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The potential of the biodegradable polymers have been realized in the orthopedic and cosmetic applications world, due to their remarkable ability to degrade in vivo, why there is no need for second surgery. Poly(lactide-co-glycolide) (PLGA) is one of the most studied and used biodegradable polymer in orthopedic field, and the aim of this study was to examine the water absorption rate of PLGA (according to ISO62:2008), and comparing different dehumidifying gases for the drying processes. The average absorption rate of the material was calculated to be for the process phase 2 samples 2.18 m-% and for the process phase 1 samples 0.51 m-% during 24 hours, in 100 % RH and at 23℃. In 50 % RH conditions the process phase 2 samples absorbed 0.74 m-% and for the process phase 1 samples 0.17 m-% during 24 hours. Since the production has under 50 % RH conditions, there should not be occurring any major degradation in these temperature values. According to the directional tests that had done, compressed dry air (CDA) could be potential alternative for the nitrogen gas in drying and preservating processes, since its capability to preserve moisture content of the samples was relatively similar to nitrogen. However, some advantages were noticed compared to nitrogen, such as, more efficient water removal capability of the CDA, due to reactivity of oxygen with the water molecules.

Diagnostic reference levels (DRLs) are pre-determined dose levels employed for patient dose management in diagnostic radiology. Cone-beam computed tomography (CBCT) has gained significant popularity in the imaging of the head and neck region eliciting the need for DRLs for CBCT examinations. The main objective of the study was to collect dose data for the establishment of indication-based national DRLs for CBCT examinations of the head and neck region in Finland as well as to form proposals for the prospective DRLs. In addition, examination frequencies, the use of optimised imaging parameters and the effect of manufacturer and model of the CBCT device on dose were studied. The study was conducted as a nation-wide dose survey among the Finnish facilities conducting CBCT examinations of the head and neck region. The response rate was 56% in terms of registered devices and 57% in terms of listed places of use. DRL proposals were made for seven indications. The values for the proposals were derived from the 75th percentile values of the indication-specific dose distributions. Very wide dose ranges were observed within all seven indications. The most common indication was pre-surgical imaging of implant treatments. A significant portion of the respondents had used default factory settings instead of optimising the imaging parameters. No particular manufacturer or device model stood out as producing exceptionally high or low doses regardless of the user. The Finnish DRLs for CBCT examinations will be issued based on the results of this study.

Lipid domains in plasma membranes are typically nanosized regions with a distinct lipid content. They have been suggested to have multiple functions. For example, the lipid environment can affect the activity of membrane proteins through direct lipid binding or by changing membrane properties. However, the detailed plasma membrane lipid organization and forces driving domain formation have remained elusive, since lipid domains are difficult to study due to their nanoscale size. In this thesis, we study the effects of three different G protein-coupled receptors (GPCRs) on the lateral and transmembrane lipid distributions. To this end, we use self-assembly through molecular dynamics (MD) simulations to study the lipid environment of the β_2-adrenergic receptor, the µ-opioid receptor, and rhodopsin proteins in plasma membrane models that account for the effect of 16 abundant lipid species. Our study reveals that GPCRs affect the lateral organization but not the transmembrane distribution of lipids. Polyunsaturated lipids and one monounsaturated lipid, phosphatidylinositol, are preferred to reside at the protein surface over other studied lipids. Thus, we conclude that GPCR proteins can induce domain formation in plasma membrane models. The results provide important insights into domain formation, which is important to better understand the functioning of GPCR proteins that are essential for human health.

Tämä diplomityö käsittelee implantoitavaa abioottista glukoosipolttokennoa, jota voidaan käyttää glukoosianturina tai ihmiseen integroidun elektroniikan teholähteenä. Polttokennon toiminta perustuu glukoosin hapettumiseen huokoisella anodilla elektrodimateriaalin katalyyttisen vaikutuksen vuoksi. Katodina toimii sileä elektrodi, ja se katalysoi hapen pelkistymistä vedeksi. Anodin ja katodin välille muodostuu jännite, mikäli ne ovat reaktiospesifejä. Tämän työn taustalla olevan polttokennon reaktiospesifiys on toteutettu huokoisen ja sileän pinnan avulla. Glukoosin hapettuminen on hidas reaktio, ja glukoosimolekyylit pääsevätkin diffuntoitumaan syvälle huokosiin. Hapen pelkistyminen on nopea reaktio, joten happi ei diffuntoidu huokosiin, vaan reagoi sileällä katodilla. Tämä työ esittelee ja selittää ilmiöitä, jotka vaikuttavat abioottisen glukoosipolttokennon toimintaan. Työn suurinta antia on se, että työssä on selvitetty niin kemialliset, fysikaaliset kuin myös fysiologiset ilmiöt niin tarkasti kuin mahdollista, sillä ne kaikki vaikuttavat polttokennoon, mikäli se implantoidaan ihmiskehoon. Työssä käsitellään lyhyesti myös polttokennoon liittyvää eettistä pohdintaa. Työn kokeellisena osuutena on reagoivien molekyylien diffuusion mallintaminen, polttokennon toimiessa anturina ja teholähteenä. Näiden kahden sovelluksen suurimpana erottavana tekijänä on elektrodien koko: Anturin tulee olla mahdollisimman pieni siten, että vielä saadaan luotettavia tuloksia glukoosikonsentraatiosta, kun taas teholähteen tuottaman tehon tulee olla riittävä. Riittävä tehontuotto varmistetaan riittävän suurella pinta-alalla. Lopputuloksena voidaan todeta, että reaktiota rajoittava tekijä on vereen liuenneen hapen konsentraatio. Molekyylien diffuusio rajoittaa reaktiota vielä lisää, kun hapen diffuusio katodille on hidasta. Anturikonfiguraatiossa käytettävät hyvin pienet elektrodit eivät rajoita diffuusiota juurikaan, mutta elektrodien kokoa kasvatettaessa huomataan, että diffuusionopeuteen vaikuttaa se, etteivät molekyylit pysty läpäisemään elektrodia, ja sen kiertäminen diffuusion avulla on hidasta. Voidaan myös todeta valitun mallinnusohjelman soveltuvan polttokennon tarkasteluun myös jatkossa. This is a Master’s Thesis about an implantable abiotic glucose fuel cell, which can be used as a glucose sensor or as a power source for bio-integrated electronics. The functionality of the fuel cell is based on oxidation of glucose molecules on the porous anode, which catalyzes the reaction. In this particular case, the cathode is a smooth surface, which catalyzes oxygen reduction reaction (ORR). If the anode and the cathode are reaction spesific, there will be a voltage between them. In this case the reaction specificity is achieved with a porous and a smooth electrode surface. Glucose oxidation reaction is a sluggish reaction, and the glucose molecules can diffuse into the pores of the anode. Oxygen reduction reaction is a fast reaction, which means that oxygen molecules cannot reach the pores, but reacts on the smooth surface. This thesis presents and explains phenomena affecting the function of the abiotic glucose fuel cell. The main point is to consider chemical, physical and physiological phenomena as precisely as possible, because the fuel cell is supposed to be implantable. There is also an ethical briefing to the bio-integrated electronics. The experimental part of the thesis is modeling the diffusion of the reactive molecules when the fuel cell acts as a sensor and as a power source. The main physical difference between these two applications is the size of the electrodes: The sensor should be as small as possible so that we can still get reliable information about the glucose concentration. When used as a power source, the area of the fuel cell must be large enough to provide enough energy to the electronics. As a result we can say that the most restraining factor is the concentration of oxygen dissolved into the blood. The next limiting phenomenon is diffusion of oxygen molecules to the cathode. The small electrodes used in sensor configuration do not limit the diffusion, but when the area of the electrodes is increased, we noticed that the molecules can no more diffuse past the electrode as quickly as with the smaller electrode. We can also recommend the modeling software for future modeling of glucose fuel cell.

Tutkimuksessa selvitettiin diabeteksen hoitoa tukevien ohjelmien käytettävyyttä sekä käytön vaikutusta koettuun omahoidon laatuun. Tutkimuksessa oli kaksi eri ryhmää, joista toinen ryhmä käytti kehitysvaiheessa olevaa Sensotrendin diabetespäiväkirjaa ja toinen ryhmä jatkoi aiemmin käyttämiensä ohjelmien käyttöä. Tutkimus toteutettiin yhteistyössä Tampereen teknillisen yliopiston, Sensotrend Oy:n sekä Tampereen kaupungin diabetesvastaanoton kanssa, ja siihen rekrytoitiin Tampereen diabetesvastaanotolta henkilöitä, joilla oli insuliinipumppuhoitoinen tyypin 1 diabetes. Tutkimukseen osallistuvat vastasivat tutkimuksen alussa, keskivaiheilla ja lopussa käytettävyyteen liittyvään kyselyyn. Molemmat ryhmät saivat myös tutkimuksen alussa ja lopussa täytettäväkseen diabeteksen omahoitoon liittyvän kyselylomakkeen. Tutkimuksen alussa ja lopussa mitattiin pituus, paino, vyötärönympärys sekä verestä HbA1c (mmol/mol). Teemahaastatteluita tehtiin yhdelle hoitohenkilökuntaan kuuluvalle sekä tavanomaisia ohjelmia käyttävälle tutkittavalle. Lisäksi päiväkirjasovelluksen kautta kerättiin lokitietoja päiväkirjan käytöstä. Tutkimus kesti kokonaisuudessaan 6-7 kuukautta. Yleisimmät käytössä olevat ohjelmat olivat Medtronic CareLink, AccuChek SmartPix ja Mendor Balance. Tutkimusryhmäläisillä oli käytössä Sensotrendin diabetespäiväkirja. Erilaisia laitteita tutkittavilla oli käytössä vain vähän, ja omahoidon seurannassa paperikirjanpitokin oli yleistä. Ohjelmien käytössä koettiin erilaisia vaikeuksia muun muassa laitteiden, yhteensopivuuden ja itse ohjelman käytön kanssa. Suurin osa tutkittavista molemmista ryhmistä antoi ohjelmille SUS-pisteitä alle 68, joka kertoo ohjelman olevan keskivertoa huonompi käytettävyydeltään. Diabeteksen hoito-ohjelmat eivät siis ole käytettävyydeltään käyttäjien haluamalla tasolla. Ideaaliseen omahoito-ohjelmaan toivottiin reaaliaikaisuutta, palautteen antamista, neuvoja sekä eri verensokeritasoon vaikuttavien tekijöiden seurantaa. Omahoidon laadussa tutkimuksen aikana tutkimusryhmässä tapahtui paranemista tuloksissa terveydenhuollon ja omahoidon aihealueisiin liittyvissä kysymyksissä. Transteoreettisessa muutosvaihemallissa tutkimusryhmäläiset siirtyivät tutkimuksen aikana kohti pysyvää omahoidon muutosta. HbA1c-arvot laskivat tutkimuksen aikana tutkimusryhmän naisilla sekä vertailuryhmän miehillä. Tutkimusryhmän miehillä sekä vertailuryhmän naisilla arvot puolestaan nousivat. Yhteenvetona voidaan todeta, että diabeteksen omahoidon ohjelmat vaativat vielä kehitystä monella osa-alueella. Tämänhetkisten omahoitosovellusten avulla voidaan seurata, millaiset verensokeritasot ovat olleet menneisyydessä ja useita eri verensokeriin vaikuttavia tekijöitä on hankala seurata yhdenaikaisesti. Tulevaisuudessa diabeetikoiden hoito-ohjelmien tulisi keskittyä nykyhetkeen sekä tulevaan hoitotasapainoon, joihin diabeetikko voi vaikuttaa senhetkisillä valinnoilla.

Bioactive glasses are a promising, relatively new material group to be utilized in regenerative medicine. The main target area of these materials is in bone tissue engineering, where commercial products are already used; in the form of as granules and pastes. In future, there is an ever increasing demand for artificial bone grafts having 3-D structure and high mechanical strength. Traditional bioactive glasses are silica-based. Despite their known biological properties, they tend to crystallize easily upon heat processing. In clinical studies it has been found that they do not completely convert to hydroxyapatite (HA); the substance which is replaceable by bones’ remodeling mechanism. Boron containing glasses may overcome these problems as they convert faster to HA and have enhanced thermal properties. The objective of the study was to assess if magnesium and strontium could further improve the properties of a borosilicate glass composition. In the study, part of the calcium was substituted with magnesium and/or strontium and the impact on (i) sintering and (ii) dissolution of the produced glasses studied. Six different glass compositions were prepared. A borosilicate glass with composition 47.1 SiO2 - 6.7 B2O3 - 22.7 Na2O - 21.8 CaO - 1.7 P2O5 in mol-% (labelled B12.5) was prepared and used as reference. This glass composition corresponds to the current commercial silicate bioactive glass, S53P4, where 12.5% of the SiO2 is replaced with B2O3. From this composition, glasses with different amounts of Mg and/or Sr were produced. The change in physical, structural and thermal properties, induced by the substitution, were studied. Sintering and crystallization was studied at various temperatures and analyzed with scanning electron microscopy (SEM) and X-ray diffraction (XRD). The bioactivity of the glasses was assessed by dissolution in simulated body fluid (SBF), for immersion time from 6 hours to 2 weeks. Upon dissolution the pH of the solution and the amount of ions leached out from the glass to the SBF was measured. Fourier transform infrared (FTIR) spectroscopy was utilized to evaluate HA formation on the glass surface. Results showed that Mg and Sr assumed similar structural role, in the glass network, as calcium. Mg and Sr seemed to improve the overall sintering behavior by decreasing the glass viscosity. Ions released from the glasses upon immersion decreased with increasing Ca substitution. The formation of HA layer appeared quite rapidly on all of the glasses. Mg and Sr are suspected to be introduced in the HA layer formed at the surface of the glass. The mixed glass (containing both Mg and Sr) seem to be the best candidate for future work on glass scaffold sintering for tissue engineering.

