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Haptoglobin (Hp) is a positive acute phase protein, synthesized in the liver, with four N-glycosylation sites carrying mainly complex type N-glycans. Its glycosylation is altered in different types of diseases but still has not been extensively studied mainly due to analytical challenges, especially the lack of a fast, efficient, and robust high-throughput Hp isolation procedure. Here, we describe the development of a high-throughput method for Hp enrichment from human plasma, based on monolithic chromatographic support in immunoaffinity mode and downstream Hp N-glycome analysis by hydrophilic interaction ultrahigh-performance liquid chromatography with fluorescent detection (HILIC-UHPLC-FLR). Chromatographic monolithic supports in a 96-well format enable fast, efficient, and robust Hp enrichment directly from diluted plasma samples. The N-glycome analysis demonstrated that a degree of Hp deglycosylation differs depending on the conditions used for N-glycan release and on the specific glycosylation site, with Asn 241 being the most resistant to deglycosylation under tested conditions. HILIC-UHPLC-FLR analysis enables robust quantification of 28 individual chromatographic peaks, in which N-glycan compositions were determined by UHPLC coupled to electrospray ionization quadrupole time of flight mass spectrometry. The developed analytical approach enables fast evaluation of total Hp N-glycosylation and is applicable in large-scale studies.

S. epidermidis is an important opportunistic pathogen causing chronic prosthetic joint infections associated with biofilm growth. Increased tolerance to antibiotic therapy often requires prolonged treatment or revision surgery. Phage therapy is currently used as compassionate use therapy and continues to be evaluated for its viability as adjunctive therapy to antibiotic treatment or as an alternative treatment for infections caused by S. epidermidis to prevent relapses. In the present study, we report the isolation and in vitro characterization of three novel lytic S. epidermidis phages. Their genome content analysis indicated the absence of antibiotic resistance genes and virulence factors. Detailed investigation of the phage preparation indicated the absence of any prophage-related contamination and demonstrated the importance of selecting appropriate hosts for phage development from the outset. The isolated phages infect a high proportion of clinically relevant S. epidermidis strains and several other coagulase-negative species growing both in planktonic culture and as a biofilm. Clinical strains differing in their biofilm phenotype and antibiotic resistance profile were selected to further identify possible mechanisms behind increased tolerance to isolated phages.

The rise of biosimilar monoclonal antibodies has renewed the interest in monoclonal antibody (mAb) charge variants composition and separation. The sample displacement chromatography (SDC) has the potential to overcome the low separation efficiency and productivity associated with bind-elute separation of mAb charge variants. SDC in combination with weak cation exchanging macroporous monolithic chromatographic column was successfully implemented for a separation of charge variants and aggregates of monoclonal IgG under overloading conditions. The charge variants composition was at-line monitored by a newly developed, simple and fast analytical method, based on weak cation exchange chromatography. It was proven that basic charge variants acted as displacers of IgG molecules with lower pI, when the loading was performed 1 to 1.5 pH unit below the pI of acidic charge variants. The efficiency of the SDC process is flow rate independent due to a convection-based mass transfer on the macroporous monolith. The productivity of the process at optimal conditions is 35 mg of purified IgG fraction per milliliters of monolithic support with 75-80% recovery. As such, an SDC approach surpasses the standard bind-elute separation in the productivity for a factor of 3, when performed on the same column. The applicability of the SDC approach was confirmed for porous particle-based column as well, but with 1.5 lower productivity compared to the monoliths.

Human plasma transferrin (Tf) N-glycosylation has been mostly studied as a marker for congenital disorders of glycosylation, alcohol abuse, and hepatocellular carcinoma. However, inter-individual variability of Tf N-glycosylation is not known, mainly due to technical limitations of Tf isolation in large-scale studies. Here, we present a highly specific robust high-throughput approach for Tf purification from human blood plasma and detailed characterization of Tf N-glycosylation on the level of released glycans by ultra-high-performance liquid chromatography based on hydrophilic interactions and fluorescence detection (HILIC-UHPLC-FLD), exoglycosidase sequencing, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). We perform a large-scale comparative study of Tf and immunoglobulin G (IgG) N-glycosylation analysis in two human populations and demonstrate that Tf N-glycosylation is associated with age and sex, along with multiple biochemical and physiological traits. Observed association patterns differ compared to the IgG N-glycome corroborating tissue-specific N-glycosylation and specific N-glycans’ role in their distinct physiological functions.

V diplomski nalogi predstavimo problem LWE in šifrirno shemo osnovano na tem problemu. Pokažemo pravilnost in delno tudi varnost sheme pred napadi s klasičnimi računalniki. Opišemo napad s prevodom iskalnega problema LWE na problem najkrajšega vektorja na rešetkah in uporabo algoritma za reševanje tega problema. We define LWE search problem and describe public key-cryptosystem based on such problem, for which we prove correctness and partially prove security against attacks with classical computers. We also describe an attack on such cryptosystem by reducing it to shortest vector problem on lattices and describing an algorithm for solving it.

The COVID-19 pandemic triggered an unprecedented rate of development of mRNA vaccines, which are produced by in vitro transcription reactions. The latter has been the focus of intense development to increase productivity and decrease cost. Optimization of IVT depends on understanding of the impact of individual reagents on the kinetics of mRNA production and the consumption of building blocks, which is hampered by slow, low-throughput, end-point analytics. We implemented a workflow based on rapid at-line HPLC monitoring of consumption of NTPs with concomitant production of mRNA, with a sub-3 min read-out, allowing for adjustment of IVT reaction parameters with minimal lag. IVT was converted to fed-batch resulting in doubling the reaction yield compared to batch IVT protocol, reaching 10 mg/mL for multiple constructs. When coupled with exonuclease digestion, HPLC analytics for quantification of mRNA was extended to monitoring capping efficiency of produced mRNA. When HPLC monitoring was applied to production of an ARCA-capped mRNA construct, which requires an approximate 4:1 ARCA:GTP ratio, the optimized fed-batch approach achieved productivity of 9 mg/mL with 79% capping. The study provides a methodological platform for optimization of factors influencing IVT reactions, converting the reaction from batch to fed-batch mode, determining reaction kinetics, which are critical for optimization of continuous addition of reagents, thereby paving the way towards continuous manufacturing of mRNA. This article is protected by copyright. All rights reserved.

Haptoglobin (Hp) is a positive acute phase protein, synthesized in the liver, with four N-glycosylation sites carrying mainly complex type N-glycans. Its glycosylation is altered in different types of diseases but still has not been extensively studied mainly due to analytical challenges, especially the lack of a fast, efficient, and robust high-throughput Hp isolation procedure. Here, we describe the development of a high-throughput method for Hp enrichment from human plasma, based on monolithic chromatographic support in immunoaffinity mode and downstream Hp N-glycome analysis by hydrophilic interaction ultrahigh-performance liquid chromatography with fluorescent detection (HILIC–UHPLC–FLR). Chromatographic monolithic supports in a 96-well format enable fast, efficient, and robust Hp enrichment directly from diluted plasma samples. The N-glycome analysis demonstrated that a degree of Hp deglycosylation differs depending on the conditions used for N-glycan release and on the specific glycosylation site, with Asn 241 being the most resistant to deglycosylation under tested conditions. HILIC–UHPLC–FLR analysis enables robust quantification of 28 individual chromatographic peaks, in which N-glycan compositions were determined by UHPLC coupled to electrospray ionization quadrupole time of flight mass spectrometry. The developed analytical approach enables fast evaluation of total Hp N-glycosylation and is applicable in large-scale studies.

