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Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by impaired skin barrier function. Amongst the various dermal formulations that are being used and/or investigated for AD treatment, one of the advanced approaches is the use of hydrogels as film-forming systems that are applied directly to the skin and have the added value of providing a physical barrier, which is lacking in atopic skin. Novel film-forming hydrogels based on two different nanocrystalline celluloses (NCCs) in combination with one of two natural polymers (alginate or pectin) were developed for incorporation of betamethasone dipropionate (BDP). Initially, the low water solubility of BDP was resolved by prior dissolution in a self-microemulsifying drug delivery system (SMEDDS). The mixture of Kolliphor® EL/Capryol® 90 in a ratio of 8/2 was chosen on the merit of its high BDP-saturated solubility and no BDP precipitation upon water dilution, enabling BDP to remain dissolved after incorporation into hydrogels. The solvent evaporation method was used to prepare the films, and their high water retention capacity was confirmed in vitro on artificial membranes and pig ear skin. The presented results thus confirm NCC-based film-forming hydrogels as a very promising drug delivery system for AD treatment. Nasl. z nasl. zaslona. Opis vira z dne 10. 7. 2023. Št. članka: 1918. Bibliografija: str. 20-21. Abstract. ARRS

Uvod: Poglavitni namen doktorskega dela je bil pridobiti dodaten vpogled v patogenetske mehanizme gigantoceličnega arteritisa (GCA) in povezati le-te z ultrazvočno preiskavo arterij (danes preiskavo izbora v diagnostiki gigantoceličnega vaskulitisa). V raziskavah, opravljenih v okviru doktorskega dela, smo preverili izražanje mikro RNA (miRNA) v temporalnih arterijah (TA) bolnikov in njihove genske tarče. Fenotipizirali in kvantitativno smo ocenili imunske celice, ki sestavljajo vnetni infiltrat v biopsijah temporalnih arterij (TAB) bolnikov z GCA, ter pridobljene histopatološke podatke povezali z izražanjem miRNA. Poleg tega smo ocenili medsebojno povezanost med izsledki ultrazvočne (UZ) preiskave TA in histopatološkimi podatki ter izražanjem izbranih miRNA na drugi strani. Metode: V prvem delu doktorskega dela smo z analizo izražanja miRNA pri bolnikih z GCA ocenili izražanje 28 miRNA v vzorcih TAB 30 bolnikov s histološko potrjeno boleznijo in izsledke primerjali z izražanjem miRNA v vzorcih TAB 16 bolnikov, ki niso imeli histološko potrjene bolezni, ter vzorcih TAB 22 oseb brez GCA. V drugem delu raziskave smo kvantitativno ocenili histološke spremembe v temporalnih arterijah bolnikov z GCA s histopatološkimi in imunohistokemičnimi tehnikami. V tretjem delu raziskave smo preverili, kako so najdbe UZ pregleda TA povezane s histolopatološkimi spremembami TA in z izražanjem miRNA. Rezultati: Z raziskavo izražanja miRNA smo ugotovili aberantno (več kot dvakratno) povišano ali znižano izražanje 28 miRNA. Aberantno izražene miRNA so vpete v patogenetske mehanizme, ki sodelujejo pri preoblikovanju arterij in uravnavanju odziva imunskega sistema. V raziskavi povezanosti histopatoloških lastnosti infiltrata in izražanjem miRNA smo ugotovili, da je vnetnocelični infiltrat sestavljen pretežno iz CD3+, CD4+, CD8+ limfocitov T, CD68+ makrofagov, ki jih je spremljalo povečano izražanje citoplazemskega jedrnega faktorja aktiviranih T celic 1 (NFATC). Pokazali smo, da bi spremenjeno izražanje izbranih miRNA lahko bilo povezano z infiltracijo žilne stene z limfociti in makrofagi ter aktivacijo limfocitov T. V raziskavi povezanosti histopatoloških, epigenetskih, kliničnih in slikovnih podatkov smo ugotovili močno povezanost prisotnosti halo znaka in debeline kompleksa intime-medije z intenzivnostjo vnetnega infiltrata, spremenjenim profilom izražanja miRNA in prisotnostjo klinične spremembe TA ter bolečine v žvečnih mišicah. Zaključki: Opredelitev profilov