The cornea is a transparent tissue protecting the most anterior part of the eye and if it is damaged, it may result in a partial or complete loss of vision. Millions of patients are in need of donor cornea, but there is a substantial shortage of good-quality corneas and there is always a risk of tissue rejection when allogeneic tissues are transplanted. Tissue engineering offers a way to develop an artificial cornea using biomaterials together with the patients’ own cells to provide an alternative for conventional tissue transplantation. The objective of this thesis was to study and comprehend the current state of corneal tissue engineering research and investigate which biomaterials could be suitable for the purpose of creating a biomaterial-based corneal substitute. In this thesis, the experimental part concentrated on studying hydrogels with corneal cells. In the experimental part, the behaviour of immortalised human corneal epithelial cells (HCE) and stromal keratocytes (HCK) in 2D and 3D cultures were studied. The aim of the 2D cell culture study was to test how these two cell lines behave in chosen different culture medium options, three commercially available media (CnT-20, CnT-30 and CnT-Prime-CC) and HCE and HCK maintenance media. The best suited media were then used in 3D cell culture with hydrogels. There were hydrogels A, B, C and D and one commercial hydrogel. These were tested in 3D cell culture where keratocytes were mixed with the hydrogel and epithelial cells were seeded on top. The hydrogel-cell samples were cultured in CnT media for 7 to 14 days and air-lifted for 7 days for epithelial cell stratification. Cell proliferation in 2D culture was evaluated with PrestoBlue™ assay and cell behaviour and expression of relevant corneal proteins were analysed with immunofluorescence staining (IF). These results indicated CnT-20 to be the best option for both cell lines, but all three CnT media were tested also in 3D cell culture with hydrogels. The hydrogel experiments were also evaluated with IF staining. Interestingly, the most suitable medium for 3D cell culture was CnT-Prime-CC. The best suited hydrogel option in this study was hydrogel A and resulting cell-hydrogel samples were thin and soft but had clear, transparent and even structure.

Dimensionality reduction is a commonly used method in engineering sciences, such as control theory, for improving computational efficiency of simulations of complex nonlinear mathematical models. Additionally, it is a way of surfacing the most important factors that drive the dynamics of the system. In the field of neuroscience, there is a great demand to incorporate molecular and cellular level detail in large-scale models of the brain in order to produce phenomena such as learning and behavior. This cannot be achieved with the computing power available today, since the detailed models are unsuitable for large-scale network or system level simulations. In this thesis, methods for mathematical model reduction are reviewed. In the field of systems biology, models are typically simplified by completely eliminating variables, such as molecules, from the system, and making assumptions of the system behavior, for example regarding the steady state of the chemical reactions. However, this approach is not meaningful in neuroscience since comprehensive models are needed in order to increase understanding of the target systems. This information loss problem is solved by mathematical reduction methods that strive to approximate the entire system with a smaller number of dimensions compared to the original system. In this study, mathematical model reduction is applied in the context of an experimentally verified signaling pathway model of plasticity. The chosen biophysical model is one of the most comprehensive models out of those that are currently able to explain aspects of plasticity on the molecular level with chemical interactions and the law of mass action. The employed reduction method is Proper Orthogonal Decomposition with Discrete Empirical Interpolation Method (POD+DEIM), a subspace projection method for reducing the dimensionality of nonlinear systems. By applying these methods, the simulation time of the plasticity model was radically shortened although approximation errors are present if the model is reviewed on large time scales. It is up to the final application of the model whether some error or none at all is tolerated. Based on these promising results, subspace projection methods are recommended for dimensionality reduction in computational neuroscience.

In this MSc thesis we studied how deep learning methods can be applied to class prediction of complex disorder tasks with gene expression data. Microarrays and sequencing can generate representations of expression of different biomolecular molecules in samples. With appropriate machine learning methods and data we can build classifiers that can handle various classisscation tasks with many practical applications. Deep belief networks are our principal deep learning models. We carried out tests to see how they perform alone and with support vector machines combined. We compared two different optimization algorithms, backpropagation and resilient backpropagation, that are used at the fine-tuning stage of learning. The three example data sets are composed of lung cancer, breast cancer and inflammatory bowel disease samples. For assessment of performance we used leave-one-out cross-validation with accuracy, sensitivity and specificity as performance metrics. Moreover, we computed the standard error of the mean for each metric. In order to make the results more credible and interesting, we compared them with similar previous studies. Our cross-validation results and comparison with previous studies show that we achieved good or excellent performances for most of the tasks. A remarkable aspect is that we in general omitted prior use of feature selection and dimensionality reduction that have been used previously almost invariably. The resilient backpropagation algorithm worked with whole microarray data sets, whereas the basic backpropagation algorithm worked well with whole RNA-Seq data sets and feature selected data.

Hydrogels differ from many other cell culture substrates in that they provide a three-dimensional, soft environment for cells, opposed to a two-dimensional surface on which cells are traditionally cultured. This geometry better represents the in vivo environment of cells. In addition, hydrogels are easily modified for enhanced cell viability and growth. Therefore, new hydrogels are continuously developed for cell culture and tissue engineering purposes. Applicability of a hydrogel as cell culture substrate depends on several chemical and physical attributes. Together these attributes affect cytocompatibility of the hydrogel, i.e. cell attachment, viability and cell specific response. The objective of this thesis was to create a protocol for first-step, short-term cell culture screening of new hydrogels. Cytocompatibility testing and analysis methods need to be adjusted for macroscopic 3D hydrogels compared to 2D culturing systems. Another challenge in this screening is adequate stability of cells and their representativeness of the cells intended to be used in the final application. An interview was conducted with Regenerative Medicine professionals in BioMediTech Institute (University of Tampere) who are working with cardiomyocytes, bone and cartilage cells, corneal and retinal epithelial cells, and neuronal cells. Knowledge was gathered on the properties of representable cell models of each cell type and on tissue-specific aspects of analyzing. A commercial human embryonic fibroblast cell line WI-38 was chosen as a representative first-step cell model and acquired for the first-step cytocompatibility testing. In addition, tissue specific cell lines were recommended for further testing. Cells were cultured ontop and encapsulated in PuraMatrix® and gellan gum hydrogels, and on poly(dimethylsiloxane) (PDMS) and uncoated well plate. Cells were stained with Live/dead viability/cytotoxicity kit. The suitability of Nikon BioStation CT cell culture observation system was evaluated for the screening of hydrogels and compared with two other imaging systems. It was possible to assess cell attachment and viability from fluorescence images taken with BioStation CT, even though it was not an optimal imaging system for 3D hydrogel screening. As the result of this thesis, a first-step cytocompatibility testing protocol was created for assessing attachment and viability of cells that were cultured on top and encapsulated in hydrogels. Hydrogeelit eroavat monista muista soluviljelyalustoista niin, että ne tarjoavat soluille kolmiulotteisen, pehmeän kasvuympäristön. Perinteinen kasvualusta on sen sijaan kaksiulotteinen ja kova pinta. Hydrogeelien kolmiulotteinen (3D) ympäristö kuvastaa paremmin ympäristöä, jossa solut kasvavat kehossa. Lisäksi hydrogeelit ovat helposti kemiallisesti muokattavissa, jolloin solujen kasvua ja elinkykyisyyttä materiaalissa voidaan parantaa. Uusia hydrogeelejä kehitetäänkin jatkuvasti soluviljelyn ja kudosteknologian tarpeisiin. Hydrogeelin soveltuvuus soluviljelyn kasvualustaksi on riippuvainen monista sen kemiallisista ja fysikaalisista ominaisuuksista. Nämä ominaisuudet vaikuttavat yhdessä hydrogeelin sytokompatibiliteettiin, eli solujen kiinnittymiseen, elinkykyisyyteen ja solutyypistä riippuvaan vasteeseen. Tämän diplomityön tarkoituksena oli kehittää lyhytaikaiseen soluviljelyyn perustuva protokolla uusien hydrogeelien alkuvaiheen seulontaan. Koe- ja analysointimenetelmiä on säädettävä sytokompatibiliteetin testaamiseksi, kun soluja kasvatetaan 3D-materiaalissa verrattuna kaksiulotteisella materiaalilla kasvattamiseen. Lisäksi seulonnan haasteena on solujen riittävä stabiilius sekä edustavuus verrattuna soluihin, joita on tarkoitus käyttää lopullisessa sovelluksessa. Diplomityössä haastateltiin BioMediTech -instituutin (Tampereen yliopisto) Regeneratiivisen lääketieteen tutkimusryhmiä, jotka tutkivat kardiomyosyyttejä, luu- ja rustosoluja, verkkokalvon ja sarveiskalvon soluja sekä hermoston soluja. Haastattelussa kerättiin tietoa kutakin solutyyppiä edustavien solujen ominaisuuksista sekä näiden solutyyppien analysoimisesta. Kaupallinen ihmisen embryonaalinen fibroblastisolulinja WI-38 valittiin edustavaksi ensimmäisen vaiheen solumalliksi ja se hankittiin ensimmäisen vaiheen sytokompatibiliteettitestausta varten. Lisäksi jatkotutkimuksia varten ehdotettiin kudoskohtaisia solulinjoja. Soluja kasvatettiin PuraMatrix®- ja gellaanikumihydrogeelien päällä ja sisällä, sekä polydimetyylisiloksaanilla (PDMS) ja päällystämättömällä kuoppalevyllä. Solut värjättiin Live/dead viabiliteetti/sytotoksisuus -kitillä. Nikon BioStation CT -kuvantamisjärjestelmän soveltuvuutta hydrogeelien seulontaan arvioitiin, ja sitä verrattiin kahteen muuhun kuvantamisjärjestelmään. BioStation CT:n avulla saaduista fluoresenssikuvista oli mahdollista määrittää solujen elinkykyisyys ja kiinnittyminen, mutta se ei kuitenkaan ollut optimaalinen 3D-hydrogeelien seulontaan. Diplomityön tuloksena tuotettiin sytokompatibiliteetin testausprotokolla, jota voidaan käyttää hydrogeelien päällä ja sisällä kasvatettujen solujen kiinnittymisen ja elinkykyisyyden määrittämiseksi.

Microcopy has always been an important tool to increase our knowledge about structural elements that are responsible for the functional outcome of living organisms. Unraveling the relationship between the structure and function is of crucial importance for our understanding e.g. normal and pathophysiological events. “Structure without function is dead and a function without a structure is a ghost”, summarizes the importance of this relationship. In this thesis, the main principles of the microscopes used nowadays in life sciences are described followed by a discussion about the needs met by now and those that can be predicted to be also major issues in the future. The final aim is to find ideas to develop a new microscopic imaging system. It is already 400 years since the first microscope was used. However, the major innovations and development in microscopy were done during the last 50-60 years. Highlights can be set on novel fluorescent probes and innovative methods utilizing signal processing and computation with which light microscopy has been pushed over the diffraction limit of light i.e. 200 nm. Super resolution microscopy provides detections of structures, complexes and individual proteins by fluorescent probes with a resolution around 10 nm. It is a powerful tool for both spatial and temporal imaging of living organisms. However, the major limitation is that laser beam techniques require structures to be labeled with fluorescence probes and, thus, visualizes the probe and not the structural element itself. Transmission electron microscopy, on the other hand provides super resolution (up to 0.14 nm) images about the structure and its components directly. Unfortunately, it is still practically impossible to image temporal events in living cells with electron microscope. Thus, we still have a big gap between light and electron microscopy. It is suggested in the current thesis that the microscopic systems that are based on second or third harmonic generation combined with lens free tomographic imaging and/or 4D ultrafast electron microscopy could be building blocks for a new type of microscope.

