes. Results: We identified three genes G6PDH, PGI1, and TKL1 as suitable regulation targets on the tryptophan pathway. In two screening rounds we derived altogether 268 strains of S. cerevisiae that produce different levels of tryptophan, the highest having a 4.5 fold change in its absorbance values in comparison to the parental strain. With genotypes derived from simulated data we implemented three regression models on the data to mediate a machine learning program capable of predicting the best promoter combinations once inputted real genotypes. Conclusions: Based on our experiments, the FBA derived predictions of genes to regulate seemed to be valid, and under current knowledge we managed to implement three simulatenous CRISPR/Cas9 mediated genome editions to modify the promoters of the selected genes. Generalized additive models described the absorbance values well with the simulated genotypes. In future, the workflow must nonetheless be further fine-tuned by the validation and elucidation of the genotypes derived from the CRISPR/Cas9 experiments, using the machine learning models to predict the genotypes for the next round of screening.

In this paper, we study the problem of feature selection in cancer-related machine learning tasks. In particular, we study the accuracy and stability of different feature selection approaches within simplistic machine learning pipelines. Earlier studies have shown that for certain cases, the accuracy of detection can easily reach 100% given enough training data. Here, however, we concentrate on simplifying the classification models with and seek for feature selection approaches that are reliable even with extremely small sample sizes. We show that as much as 50% of features can be discarded without compromising the prediction accuracy. Moreover, we study the model selection problem among the ℓ1 regularization path of logistic regression classifiers. To this aim, we compare a more traditional cross-validation approach with a recently proposed Bayesian error estimator.

Formate dehydrogenases (FDHs) are a set of mostly homodimeric oxidoreductases that catalyze the oxidation of formate to carbon dioxide with the concurrent reduction of NAD+ to NADH. These enzymes are common to methylotropic yeasts and bacteria in which they are vital in the catabolism of methanol and methane. FDHs catalyze relatively simple hydride transfer reaction in which formate donates a hydride to the C4N atom of the nicotinamide ring of NAD+, yielding carbon dioxide and NADH. During the recent decades, FDHs have been utilized in coenzyme regeneration systems that produce NADH which would be expensive to manufacture by conventional chemical synthesis. Another promising application of FDHs is carbon fixation from the atmosphere and the oceans using the reverse reaction, the reduction of carbon dioxide to formate. Recent experimental evidence suggests that formate dehydrogenases are able to reduce bicarbonate to formate as well. The aim of this thesis was to develop a model for the catalytic mechanism for the reduction of bicarbonate in the active site of FDH from bacterium Pseudomonas sp. strain 101. For this purpose, three different initial models for classical molecular dynamics simulations were constructed. The PDB structure 2NAD was used as a basis for all models. In addition to the main simulation which included bicarbonate and NADH in the active site, two reference simulations were performed as well. In the first one, formate and NAD+ were simulated in the active site to ensure that the results would be comparable to earlier computational studies on the same subject. The purpose of the second reference simulation was to observe if carbon dioxide could react to formate in the presence of NADH. It was discovered that bicarbonate settled in a conformation ideal for hydride transfer during the simulation. However, the process itself was a time-consuming process, taking up to 3 nanoseconds of simulation time. Experimental results seemed to support this observation. Based on the optimal conformation of bicarbonate, a model for the catalytic mechanism was developed. In the model, the hydride donated by NADH would form a covalent bond to the carbon atom of bicarbonate, resulting in a surplus of electrons within the molecule. This would cause the complex formed by bicarbonate and the hydride to break into formate and hydroxide ions. In the reference simulation with formate the optimal conformations for hydride transfer were not observed, but this was probably due to the inaccuracies of the classical molecular dynamics method described in earlier studies. Carbon dioxide seemed to have a destabilizing effect on the active site. The reverse reaction could still be possible as carbon dioxide occasionally drifted close to the hydride of NADH bound to the C4N.