izražanja miRNA v steni temporalnih arterij bolnikov z GCA, ki potencialno sodelujejo pri uravnavanju vnetne infiltracije, imunskega odziva in fenotipa gladkih mišičnih celic, omogoča dodaten vpogled v zapleteno patogenezo GCA. Pokazali smo, da bi spremenjeno izražanje izbranih miRNA lahko bilo povezano z infiltracijo žilne stene z limfociti in makrofagi ter aktivacijo limfocitov T, ki so povečano izražali NFATC v TAB bolnikov z GCA. Naša raziskava omogoča vpogled v medsebojno povezanost parametrov UZ preiskave z ugotovljenimi histopatološkimi, epigenetskimi, kliničnimi in laboratorijskimi lastnostmi žilnih lezij pri bolnikih z GCA. Introduction: The main aim of this PhD thesis was to gain further insight into the pathogenetic mechanisms of giant cell arteritis (GCA) and to relate these to arterial ultrasound (nowadays the test of choice in the diagnosis of giant cell vasculitis). In the studies carried out as part of this PhD, we examined the expression of microRNAs (miRNAs) in temporal arteries (TA) of patients and their target genes. We phenotyped and quantified immune cells constituting the inflammatory infiltrate in temporal artery biopsies (TAB) from GCA patients and correlated the histopathological data obtained with miRNA expression. In addition, we evaluated the correlation between TA ultrasound (US) findings and, histopathological data and the expression of selected miRNAs on the other hand. Methods: In the first part of the study, we evaluated the expression of 28 miRNAs in TAB specimens from 30 patients with histologically confirmed disease and compared the results with the expression of miRNAs in TAB specimens from 16 patients without histologically confirmed disease and 22 subjects without GCA. In the second part of the study, we quantitatively assessed histological changes in temporal arteries of patients with GCA using histopathological and immunohistochemical techniques. In the third part of the study, we examined how findings on US examination of the TA correlated with histopathological changes of the TA and with miRNA expression. Results: The miRNA expression study revealed aberrant (more than twofold) elevated or decreased expression of 28 miRNAs. Aberrantly expressed miRNAs are involved in pathogenetic mechanisms involved in arterial remodelling and regulation of the immune response. In a study of the association between histopathological characteristics of the infiltrate and miRNA expression, we found that the inflammatory cell infiltrate consisted predominantly of CD3+, CD4+, CD8+ T lymphocytes, CD68+ macrophages, accompanied by increased expression of cytoplasmic nuclear factor of activated T cells 1 (NFATC). We showed that altered expression of selected miRNAs could be associated with infiltration of the vascular wall by lymphocytes and macrophages and activation of T lymphocytes. In a study of the association of histopathological, epigenetic, clinical and imaging data, we found a strong correlation between the presence of halo sign and the thickness of the intima-media complex with the intensity of the inflammatory infiltrate, the altered miRNA expression profile, the change in clinical appearance of TA and pain in the masticatory muscles. Conclusions: Identification of miRNA expression profiles in the temporal artery wall of GCA patients, potentially involved in the regulation of inflammatory infiltration, immune response and smooth muscle cell phenotype, provides new insights into the complex pathogenesis of GCA. We showed that altered expression of selected miRNAs could be associated with infiltration of the vascular wall by lymphocytes and macrophages and activation of T lymphocytes, which upregulated NFATC expression in the TAB of GCA patients. Our study provides insight into the correlation of ultrasound examination parameters with the histopathological, epigenetic, clinical and laboratory features of vascular lesions found in GCA patients.