Elution of strong and weak anion exchangers with sodium chloride gradients is commonly employed for analysis of sample mixtures containing different isomers of plasmid DNA. Gradient elution of a weak anion exchanger (diethylaminoethyl) in the presence of guanidine hydrochloride (Gdn) roughly doubles resolution between open-circular (oc) and supercoiled (sc) isomers. It also improves resolution among sc, linear, and multimeric/aggregated forms. Sharper elution peaks with less tailing increase sensitivity about 30%. However, elution with an exclusively Gdn gradient to 900 mM causes more than 10% loss of plasmid. Elution with a sodium chloride gradient while maintaining Gdn at a level concentration of 300 mM achieves close to 100% recovery of sc plasmid while maintaining the separation improvements achieved by exclusively Gdn elution. Corresponding improvements in separation performance are not observed on a strong (quaternary amine) anion exchanger. Other chaotropic salts do not produce a favorable result on either exchanger, nor does the inclusion of surfactants or EDTA. Selectivity of the diethylaminoethyl-Gdn method is orthogonal to electrophoresis, but with better quantification than agarose electrophoresis, better quantitative accuracy than CE, and resolution approaching CE.

Nanotechnologies hold great promise for various applications. To predict and guarantee the safety of novel nanomaterials, it is essential to understand their mechanism of action in an organism, causally connecting adverse outcomes with early molecular events. This is best investigated using noninvasive advanced optical methods, such as high-resolution live-cell fluorescence microscopy, which require stable labeling of nanoparticles with fluorescent dyes. However, as shown here, when the labeling is performed inadequately, unbound fluorescent dyes and inadvertently altered chemical and physical properties of the nanoparticles can result in experimental artefacts and erroneous conclusions. To prevent such unintentional errors, we introduce a tested minimal combination of experimental methods to enable artefact-free fluorescent labeling of metal-oxide nanoparticles-the largest subpopulation of nanoparticles by industrial production and applications-and demonstrate its application in the case of TiO2 nanotubes. We (1) characterize potential changes of the nanoparticles' surface charge and morphology that might occur during labeling by using zeta potential measurements and transmission electron microscopy, respectively, and (2) assess stable binding of the fluorescent dye to the nanoparticles with either fluorescence intensity measurements or fluorescence correlation spectroscopy, which ensures correct nanoparticle localization. Together, these steps warrant the reliability and reproducibility of advanced optical tracking, which is necessary to explore nanomaterials' mechanism of action and will foster widespread and safe use of new nanomaterials.

Impact of Covid-19 epidemic on the lives of residents of homes for old people The year 2020 was marked by the Covid-19 epidemic, which had the greatest impact on socially vulnerable groups, especially old people. Based on qualitative research, the article explains the impact of measures to curb the Covid-19 epidemic on the lives of old people in the Ajdovščina Home for the Old People. During this period, social work found itself in a difficult situation and was forced to respond. In institutions for old people, it found itself in an unenviable position, as it had to adapt very quickly to new challenges - to maintain contact in a time that severely limited physical contact, and to enable communication between residents and their loved ones. The article connects homes for old people with the concept of a total institution and shows how social work in the home for old people coped with challenges during the Covid-19 epidemic. The impact of epidemiological measures and social isolation on people's mental health and everyday life is shown. The fundamental tasks of social workers in crisis situations are listed.

Zdrav organizem v telesu ves čas vzdržuje ravnovesje vnetnih procesov s protivnetnimi, s čimer zmanjšuje možnosti škodljivih učinkov prekomernega sistemskega vnetja. Med pomembne dejavnike uravnavanja vnetja in imunosti sodita endokrina odziva preko hipotalamo-hipofizo-suprarenalne poti in simpatično-medularno-adrenalne poti ter nevrogeni odziv preko vagusa. 25 let raziskovanja je vodilo do podrobnejšega razumevanja delovanja in funkcije novo odkritega holinergičnega protivnetnega odziva. Njegovo aktivacijo v nucleus tractus solitarius sprožijo začetni mediatorji vnetja, čemur sledi aktivacija eferentnega vagusa in vraničnega živca, iz katerega se v belo pulpo vranice sprošča noradrenalin. Slednji preko adrenergičnih receptorjev aktivira specifične limfocite T v vranici, ki sintetizirajo in sproščajo acetilholin, ki se veže na podenote α7 nikotinskega receptorja na bližnjih monocitih. V njih se sprožijo signalne poti, ki zmanjšajo nastajanje in izločanje vnetnih citokinov. Določanje izražanja nikotinskega (CHRNA7) in adrenergičnega (ADRB2) receptorja ter encimov holin acetiltransferaze (CHAT) in acetilholinesteraze (ACHE) odpira nove možnosti za razumevanje poteka holinergičnega protivnetnega odziva in zdravljenje bolezni. V eksperimentalnem delu je preizkušen in analiziran potek dela, ki zajema izolacijo imunskih celic iz periferne človeške krvi, izolacijo RNA z dodatkom DNaze in merjenje izražanja različnih genov holinergičnega sistema s qPCR v realnem času. Izražanje CHRNA7 smo potrdili v vseh vrstah levkocitov, pri čemer je raven izražanja najvišja v izoliranih monocitih in praktično zanemarljiva v nevtrofilcih. Zaradi slednjega menimo, da granulociti, prisotni v izolatih monocitov, ne vplivajo pomembno na določanje ravni izražanja tega gena v monocitih. Uspešno smo določili izražanje ACHE v limfocitih T, zanesljivih rezultatov izražanja CHAT in ADRB2 v limfocitih T pa nismo dobili. Na manjši skupini bolnikov z zmerno hudim potekom akutnega COVID-19 smo potrdili trend znižanega izražanja CHRNA7 v monocitih in značilno povečano izražanje ACHE v limfocitih T. To nakazuje, da bi bil lahko pri teh bolnikih oslabljen vagalni protivnetni odziv. Kljub temu, da določanje izražanja CHAT in ADRB2 ni dovolj zanesljivo za nadaljnjo kvantitativno analizo, smo z vzpostavitvijo metodologije postavili temelj za nadaljnje raziskave in spremljanje izražanja bioloških markerjev v levkocitih periferne krvi, ki dajejo vpogled v stanje in delovanje protivnetnega holinergičnega sistema. Healthy organisms constantly maintain a balance between inflammatory and anti-inflammatory processes, thus preventing the harmful effects of excessive systemic inflammation. Important factors in the regulation of inflammation include the endocrinic hypothalamic-pituitary-suprarenal pathway and the sympathetic-medullary-adrenal pathway, as well as the neurogenic vagal. 25 years of research has led to a more detailed understanding of the action and function of the newly discovered cholinergic anti-inflammatory response. Its activation in the nucleus tractus solitarius is triggered by initial mediators of inflammation, followed by activation of the efferent vagus and the splenic nerve, from which noradrenaline is released into the white pulp of the spleen. Noradrenaline activates specific T lymphocytes in the spleen, which synthesize and release acetylcholine. It binds to α7 subunits of the nicotinic receptor on nearby monocytes and affects signal pathways in monocytes to reduce inflammation. Determining the expression of the nicotinic (CHRNA7) and adrenergic (ADRB2) receptors, as well as the choline acetyltransferase (CHAT) and acetylcholinesterase (ACHE) opens new possibilities for understanding the course of the cholinergic anti-inflammatory response. The experimental process, that has been critically tested and analysed, includes the isolation of immune cells from peripheral human blood, the isolation of RNA with DNase and the measurement of the expression of various genes of the cholinergic system by qPCR in real time. The expression of CHRNA7 was confirmed in all types of leukocytes it is the highest in monocytes and negligible in neutrophils. We successfully determined the expression of ACHE in T lymphocytes, but we did not get reliable results of the expression of CHAT and ADRB2 in T lymphocytes. In a small group of patients with a moderately severe acute COVID-19, we confirmed decreased expression of CHRNA7 in monocytes and characteristically increased expression of ACHE in T lymphocytes. This is in accordance with the results of other studies and suggests weakened anti-inflammatory system in these patients. Even though the measurement of expression of CHAT and ADRB2 is not reliable enough for further quantitative analysis, we laid the foundation for further research and monitoring the expression of biological markers in peripheral blood leukocytes, which provide insight into the state and function of the anti-inflammatory cholinergic system.