Healthy human heart rate fluctuates overtime showing long-range fractal correlations. In contrast, various cardiac diseases and normal aging show the breakdown of fractal complexity. Recently, it was shown that human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) intrinsically exhibit fractal behavior as in humans. Here, we investigated the fractal complexity of hiPSC-derived long QT-cardiomyocytes (LQT-CMs). We recorded extracellular field potentials from hiPSC-CMs at baseline and under the effect of various compounds including β-blocker bisoprolol, ML277, a specific and potent IKs current activator, as well as JNJ303, a specific IKs blocker. From the peak-to-peak-intervals, we determined the long-range fractal correlations by using detrended fluctuation analysis. Electrophysiologically, the baseline corrected field potential durations (cFPDs) were more prolonged in LQT-CMs than in wildtype (WT)-CMs. Bisoprolol did not have significant effects to the cFPD in any CMs. ML277 shortened cFPD in a dose-dependent fashion by 11 % and 5–11 % in WT- and LQT-CMs, respectively. JNJ303 prolonged cFPD in a dose-dependent fashion by 22 % and 7–13 % in WT- and LQT-CMs, respectively. At baseline, all CMs showed fractal correlations as determined by short-term scaling exponent α. However, in all CMs, the α was increased when pharmacological compounds were applied indicating of breakdown of fractal complexity. These findings suggest that the intrinsic mechanisms contributing to the fractal complexity are not altered in LQT-CMs. The modulation of IKs channel and β1-adrenoreceptors by pharmacological compounds may affect the fractal complexity of the hiPSC-CMs. Electronic supplementary material The online version of this article (doi:10.1007/s12015-016-9686-0) contains supplementary material, which is available to authorized users.

BACKGROUND: Epidemiological studies have observed a positive association between an earlier age at sexual development and prostate cancer, but markers of sexual maturation in boys are imprecise and observational estimates are likely to suffer from a degree of uncontrolled confounding. To obtain causal estimates, we examined the role of pubertal development in prostate cancer using genetic polymorphisms associated with Tanner stage in adolescent boys in a Mendelian randomization (MR) approach. METHODS: We derived a weighted genetic risk score for pubertal development, combining 13 SNPs associated with male Tanner stage. A higher score indicated a later puberty onset. We examined the association of this score with prostate cancer risk, stage and grade in the UK-based ProtecT case-control study (n = 2,927), and used the PRACTICAL consortium (n = 43,737) as a replication sample. RESULTS: In ProtecT, the puberty genetic score was inversely associated with prostate cancer grade (odds ratio (OR) of high- vs. low-grade cancer, per tertile of the score: 0.76; 95 % CI, 0.64-0.89). In an instrumental variable estimation of the causal OR, later physical development in adolescence (equivalent to a difference of one Tanner stage between pubertal boys of the same age) was associated with a 77 % (95 % CI, 43-91 %) reduced odds of high Gleason prostate cancer. In PRACTICAL, the puberty genetic score was associated with prostate cancer stage (OR of advanced vs. localized cancer, per tertile: 0.95; 95 % CI, 0.91-1.00) and prostate cancer-specific mortality (hazard ratio amongst cases, per tertile: 0.94; 95 % CI, 0.90-0.98), but not with disease grade. CONCLUSIONS: Older age at sexual maturation is causally linked to a reduced risk of later prostate cancer, especially aggressive disease. BioMed Central open access