Kudosteknologia pyrkii korjaamaan tai luomaan uusia kudoksia ja elimiä vaurioituneiden tilalle. Se hyödyntää biohajoavia tukirakenteita (skaffoldeja), soluja ja kasvutekijöitä yksinään tai yhdessä. Eräs lupaava kudosteknologian menetelmä alkaa solunsyötöllä, jossa huokoiseen skaffoldiin istutetaan soluja ja rakennetta kasvatetaan in vitro ennen sen implantoimista vaurioalueelle. Solunsyötön pitäisi johtaa tasaiseen solujakaumaan skaffoldin sisällä - muuten rakennetun kudoksen toiminnollisuus ja mekaaniset ominaisuudet saattavat jäädä odotettua huonommiksi. Jotta arvokkaita soluja ei hukattaisi, solunsyöttöprosessin tulisi olla myös tehokas. Tämä edesauttaa myös nopeampaa kudoksen muodostumista. Työssä testattiin kuutta erilaista solunsyöttömenetelmää tarkoituksena optimoida syöttömenetelmä kahdelle ylikriittisellä hiilidioksidilla prosessoidulle skaffoldityypille. Näistä toinen oli poly(L-laktidi-ko-ε-kaprolaktoni) 70/30 (PLCL) -kopolymeeristä valmistettu ja toinen PLCL:n sekä β-trikalsiumfosfaattikeraamin komposiitti. Solunsyöttömenetelmien toimivuus varmistettiin kahdella poly-L/D-laktidi 96L/4D (PLDLA 96/4) kuiduista valmistetulla skaffoldityypillä. Lisäksi tutkittiin uudenlaista solunsyöttömallia, joka pohjautuu rautaa sisältäviin mikropartikkeleihin. Mallissa soluja jäljiteltiin halkaisijaltaan 15 µm ja 100 µm kokoisilla partikkeleilla. Aikuisen rasvan kantasoluja käytettiin solukokeissa, koska niillä on valtava potentiaali ruston ja luun kudosteknologiassa. Tutkimuksissa mikropartikkelisolunsyöttömallin huomattiin toimivan ja tarjoavan hyödyllistä tietoa skaffoldien rakenteesta ja sen soveltuvuudesta solujen istutukseen. Rautapartikkelien jakaumat vaikuttivat tosin tasaisemmilta kuin vastaavien solunsyöttökokeiden tulokset. Solunsyöttömalli osoitti myös komposiittiskaffoldien rakenteen olevan solunsyötön suhteen haasteellisempi kuin vastaavien PLCL skaffoldien. Mikro-CT kerroskuvauksen avulla pystyttiin havaitsemaan myös nanokokoluokan rautaoksidipartikkeleilla leimattujen solujen sijainti skaffoldien sisällä. Tämän perusteella huomattiin, että tavallisella ruiskulla voitiin pakottaa solususpensiota PLCL skaffoldien sisään ja parantaa siten solujen tasaista jakautumista verrattuna muihin menetelmiin. Johtuen teknologisista rajoitteista solunsyötön onnistumisen arviointi komposiittiskaffoldeissa oli haasteellista. Solunsyöttöä rautapartikkeleilla mallinnettaessa huomattiin niiden rakenteen olevan kuitenkin hieman vaikeampi solujen istutusta ajatellen. Perinteinen staattinen solujen pipetointi skaffoldin pinnalle oli riittävä PLDLA kuituskaffoldeissa, tuottaen tasaisen solujakauman ja tyydyttävän solumäärän skaffoldeissa. Tissue engineering aims to regenerate or create damaged or lost organs and tissues by utilizing biodegradable scaffolds, cells and growth factors. One promising tissue engineering strategy involves seeding cells into a porous scaffold and culturing it in vitro, which is followed by implantation into the defect site. Cell seeding should result in a uniform distribution of cells inside the scaffold - otherwise the functionality and mechanical properties of the engineered construct can be compromised. Also a high seeding efficiency is appreciated to avoid wasting valuable cells and to enable faster tissue formation. The aim of this study was to test six different cell seeding methods in order to find an optimal method for supercritical carbon dioxide (ScCO2) processed scaffolds. Of these scaffolds, one was a copolymeric poly(L-lactide-co-ε-caprolactone) 70/30 (PLCL) scaffold, whereas the other one was a composite of PLCL and β-tricalcium phosphate. The functionality of the cell seeding methods was verified with two scaffold types that were manufactured from poly-L/D-lactide 96L/4D (PLDLA 96/4) fibers. In addition, a novel cell seeding model utilizing iron-labeled microspheres with diameters of 15 µm and 100 µm was proposed. Adipose-derived stem cells (ASC) were used in the experiments due to their potential in hard-tissue engineering. The study revealed that the microsphere seeding model is functional, offering useful information about the seedability of the ScCO2 processed scaffolds. The microsphere distributions were noticed to be more uniform compared to the corresponding cell seeding results. The microsphere model also suggested more challenging seedability of the composite scaffolds compared to the PLCL substrates. Applying micro-computed tomography (micro-CT) imaging and seeding ASCs fed with iron oxide nanoparticles, it was noted that the uniformity of the cell distribution in ScCO2 processed PLCL scaffolds can be enhanced by forcing the cell suspension into the scaffold with a syringe. Due to technological limitations, evaluating cell seeding in the composite scaffolds was more challenging. However, the cell experiments supported the microsphere model, indicating a more difficult seedability compared to the PLCL scaffolds. A static pipetting of cells on top of the PLDLA fabrics was enough to provide desirable cell distribution and cell numbers in those scaffold types.

Tämän diplomityön tarkoituksena oli kehittää paremmaksi jo olemassa olevaa lääkeluovutussysteemiä, joka perustuu kahden valmistusmenetelmän yhdistelmää käyttäen kehitettyihin, deksametasonia (DEX) sisältäviin, biohajoaviin poly (laktidi-co-glykolidi) (PLGA) mikropartikkeleihin. Myös gammasäteilytystä mahdollisena mikropertikkeleiden sterilointimenetelmänä tutkittiin. Työssä oli myös tarkoituksena hidastaa lääkeluovutusta entisestään ja minimoida alun nopea lääkeaineen vapautuminen kehittämällä hydrogeeli-mikropartikkelikomposiitti käyttäen aldehydi-modifioitua hyaluronihappoa (HA), ja ristisilloittajana hydrazidi-modifioitua PVA:a. Lisäksi komposiittigeelin mekaanisia ominaisuuksia tutkittiin puristuskokeilla. Deksametasonin lisäksi, myös uuden, tähän mennessä lääkeluovutussysteemeissä käyttämättömän lääkeaineen, muraglitazarin, kapselointia mikropartikkeleihin tutkittiin. PLGA-PVA-DEX mikropartikelisysteemia onnistuttiin parantamaan. Prosessin saantoprosentti nousi noin 60 %, ja mikropartikkeleiden pienestä koosta huolimatta hitaampi, neljän viikon aikana tapahtuva lääkeluovutus saavutettiin. Pidennetyt lääkeluovutuskokeet paljastivat odottamattoman ongelman, deksametasonin hajoamisen, joka johti uuden analysointimenetelmän kehittämisen korkean erotuskyvyn nestekromatografialle (HPLC). Gammasteriloinnilla oli haitallinen vaikutus mikropartikkeleiden morfologiaan ja lääkeluovutukseen. Hydrogeeli-mikropartikkelikomposiitti käyttäytyi puristuskokeessa samalla tavalla kuin kontrolligeeli. Lääkeluovutusprofiilin muuttaminen kuitenkin epäonnistui. Sen sijaan mahdollinen hydrogeelin deksametasonia stabiloiva vaikutus paljastui. Myös muraglitazarin kapselointi PLGA-PVA mikropartikkeleihin onnistui täysin samaa valmistusmenetelmää käyttäen. Lääkeluovutus mikropartikkeleista tapahtui viikon kuluessa. Johtopäätöksenä voidaan todeta, että hieman muunneltu, emulsio/liuottimen haihdutus- ja suihkukuivausmenetelmän yhdistelmä on sopiva lääkeainetta sisältävien mikropartikkeleiden valmistukseen. Gammasterilointia ei suositella sen haitallisten vaikutusten vuoksi. In vitro lääkeluovutus mikropartikkeleista on suhteellisen nopea, joten lisätutkimuksia lääkeluovutusprofiilin hidastamiseksi tarvitaan. Erityisesti hydrogeelin käyttöä lääkeluovutusprofiilin muuttamiseen suositellaan, sillä tässä tutkimuksessa saatiin kehitettyä hydrogeeli-mikropartikkelikomposiittimateriaali hyvillä ominaisuuksilla ja suuremmalla mikropartikkelipitoisuudella. The aim of this thesis was to improve already developed drug release system for model drug dexamethasone (DEX), in which biodegradable, poly (lactide-co-glycolide) (PLGA) – polyvinyl alcohol (PVA) microparticles for drug release was designed, using combination of two preparation methods. Gamma irradiation as a sterilization method for the microparticles was also studied. There were also attempt to sustain the release rate even more and minimize the burst release of DEX by designing hydrogel-microparticle composite using aldehyde-modified hyaluronic acid (HA) crosslinked by hydrazide-modified PVA. Also mechanical properties of the composite were determined with compression test. In addition to DEX, encapsulation of a second drug, muraglitazar was investigated. Until now, muraglitazar has not been used in any drug delivery device. Improvement of the existing PLGA-PVA-DEX microparticles succeeded. Yield of the spray dried microparticles increased approximately 60 %. Despite of smaller size of the microparticles, slower drug release rate, during four weeks, was achieved. However, prolonged drug delivery tests revealed unexpected problem, degradation of the DEX in aqueous solution, and analytical problems. It resulted in a need to design the new method for analyzing DEX with high performance liquid chromatography (HPLC). Gamma irradiation was proved to have adverse effects to the microparticle morphology and also drug release profile. Hydrogel-microparticle composite showed very similar mechanical properties with control hydrogel. Unfortunately, modification of the drug release profile of DEX failed. Still, analysis of drug release samples from composite gel denoted possible stabilization of the DEX in presence of hydrogel or one of its components. Hydrophobic drug, muraglitazar was successfully encapsulated in PLGA-PVA microparticles using the same method. Drug was released from the microparticle within one week. As a conclusion, slightly modified preparation method combining emulsion method and spray drying is suitable for producing drug loaded microparticles with improved properties. Gamma sterilization has adverse effects to the microparticles and is therefore not recommended to use for sterilization. In vitro drug release from the particles is relatively fast, so further investigation is needed to modify drug release profiles. Especially modifying with hydrogel is recommended, as hydrogel-microparticle composite with good properties and larger amount of microparticles was developed in this study.

Tämän diplomityön tavoitteena oli etsiä keinoja elintarvikealan yrityksen jätehuollon kehittämiseen, tutkia jätelainsäädännön muutosten vaikutuksia jätehuoltoon ja selvittää vaihtoehtoisia käsittelymenetelmiä kohdeyrityksessä muodostuville jätteille kaatopaikkasijoituksen ja energiahyödyntämisen sijaan. Tarpeen jätehuollon kehittämiselle antoivat muutokset jätelainsäädännössä, joista työn kannalta oleellisimpana ovat vuonna 2016 voimaan astuvat rajoitukset biohajoavan ja muun orgaanisen jätteen sijoittamisesta kaatopaikalle. Työn tarkoituksena oli tutkia näiden rajoitusten vaikutuksia jätehuoltoon ja kehittää jätehuoltoa etusijajärjestyksen mukaisesti lisäämällä jätteiden materiaalihyödyntämistä. Vaaralliset jätteet on jätetty tarkastelun ulkopuolelle. Kehitystarpeiden määrittelemistä varten tarkasteltiin kohdeyrityksen jätehuoltoa tutustumalla tuotantolaitoksiin, tutkimalla jätteen määrää ja lajittelua, suorittamalla henkilökunnan haastatteluja sekä tutkimalla jätehuollon kirjallista aineistoa. Alkukartoituksen tietojen pohjalta arvioitiin jätehuollon nykytilaa ja annettiin kehitysehdotuksia. Lisäksi arvioitiin, miten jätehuollon etusijajärjestyksen toteutumista voitaisiin tehostaa ja miten se vaikuttaisi lajitteluun ja jätehuollon toimintaan käytännössä. Muovijätteen erilliskeräys ja sen toimittaminen kierrätykseen on tärkein jätteiden materiaalihyödyntämisen astetta nostava tekijä. Työhön kuului muovijätteen erilliskeräyksen kokeilu, jonka avulla saatiin tietoa kohdeyrityksessä muodostuvan muovijätteen määrästä ja laadusta. Olennaisena osana työhön kuului lisäksi jätehuollon muutoksista aiheutuvien kustannusten arviointi. Arvioitiin, että kohdeyrityksen jätehuolto toimii pääsääntöisesti hyvin. Kehitysehdotuksia annettiin muun muassa jätteiden sisäiseen logistiikkaan, jäteastioiden merkintöihin ja lajitteluohjeisiin liittyen. Kustannuslaskelmien mukaan kohdeyritykselle edullisin vaihtoehto toimia jätehuollon uudistuksissa on toimittaa nykyinen kaatopaikalle ohjattava jäte jätteenpolttolaitokseen ja jatkaa energiajätteen lajittelua toimitettavaksi kierrätyspolttoaineen raaka-aineeksi. Muovijäteselvityksen ja kustannuslaskelmien perusteella on kannattavaa aloittaa LDPE-, HDPE- ja muovilaminaattien erilliskeräys ja toimittaminen materiaalihyötykäyttöön. Selvityksen mukaan kohdeyrityksessä muodostuu arviolta vähintään 83 tonnia muovijätteitä vuodessa, josta lähes kaikki pystytään kierrättämään. Ongelmia muovijätteen kierrättämiselle saattavat aiheuttaa jätevirran heterogeenisyys ja likaisuus, jonka vuoksi oikeanlainen syntypaikkalajittelu on erittäin tärkeää. Erilliskeräyksen aloittaminen lisää aluksi jätehuollon kustannuksia, mutta pitkällä aikavälillä tuo säästöä, sillä jätteen kierrättäminen materiaalina on edullisempaa kuin hyödyntäminen energiana.