Type I metacaspases are the most ubiquitous of the three metacaspase types and are present in representatives of prokaryotes, unicellular eukaryotes including yeasts, algae, and protozoa, as well as land plants. They are composed of two structural units: a catalytic so-called p20 domain with the His-Cys catalytic dyad and a regulatory p10 domain. Despite their structural homology to caspases, these proteases cleave their substrates after the positively charged amino acid residues at the P1 position, just like the metacaspases of type II and type III. We present a protocol for expression and purification of the only type I protease from a secondary endosymbiosis Guillardia theta , GtMCA-I by overexpression of its gene in BL21 (DE3) E. coli cells and one-day sequential purification using nickel-affinity, ion-exchange, and size-exclusion chromatography.

Nanoparticle toxicity assessments have moved closer to physiological conditions while trying to avoid the use of animal models. An example of new in vitro exposure techniques developed is the exposure of cultured cells at the air–liquid interface (ALI), particularly in the case of respiratory airways. While the commercially available VITROCELL® Cloud System has been applied for the delivery of aerosolized substances to adherent cells under ALI conditions, it has not yet been tested on lung surfactant and semi-adherent cells such as alveolar macrophages, which are playing a pivotal role in the nanoparticle-induced immune response. Objectives: In this work, we developed a comprehensive methodology for coating semi-adherent lung cells cultured at the ALI with aerosolized surfactant and subsequent dose-controlled exposure to nanoparticles (NPs). This protocol is optimized for subsequent transcriptomic studies. Methods: Semi-adherent rat alveolar macrophages NR8383 were grown at the ALI and coated with lung surfactant through nebulization using the VITROCELL® Cloud 6 System before being exposed to TiO2 NM105 NPs. After NP exposures, RNA was extracted and its quantity and quality were measured. Results: The VITROCELL® Cloud system allowed for uniform and ultrathin coating of cells with aerosolized surfactant mimicking physiological conditions in the lung. While nebulization of 57 μL of 30 mg/mL TiO2 and 114 μL of 15 mg/mL TiO2 nanoparticles yielded identical cell delivered dose, the reproducibility of dose as well as the quality of RNA extracted were better for 114 μL.

Uvod: Program ambulantne kardiološke rehabilitacije bolnikov po akutnem miokardnem infarktu je vsebinsko strukturiran in kardiološko nadzorovan program, ki sloni na redni telesni vadbi in dokazano zmanjša umrljivost zaradi bolezni srca in žilja. Pomembno vlogo pri presojanju kakovosti programa ambulantne kardiološke rehabilitacije ima tudi bolnikovo mnenje. Merjenje zadovoljstva bolnikov nam pomaga pri izboljševanju programa ambulantne kardiološke rehabilitacije. Namen: Namen magistrskega dela je predstaviti oblike, potek in razvoj programov kardiološke rehabilitacije v Sloveniji in ugotoviti, kako zadovoljni so bolniki po prebolelem akutnem miokardnem infarktu s programom ambulantne kardiološke rehabilitacije v Splošni bolnišnici Murska Sobota. Metode dela: Izvedena je bila neeksperimentalna presečna raziskava. V teoretičnem delu smo uporabili metodo pregleda domače in tuje strokovne literature, v empiričnem delu pa smo uporabili neeksperimentalno kvantitativno metodo z uporabo anketnega vprašalnika. Zbrane podatke smo obdelali in analizirali s programoma Microsoft® Excel in SPSS. Vzorec je predstavljalo 140 bolnikov po prebolelem akutnem miokardnem infarktu, od tega je bilo 77,9 % moških in 22,1 % žensk, s povprečno starostjo 63 let. Rezultati: Raziskava je pokazala, da je bilo 80,7 % bolnikov zelo zadovoljnih s programom ambulantne kardiološke rehabilitacije, 83,6 % bolnikov je bilo zelo zadovoljnih z vadbenim delom programa, 76,4 % bolnikov pa je bilo zelo zadovoljnih s svetovanjem o spremembi življenjskega sloga v sklopu programa. Med različnimi vidiki in koristmi programa ambulantne kardiološke rehabilitacije so bili bolniki najbolj zadovoljni z odnosom medicinskih sester in fizioterapevtov (4,95 ± 0,219 in 4,95 ± 0,219) ter odnosom zdravnikov (4,79 ± 0,424). Razprava in zaključek: Naša raziskava je prva na področju preučevanja zadovoljstva bolnikov s programom ambulantne kardiološke rehabilitacije, ki v Sloveniji poteka od leta 2017, v Splošni bolnišnici Murska Sobota pa od leta 2018. Pokazala je, da so povprečne vrednosti vseh trditev, ki se nanašajo na zadovoljstvo s programom ambulantne kardiološke rehabilitacije v Splošni bolnišnici Murska Sobota, zelo visoke. Rezultati raziskave ponujajo izhodišča za oblikovanje organizacijskih in vsebinskih dopolnitev programa ambulantne kardiološke rehabilitacije, ki bo na ta način še bolj prilagojen potrebam bolnikov. Introduction: The outpatient hospital-based cardiac rehabilitation program after acute myocardial infarction is content-structured and cardiologically controlled program that is based on regular physical exercise and has been proven to reduce mortality of cardiovascular disease. The patient`s opinion plays an important role in assessing the quality of outpatient hospital-based cardiac rehabilitation program. Measuring patient satisfaction can help us to improve the outpatient hospital-based cardiac rehabilitation program. Purpose: The purpose is to determine the form, course and development of cardiac rehabilitation programs in Slovenia and find out the satisfaction of acute myocardial infarction patients with outpatient hospital-based cardiac rehabilitation program in General Hospital Murska Sobota. Methods: A non-experimental cross-sectional study research methodology was used. For the theoretical part, we analysed Slovenian and foreign professional literature. For empirical part, we used the non-experimental quantitative method. With survey questionnaire, we collected the opinions of patients and statistically analysed the data with descriptive statistics, Microsoft® Excel and SPSS. The questionnaire was answered by 140 respondents, 77,9 % of men and 22,1 % of women. Average age of respondents was 63. Results: The results of the study showed that 80,7 % of patients were generally very satisfied with the outpatient hospital-based cardiac rehabilitation program. We found that 83,6 % of patients were very satisfied with the exercise part of program and 76,4 % of patients were very satisfied with lifestyle counsellig. Among the various aspects and benefits of the outpatient hospital-based cardiac rehabilitation program, patients were most satisfied with the attitude of nurses and physiotherapists (4.95 ± 0.219 and 4.95 ± 0.219) and the attitude of physicians (4.79 ± 0.424). Discussion and conclusion: Our research is the first survey in the field of measuring patient satisfaction with the outpatient hospital-based cardiac rehabilitation program, which has been taking place in Slovenia since 2017 and at General Hospital Murska Sobota since 2018. Our research showed that the average values of all statements relating to satisfaction with the outpatient hospital-based cardiac rehabilitation program are very high. The identified views of patients represent the starting points for organizational and content updates to the outpatient hospital-based cardiac rehabilitation program, which will become even more tailored to the perceived needs of patients.