Background and Aims Humans are able to quickly recognize and evaluate visual patterns, thus this thesis aims to apply this feature to the analysis of aspects of the conservation of carbonic anhydrase proteins. This was facilitated through the creation of two pipelines: * One to create a publically available specialized database to service the CA research world named CAbase, and, * One to create a visual display of the aligned exons of the cDNA transcripts contained within CAbase with indicators to show the positions of start and stop codons along with the locations of the predicted signal and mitochondrial targeting peptides. This pipeline was named Exon_Analysis. Carbonic anhydrases (CAs) are ubiquitous proteins that reversibly catalyse carbon dioxide into carbonic acid. Through the events of duplication, the CAs exist in at least 16 different isoforms and potentially up to 17 different isoforms. Methods The pipelines were created using freely available tools that included python, MySQL, various bioinformatic tools such as Clustal Omega, PRANK, BLAST and Pal2Nal. The data for CAbase was extracted from Ensembl, NCBI, UniProt, RSCB PDB, UniGene and FlyBase. Additionally, calculated data from using SignalP and TargetP was also included. CAbase is hosted on the Amazon Web Server and can be accessed using any computer that has access to the Internet and has MySQL installed. Exon_Analysis draws a scaled exon MSA schematic based on a PRANK MSA of the cDNA transcripts for a CA isoform. The exons and other indicators such as the start and stop codon, and the signal and target peptides are all drawn in different colours and in their scaled locations. Thus it is possible to see the conserved nature of the exons within the coding regions and the aligned start and stop codons and the peptides for each CA isoform. Results CAbase is now publically available for anyone to use. However, it is still somewhat user unfriendly due to the requirement that user be familiar with SQL. CAbase facilitated the use of Exon_Analysis. This pipeline has enabled the quality control of the predicted genes one exon at a time. Evolutionary events such as the conservation of short exons within CAs VI, IX, XII, XIV, X and XI has been shown in the exon MSA schematics. Both tools could be adapted for use with other proteins. Conclusion CAbase is a specialized database for CA researchers that can continue to be adapted to their needs. It allows researchers to create specific queries without having to filter for unwanted proteins. Exon_Analysis creates an exon MSA schematic to visualise the relationship between the exons and other features of interest. This facilitates the quality control of predicted genes one exon at a time.

Circulating insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) are associated with prostate cancer. Using genetic variants as instruments for IGF peptides, we investigated whether these associations are likely to be causal. We identified from the literature 56 single nucleotide polymorphisms (SNPs) in the IGF axis previously associated with biomarker levels (8 from a genome-wide association study [GWAS] and 48 in reported candidate genes). In ∼700 men without prostate cancer and two replication cohorts (N ∼ 900 and ∼9,000), we examined the properties of these SNPS as instrumental variables (IVs) for IGF-I, IGF-II, IGFBP-2 and IGFBP-3. Those confirmed as strong IVs were tested for association with prostate cancer risk, low (< 7) vs. high (≥ 7) Gleason grade, localised vs. advanced stage, and mortality, in 22,936 controls and 22,992 cases. IV analysis was used in an attempt to estimate the causal effect of circulating IGF peptides on prostate cancer. Published SNPs in the IGFBP1/IGFBP3 gene region, particularly rs11977526, were strong instruments for IGF-II and IGFBP-3, less so for IGF-I. Rs11977526 was associated with high (vs. low) Gleason grade (OR per IGF-II/IGFBP-3 level-raising allele 1.05; 95% CI: 1.00, 1.10). Using rs11977526 as an IV we estimated the causal effect of a one SD increase in IGF-II (∼265 ng/mL) on risk of high vs. low grade disease as 1.14 (95% CI: 1.00, 1.31). Because of the potential for pleiotropy of the genetic instruments, these findings can only causally implicate the IGF pathway in general, not any one specific biomarker.

Background and aims: Limbal stem cell deficiency (LSCD) is a potentially blinding corneal disease caused by trauma, such as chemical burn, inflammation, or hereditary factors. LSCD is characterized by dysfunction of limbal epithelial stem cells (LESC) and failure of barrier function of limbus. Surrounding conjunctival tissue invades transparent and avascular corneal epithelium, which leads to vascularization and haziness. In LSCD, the surface of the eye cannot recover itself due to lack or dysfunction of LESCs so the treatment needs to bring new stem cells to continue tissue regeneration. Various approaches have been proposed to treat LSCD, some using limbal tissue transplantations from healthy eye and some have been cell-based therapies. This study presents a method developed in the Eye group at the University of Tampere, which uses human pluripotent stem cells (hPSCs) to differentiate corneal epithelial-like cells. This study's aim was to further optimize this method and test enzymatic dissociation method for stem cells. Methods: In this study, cell differentiation was repeated six times. In every experiment, hPSCs were dissociated from feeder cells either mechanically or enzymatically. The cells were suspension cultured in small molecule and growth factor supplemented induction medium as embryoid bodies (EBs). After seven-day induction, the cells were plated to well plates, which were coated with collagen IV or surface x. The adherent cell culture lasted 21 days. Cells were analyzed with immunofluorescence at midpoint and end point of the experiments, to identify either LESC-like cells or corneal epithelial cells. Results: Mechanical and enzymatic dissociation methods both worked as detaching methods. Enzymatic dissociation gave partially better cell numbers than mechanical dissociation. EBs survived better in suspension culture when the growth area was smaller. Collagen IV and surface x both worked as growth surface but cell numbers were better with surface x. In adherent cell culture, the growth area proved to be important, because cells seemed to suffer from overcrowding. At the end point of the experiments, there were cells positive for both LESC and cornea epithelium markers. Conclusions: Enzymatic dissociation method worked as a dissociation method alongside the mechanical dissociation. Enzymatically dissociated EBs survived and outgrowths were better than mechanically dissociated EBs. Surface x improved cell attachment and enhanced their differentiation towards corneal epithelium.