The modern neuroscience aims for more and more detailed understanding of the reasons, which form the basis for search for cure or diseases related to the brain. Today we know that brain consists of multiple different cell types and functional organs ranging from blood vessels to glia cells and neurons. Each cell type has its specific functions which can be for example structural, immunological or signaling. To study the functions of different living systems, it is often necessary to disturb the normal function to understand the underlying mechanisms. For this reason, methods for avoiding the use of model animals have been developed. One of the tools available for this is in silico modeling of the systems. For this work, I have developed a computational model of a joint function of two cells types by combining existing models. Neurons form a neuronal network which is modeled using INEX by Kerstin Lenk (Lenk, 2011). The other cell type called astrocytes are modeled by Lallouette’s UAR model (Lallouette, De Pittà, Ben-Jacob, & Berry, 2014) and a near synapse simulator interface together with De Pittá’s presynapse model (De Pittà, Volman, Berry, & Ben-Jacob, 2011). The combined model is called INEXA. We present this simulation scheme in an attempt to model the interactions of astrocyte network and neuronal network. Eighteen simulations were run to test different stages of the simulator. In phase one, the simulator INEX was run so that neurons had no synaptic strengths to get noise output of the basic activity driving the network. In phase two, Tsodyks-Markram presynapses were added to the neuronal network. In phase three, the presynapse area simulation including local astrocytes was added, and in phase four, the full INEXA was tested with 10%, 20% and 30% of the simulated culture being astrocytes. The results show that the interconnected simulated astrocyte-neuron networks have properties that induce population bursting behavior while restricting hyperactivity. As such the astrocytes could be related to modulating signaling patterns towards bursting and restricting epileptic behavior.

Tutkimustulosten kaupallistamista pidetään yhtenä yliopistojen perusfunktioista. Tällä funktiolla tarkoitetaan prosessia, jossa yliopistotutkija osallistuu yhteistyöhankkeeseen tuottamallaan uudella tiedolla, joka myöhemmin materialisoituu kaupalliseksi hyödykkeeksi. Tutkimustulosten kaupallistamisprosessissa sekä yliopistoilla että yrityksillä on ollut selkeät roolit, mutta raja tutkimuksen, kehittämisen sekä kaupallistamisen välillä on häviämässä. Molemminpuolista ymmärrystä yliopistojen ensisijaisesta tehtävistä sekä yksityisen ja julkisen toiminnan välisistä eroista pidetään yhteistyön onnistumisen edellytyksenä. Tässä diplomityössä yliopistojen sekä yritysten muuttunutta työnjakoa tutkittiin lääkinnällisiä laitteita sekä in vitro -diagnostiikkaan tarkoitettuja lääkinnällisiä laitteita (IVD -laite) koskevien tutkimustulosten kaupallistamisprosessin kautta. Tutkimuksen tarkoituksena oli selvittää, minkälaista laatuun ja viranomaisvaatimuksiin liittyvää dokumentaatiota yliopistojen tulisi tuottaa, jotta yliopistossa kehitetty lääkinnällisen laitteen tai IVD -laitteen tuoteaihio olisi entistä otollisempi kaupallistettavaksi erityisesti Euroopan talousalueella. Tutkimus suoritettiin haastattelemalla lääkinnällisten laitteiden sekä IVD-laitteiden alan tutkijoita sekä alan yrityksiä, jotka olivat joko yliopistojen yhteistyökumppaneita tai yliopistosta irtaantuneita spin-off -yrityksiä. Haastattelun otos koostui yhteensä 12 haastateltavasta organisaatiosta. Haastattelututkimuksen tulosten mukaan lääkinnällistä laitetta tai IVD -laitetta kaupallistavat organisaatiot arvostaisivat eniten asiakasvaatimuksia sekä teknisiä vaatimuksia kuvaavaa dokumentaatiota, sillä ne kertovat asiakaskysynnästä sekä laitteen yleisestä toteutettavuudesta. ISO 13485 laadunhallintastandardin noudattamattomuutta ei pidetty esteenä yliopistossa kehitetyn lääkinnällisen laitteen tai IVD -laitteen tuoteaihion kaupallistamiselle. Tuoteaihion kaupallistamista voitaisiin kuitenkin edesauttaa harjoittamalla erityisesti riskinhallintaa, sillä sitä pidetään yhtenä tärkeimpänä laadunhallinnan osiona ja sen merkitys tulee kasvamaan uudistuvien viranomaisvaatimusten myötä.

This thesis is a numerical evaluation of SAR compliance assessment with realistic exposure conditions using accurate human and mobile phone models. Four hand models with four adult and five child head models were simulated with two accurate Nokia mobile phone models, 6630 and 8310. In total over 400 simulations were run with comparable settings to achieve possibility to consider different aspects appearing in the results. No systematic differences between models are observed, however some differences between different anatomical models, which in some cases seem to be related to the distance of the source and tissues in the cheek area, are observed. Most of the differences seen in the results are also related to the hand holding the device. Our results do generally confirm the SAM phantom conservativeness in the tested configurations. Important observations about phone material assignment and the connection of head and hand voxels in the simulation domain were made during the process.

Prostate cancer is the most common cancer in Western countries. As the disease proceeds to castration resistant phase, it often leads to death due to lack of effective treatment methods and lack of knowledge of molecular mechanisms leading to it. Migration and invasion inhibitory protein (MIIP) has been shown to have tumor suppressing effects in other cancers. The effect of it in prostate cancer has not been previously studied. The aims of this study were to discover the effect of MIIP on growth and migration of prostate cancer cells and to detect possible mutations and a single nucleotide polymorphism (SNP) rs2295283 previously shown to affect breast cancer risk from the gene. DU145 and LAPC-4 prostate cancer cell lines were used. DU145 cells were stably transfected with a plasmid containing MIIP and an empty control plasmid. LAPC-4 cells were transfected with small interfering RNAs (siRNAs) targeting MIIP and a control siRNA. Proliferation of the cells was studied with image analysis and alamarBlue methods. Migration was studied with scratch assay. Mutations and SNPs were studied by sequencing. Clear results from the effects of silencing MIIP in LAPC-4 cells were not achieved. In the proliferation assays, image analysis and alamarBlue methods gave contradictory result and due to difficulties in image analysis, results from alamarBlue should be considered more reliable. AlamarBlue assay showed the metabolic activity of DU145 cells overexpressing MIIP to be significantly lower than in cells transfected with control vector. However, DU145 cells overexpressing MIIP migrated significantly faster that cells transfected with control vector. Mutations in the gene were not detected and SNP rs2295283 was not found to be associated with prostate cancer risk. It seems unlikely that MIIP would have a significant effect in most prostate cancer cases. However, it is possible that the role of MIIP is more notable in some of prostate cancer subtypes. More experiments are needed to fully uncover the effect of MIIP in prostate cancer cells.

Native cartilage has very little capacity for self-healing and even the current clinical methods have limited ability to regenerate functional cartilage. In the recent decade research related to cartilage repair has been increasingly focused on tissue engineering solutions that offer scaffold-based strategies for new cartilage formation. The combination of collagen (COL) with two glycosaminoglycans, chondroitin sulfate (CS) and hyaluronic acid (HA) has received widespread interest because all three are naturally abundant in the native cartilage tissue. The objective of this thesis was to fabricate and characterize COL, HA and/or CS containing scaffolds applicable for cartilage tissue engineering. Porous 3D scaffolds were fabricated by freeze-drying and cross-linked with either 1-ethyl-3-(3-dimethylaminopro-pyl)-carbodiimide hydrochloride / N-hydroxysuccinimide (EDC/NHS) or genipin (GP). Fabricated COL+CS/HA and COL+CS+HA scaffolds were characterized by compression and water uptake testing, Fourier transform infrared (FTIR) spectroscopy and micro-computed tomography (micro-CT) imaging. In general, the 80 wt.% COL containing composite scaffolds endured fabrication and both cross-linking procedures better than the 60 wt.% COL containing composite scaffolds. Water uptake ability was higher in GP cross-linked versus EDC/NHS cross-linked scaffolds, in the 80 wt.% COL containing versus 60 wt.% COL containing scaffolds and in COL+HA versus COL+CS composite scaffolds. The swelling/shrinkage upon water uptake of all of the scaffolds was below 20%; in most cases around 10%. FTIR spectra confirmed successful cross-linking with both GP and EDC/NHS and micro-CT images revealed highly porous microstructure (88-94%) with interconnected pores (pore sizes 26-57 μm) in all the scaffolds. The dry scaffolds had significantly higher compressive modulus than the corresponding wet scaffolds; the difference being bigger with GP cross-linked scaffolds. In case of both dry and wet scaffolds the three highest compressive modulus values were measured from CS containing scaffolds. Two scaffold groups with the lowest compressive modulus contained COL and HA. Both wet and dry scaffolds recovered well from compression. This thesis demonstrated successful incorporation of CS and/or HA to COL in order to fabricate a highly porous freeze-dried 3D scaffold applicable for cartilage TE. In addition, the novel crosslinker GP proved to be a promising alternative to the conventional EDC/NHS crosslinker.

Prostate cancer is a highly common disease in the Western countries with its prevalence growing every year with the standard of living and life expectancy. Despite extensive studies prostate cancer presents still considerable challenges to patient care, scientists and societies. As still no curing treatment exists for the recurrent form of the disease prostate cancer is likely to remain an intensive subject for study also in the future. Even though numerous studies exists that try to characterize individual components and pathways of regulation, no real attempts have been made to characterize the regulation profile of prostate cancer in a genome-wide level. This thesis forms a basis for this ambitious feat by using computational methods in transcription factor mediated regulation prediction and network inference. DNase I hypersensitivity guided genome scanning with transcription factor specific position weight matrices is performed in several manners, producing a catalogue of putative regulation connections in LNCaP cells. The predicted connections are subjected to thorough evaluation and several applications of the first method are presented, forming a strong set of basic guidelines for computational prediction based regulome assembly for future studies. In addition, as a further application gene expression data is applied to selectively extract a subnetwork of regulation present exclusively in the studied LNCaP cells. As a conclusion the genome-wide computational regulome assembly is presented as a functional but challenged approach with enormous potential due to its possible applications as cell type or disease specific network profiling tool.

Rikastushiekka-alueelta suotautuvat sulfaattipitoiset vedet ovat aiheuttaneet rehevöitymistä Siilinjärven fosfaattikaivoksen lähijärvissä. Yksi tapa puhdistaa sulfaattipitoisia vesiä on käyttää biologiseen sulfaatinpelkistykseen perustuvia reaktiivisia seinämiä. Hapettomissa oloissa sulfaatinpelkistäjäorganismit pelkistävät sulfaattia sulfidiksi, joka muodostaa metallien kanssa niukkaliukoisia saostumia. Näin nämä rikkiä sisältävät yhdisteet jäävät seinämärakenteen sisään ja saadaan poistettua vedestä. Tässä työssä tutkittiin mahdollisuutta puhdistaa Siilinjärven kaivoksen rikastushiekka-alueilta suotautuvia vesiä biologisella reaktiivisella seinämällä. Tutkimus suoritettiin paikan päällä käyttäen pilot-mittakaavan reaktoria, joka täytettiin sulfaatinpelkistäjien elektronidonoriksi soveltuvalla reaktiivisella materiaalilla. Reaktiivisen materiaalin koostumus oli: 45 % soraa vedenläpäisevyyden varmistamiseksi, 45 % elektronidonorien seosta (1/3 säilörehua, 1/3 puuhaketta ja 1/3 kompostimateriaalia) ja 10 % mikrobisiirrosteena toimivaa järven pohjamutaa. Reaktorin läpi pumpattiin sulfaattipitoista (keskimääräinen pitoisuus 124,8 mg/l) suotovettä siten, että veden viipymä reaktorissa oli 15 päivää. Reaktoria ylläpidettiin kolmen kuukauden ajan ja sen toimintaa tarkkailtiin ottamalla näytteitä sisään menevästä ja ulostulevasta vedestä kahdesti viikossa. Reaktorissa saavutettiin täydellinen sulfaatinpoisto syöttöveden lämpötilan ollessa vähintään 12 °C. Lämpötilan laskiessa sulfaatinpoistotehokkuus kuitenkin heikkeni siten, että lämpötilan ollessa 8 °C vain n. 50 % ja lämpötilan ollessa 5 °C vain n. 25 % sulfaatista poistui. Näiden tulosten ja lämpötilatilastojen perusteella arvioitiin, että 15 päivän viipymällä saataisiin keskimäärin 52,5 % vuoden aikana reaktoriin sisään menevästä sulfaatista poistettua. Tavoitteena oli vuotuinen 2/3 sulfaatinpuhdistusteho, joka laskelmien mukaan saavutettaisiin nostamalla viipymä 30 päivään. Reaktorista kuitenkin huuhtoutui orgaanista ainesta ja typpeä puhdistettuun veteen, mikä on huomioitava jatkosuunnittelussa. Reaktorissa käytettyjen elektronidonoreiden tehokkuutta tutkittiin kasvattamalla käytettyä sulfaatinpelkistäjäviljelmää eri elektronidonoreilla erikseen ja yhdessä panospullokasvatuksissa. Kasvatusten sulfaattipitoisuutta ja liukoisen orgaanisen aineen määrää tarkkailtiin kokeen edetessä. Erityisesti säilörehu ja puuhake vaikuttivat kokeiden perusteella lupaavilta elektronidonoreilta sulfaatinpelkistykseen. Kaiken kaikkiaan biologinen sulfaatinpelkistys osoittautui toimivaksi tavaksi vähentää vesistöihin kohdistuvaa sulfaattikuormaa. Tulosten perusteella reaktiivisen seinämän käyttöönottoon liittyvien käytännön järjestelyjen selvittämistä suositellaan. Sulfate containing seepage waters from tailing ponds have caused eutrofication in nearby lakes of Siilinjärvi mining site. One way to treat these sulfate containing waters is to use a permeable reactive barrier exploiting biological sulfate reduction. In anaerobic conditions, sulfate reducing organisms can reduce sulfate into sulfide, which forms sparingly soluble precipitates with metals. This way, sulfur compounds are trapped within the barrier and removed from the water stream. In this work, the utilization of a biological reactive barrier for purification of seepage waters at the Siilinjärvi mining site was studied. The study was performed on site using a pilot-scale reactor filled with reactive material. The reactive material consisted of: 45% of gravel for ensuring the permeability of the barrier, 45% of electron donor mixture (1/3 silage, 1/3 wood chips and 1/3 compost material) and 10% of bacterial inoculum, which was bottom sediment of a nearby lake. Sulfate containing (average concentration 124.8 mg/l) seepage water was pumped into the reactor so that its hydraulic retention time was 15 days. The reactor was operated for three months and its influent and effluent quality was monitored by taking water samples twice a week. Complete removal of sulfate was obtained in the reactor when the temperature of the influent was at least 12 °C. However, when the temperature decreased to 8 °C, only approximately 50% of the sulfate was removed and only around 25% when the temperature was 5 °C. Based on these results and temperature statistics it was estimated that with a retention time of 15 days, 52.5 % of sulfate entering the reactor within a year could be removed. The goal was a yearly sulfate removal efficiency of 2/3, which was calculated to be achievable by increasing the retention time to 30 days. However, organic matter and nitrogen was released from the reactor, which needs to be taken into account in future planning. The electron donors used in the reactor were studied by growing the used sulfate reducing community with different electron donors as batch bottle incubations. Sulfate concentration and concentration of organic matter in the bottles were monitored during the experiment. According to the results, especially silage and wood chips seemed to be promising electron donors for sulfate reduction. All in all, biological sulfate reduction proved to be a functional way to reduce the sulfate load of nearby lakes. Based on the results, it is advised to start examining the practical arrangements related to the utilization of a permeable reactive barrier.