Metacaspases are essential cysteine proteases present in plants, fungi, and protists that are regulated by calcium binding and proteolytic maturation through mechanisms not yet understood. Here, we developed and validated activity-based probes for the three main metacaspase types, and used them to study calcium-mediated activation of metacaspases from their precursors in vitro. By combining substrate-inspired tetrapeptide probes containing an acyloxymethylketone (AOMK) reactive group, with purified representatives of type-I, type-II, and type-III metacaspases, we were able to demonstrate that labeling of mature metacaspases is strictly dependent on calcium. The probe with the highest affinity for all metacaspases also labels higher molecular weight proteoforms of all three metacaspases only in the presence of calcium, displaying the active, unprocessed metacaspase intermediates. Our data suggest that metacaspase activation proceeds through previously unknown active intermediates that are formed upon calcium binding, before precursor processing.

Although electrostatic modification of bacterial surfaces using polyelectrolytes (PEs) is a convenient and versatile tool for biotechnological processes, the ambiguities in toxicity of PEs between various bacteria and the insufficient understanding of the mechanism of action of cationic PEs and their nano-thick shells formed around the bacteria create a bottleneck of the approach. Here, we show how the viability of two bacterial strains, Escherichia coli and Pseudomonas stutzeri, both from the Gram-negative group differs, when the cells are exposed to cationic PEs under different conditions. Although the cell wall architecture of the strains should be structurally similar, we found that the viability of E. coli was not affected by the electrostatic deposition of polyethyleneimine (PEI) or poly(allylamine) hydrochloride (PAH), whereas for P. stutzeri the deposition resulted in high death rates. The cells of E. coli proved to be suitable templates for Layer-by-Layer (LbL) modification, while in P. stutzeri a modified protocol with mild conditions had to be used to ensure the viability of the cells. Super resolution stimulated emission depletion (STED) microscopy allowed us to clearly visualize that after PE deposition onto the surface of the cells, the PEs could penetrate inside the cells of P. stutzeri, while forming a capsule around E. coli as expected. Therefore, this knowledge will help us select the most appropriate combinations of strains and PEs, for biotechnological processes or biomedical application, preventing unwanted toxicity.

V magistrskem delu sem raziskoval, kako je epidemija covida-19, in z njo povezani ukrepi za preprečevanje širjenja okužbe, vplivala na življenje stanovalcev v Domu starejših občanov Ajdovščina. Zanimalo me je predvsem življenje stanovalcev v času omejevanja obiskov in izhodov iz doma. Pozornost sem namenil tudi času izolacij med aktivno okužbo v domu. Zanimale so me stiske in strahovi stanovalcev, ki so se soočili z valom informacij ter opozoril o nevarnosti covida-19 za stare ljudi, večjo pozornost pa sem namenil raziskovanju posledic socialne izolacije in prekinitve stikov z družino ter drugimi bližnjimi in s tem povezanimi občutki ter stiskami stanovalcev. Želel sem izvedeti, kako in v kakšnem obsegu je bilo stanovalcem v pomoč in oporo osebje doma s poudarkom na socialni službi. Zanimalo me je, kako so stanovalci sprejeli stike z bližnjimi prek informacijsko-komunikacijske tehnologije. Ugotovitve iz raziskave so precej dramatične in težke. Stanovalci so povedali, kako težko so živeli s socialno izolacijo in ostalimi ukrepi ter kako grozne občutke in doživljanja so pretrpeli v času od začetka skozi različne faze epidemije covida-19. For my master thesis I have researched how the covid-19 epidemic and related measures for prevention of spreading of the disease has affected the lives of the residents of the Home for the Older People Ajdovščina. I mainly focused on the lives of the residents at the time of restricted visits and exists from the home. The other focus was paid to the isolation during active disease in the home. I was interested in the hardships and fears residents faced with a wave of information and warnings about the dangers of covid-19 for the older people however, the main focus was kept on the feelings and distress related to social isolation and severing contact with family members and other loved ones. The objective was finding out how and to what extent the staff at the home was helpful and supportive to the residents with the emphasis being on social work. I was also interested in how the residents' faced with being in contact with their relatives through information and communication technology. The findings of this master thesis are quite dramatic and serious. The residents have elaborated on how hard living in social isolation and with other measures has been as well as what horrible feelings and experiences they have been through from the start and throughout different stages of covid-19 epidemic.

Biological functions of cell membranes and their correlation to the heterogeneity of the latter%s lipid composition are still poorly understood. Fluorescence provides one of the most versatile tools for studying biological membranes. However, few bright and photostable fluorescent probes for labeling plasma membranes are available. We have designed and synthesized two such probes, 8 and 9, that are based on the thiazole-coumarin scaffold. Both are environment sensitive and exhibit similar shifts of emission spectra in a variety of solvents as probes based on 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD). In particular, the second, positively charged probe 9 labels the plasma membrane selectively with limited redistribution to other membranes of the cell. Unfortunately, compared to the other two probes tested, 8 and 6-NBD-PC, it exhibits the highest rate of photobleaching. Nevertheless, these new thiazole-coumarin based membrane probes provide a viable approach to the design of novel membrane probes. Biološke funkcije celičnih membran in njihove korelacije s heterogenostjo njihove lipidne sestave so še vedno slabo raziskane. Fluorescenca omogoča enega izmed najbolj vsestranskih pristopov k raziskovanju bioloških membran, vendar je za označevanje plazemskih membran na voljo le malo svetlih in fotostabilnih fluorescenčnih označevalcev. Načrtovali in sintetizirali smo dve novi tovrstni fluorescenečni barvili, spojini 8 in 9, ki temeljita na tiazolo-kumarinskem ogrodju. Obe izkazujeta občutljivost na okolje in kažeta podobne premike v emisijskih spektrih v različnih topilih kot je bilo opaženo pri 7-nitrobenz-2-oksa-1,3-diazol-4-ilu (NBD). Drugo, pozitivno nabito barvilo 9, obarva plazemsko membrano selektivno in z omejenim prerazporejanjem v ostale celične membrane. Žal pa 9, za razliko od preostalih dveh testiranih barvil, torej 8 in 6-NBD-PC, zelo hitro fotobledi. Ne glede na to, razvoj dveh novih tiazolo-kumarinskih membranskih barvil predstavlja smiselen pristop k načrtovanju novih membranskih barvil.