Microseminoprotein-beta (MSMB, MSMB) is an abundant secretory protein contributed by the prostate, and is implicated as a prostate cancer (PC) biomarker based on observations of its lower expression in cancerous cells compared with benign prostate epithelium. However, as the current literature on MSMB is inconsistent, we assessed the expression of MSMB at the protein and mRNA levels in a comprehensive set of different clinical stages of PC. Immunohistochemistry using monoclonal and polyclonal antibodies against MSMB was used to study protein expression in tissue specimens representing prostatectomies (n = 261) and in diagnostic needle biopsies from patients treated with androgen deprivation therapy (ADT) (n = 100), and in locally recurrent castration-resistant PC (CRPC) (n = 105) and CRPC metastases (n = 113). The transcript levels of MSMB, nuclear receptor co-activator 4 (NCOA4) and MSMB-NCOA4 fusion were examined by qRT-PCR in prostatectomy samples and by RNA-sequencing in benign prostatic hyperplasia, PC, and CRPC samples. We also measured serum MSMB levels and genotyped the single nucleotide polymorphism rs10993994 using DNA from the blood of 369 PC patients and 903 controls. MSMB expression in PC (29% of prostatectomies and 21% of needle biopsies) was more frequent than in CRPC (9% of locally recurrent CRPCs and 9% of CRPC metastases) (p

Background and aims: Breast cancer is the second most common cancer in the world after lung cancer, and the most common cancer among women. The risk factors for breast cancer include different hormonal factors and consequently, estrogen has been found to promote breast cancer pathogenesis. Bone morphogenetic proteins (BMP) are a group of growth factors that have been connected to different cancers, including breast cancer. BMPs have been noted to have different effects on breast cancer cells, decreasing proliferation in some, and in contrast, increasing migration and invasion in others. The estrogen and BMP signaling pathways have been shown to have a connection, and they can influence each other's functions. In this study, the objectives are to examine the effect of estrogen on BMP gene expression in six breast cancer cell lines, and to study the effects of estrogen and BMP4 on cell proliferation in two breast cancer cell lines. Methods: The breast cancer cell lines (estrogen receptor positive BT-474, MCF-7, MDA-MB-361, T-47D and ZR-75-30, and the estrogen receptor negative MDA-MB-231) were cultured in estrogen free medium for three days before treatment with 17ẞ-estradiol (E2, 100 nM) or vehicle control for 24 and 48 hours. The gene expression of BMP4 and BMP7, and the positive control genes GREB1 and TFF1 was assessed with qRT-PCR by using a SYBR Green I dye based assay. For examining the cell proliferation, BT-474 and T-47D cells were cultured for three days in estrogen free conditions, and treated with E2 (100 nM), human recombinant BMP4 (100 ng/ml), both, or vehicle control. The cell proliferation was followed by microscopy and imaging, and finally by counting the cells at six (T-47D) and seven (BT-474) days after the treatments. Results: The expression of positive control genes GREB1 and TFF1 increased after estrogen treatment in most estrogen receptor positive cell lines, as expected. Treatment with E2 had a variable effect on BMP4 and BMP7 expression depending on the cell line. BMP4 expression was notably decreased in BT-474, MCF-7 and MDA-MB-361 cell lines, but not in others. The expression of BMP7 was notably decreased in BT-474, T-47D and ZR-75-30, but again, there was no significant change in the others. The cell proliferation experiment showed that as expected, E2 increased cell growth both in BT-474 and T-47D cell line, and that BMP4 was able to inhibit the effect of E2 on cell growth. Conclusions: Estrogen has different effects on BMP4 and BMP7 expression depending on the cell line. In some it decreases BMP expression, and in others it has no significant effect. BMP4 was able to diminish the estrogen induced cell proliferation. This is an important finding that may have clinical applicability for the treatment of breast cancer patients.