In anaerobic wastewater treatment micro-organisms convert the organic compounds in the wastewater in the absence of oxygen and produce energy-rich biogas. Biogas consists mainly of methane and carbon dioxide and it may be used in electricity and heat production. Up-flow anaerobic sludge bed reactor (UASB) is one of the most used anaerobic wastewater treatment technologies. Sludge bed inside the reactor consist of microbial aggregates called granules. Micro-organisms require trace elements to sustain their vital functions. Laboratory experiments have confirmed the positive effects of trace element additions in the UASB reactor's performance. The objective of this experiment was to study the effects of added trace element based nutrient solutions on brewery wastewater fed UASB reactor’s performance. Five UASB reactors were operated for 83 days. One reactor was kept as a control reactor, and the other four were fed with nutrient solutions: Solution BDP-881 (containing Fe, Ni, Co, Se, and Mo), Solution A (containing BDP-881 and Zn), Solution B (containing BDP-881, Zn and Mn) and Solution C (containing BDP-881, Zn, Mn and Ca). The methane production in the control reactor dropped significantly, when organic loading rate (OLR) was 14 g SCOD/l/d (operation day 64). At OLR 16 g SCOD/l/d (operation day 75) the methane production halted. At the same OLR the trace element fed reactors kept working properly. Trace element addition allowed 37 % higher OLR compared to the control reactor. After the methane production halted in the control reactor, it was also fed with BDP-881 solution. In a couple of days, the reactor recovered and methane production and COD removal rose from 0 l/d to 1.7 l/d and from 7 % to 43 %, respectively. According to this study, it is likely that BDP-881 alone improves the reactor performance, because there were no differences in reactor performance between four nutrient solutions fed reactors. However, there could have been differences, if OLR would have been elevated even more. Maximum OLR for trace element fed reactors should be determined, before the solutions are used in industrial applications.

Öljypohjaisille muoveille on pyritty löytämään uusia materiaalivaihtoehtoja, sillä raakaöljyn hinnan nousu on vaikuttanut myös muovien hintoihin. Puumuovikomposiiteissa osa muovista on korvattu puulla, joka on edullinen ja nopeasti uusiutuva biokomposiitti. Tämän työn tarkoituksena oli selvittää, miten luonnonperäisen puutislefraktion lisäys vaikuttaa puumuovikomposiittien ominaisuuksiin. Puu ja muovit ovat kemiallisesti hyvin erilaisia materiaaleja. Puumuovikomposiittien ominaisuudet voivat olla riittämättömät sovelluksissa, joissa vaaditaan esimerkiksi suurta mekaanista kestävyyttä. Puuta voidaan kuitenkin muokata kemiallisesti lämpökäsittelyllä, joka perustuu korkean lämpötilan ja väliaineen, kuten vesihöyryn, aiheuttamiin muutoksiin. Puumuovikomposiitteja on tutkittu runsaasti viime aikoina. Tutkimuksissa on muun muassa selvitetty, miten puukuitupitoisuuden muutokset, kuituorientaatio, -koko ja kytkentäaineiden käyttö vaikuttavat puumuovikomposiittien ominaisuuksiin. Kirjallisuusselvityksen perusteella vielä ei ole kuitenkaan tehty tutkimuksia siitä, miten puun hidaspyrolyysistä saadun tislefraktion lisäys vaikuttaa puumuovikomposiittien ominaisuuksiin. Työn kokeellisessa osuudessa tutkittiin kahden suomalaisyrityksen, LunaCompin ja UPM:n, puumuovikomposiittien ominaisuuksia, kun niihin oli lisätty hidaspyrolyysistä saatua puutislettä tai puutisleellä käsiteltyä materiaalia. Referenssimateriaaleina olivat näytteet, joihin puutislettä ei lisätty. Tisleen lisäyksen aiheuttamia muutoksia arvioitiin mekaanisten testien, mikroskooppi- ja protoninsiirtoreaktio-massaspektrometritutkimusten perusteella. Lisäksi näytteiden veden absorptioita verrattiin keskenään. Tutkimukset tehtiin ruiskuvalulla valmistetuille koesauvoille. Kaikissa tutkimuksissa käytetyissä puumuovikomposiiteissa muovimatriisina oli polypropeeni. Tulosten perusteella käytetyn puutislefraktion lisäys ei paranna puumuovikomposiittien mekaanisia ominaisuuksia, mutta helpottaa granulaattien prosessointia. Puutisleen lisäys vähensi joidenkin näytteiden veden absorptioita. Valo- ja pyyhkäisyelektronimikroskooppikuvissa materiaalien välillä huomattiin vain pieniä eroja. Protoninsiirtoreaktio-massaspektrometritutkimusten perusteella puutisle sisälsi muita näytteitä enemmän suuren moolimassan yhdisteitä. Kun tislettä lisättiin LunaGrainiin, tutkittuja yhdisteitä vapautui näytteistä enemmän. LunaGrainin tai tislekäsitellyn LunaGrainin lisäys UPM ForMi 50:een ei vaikuttanut vapautuvien yhdisteiden määrään samalla tavalla kuin tisleen lisäys LunaGrainiin. Tislekäsittelyn aiheuttamat muutokset olivat Mann-Whitneyn U-testin perusteella pääosin tilastollisesti merkitseviä. Tulevissa tutkimuksissa tislettä kannattaa lisätä puumuovikomposiitteihin eri määriä, jolloin puutisleen todellisista vaikutuksista puumuovikomposiitteihin saadaan parempi käsitys. Myös muiden tislefraktioiden käyttömahdollisuutta olisi hyvä selvittää.

Työssä tutkittiin paperiteollisuuden jätevedenpuhdistamon lietteiden metaanintuottoa anaerobisessa hajoamisessa. Sellu- ja paperiteollisuuden jätevedenpuhdistamon lietteitä päätyy Suomessa kaatopaikoille vuosittain tuhansia tonneja. Kaatopaikkalainsäädännön tiukentuminen ja mahdollisuus jätteissä olevan potentiaalin hyödyntämiseen ovat lisänneet kiinnostusta lietteiden käyttöön energiantuotannossa. Biokaasuntuottoa pidetään kannattavana vaihtoehtona energian tuottamiseen metsäteollisuuden lietteistä niiden alhaisen kuiva-ainepitoisuuden vuoksi. Paperiteollisuuden biolietettä sekä bio- ja primäärilietteen seosta tutkittiin puolijatkuvatoimisissa reaktoreissa 260 päivää kestäneissä reaktorikoeajoissa mesofiilisissä olosuhteissa. Bio- ja primäärilietteiden metaanipotentiaalit myös määritettiin panoskokeissa. Lisäksi tutkittiin ultraäänikäsittelyn sekä hydrotermisen esikäsittelyn vaikutusta biolietteen metaanisaantoon. Panoskokeissa biolietteen metaanipotentiaali oli 96 l CH4/kg VS ja primäärilietteen 127 l CH4/kg VS. Reaktorikokeissa biolietteen metaanisaanto viipymäajalla (HRT) 20 d ja kuormituksella (OLR) 0,5–1,6 kg VS/l d oli 73 l CH4/kg VS ja bio- ja primäärilietteen seoksen (HRT 20 d, OLR 1,2–1,7 kg VS/l d) hieman korkeampi, 87 l CH4/kg VS. Käytetyillä kuormituksilla ja viipymäajalla prosessi toimi tasapainoisesti. Esikäsittelymenetelmistä hydroterminen esikäsittely oli tehokkaampi nostaen metaanisaantoa jopa 73 %. Ultraäänikäsittely nosti metaanisaantoa 46 %. Bio- ja primäärilietteiden määritetyt metaanisaannot ovat alhaisia, johtuen raaka-aineen lignoselluloosapitoisuudesta ja lignoselluloosan huonosta hajoamisesta prosessissa. Esikäsittelymenetelmillä voidaan kuitenkin nostaa lietteiden metaanisaantoja. In this work the methane production of paper mill wastewater sludges was studied in anaerobic digestion. Thousands of tons of waste sludges from pulp and paper mills are landfilled every year in Finland. Due to legislation and possibility to take advantage of the potential of the waste sludges, interest towards energy production from waste sludges has arisen. Production of biogas is one of the promising ways to produce energy from waste sludges because of their low dry matter content. Biosludge and co-digestion of bio and primary sludges from paper mill were studied in semicontinuous reactors for 260 days in mesophilic conditions. Methane potential of bio and primary sludges were also measured in batch experiments. In addition the effect of ultrasound and hydrothermal pretreatment on methane yields were studied for biosludge. In batch experiments the methane potential of 96 l CH4/kg VS was obtained for biosludge and 127 l CH4/kg VS for primary sludge. In reactor experiments the methane yield of biosludge with hydraulic retention time (HRT) of 20 days ja organic loading rate (OLR) of 0,5–1,6 kg VS/l d was 73 l CH4/kg VS. For co-digestion (HRT 20 d, OLR 1,2–1,7 kg VS/l d) of primary and bio sludge methaneyield of 87 l CH4/kg VS was obtained. Anaerobic digestion was shown to be feasible for the studied sludges with used loading rates and retention times. Hydrothermal pretreatment was more effective of the pretreatment methods resulting in 73 % increase in methane yield. Ultrasound pretreatment increased the methane yield with 46 %. Due to low biodegradability of lignocellulose in anaerobic digestion the obtained methane yields were relatively low for bio and primary sludge. The methane yields for sludges with high lignocellulose content can be increased by different pretreatment methods.

CD44 is a transmembrane receptor protein binding its carbohydrate ligand, hyaluronic acid (HA), in a reversible fashion. In addition to enabling normal cell migration, such as the rolling of white blood cells, these carbohydrate-protein interactions are exploited by malignant cancer cells metastasizing through the blood stream. A normal cell therefore requires effective regulatory mechanisms for controlling the binding affinity between CD44 and HA. Earlier studies addressing this topic have, however, been unable to identify these regulatory mechanisms. More precisely, traditional wet-lab experiments, limited by their spatial and temporal resolution, have been inadequate in describing transient nanoscale phenomena, such as the ligand binding. Previous computer simulations have, on the other hand, been limited by both veracity of simulation models and availability of computational resources. In this Thesis, we use all-atom explicit-solvent molecular dynamics (MD) simulations to study the adsorption of HA oligomer to a human wild-type CD44 HA binding domain (HABD). In practice, we explore the role of three potential regulation mechanisms: size of the ligand, glycosylation of the protein, and conformation of the protein. First, free energy profiles for the adsorption of HA octamer, revealing the strength of an individual CD44-HA interaction to be over 25 kJ/mol, suggest ligand binding to be irreversible. Second, by glycosylating the HABD at residues Asn25, Asn100, and Asn110, we show the first of these residues to block most of the native binding interactions. As a result, the strength of the adsorption is, at least when using charge-neutral core pentasaccharides as the attached carbohydrates, decreased by 40 %. More surprisingly, the simulation data reveal a conformation transition previously correlated with high-affinity binding to, in fact, act as a molecular mechanism repelling the bound HA oligomer, and thereby dynamically regulating the biological activity of the CD44. The findings of this study uncover how the binding of HA to its CD44 receptor is regulated. This information may facilitate the design and targeting of novel drugs and therapies against conditions, such as cancer. Lastly, the insight from this study is of potential value when considering the carbohydrate-protein interactions of other cell surface receptors.