The potential of live-cell stimulated emission depletion (STED) nanoscopy has not yet been fully exploited. Currently, the main limitation is the small number of fluorophores and probes that can sustain high light intensity/high dose employed in STED. Namely, fluorophores suitable for STED nanoscopy must be bright and highly photostable and exhibit a large Stokes shift. To expand the list of available probes, we synthesized and evaluated several new membrane probes for live-cell STED nanoscopy. Of the tested probes, probes MePyr500, ThiaCN545 and NB640 not only allow high-resolution STED images, but also partition into the intracellular membranes relatively quickly, thus lacking the selectivity of labelling solely the plasma membrane. During experiments, cytotoxicity was observed merely with the probe ThiaCN545, which blebs the plasma membrane. In comparison with commercially available CellMask Orange and STAR RED (KK114) DPPE, all our tested probes exhibited better photostability with the exception of NB640, which had the fastest bleaching rate of all tested probes. The best overall results can be assigned to the probe MePyr500, providing high-resolution STED images as well as high photostability with no noticeable cytotoxicity, making it an excellent candidate for further development.

To avoid a devastating effect of eye vision impairment on the information flow from the eye to our brain, enormous effort is being put during the last decades into the development of more sensitive diagnostics and more efficient therapies of retinal tissue. While morphology can be impressively imaged by optical coherence tomography, molecular-associated pathology information can be provided almost exclusively by auto-fluorescence-based methods. Among the latter, the recently developed fluorescence lifetime imaging ophthalmoscopy (FLIO) has the potential to provide both structural information and interacting pictures at the same time. The requirements for FLIO laser sources are almost orthogonal to the laser sources used in phototherapy that is expected to follow up the FLIO diagnostics. To make theranostics more effective and cheaper, the complete system would need to couple at least the modalities of low-power high-repetition-rate FLIO and precision high-pulse energy-adjustable repetition rate phototherapy. In addition, the intermediate-power high repetition rate for two-photon excitation would also be desired to increase the depth resolution. In our work, compact fiber-laser based on high-speed gain-switched laser diode has been shown to achieve adaptable/independently tunable repetition rate and energy per pulse allowing coupled fluorescence lifetime diagnostics via two-photon excitation and phototherapy via laser-induced photodisruption on a local molecular environment in a complex ex vivo retinal tissue.

Regulirana celična smrt (RCD) je evolucijsko ohranjen proces, ki je bil opažen pri posameznih predstavnikih vseh živečih organizmov. Sprva so ta pojav pripisovali le evkariontskim večceličnim organizmom, vendar so kasneje opazili, da do tega pojava prihaja tudi pri enoceličnih organizmih kot so kvasovke, alge in bakterije. Pri izvedbi RCD pri živalskih organizmih so ključne cisteinske endopeptidaze kaspaze. Pri ostalih organizmih kaspaze niso prisotne, so pa prisotni njim homologni proteini, metakaspaze. Kljub strukturni homologiji med kaspazami in metakaspazami se ti dve skupini po funkcionalnih lastnostih bistveno razlikujeta. Metakaspaze namreč cepijo substrate za pozitivno nabitimi aminokislinskimi ostanki ter za svojo katalitično aktivnost ne potrebujejo oligomerizacije. Večina metakaspaz je katalitično aktivna le v prisotnosti kalcijevih ionov. Glavni namen tega dela je bil izraziti, očistiti in okarakterizirati predstavnice vseh treh tipov metakaspaz. Metakaspazi GtMC1 (tip I) in GtMC2 (tip III) izvirata iz sekundarnega endosimbionta, alge Guillardia theta. CrMC2 je metakaspaza tipa II iz zelene alge Chlamydomonas reinhardtii. Proteolitična aktivnost vseh treh metakaspaz je podobna, saj proteinske substrate cepijo le v prisotnosti kalcijevih ionov. Vse tri proteaze cepijo majhna sintetična substrata Z-FR-AMC in Z-RR-AMC, vendar se njihove Michaelisove konstante precej razlikujejo. V delu smo preverjali tudi inhibitorno sposobnost proteinskega inhibitorja CrSERPIN-a ob spremljanju hidrolize majhnega sintetičnega substrata. CrSERPIN najbolj inhibira CrMC2, najmanj pa GtMC2. Testirali smo tudi štiri različne tetrapeptidne sonde za detekcijo metakaspazne aktivnosti, ki se ireverzibilno vežejo v aktivno mesto metakaspaz. Sonde EKAK-AOMK, EKTK-AOMK, IRSK-AOMK in IISK-AOMK so bile načrtovane tako, da so specifične za metakaspaze. Njihov peptidni del je prilagojen tako, da je čimbolj podoben substratni specifičnosti že okarakteriziranih metakaspaz. Konstante inaktivacije sond so najvišje za metakaspazo CrMC2, iz česar sklepamo, da so sonde najbolj primerne za detekcijo metakaspaz tipa II. Sonda z najvišjimi konstantami inaktivacije za posamezno metakaspazo je IRSK-AOMK, kar kaže na to, da imajo vse tri metakaspaze podobno kemijsko okolje vezavnega žepa za substrat. Regulated cell death (RCD) is an evolutionarily conserved process that can be observed in representatives of all living organisms. It was first described in eukaryotic multicellular organisms, but was later also observed in unicellular organisms such as algae and bacteria. Main executioners of metazoan RCD are cysteine endopeptidases called caspases. Caspases are not present in non-metazoan organisms. Instead, other cysteine proteases homologous to caspases are found in these organisms. They are termed metacaspases. Despite structural homology to caspases, metacaspases display different characteristics: they cleave their substrates after positively charged amino acids, do not require dimerization for catalytic activity, and most of them require calcium ions for their proteolytic activity. The main objective of this thesis was the expression, purification and characterization of three metacaspases, each representative of the three types. The metacaspases GtMC1 and GtMC2, a type I and type III metacaspase, respectively, originate from the algal organism Guillardia theta, which is a secondary endosymbiont. CrMC2 is a type II metacaspase from a green algal organism Chlamydomonas reinhardtii. Proteolytic activities of the three metacaspases are similar: they cleave protein substrates only in the presence of calcium ions. Cleavage of the small synthetic substrates Z-FR-AMC and Z-RR-AMC is also successful, however Michaelis constants for their hydrolysis are substantially different. The inhibitory efficiency of the protein inhibitor CrSERPIN was also tested using a small synthetic fluorogenic substrate. CrSERPIN inhibits CrMC2 most effectively and GtMC2 least effectively. We also tested four different tetrapeptide probes for the detection of metacaspases that irreversibly bind to their active sites. The probes EKAK-AOMK, EKTK-AOMK, IRSK-AOMK and IISK-AOMK were specifically designed for the detection of metacaspases and their peptide moieties were adjusted according to the substrate specificity of other characterized metacaspases. The inactivation constants of the probes are highest for the metacaspase CrMC2, indicating that these probes are most suitable for the detection of type II metacaspases. The probe with the highest inactivation constant for each metacaspase is IRSK-AOMK, indicating that all three metacaspases have a similar chemical nature of the substrate binding pocket.