Common variation on 14q24.1, close to RAD51B, has been associated with breast cancer: rs999737 and rs2588809 with the risk of female breast cancer and rs1314913 with the risk of male breast cancer. The aim of this study was to investigate the role of RAD51B variants in breast cancer predisposition, particularly in the context of familial breast cancer in Finland. We sequenced the coding region of RAD51B in 168 Finnish breast cancer patients from the Helsinki region for identification of possible recurrent founder mutations. In addition, we studied the known rs999737, rs2588809, and rs1314913 SNPs and RAD51B haplotypes in 44,791 breast cancer cases and 43,583 controls from 40 studies participating in the Breast Cancer Association Consortium (BCAC) that were genotyped on a custom chip (iCOGS). We identified one putatively pathogenic missense mutation c.541C>T among the Finnish cancer patients and subsequently genotyped the mutation in additional breast cancer cases (n = 5259) and population controls (n = 3586) from Finland and Belarus. No significant association with breast cancer risk was seen in the meta-analysis of the Finnish datasets or in the large BCAC dataset. The association with previously identified risk variants rs999737, rs2588809, and rs1314913 was replicated among all breast cancer cases and also among familial cases in the BCAC dataset. The most significant association was observed for the haplotype carrying the risk-alleles of all the three SNPs both among all cases (odds ratio (OR): 1.15, 95% confidence interval (CI): 1.11–1.19, P = 8.88 x 10−16) and among familial cases (OR: 1.24, 95% CI: 1.16–1.32, P = 6.19 x 10−11), compared to the haplotype with the respective protective alleles. Our results suggest that loss-of-function mutations in RAD51B are rare, but common variation at the RAD51B region is significantly associated with familial breast cancer risk. Public Library of Science open access

To understand how Toll signaling controls the activation of a cellular immune response in Drosophila blood cells (hemocytes), we carried out a genetic modifier screen, looking for deletions that suppress or enhance the mobilization of sessile hemocytes by the gain-of-function mutation Toll10b (Tl10b). Here we describe the results from chromosome arm 3R, where five regions strongly suppressed this phenotype. We identified the specific genes immune response deficient 1 (ird1), headcase (hdc) and possibly Rab23 as suppressors, and we studied the role of ird1 in more detail. An ird1 null mutant and a mutant that truncates the N-terminal kinase domain of the encoded Ird1 protein affected the Tl10b phenotype, unlike mutations that affect the C-terminal part of the protein. The ird1 null mutant suppressed mobilization of sessile hemocytes, but enhanced other Tl10b hemocyte phenotypes, like the formation of melanotic nodules and the increased number of circulating hemocytes. ird1 mutants also had blood cell phenotypes on their own. They lacked crystal cells and showed aberrant formation of lamellocytes. ird1 mutant plasmatocytes had a reduced ability to spread on an artificial substrate by forming protrusions, which may explain why they did not go into circulation in response to Toll signaling. The effect of the ird1 mutation depended mainly on ird1 expression in hemocytes, but ird1-dependent effects in other tissues may contribute. Specifically, the Toll receptor was translocated from the cell membrane to intracellular vesicles in the fat body of the ird1 mutant, and Toll signaling was activated in that tissue, partially explaining the Tl10b-like phenotype. As ird1 is otherwise known to control vesicular transport, we conclude that the vesicular transport system may be of particular importance during an immune response. Public Library of Science open access