One of the main challenges in the mining industry is the emission of harmful components to the environment. Explosive residues and cyanide-containing leaching solutions release ammonium and nitrate to the environment, which can cause eutrophication, formation of nitrous oxide and acid rain. Biological nitrogen removal in municipal wastewater treatment has lower cost and is more efficient than chemical and physical removal methods. Mining wastewaters are usually acidic and contain toxic heavy metals, which can inhibit the biological processes. Therefore, in the present study, the suitability of biological nitrogen removal for mining wastewaters was studied. Classical biological nitrogen removal generally consists of two processes. Through nitrification ammonium is oxidized to nitrate, which is then reduced to nitrogen gas through denitrification. In this study, two different wastewater treatment plant sludges were used as source of microbial biomass. Main denitrifiers enriched were bacteria belonging to genera Dechloromonas, Rhodobacter and Sulphurospirillum, whereas the main nitrifier found was Candidatus Nitrospira defluvii. Limiting pH for denitrification in batch assays was 4.8. With pH 5.0, denitrification was not inhibited. However, continuous fluidized bed reactors (FBRs) were able to neutralize influent of pH 2.5 due to dilution of influent solution with efficient mixing and produced alkalinity. Denitrification was complete at 7°C and 21°C, within the detection limits, when double stoichiometric ethanol/nitrate ratio was provided. When stoichiometric ratio was provided, some nitrate and nitrite remained. All studied heavy metals decreased denitrification rate in batch assays. Addition of 50 mg/l of copper, cobalt, nickel and arsenic to batch assays at pH 7 resulted in initial dissolved concentrations of 0.8, 55, 63 and 10.5 mg/l. With these concentrations denitrification rate was 11, 15, 18 and 34% slower, respectively, compared to similar experiments without metals. Added nickel concentration of 100 mg/l resulted in 91% slower denitrification. Nitrification was efficient in both fluidized bed reactors (FBR) and membrane bioreactors (MBR). Ammonium was completely removed at 21°C when pH was maintained at 6-8.4 with sodiumbicarbonate buffer. Ammonium concentration of 100 mg/l was fed with 7 and 20 h hydraulic retention time (HRT) to FBR and MBR, respectively. In MBR, continuous stirring at 120 rpm provided sufficient sludge suspension and use of aeration directly under hollow fiber membrane prevented membrane fouling. Simultaneous nitrification and denitrification was investigated in the MBR. Reactor was operated with 60 minutes of aeration and 20-60 minutes of non-aeration intervals. Total nitrogen removal was not as efficient as in separate nitrification and denitrification reactors. The highest total nitrogen removal of 67% was achieved when the non-aeration intervals were 60 minutes. However, for future research, longer enrichment time of the bacteria and further optimization of aeration intervals could enhance nitrogen removal. In this study, no gaseous denitrification products were analyzed. This would be however essential, since previous results from literature reported increase of N2O production in low pH and in copper containing studies. It would be also interesting to study metal toxicity in continuous flow reactors. This way cells would have enough time to uptake toxic metals and the long term toxic effects on bacteria could be analysed. In addition, several different metals should be tested at the same time, since mining wastewaters contain multiple metals and their combined effects can be significantly different from effects of one metal at a time.

Tässä työssä tarkastellaan oljen hyödyntämistä biokaasulaitoksen raaka-aineena. Suomessa kasvaa vuosittain laskelmien mukaan, noin miljoona tonnia taloudellisesti hyödynnettävissä olevaa olkibiomassaa. Olkibiomassa koostuu pääosin lignoselluloosasta, joka on rakenteellisesti kestävää ja vaatii esikäsittelyä jotta se on teknis-taloudellisesti hyödynnettävissä. Anaerobisessa hajoamisessa esikäsitellystä olkibiomassasta voidaan tuottaa biokaasua, jota puhdistamisen jälkeen voidaan käyttää yhdistettyyn lämmön- ja sähköntuotantoon kaasuturbiinien avulla, paineistaa ja syöttää maakaasuverkkoon tai käyttää liikennepolttoaineena. Viljan tuotannon sivutuotteena syntyvä olkibiomassa on haasteellinen raaka-aine, johtuen sen biokemiallisesta kestävyydestä sekä alhaisesta tiheydestä. Puitu olki on kuivattava, paalattava, kuljetettava ja varastoitava ennen sen käyttöä biokaasun tuotantolaitoksella. Paalattu olki on revittävä pieneksi, jotta sitä voidaan tehokkaasti esikäsitellä. Esikäsittelyn tarkoituksena on hajottaa lignoselluloosan rakenne ja tehdä siitä mikrobeille soveltuvaa ravintoa. Eri esikäsittelymenetelmät tuottavat erilaisen hajoamistuloksen. Oleellisinta on saada ligniini irtoamaan hemiselluloosasta ja selluloosasta, jonka jälkeen edellä mainitut voivat hydrolysoitua. Anaerobinen hajoaminen koostuu neljästä päävaiheesta, entsymaattisesta hydrolyysistä, hapon muodostuksesta, asetaatin muodostuksesta ja metaanin tuottovaiheesta. Niiden vaatimat olosuhteet poikkeavat toisistaan. Optimoimalla olosuhteet jokaiselle soveltuvaksi saadaan aikaan tehokas anaerobinen hajoaminen. Tässä työssä optimaaliset olosuhteet luotiin hajottamalla olkibiomassaa kaksivaiheisilla yhdistelmäreaktoreilla. Lietepatjareaktoreissa (LBR) tapahtui entsymaattinen hydrolyysi ja hapon muodostus, jonka jälkeen effluentti johdettiin pystyvirtaus lietepatjareaktoreihin (UASB) asetaatinmuodostukseen ja metaanintuottoon. Kokeellisessa osiossa selvitettiin esikäsittelemättömän ohran oljen käyttöä raaka-aineena biokaasuntuotannossa. Koska raaka-aine on uusi ja vähän tutkittu, päädyttiin tutkimaan raaka-aineen käyttäytymistä bioreaktorissa. Kokeessa saatiin poistettua noin 15 % liukoisesta COD sekä noin 20 % raaka-aineen VS:stä. Metaanintuotoksi saatiin 0,27 Ndm3 CH4/g VS käsittelemättömästä oljesta ja liuotetusta oljesta 0,29 Ndm3 CH4/g VS. Tulokset ovat samankaltaisia kuin kirjallisuuslähteissä. Oljen liottaminen vedessä ennen kokeen aloittamista nopeutti metaanintuottoa puolella verrattuna liottamattomaan olkeen. Olkibiomassa on potentiaalinen biokaasun raaka-aine, koska sitä on saatavissa kohtuullisin kustannuksin ja se ei kilpaile ravinnontuotannon kanssa. Toisen sukupolven biokaasulaitokset, jotka hyödyntävät lignoselluloosapohjaista raaka-ainetta, teke-vät tuloaan maailmalla. In this thesis suitability of straw biomass as a feedstock for biogas production was studied. It has been estimated that about annually about one million ton of straw biomass could be economically utilized In Finland. Straw biomass consists mainly of lignocellulose, which is structurally durable and requires pre-processing so that it could be technically and economically exploitable. Pretreated straw biomass can be used as a feedstock in anaerobic digester for production of biogas. Treated biogas is suitable fuel for CHP-plant and it can be injected in natural gas grid or used as a fuel for transportation. Straw is a side product of grain harvesting. It is a complicated raw material, because it is biochemically durable and it´s density is low. Harvested straw have to be dried, baled, transported and storaged before it can be used in biogas plant. Bale of straw have to break down, so that pretreatment could be effective. Pretreatment is needed for degradation of the structure of lignocellulose and to increase bioavailability for microbes. Degradation is depended on pretreatment technics that is used. The key point of suitable pretreatment is degradation of lignin, so that hemicellulose and cellulose could be hydrolyzed. There are four main phases in anaerobic digestion; enzymatic hydrolysis, asidogenesis, asetogenesis and methanogenesis. Requirements of conditions were different in each phases. Efficient anaerobic digestion can be achieved by optimizing reaction conditions during different phases. In this study it was used two-phase combination reactors to achieve optimal reaction conditions for different phases. In leach bed reactor (LBR) occurred enzymatic hydrolysis and asidogenesis. From LBR effluent was transferred to the up-flow anaerobic sludge blanket reactor (UASB), where asetogenesis and methanogenesis occurred. In this thesis it was studied biogas production from barley straw, without pretreatment. Barley straw is a new feedstock and less studied, so that the behavior of the barley straw in the process were decided to keep on focus. 15 % of suspended COD and approximately 20 % of volatile solids (VS) were removed. Methane production of barley straw without pretreatment was 0.27 Ndm3 CH4/g VS and as soaked it was 0.29 Ndm3 CH4/g VS. The results were in the line with literature. Straw were soaking in water before anaerobic digestion, in which case there was two times faster methane production than unsoaked straw. Straw biomass is suitable feedstock for biogas production. Straw is economically obtainable and it doesn´t compete with food production. 2nd generation biogas plants that are able to utilize lignocellulosic feedstock, are on their way.

Proteins are the key factors in every living organism and they contribute to almost every biological process and structure in a cell. Proteins are formed through the process of protein synthesis, in which the genetic code of DNA in genes is first transcribed into RNA and then finally translated into a protein. Each gene in a cell is a short part of a longer DNA molecule, and each gene encodes the synthesis of a certain protein or pro-teins. Currently the state-of-the-art tool to evaluate which genes are expressed in a given biological sample is RNA-sequencing, which captures the RNA content of the cells at a specific time point. Although RNA-sequencing is often used to measure the expression of known genes in the genome, it can also be utilized to search for new genes, or novel transcripts. To date, no standard tool has been published that effectively and thoroughly identi-fies novel transcripts from RNA-sequencing data. In this work, a tool aiming to solve this issue – denoted Novellette – is presented. Novellette attempts to identify differen-tially expressed regions in the genome that do not overlap with any known genes, and then performs a full gene structure analysis to the regions. For this process, information both from processed RNA-sequencing data and the known DNA sequence of the studied organism is utilized when searching for features in the novel transcript candidates that are common for protein-coding genes. The features are then scored and the final novel transcript candidates are ranked based on their score values. In addition to developing an RNA-sequencing tool in this work, the basics of statistical testing and other mathematical methods related to RNA-sequencing data analysis are introduced and the normality of count based RNA-sequencing data is assessed with publically available data. The results from analyses performed with various input data show that Novellette is able to reliably detect novel transcripts and distinguish protein-coding regions from non-coding regions in the genome with the proposed scoring approach. In addition, the count based RNA-sequencing data is shown to very poorly follow the normal distribu-tion, hence pinpointing the importance of statistical hypothesis testing methods that do not assume data normality. In conclusion, a functional and useful bioinformatics tool has been developed in this work that has the potential to become a standard method for novel transcript identification.

Retinal diseases, especially age-related macular degeneration (AMD), are becoming a large issue as life expectancy increases. Blood-retinal barrier (BRB) is located adjacent to the retina and has many important functions in normal vision and it is the main site associated with AMD pathogenesis. BRB consists of retinal pigment epithelium (RPE), Bruch's membrane (BrM) and choriocapillaris endothelium. Mathematical modeling provides a tool to study the properties of the BRB and thus new treatment options for AMD. In experimental research, the BRB barrier function is usually measured with a so-called diffusion chamber. In this thesis, two models are constructed: 1) a passive diffusion model across the BRB based on the physicochemical properties of the BRB (BRB model) and 2) a model of a miniaturized half-perfusion chamber concept to study the effects of different parameters to the functionality of the system (chamber model). The predictive BRB model was constructed by combining physical theories from the literature to describe the hindered diffusion within a certain type of geometry. The main objectives were to construct the model, to study the behavior of the permeability of the whole BRB and its parts as a function of different molecular properties as well as to validate the model by using experimentally measured permeabilities. The chamber model was done by using finite element method (FEM) and by utilizing Comsol Multiphysics FEM modeling software. The main interest was on the effects of certain system dimensions and other chamber parameters to the error caused by the measurement system. The results of the BRB model indicate that it predicts the correct magnitudes of the BRB permeabilities, but fails to predict the more accurate behavior based on the molecular properties of the diffusing molecule, such as molecular size and lipophilicity. As assumed, the model confirms that RPE forms the most significant barrier in the BRB, as the BrM has affects only the very lipophilic molecules. With the chamber model, the results show that the functionality of this kind of half-perfusion chamber is limited by the free diffusion rate. The chamber dimensions and other parameters are less significant, as they mainly just change the diffusion distances. The proposed BRB model is the first model of this kind for the BRB. It can be utilized to study the barrier properties in normal function and in disease, especially with little refinements. There are several directions towards which the model can be taken in the future, such as dynamic regulation of the permeability. The chamber model clearly suggests that the free diffusion rate is the main issue with this kind of half-perfusion chamber. The ability of the diffusion chambers to measure the actual absolute values of permeability, and to provide reliable results, should be viewed with caution.