Nanotechnologies hold great promise for various applications. To predict and guarantee the safety of novel nanomaterials, it is essential to understand their mechanism of action in an organism, causally connecting adverse outcomes with early molecular events. They are best investigated using non-invasive advanced optical methods, such as high-resolution live-cell fluorescence microscopy, which require stable labelling of nanoparticles with fluorescent dyes. When performed inadequately, unbound fluorophores and inadvertently altered chemical and physical properties of the nanoparticles can, however, result in experimental artefacts and erroneous conclusions.To prevent such unintentional errors, we here describe a minimal combination of experimental methods to enable artefact-free fluorescent labelling of metal-oxide nanoparticles – the largest subpopulation of nanoparticles by industrial production and applications – and demonstrate its application in the case of TiO2 nanotubes. We 1) characterize potential changes of the nanoparticles’ surface charge and morphology that might occur during labelling, and 2) assess stable binding of the fluorescent dye to nanomaterial, which ensures correct nanoparticle localization. Together, these steps warrant the reliability and reproducibility of advanced optical tracking, which is necessary to explore nanomaterials’ mechanism of action and will foster widespread and safe use of new nanomaterials.Abstract Figure

Triatlon moči postaja v Sloveniji vse bolj priljubljena športna disciplina. To velja tako za tekmovalno kot za rekreativno pojavno obliko. Vsako leto se tako povečuje število oseb, ki se ukvarja s tem športom rekreativno ali tekmovalno, bodisi organizirano ali neorganizirano. Mnogi, še posebej začetniki, največkrat že pri treningu utrpijo poškodbe. Najpogostejši razlog zanje so pomanjkljivosti pri pravilni izvedbi tehnike posameznih prvin triatlona. Namen magistrskega dela je bil ugotoviti strategije za preprečevanje odsotnosti s trenažno-tekmovalnega procesa pri triatlonu moči. Raziskavo smo izvedli na vzorcu 78 anketirancev, ki so se s triatlonom moči ukvarjali rekreativno ali tekmovalno. Ugotovili smo, da je imela večina anketirancev pomanjkljivo znanje o metodah osnovnega in specialnega ogrevanja, prav tako niso poznali specialnih gimnastičnih vaj, s katerimi bi lahko izboljšali kakovost izvedbe tehnike posameznih prvin. Anketiranci poleg tega niso znali prepoznati osebnih omejitev in pomanjkljivosti, kar se je odražalo v slabši tehniki in s tem večji možnosti za nastanek poškodb. Ugotovili smo še, da so anketiranci relativno dobro poznali različne strategije za izboljšanje regeneracije po treningu, ki so jih pogosto koristili. Na osnovi opisanih ugotovitev smo za dvigalce pri triatlonu moči pripravili program, s katerim lahko izboljšajo svoje znanje o uporabi različnih strategij za preprečevanje odsotnosti s trenažno-tekmovalnega procesa. V programu so opisani testi, ki se uporabljajo za diagnosticiranje gibalnih omejitev. Za izboljšanje teh omejitev smo predstavili različne vaje. Prav tako smo v program vključili prehranska priporočila, priporočila za vnos tekočin ter druge pomembne dejavnike, s katerimi izboljšamo regeneracijo po naporu pri triatlonu moči. Predvidevamo, da lahko poznavanje in uporaba sredstev programa za preprečevanje odsotnosti s trenažno-tekmovalnega procesa pri triatlonu moči zmanjša možnost nastanka poškodb in izboljša regeneracijo po treningih in tekmah. Powerlifting popularity in Slovenia is rising fast. Every year there are more people who practise powerlifting for recreation and those who compete in powerlifting on national or international levels. Powerlifters, especially newcomers, often get injured during the training phase, wherein majority of the injuries are associated with poor lifting technique. The purpose of the master thesis was to identify the strategies for prevention of absence from competing and training in powerlifting. The study enrolled 78 participants, with recreational and competitive experiences in powerlifting. Our results have demonstrated the lack of knowledge regarding general and specific warmup, as well as specific exercises with aims of improving mobility and movement patterns of each discipline of powerlifting. On the other hand, our result suggested that participants are fairly well educated about various regeneration techniques, which they used regularly. In line with the findings of our study, we have developed a training handbook to additionally equip the powerlifters with new knowledge of various training and regeneration strategies with aims of reducing the absence from training and competition. Training handbook includes assessments of joint mobility and exercise for improving movement discrepancies. In addition, handbook also includes nutrition and hydration advice along with other important factors for optimal recovery. We assume that the knowledge of strategies included in the handbook can reduce the risk of injury and improve regeneration between trainings and competitions.

Glavni cilj magistrske naloge je obravnava opismenjevanja v učbenikih, ki se uporabljajo za poučevanje angleščine v drugi triadi osnovne šole. Ker je dandanes pismenost tako v maternem, kot tudi v tujem jeziku pomembna, če ne nujna, delo raziskuje, katere metode opismenjevanja so prisotne pri pouku angleščine. V prvemu delu je predstavljen proces branja ter razlike v pisnih sistemih angleščine ter slovenščine. Sledi pregled metod opismenjevanja v angleško-govorečih državah in v Sloveniji. Te so: sintetična, analitična in globalna metoda. Enakovredna uporaba vseh treh pa dosega najboljše rezultate pri opismenjevanju. Ker sta si angleščina in slovenščina ortografsko zelo različni, je naštetih še nekaj pogostih težav, ki se lahko pojavijo pri opismenjevanju v angleščini. V empiričnemu delu je analiziranih devet učbenikov angleškega jezika: trije za 4. razred, trije za 5. razred in trije za 6. razred. Cilj naloge je bil odgovoriti na pet vprašanj: ali so v učbeniku eksplicitna fonološka navodila ali učbeniki učijo medsebojno zvezo med kombinacijami črk in glasovi ali učbeniki učijo angleško abecedo ali učbeniki krepijo glasovno zavedanje in kako ter katera metoda opismenjevanja v teh učbenikih prevladuje. Rezultati kažejo, da imajo vsi učbeniki v vsaki učni enoti vsaj eno nalogo, ki razvija in spodbuja opismenjevanje, vendar je sistematično razvijanje pismenosti v učbenikih še vedno v veliki meri zanemarjeno. The main aim of this MA thesis is to address literacy development in English textbooks used in the second cycle of primary school. As literacy skills in the mother, as well as foreign language, are important, if not essential, the research explores which methods of literacy instruction are present in English lessons. The first part presents the reading process and the differences between the English and Slovenian writing systems. An overview of methods of literacy instruction in English speaking countries and in Slovenia follows. The methods are: the synthetic, analytical, and global method. Equal use of all three yields the best results when teaching and developing literacy. Some common problems that can occur when teaching literacy skills are listed, because the English and Slovenian languages are orthographically very different. The empirical part contains an analysis of nine English textbooks: three for 4th grade, three for 5th grade and three for 6th grade. The aim of the empirical research was to answer five questions: is there explicit phonological instruction in the textbooks whether textbooks address the letter-sound correspondence whether textbooks teach the English alphabet whether textbooks strengthen phonemic awareness and how and what method of literacy prevails in these textbooks. The results show that each unit in the textbooks analyzed has at least one task that develops and encourages the literacy process, but systematic literacy development in coursebooks is still in large part neglected.

Tissue diseases and related disorders need to be first recognized using diagnostic methods and then later treated by therapeutic methods–a joint procedure called theranostics. One of the main challenges in the field of retinal therapies remains in the success of the treatment, typically improving the local metabolism, by sparing the surrounding tissue and with the immediate information of the laser effect. In our study, we present a concept for real-time controlled tissue theranostics on a proof-of-concept study capable of using a single tunable ps laser source (in terms of irradiance, fluence, and repetition rate), done on ex-vivo human retinal pigment epithelium. We have found autofluorescence intensity and lifetime imaging diagnostics very promising for the recognition and quantification of laser effects ranging from selective non-destructive molecular tissue modification to complete tissue ablation. The main novelty of our work presents the developed algorithm for optimized theranostics based on the model function used to quantify laser-induced tissue changes through the diagnostics descriptors, fluorescence lifetime and fluorescence intensity parameters. This approach, together with the operation of the single adaptable laser source, can serve as a new theranostics method in personalized medicine in the future not only limited to treat retinal diseases.

HPLC is established as a fast convenient analytical technology for characterizing the content of empty and full capsids in purified samples containing adeno-associated virus (AAV). UV-based monitoring unfortunately over-estimates the proportion of full capsids and offers little value for characterizing unpurified samples. The present study combines dual-wavelength UV monitoring with intrinsic fluorescence, extrinsic fluorescence, and light-scattering to extend the utility of HPLC for supporting development of therapeutic AAV-based drugs. Applications with anion exchange (AEC), cation exchange (CEC), and size exclusion chromatography (SEC) are presented. Intrinsic fluorescence increases sensitivity of AAV detection over UV and enables more objective estimation of empty and full capsid ratios by comparison of their respective peak areas. Light scattering enables identification of AAV capsids in complex samples, plus semiquantitative estimation of empty and full capsid ratios from relative peak areas of empty and full capsids. Extrinsic Picogreen fluorescence enables semiquantitative tracking of DNA with all HPLC methods at all stages of purification. It does not detect encapsidated DNA but reveals DNA associated principally with the exteriors of empty capsids. It also enables monitoring of host DNA contamination across chromatograms. These enhancements support many opportunities to improve characterization of raw materials and process intermediates, to accelerate process development, provide rapid in-process monitoring, and support process validation.

Za uspešno uravnavanje vnetnih reakcij pri poškodbah ali infekcijah je pravilno delovanje vnetnih in protivnetnih mehanizmov imunskega sistema ključnega pomena. Vnetje je pri zdravem človeku uravnavano s pravilnim razmerjem v količini vnetnih in protivnetnih mediatorjev, saj tako ne pride do prekomernega sistemskega vnetja ali sepse. Izločanje vnetnih in protivnetnih molekul nadzorujeta humoralni sistem (kortizol) preko hipotalamo-hipofizo-suprarenalne osi in živčni sistem preko živca vagusa. V zadnjih dvajsetih letih so znanstveniki iz različnih laboratorijev po svetu poročali o najrazličnejših odkritjih, ki so privedle do razumevanja mehanizma in pomena novo odkritega holinergičnega protivnetnega odziva, ki ga nadzira živec vagus. Holinergični protivnetni odziv temelji na aktivaciji živca vagusa ter prenosu signala na vranični živec, pri čemer se iz perifernih živčnih končičev slednjega v vranici sprošča noradrenalin, ta pa stimulira podvrsto limfocitov T, da v vranici začnejo sintetizirati in izločati acetilholin. Acetilholin se veže na α7 nikotinske receptorje na makrofagih in preko različnih mehanizmov preprečuje izločanje vnetnih citokinov. Določanje izražanja mRNA alfa-7 nikotinskih receptorjev v periferni krvi bi bila lahko dokaj enostavna metoda, s katero bi pri človeku lahko določali aktivnost holinergične protivnetne poti v različnih bolezenskih stanjih. V metodologiji smo predlagali protokol dela za vzpostavitev rutinskega merjenja izražanja mRNA α7 nikotinskih receptorjev, ki zajema izolacijo PBMC iz človeške krvi, izolacijo RNA in dvostopenjski RT-PCR. To bi lahko postalo uporabno metodološko orodje za klinične raziskave, s časom pa potencialno tudi kot rutinski diagnostični biomarker stanja holinergičnega protivnetnega odziva. Ugotovitve glede postopkov izolacije PBMC in RT-PCR smo primerjali z metodologijo izolacije, ki so jo opisali v raziskavah, kjer so raven izražanja mRNA nikotinskih acetilholinskih receptorjev v celicah iz periferne krvi pri bolnikih že določali. Nova in do sedaj redko uporabljena je metoda imunomagnetne negativne selekcije z EasySep, ki temelji na označevanju krvnih celic z monoklonskimi protitelesi in njihovo izolacijo s pomočjo magnetnih delcev. Ta metoda se zdi še posebej uporabna, saj hitro in v zadostni količini omogoča izolacijo specifičnih podvrst limfocitov kot tudi monocitov. S predlagano metodologijo qPCR želimo ugotavljati razlike v izražanju gena za α7 nikotinski receptor pri bolnikih z različnimi oblikami in jakostjo vnetja v primerjavi z zdravimi. Normal activity of inflammatory and anti-inflammatory mechanism of immune system is crucial for successful regulation of inflammation caused by infections and injuries. Inflammation is normally regulated by the right proportion of inflammatory and anti-inflammatory cytokines, so excessive systemic inflammation and sepsis are excluded. Secretion of inflammatory and anti-inflammatory cytokines is regulated by humoral system (cortisol) through hypothalamic-pituitary-adrenal axis and nervous system through vagus nerve. In the last 20 years it has been reported about many different discoveries by laboratories around the world, which lead to understanding of the mechanism and meaning of the newly discovered cholinergic anti-inflammatory pathway, which is supervised by vagus nerve. Cholinergic anti-inflammatory pathway is based on activation of vagus nerve which transducts the signal to splenic nerve, which secretes noradrenaline, the activator of specific subset of T lymphocytes in spleen. T lymphocytes then start secreting acetylcholine, which binds to α7 nicotinic receptors on macrophages and prevents the secretion of inflammatory cytokines through different molecular mechanisms. Determination of alpha-7 nicotinic receptor mRNA expression in human peripheral blood could present a simple method to determine the activity of human cholinergic anti-inflammatory response during different diseases. We have suggested methodological protocol to establish routine measurement of alpha-7 nicotinic receptor mRNA expression, which includes isolation of PBMC from human peripheral blood, isolation of whole RNA and two-level RT-PCR. This could become a useful methodological tool for clinical researches and in time also a routine diagnostic biomarker for determining the condition of cholinergic anti-inflammatory response. Findings about protocols for isolation of PBMC and RT-PCR were compared to the methodology of isolation of PMBC, which was used for routine measurement of alpha-7 nicotinic receptor mRNA expression in peripheral blood of different patients, described in different researches. Newly discovered and rarely used method for isolation of specific cells of PBMC is immunomagnetic negative selection with EasySep, which is based on labelling blood cells with monoclonal antibodies and suspension of magnetic particles. This method seems to be very useful, because it sufficiently and quickly enables isolating of specific subsets of lymphocytes and monocytes. With the suggested method and qPCR the differences between alpha-7 nicotinic receptor mRNA expression in patients with different forms and intensities of inflammation and healthy people would like to be found.