In-vitro diagnostics is currently going through changes. Instead of using central laboratories to analyze samples, point-of-care (POC) diagnostic devices are used more and more. With these devices tests can be performed rapidly near the patient in a doctor’s office, at bedside or at a patient’s home. Most POC devices are based on microfluidics, where small fluid volumes are handled inside micro scale channels. With microfluidics it has been possible to miniaturize these devices. Thus, their sample and reagent consumption is significantly smaller than in conventional methods. What comes to the performance of POC devices’, their sensitivities and limit of detection values are still worse than in conventional methods. The objective of this research was to study how the performance of a microfluidic immunoassay cartridge would be enhanced. The aim was to study how different polystyrene grades and reaction chamber geometries affect immunoassay performance. Injection molded microfluidic cartridges were used in the research. Specific antibody coating was fabricated on the reaction chamber surface. Analyte concentration was measured with fluorescence nanoparticles containing europium chelates. These nanoparticles were used as they provide higher signal levels and better sensitivities than conventional fluorescence labels. For studying the effects of geometry, a simplified finite element method (FEM) model was created to simulate the immunoreactions occurring inside a chamber. Results show that signal levels caused by unspecific binding of labels are similar in all materials, but with Empera 124N polystyrene grade signal levels of specific binding were higher than with other materials. FEM models of original reaction chambers showed that the system is fully diffusion limited. New smaller geometries were designed with the help of the FEM model. The main difference was that sample was moved back and forth over the reaction area during incubation. Measurement sensitivity was increased about 1.4-fold and the limit of detection was decreased from 1 mU/L to 0.52 mU/L. Micro structures on the reacting surface, were also tested and they increased measurement sensitivity by 3.8–fold compared to a smooth surface. As a conclusion it can be said that already a simple FEM model can be used to describe what happens in a system and how the system can be optimized. As a smaller reaction chamber was used, sensitivity and the limit of detection were enhanced and reagent consumption during fabrication was decreased. There are still some problems concerning the control of reciprocating flow and its repeatability which need to be solved.

The number of people affected by degenerative diseases of the retina increases notably due to the longer life expectancy. The weakening of the sight concerns especially older people, who suffer from degeneration of the retina and its retinal pigment epithelium (RPE) monolayer. Currently, these degenerative diseases of the retina are incurable. Cell transplantation of healthy RPE cells is considered to be a potential treatment for these diseases. Due to the unlimited self-renewal capacity and pluripotency human pluripotent stem cells (hPSCs) are considered as possible cell sources for transplantation purposes. However, the current differentiation protocols for RPE cells from hPSCs are inefficient and result in low yields. The aim of this thesis was to study whether biodegradable poly(L-lactide) (PLLA) microparticles and commercial biostable cellulose anion exchange diethylaminoethyl (DE) 53 microparticles could be exploited in increasing the efficacy of differentiation of hPSCs into RPE cells. In addition, the potential of the microparticle culture system for stem cell maintenance in feeder-free conditions was evaluated. The attachment and proliferation of hPSCs on microparticles were analyzed by immunofluorescence analysis and microscopy. The success of differentiation of RPE from stem cells in microparticle cultures was evaluated by inspecting the appearance of pigmentation in cultures as well as by investigating the expression of RPE maturation markers on gene expression levels by RT-PCR. The PLLA microparticles did not support the attachment and proliferation of hPSCs in feeder-free conditions. The poor performance of PLLA particles in cell culture studies might be due to the hydrophobic nature of the particle surfaces. In contrast, hPSCs attached, proliferated and maintained their pluripotency on DE 53 particles. Furthermore, the hPSCs were successfully differentiated into RPE cells on these DE 53 particles. However, the differentiation efficacy did not outperform the conventional differentiation protocol. This was the first study in which the hPSCs were successfully differentiated into RPE cells on microparticles. Microparticles are far from being routinely used in hPSC maintenance and differentiation to mature cell types. More long-term and large-scale studies need to be conducted in the future. Nevertheless, at the moment they seem appealing and promising with a possibility to grow stem cells in large-scale and cost effectively. The microparticle culture systems set challenges for the cell analysis due to their 3-dimensional structure and autofluorescence property. Therefore, novel methods for characterization, analysis and imaging of microparticle culture systems should be developed. /Kir12

Dry eye syndrome (DES) is disease of the eye surface, where tear film is ruptured and lubrication or moistness of the corneal and conjunctival epithelia do not work properly. DES is most common in older people, but also people with rheumatoid diseases, damaged corneal epithelia and certain medications are more likely to have DES. There are lubricating and moisturizing eye drops to relieve the symptoms of DES, administered at least twice a day depending on the severity, but they are only palliative. Eye drops for DES are composed of natural and synthetic origin polymers and their blends. Retaining methods are mainly viscosity and adherence to surfaces by weak interactions. In situ gelation by temperature, pH and osmotic change have been under research in recent years. Many studies have concentrated on ambiphilic polymer conjugates, which fit to the oily and aqueous layer of the tear film and have a better ability to carry a hydrophobic therapeutic drug molecule. Interest has also been pointed to mucoadhesive polymers, which are adhering to mucin by covalent bond. Thiolation of the polymer has been seen as a very promising method to achieve better muchoadhesion and thus has been extensively studied. A chitosan and hyaluronan are natural origin polymers, which have been thiolated and these polymer matrixes can be loaded with trehalose or albumin as therapeutic agent. In this thesis the anatomy of the eye, and especially the corneal and precorneal area and the conjunctiva is discussed. Ethiology of DES and commercial available therapies are covered in this thesis, but also therapies under research and corneal simulation methods in vitro. Kuivasilmäisyys on sairaus, jossa silmän sarveiskalvon pinnalla oleva kyynelkalvo rikkoutuu. Sarveis- ja sidekalvon kosteutus ja voitelu häiriintyvät kyynelkalvo epätasapainosta. Kuivasilmäisyys on ikääntyvien ihmisten sairaus, mutta sitä tavataan myös sarveiskalvovaurio- ja reumapotilailla. Joidenkin lääkkeiden käyttöön liittyy sivuoireina kuivasilmäisyyttä. Kuivasilmäisyyteen on saatavilla kaupallisia oireita lievittäviä silmätippoja, jotka kosteuttavat ja voitelevat silmän pintaa, mutta eivät hoida sairautta. Kuivan silmän tipat on valmistettu luonnon polymeereistä tai synteettisistä polymeereistä, joita annostellaan vähintään kahdesti päivässä. Terapeuttisen vaikutuksen kesto perustuu viskositeettiin tai tarttumiseen silmän pintaan heikoilla sidoksilla. In situ geeliytyminen lämpötilan, pH:n tai suolapitoisuuden muutoksesta on ollut tutkijoiden mielenkiinnon kohteena viime vuosina. Myös polymeerien tarttumista limakalvon pintaan (mukoadheesio) heikoilla vuorovaikutuksilla tai vahvemmilla kovalenttisilla sidoksilla on tutkittu. Amfifiliset polymeerit ovat myös vallanneet alaa, sillä niiden oletetaan sopivan kyynelkalvon pinnan öljykerrokseen ja edelleen sen alla olevaan vesikerrokseen. Amfifilisella polymeerillä on myös kyky kantaa hydrofobista lääkemolekyyliä. Polymeerien tiolointiin ja sen tutkimiseen on panostettu viime aikoina paljon. Tiolointi on menetelmä, jolla parannetaan polymeerin mukoadheesiota. Erityistä mielenkiintoa on herättänyt kitosaanin ja hyaluronihapon tiolointi, jolloin myös terapeuttinen molekyyli, kuten trehaloosi tai albumiini voitaisiin ladata polymeerimatriisiin. Tässä kirjallisuusselvityksessä käsitellään silmän anatomiaa erityisesti kyynelkalvon, sarveiskalvon ja sidekalvon osalta. Tässä opinnäytetyössä on käsitelty kuivasilmäisyyteen johtavia syitä ja nykyisiä sekä kehityksen kohteena olevia hoitomenetelmiä. Lopuksi esitellään sarveiskalvon simulointimenetelmiä, joita on käytetty laboratorio-olosuhteissa.

The aim of this project was to study whether it is possible to identify histopathological changes in liver based on gene expression profiles, regardless of the toxic causing the change. Another goal of the project was to study whether gene expression data can be used as a reference when predicting possible histopathologies in future samples treated with possible drug candidates. For this purpose, data from in vivo microarray gene expression studies of rat liver from Japanese Toxicogenomics Project database (TG-GATEs, published in February 25, 2011) was used. Possibility of using smaller doses and shorter experiment times in order to save resources was studied by comparing different exposure times and dose levels. Separation between different histopathological groups was studied using Principal Component Analysis and K-nearest neighbor cross-validation. The differentially expressed genes and enriched gene ontologies from each group were listed, and the gene lists were studied further and compared to observe possible causes and mechanisms of toxicity. Time and dose responses were analyzed similarly. Some comparisons using well-known and possible candidate drug molecules were made in order to test the predictive use of the data. In the theory section microarray technology and data analysis are explained, and the field of toxicogenomics and TG-GATEs -database are presented based on articles from scientific journals and books. Analyses are performed using R-language, Bioconductor-packages and Ingenuity IPA -software. Customized CDF-fi les were used in analysis to provide most current and accurate results. It was noticed that while other histopathologies are clearly separated from the controls, others are more spread out. Spreading is likely to be caused by number of different mechanisms causing the toxicity in some groups, but also due to different analysis factors (severities of the findings, exposure times and dose levels). It seems that the data and gene expression pro les can be used as references when estimating possible toxicity of a drug molecule and the mechanisms of it. Some dose and time response was noticed. Due to the complex nature of gene expression, samples treated with different doses and exposure times are needed to see the direction of the development: single values from a single data point can be misleading.

Escherichia coli is one of the most studied microorganisms and has found many uses in biotechnological applications. In order to make these applications economically feasible, it is necessary to get sufficiently high yields of the product molecules. Due to its popularity in bacteriological studies, a lot of knowledge about factors that are important to consider when optimizing E. coli bioprocesses has accumulated. This knowledge has been used to construct models about genetics and metabolism of this bacterium making genetic modification to improve bioprocess yields easier. Factors important in bioprocesses using E. coli are discussed in this work. While most of the work is concentrated on basic properties common to most bioprocesses, special emphasis is put on bioprocesses related to protein production as it was considered to be one of the most important bioprocess application. First, a very brief introduction to biology of E. coli is given. Then the basic parameters’ effects on bioprocesses are discussed, and finally some aspects of product formation and collection are discussed. It was shown in this work that, although a lot of progress has been made in optimization of bioprocesses where E. coli is used, in many aspects these processes still remain to be far from optimal. Therefore, it is clear that great amount of research on bioprocesses using this bacterium remains to be done.

Lateral flow assay is an antibody-based, fast, easy to use, affordable and accurate measurement method for the detection of small quantities of substances. The analytes are detected with antibody-label complex and the label can be detected. In this research the relatively new label, a paramagnetic iron oxide particle, is examined and its behaviour in lateral flow assay is compared with a commonly used label, colloidal gold. At the same time, the performance of a magnetic particle reader, MIAU (Magnasense Technologies, Finland), is evaluated. Tests were prepared for ricin, which is a toxic protein concentrated on the seed of Ricinus communis plant. In this study, the lateral flow test strips were prepared and used to measure different concentrations of two types of ricin samples: the ricin preparation extracted from the seeds and a pure ricin A-chain. The detection limits for both label types were almost the same: for the ricin preparation and A-chain the paramagnetic particles showed 56 ng/ml and 600 ng/ml and the colloidal gold 31 ng/ml and 480 ng/ml, respectively. The linear range of magnetic measurement was significantly wider than in colloidal gold measurements. Also, the re-measurements after four weeks with paramagnetic particles resulted in practically the same as immediately after the test run.