Purification of high-quality plasmid DNA in large quantities is a crucial step in its production for therapeutic use and is usually conducted by different chromatographic techniques. Large-scale preparations require the optimization of yield and homogeneity, while maximizing removal of contaminants and preserving molecular integrity. The advantages of Convective Interaction Media

Purification of high-quality plasmid DNA in large quantities is a crucial step in its production for therapeutic use and is usually conducted by different chromatographic techniques. Large-scale preparations require the optimization of yield and homogeneity, while maximizing removal of contaminants and preserving molecular integrity. The advantages of Convective Interaction Media® (CIM®) monolith stationary phases, including low backpressure, fast separation of macromolecules, and flow-rate-independent resolution qualified them to be used effectively in separation of plasmid DNA on laboratory as well as on large scale. A development and scale-up of plasmid DNA downstream process based on chromatographic monoliths is described and discussed below. Special emphasis is put on the introduction of process analytical technology principles and tools for optimization and control of a downstream process.

Blood coagulation mechanisms forming a blood clot and preventing hemorrhage have been extensively studied in the last decades. Knowing the mechanisms behind becomes very important particularly in the case of blood vessel diseases. Real-time and accurate diagnostics accompanied by the therapy are particularly needed, for example, in diseases related to retinal vasculature. In our study, we employ for the first time fluorescence hyperspectral imaging (fHSI) combined with the spectral analysis algorithm concept to assess physical as well as functional information of blood coagulation in real-time. By laser-induced local disruption of retinal vessels to mimic blood leaking and subsequent coagulation and a proper fitting algorithm, we were able to reveal and quantify the extent of local blood coagulation through direct identification of the change of oxyhemoglobin concentration within few minutes. We confirmed and illuminated the spatio-temporal evolution of the essential role of erythrocytes in the coagulation cascade as the suppliers of oxygenated hemoglobin. By additional optical tweezers force manipulation, we showed immediate aggregation of erythrocytes at the coagulation site. The presented fluorescence-based imaging concept could become a valuable tool in various blood coagulation diagnostics as well as theranostic systems if coupled with the laser therapy.

Many chronic diseases manifest themselves in prolonged inflammation and often ignored dysregulated lipid metabolism, both also associated with inhalation of certain nanomaterials. Limited knowledge of involved molecular events and their causal connections prevents reliable prediction of outcomes by efficient testing strategies. To unravel how acute nanomaterial exposure leads to chronic conditions, we employed advanced microscopy and omics in vitro , in vivo and in silico . For selected metal-oxide nanomaterials, we show that epithelial cells survive the exposure by excreting internalized nanomaterials and passivating them on the surface, employing elevated lipid synthesis. Macrophages, on the contrary, attack the defending epithelium but die degrading passivized complexes, releasing nanomaterial, which is reuptaken by epithelial cells. Constant proinflammatory signalling recruits new phagocytes that feed the vicious cycle of events resulting in a long-lasting response to a single exposure. The discovered mechanism predicts the nanomaterial-associated in vivo chronic outcomes based on simple in vitro measurements and potentially enlightens other chronic diseases. Graphical abstract

Knowing the temperature distribution within the conducting walls of various multilayer-type materials is crucial for a better understanding of heat-transfer processes. This applies to many engineering fields, good examples being photovoltaics and microelectronics. In this work we present a novel fluorescence technique that makes possible the non-invasive imaging of local temperature distributions within a transparent, temperature-sensitive, co-doped Er:GPF1Yb0.5Er glass-ceramic with micrometer spatial resolution. The thermal imaging was performed with a high-resolution fluorescence microscopy system, measuring different focal planes along the z-axis. This ultimately enabled a precise axial reconstruction of the temperature distribution across a 500-µm-thick glass-ceramic sample. The experimental measurements showed good agreement with computer-modeled heat simulations and suggest that the technique could be adopted for the spatial analyses of local thermal processes within optically transparent materials. For instance, the technique could be used to measure the temperature distribution of intermediate, transparent layers of novel ultra-high-efficiency solar cells at the micron and sub-micron levels.

Fluorescent carbon dots (CDs) are potential tools for the labeling of cells with many advantages such as photostability, multicolor emission, small size, rapid uptake, biocompatibility, and easy preparation. Affinity towards organelles can be influenced by the surface properties of CDs which affect the interaction with the cell and cytoplasmic distribution. Organelle targeting by carbon dots is promising for anticancer treatment; thus, intracellular trafficking and cytotoxicity of cationic CDs was investigated. Based on our previous study, we used quaternized carbon dots (QCDs) for treatment and monitoring the behavior of two human cancer cell MCF-7 and HeLa lines. We found similarities between human cancer cells and mouse fibroblasts in the case of QCDs uptake. Time lapse microscopy of QCDs-labeled MCF-7 cells showed that cells are dying during the first two hours, faster at lower doses than at higher ones. QCDs at a concentration of 100 µg/mL entered into the nucleus before cellular death; however, at a dose of 200 µg/mL, blebbing of the cellular membrane occurred, with a subsequent penetration of QCDs into the nuclear area. In the case of HeLa cells, the dose-depended effect did not happen; however, the labeled cells were also dying in mitosis and genotoxicity occurred nearly at all doses. Moreover, contrasted intracellular compartments, probably mitochondria, were obvious after 24 h incubation with 100 µg/mL of QCDs. The levels of reactive oxygen species (ROS) slightly increased after 24 h, depending on the concentration, thus the genotoxicity was likely evoked by the nanomaterial. A decrease in viability did not reach IC 50 as the DNA damage was probably partly repaired in the prolonged G0/G1 phase of the cell cycle. Thus, the defects in the G2/M phase may have allowed a damaged cell to enter mitosis and undergo apoptosis. The anticancer effect in both cell lines was manifested mainly through genotoxicity.

Tryptic hydrolysis of β-Lactoglobulin (β-Lg) is attracting more and more attention due to the reduced allergenicity and the functionality of resulting hydrolysates. To produce hydrolysates in an economically viable way, immobilized trypsin reactors (IMTRs), based on polymethacrylate monolith with pore size 2.1 μm (N1) and 6 μm (N2), were developed and used in a flow-through system. IMTRs were characterized in terms of permeability and enzymatic activity during extensive usage. N1 showed twice the activity compared with N2, correlating well with its almost two times higher amount of immobilized trypsin. N2 showed high stability over 18 cycles, as well as over more than 30 weeks during storage. The efficiency of IMTRs on hydrolyzing β-Lg was compared with free trypsin, and the resulting hydrolysates were analyzed by MALDI-TOF/MS. The final hydrolysis degree by N1 reached 9.68% (86.58% cleavage sites) within 4 h, while only around 6% (53.67% cleavage sites) by 1.5 mg of free trypsin. Peptides analysis showed the different preference between immobilized trypsin and free trypsin. Under the experimental conditions used in this study, the potential cleavage site Lys135-Phe136 was resistant against the immobilized trypsin in N1. This article is protected by copyright. All rights reserved