Polttoaineiden hiilivetyjen on tiedetty ylläpitävän mikrobien kasvua jo yli 70 vuoden ajan. Tämän diplomityön yhtenä tavoitteena oli tehdä kirjallisuuskatsaus erilaisten polttoaineiden mikrobiologisesta kontaminaatiosta. Kokeellisen osuuden tavoitteena oli tutkia polttoaineiden soveltuvuutta mikrobien ravinnoksi, mikrobien määrää ja diversiteettiä eri polttoaineissa, polttoaineiden pilaantumisen etenemistä sekä mikrobikasvun hillitsemistä ja estoa. Diplomityön kokeellisessa osuudessa tutkittiin kahta polttoainetta. Polttoaineiden soveltuvuutta mikrobien ravinnoksi tutkittiin kolmella eri mikrobien kasvutavalla: kiinteällä kasvatusalustalla, liukoisessa vesifaasissa ravistelupullokokein sekä biofilmissä. Polttoainetta 1 ei voida käyttää sellaisenaan hiilenlähteenä kiinteässä kasvatusalustassa, sillä se ei absorboitunut alustaan. Ravistelupullokokeissa saatiin rikastettua polttoaineessa 1 esiintyviä mikro-organismeja ja molempien tutkittujen polttoaineiden havaittiin soveltuvan mikrobien ravinnoksi. Mineraalisuolaliuoksen havaittiin edistävän ja matalan pH:n rajoittavan mikrobikasvua polttoaineissa. Biofilmin mikrobikasvua havaittiin polttoaineen 1 varastosäiliöiden pinnoitemateriaalilla päällystetyissä mikroskoopin objektilaseissa sekä polttoaineen 1 että vesifaasien rajapinnoilla. Veden saatavuuden ja matalan pH:n havaittiin rajoittavan biofilmin muodostusta. Lämpötilan vaikutusta mikrobikasvun kinetiikkaan polttoaineessa 1 tutkittiin ravistelupullokokein kolmessa lämpötilassa. Mikrobikasvun havaittiin olevan nopeinta 27°C lämpötilassa sekä tapahtuvan viiveellä ja hitaammin 37°C lämpötilassa. 6°C lämpötilassa ei havaittu mikrobikasvua tutkimusjakson aikana. Mikrobikasvun hillintää tutkittiin anaerobisissa olosuhteissa polttoaineessa 1. Mikrobikasvua ei havaittu tutkimusjakson aikana. Mikrobikasvua polttoaineessa voidaan hillitä veden määrää vähentämällä, matalalla pH:lla ja lämpötilalla sekä anaerobisilla olosuhteilla. Veden saatavuuden rajoitus polttoaineen varastosäiliössä on toteutettavissa säännöllisellä ja huolellisella vedenpoistolla. Anaerobisten olosuhteiden teknisen toteutuksen mahdollisuutta ja taloudellisuutta tulisi tutkia täydessä mittakaavassa todellisessa polttoaineen varastointiympäristössä. Mikrobien lukumäärää polttoaineessa 1 tutkittiin standardin IP 385/99 mukaisen maljakasvatusmenetelmän ja mikroskopoinnin avulla. Sekä pesäkkeitä muodostavien yksiköiden määrä (11 – 15 PMY/ml) että kokonaissolumäärä (453 – 1271 solua/ml) oli tutkituissa näytteissä hyvin pieni. Mikrobien diversiteettiä polttoaineessa 1 tutkittiin molekulaaristen menetelmien avulla. Näytteiden prokarioottidiversiteetti oli laaja, mutta eukarioottidiversiteetti oli pieni. Mikro-organismien selektiivinen rikastuminen ravistelupullokokeissa havaittiin rikastetun biomassan mikrobidiversiteetin suppeudesta alkuperäiseen näytteeseen verrattuna. Maljakasvatusmenetelmän avulla eristettiin vain viisi todennäköisesti erilaista pesäkettä, joka havainnollistaa selkeästi menetelmän valikoivuuden ja sen antaman puutteellisen kuvan mikrobiston monimuotoisuudesta. /Kir11

Most severe degenerative diseases of retina are often due to malfunctions of retinal pigment epithelium (RPE). Absence of effective treatments has led to development of cell-biomaterial constructs with the aim of creating RPE equivalents for transplantation. Presently, the poor biocompatibility of allologous and xenologous culture substrata in addition with limited amount of source tissue poses the major issues. Well-defined synthetic substrata together with utilization of human embryonic stem cell-derived RPE cells (hESC RPE) are suggested to be potential solutions. In addition, need exists for an effective method to determine the developmental status of cells during the culturing period. This need could be addressed with automated image analysis. The aim of this thesis was to examine the capability of a few specific cell culture substrata to enable attachment, proliferation and maturation of hESC RPE cells. Study included total of 17 xeno-free synthetic materials including 12 BioMaDe Gelators, Purecoat amine and carboxyl, poly(D,L-lactic-co-glycolic acid) (75:25), poly(D,L-lactic acid) (96:4) and poly(L-lactic acid-co-?-caprolactone) (70:30). In addition five materials with natural-origin were studied including chitosan, type I collagen, Matrigel and Substrate X. Type IV collagen was used as control. Growth and maturation were monitored by taking images with specific time intervals. At the end point cellular developmental status was determined by assessing the expression of maturation specific mRNAs by PCR techniques and proteins by immunofluorescence microscopy. In addition, images were used to determine the potential of ImageJ-software as user-friendly image analysis tool for RPE cell analysis. Study demonstrated poor attachment and cell survival on every xeno-free synthetic substrate with cells retaining their initial developmental phase throughout the culturing period, which was supported by gene expression analysis. On the contrary, cells on natural materials attached and proliferated readily. Maturity was further confirmed with immunofluorescence labeling. Image analysis with ImageJ, in turn, confronted many problems mainly arising from heterogeneity of the images. As a conclusion, xeno-free synthetic materials tested in this study show low potential as RPE cell substrata. However, means to enhance their performance are suggested. Despite the good results obtained with natural materials, their ill-defined structure prone to alterations in physiological conditions remains an obstacle for entering clinical experiments. Further experiments should concentrate on combining the strengths of both approaches, that is, incorporation of attachment-related functional groups into well-defined xeno-free synthetic body. In order to increase image homogeneity imaging conditions should be more carefully considered. This way the benefits of automated image analysis could be more effectively exploited. /Kir11

During the development of the neural tissue of the nervous system, supporting neuroglial cells and the extracellular matrix (ECM) guide the migration of the immature functional neurons, provide them with a scaffold to grow on and aid in the formation of synapses. ECM provides the neurons with various guidance cues that guide the migration of the neurons and the extension of the neurites. The micrometre and even nanometre guidance cues can also be incorporated into neuronal cultures in vitro to study the effect of the guidance cues and to develop small neuronal networks with a desired architecture. Three-dimensional neurocage structures were fabricated from Ormocomp®, a polymer-ceramic hybrid material, by two-photon polymerisation for this study. In this study these structures were tested with neuronal cells differentiated from human embryonic stem cells (hESC). The neurocages were attached to microscope glass slide samples, each sample containing approximately ten neurocages. The neurocages were first coated with laminin, an ECM protein, to enable the adhesion of the cells to the glass surface. The cell suspension was then applied and the cells were cultured to observe their growth and to study the guidance effects of the neurocages. During the study eight individual experiments were carried out to optimise both the application methods for the laminin solution and the cell suspension and the growth of the cells. This study utilised both simple manual methods and two different micromanipulator set-ups in the application of the laminin solution and the cell suspension. It was concluded that the best method to apply the laminin solution was the micromanipulator set-up SU2, which utilised automated micromanipulator placement and pressure regulation. The cell suspension, on the other hand, could be easily applied onto the samples by placing droplets of the solution onto the medium covering each sample. As the cell population in the culture was small, conditioned medium taken from another neuronal cell culture was tested to increase the viability of the cell cultures of this study. The conditioned medium had a clear positive effect and the use of conditioned medium in future studies is therefore recommended. During the study it was found that the cells initially inside the neurocages did not attach to the glass bottom even when the insides of the neurocages were accurately coated with the laminin solution. In contrast, the cells outside the neurocages generally attached to the glass bottom well and had a tendency to migrate towards the neurocages and over the structure walls into them. Therefore, it was concluded that the material Ormocomp® was not harmful to neuronal cells and it even seemed to attract them. Furthermore, the cells that had migrated into the neurocages readily stayed inside them and extended neurites along the edges of the neurocages. This indicated that the neurite guidance properties of the structures were very promising. /Kir11

In prokaryotes, transcription and translation are dynamically coupled, as the latter starts before the former is completed. Also, from one transcript, several translation events occur in parallel. To study how events in transcription elongation affect translation elongation and fluctuations in protein levels, we propose a delayed stochastic model of prokaryotic transcription and translation at the nucleotide and codon level that includes the promoter open complex formation and alternative pathways to elongation, namely pausing, arrests, editing, pyrophosphorolysis, RNA polymerase traffic, and premature termination. Stepwise translation can start after the ribosome binding site is formed and accounts for variable codon translation rates, ribosome traffic, back-translocation, drop-off, and trans-translation. The recent development of measurement techniques in genetics promises better un-derstanding of the functioning of biological systems. To attain the most out of these techniques, new methods are needed of interpreting the data, since most existent me-thods have been developed to analyze population level measurements, rather than ex-tracting information from single cell dynamics. For example, one needs accurate estima-tion of the measurement noise from single cell measurements of gene expression. We use recently developed methods to measure gene expression in vivo in individual cells, at the single RNA and protein molecule levels. Such measurements of gene expression, attained in various conditions, as well as the proposed modeling strategy, are used to study and model the dynamics of gene expression at the single event level and to esti-mate noise sources in the processes. First, the model is shown to accurately match the measurements of sequence-dependent translation elongation dynamics. Next, the degree of coupling between fluc-tuations in RNA and protein levels, and its dependence on the rates of transcription and translation initiation is characterized. Finally, sequence-specific transcriptional pauses are found to have an effect on protein noise levels. For parameter values within realistic intervals, transcription and translation are found to be tightly coupled in Escherichia coli, as the noise in protein levels is mostly determined by the underlying noise in RNA levels. Sequence-dependent events in transcription elongation, e.g. pauses, are found to cause tangible effects in the degree of fluctuations in protein levels, implying that these are evolvable. /Kir11

Acinetobacter baylyi sp. ADP1 is a nutritionally versatile bacterial strain capable of natural transformation and homologous recombination. It has a unique metabolism, as it is capable of using a vast collection of compounds as a sole carbon source. It is non-pathogenic and easy to work with in the laboratory. The great potential of ADP1 lies in its usage as a laboratory model organism, as well as in bioremediation. The strain is well characterized, as full genomic sequence and metabolic model exist already. However, no metabolomic analyses have been performed so far. Metabolomics has recently developed into an important field in biotechnology with many potential new applications. As a new area of interest, it is only in early stages of development, but nevertheless many powerful techniques already exist. However, lots remain to be discovered. As every bacterial species can be very different, the methods of metabolomics need to be designed for every study independently. This is why it is of great importance to optimize the metabolite extraction protocol specifically for each project. The purpose of this work was to optimize a new extraction protocol of intracellular metabolites from ADP1, for the purpose of metabolomic analysis with high resolution mass spectrometry. An initial protocol, set up and Genoscope, and based on literature and some previous testing, was used as a basis for the optimization. From this protocol, the culture conditions, quenching procedure, sample storage and data analysis were reassessed. In the end, the final protocol was tested with the cells cultivated in two different carbon sources. The final protocol improved the quality of acquired data significantly, providing a better efficiency in quenching of the metabolism and metabolite extraction, leading to more detected metabolites in the final data. In addition, the extraction process was made more organized and faster to perform. /Kir11

Age-related macular degeneration (AMD) and some other retinal diseases are associated with degeneration of retinal pigment epithelium (RPE) cells. A potential cure could involve cell transplantation therapy using RPE cells differentiated from pluripotent human embryonic stem cells (hESCs). A big challenge is to produce a homogeneous population of hESC-derived RPE (hESC-RPE) cells, and coating materials are one potential alternative to promote the differentiation. The aim of this study was to evaluate the cell attachment and differentiation of hESC-RPE cells on different coating materials and culture conditions. hESCs were derived and maintained without serum and animal feeder cells. This study consisted of three parts. In the first part, hESCs were cultured on different xeno-free coating materials and in a culture medium that stimulated the differentiation towards RPE cells. CELLstart™, collagen IV, and VitroCol™ were the best coating materials to support the cell attachment. The differentiation of the hESCs towards RPE cells was analyzed by visual pigment observation, immunocytochemistry, and quantitative real time PCR. There were no significant differences in the differentiation of the cells cultured on CELLstart™, collagen IV, or VitroCol™. In the second part, confocal microscopy wells were tested. hESCs differentiating to RPE cells and enzymatically degraded hESC-RPE cells attached and grew best in collagen IV coated Ibidi wells. In the third part, hESC-RPE cells were cultured in BD BioCoat™ collagen IV cell culture inserts in culture mediums containing basic fibroblast growth factor (bFGF) or not. RPE cells attached and grew well in the inserts with or without bFGF. The good attachment of hESCs differentiating towards RPE cells to CELLstart™, collagen IV, and VitroCol™ coatings was a remarkable result, since to my knowledge no publication of hESC differentiation towards RPE cells with a xeno-free coating material and a serum-free culture medium in adherent cell culture exists. Collagen IV can be recommended as a coating material for hESCs differentiating towards RPE cells, since it is cheaper than CELLstart™ and VitroCol™ and easy to use. In the future, the effects of growth factors and other supplements on RPE differentiation could be studied. Collagen IV can also be recommended for Ibidi confocal microscopy wells for hESC-RPE cells, at least for short-term experiments. Longer-term effects should be further studied. BD BioCoat™ collagen IV cell culture inserts can be utilized in research purposes, but they are not optimal since they contain xeno-products. In the future, the ability of bFGF to promote RPE cell cluster distribution and to transdifferentiate RPE cells could be further studied. /Kir10