Imunski sistem ščiti telo pred okužbami, boleznimi in škodljivimi snovmi, kot so virusi, bakterije in paraziti ter ohranja ravnovesje med obrambo telesa in preprečevanjem prekomernega vnetja ali avtoimunskih odzivov. To doseže z zaznavanjem in nevtraliziranjem patogenov ter nadzorovanjem popravila tkiv in celjenja. Prehranska podpora imunskemu sistemu je ključnega pomena za vzdrževanje optimalnega zdravja in preprečevanje bolezni. Osredotočanje na makrohranila, kot so beljakovine, ogljikovi hidrati in maščobe, pomaga zagotavljati temelje uravnotežene prehrane, ki podpira imunsko funkcijo. Beljakovine igrajo pomembno vlogo pri podpori imunskega sistema, saj so osnovna gradbena enota za rast in obnovo tkiv ter proizvodnjo protiteles in imunskih celic. Uživanje kakovostnih virov beljakovin, kot so pusto meso, ribe, mlečni izdelki, stročnice in oreščki, zagotavlja esencialne aminokisline, ki so potrebne za vzdrževanje zdravega imunskega sistema. Ogljikovi hidrati so glavni vir energije za imunske celice, ki se borijo proti okužbam. Dobra prehranska izbira ogljikovih hidratov vključujejo živila z visoko vsebnostjo vlaknin, kot so polnozrnate žitarice, stročnice, sadje in zelenjava. Prehranske vlaknine prispevajo k zdravju črevesja, ki je pomemben dejavnik pri podpori imunske funkcije, saj je velik del imunskega sistema lociran v črevesju. Maščobe, zlasti nenasičene maščobne kisline, igrajo pomembno vlogo pri uravnavanju imunskega odziva in zmanjševanju vnetja. Omega-3 in omega-6 maščobne kisline, ki jih najdemo v mastnih ribah, oreščkih, semenih in rastlinskih oljih, so bistvene za delovanje imunskih celic in tvorbo protivnetnih spojin. Vzdrževanje uravnotežene prehrane, bogate s kakovostnimi beljakovinami, kompleksnimi ogljikovimi hidrati in zdravimi maščobami, je ključnega pomena za optimalno delovanje imunskega sistema in ohranjanje splošnega zdravja. The immune system protects the body from infections, diseases, and harmful substances such as viruses, bacteria, and parasites, and maintains a balance between the body's defense and preventing excessive inflammation or autoimmune responses. It achieves this by detecting and neutralizing pathogens and controlling tissue repair and healing. Nutritional support for the immune system is essential for maintaining optimal health and preventing disease. Focusing on macronutrients such as proteins, carbohydrates, and fats helps provide the foundation for a balanced diet that supports immune function. Proteins play an important role in supporting the immune system, as they are the basic building block for growth and tissue repair and the production of antibodies and immune cells. Consuming quality protein sources such as lean meat, fish, dairy products, legumes, and nuts, provides essential amino acids needed to maintain a healthy immune system. Carbohydrates are the main source of energy for immune cells fighting infections. Good dietary choices for carbohydrates include high-fiber foods such as whole grains, legumes, fruits, and vegetables. Dietary fiber contributes to gut health, which is an important factor in supporting immune function, as a large part of the immune system is located in the gut. Fats, especially unsaturated fatty acids, play an important role in regulating the immune response and reducing inflammation. Omega-3 and omega-6 fatty acids, found in fatty fish, nuts, seeds, and vegetable oils, are essential fort he functioning of immune cells and the formation o fanti-inflammatory compounds. Maintaining a balanced diet rich in quality proteins, complex carbohydrates, and healthy fats is essential for optimal immune system function and overall health.

Vnetna bolezenska stanja so razširjena skupina akutno in kronično pogojenih bolezni. Na voljo imamo širok nabor zdravil, s katerimi v večini primerov vnetje ustavimo ali ustrezno nadziramo. Kljub temu ta zdravila izkazujejo določene neželene učinke, ki otežujejo njihovo dolgoročno rabo. Z namenom razvoja novih protivnetnih zdravil so v ospredju raziskave protein kinaz, novejših tarč, ki so udeležene v znotrajceličnem vnetnem signaliziranju in v poteh sinteze vnetnih mediatorjev. Z njihovim zaviranjem bi lahko na drugačen način omejili vnetni odziv telesa in bolj specifično ciljali prizadeti del organizma. S transformirajočim rastnim dejavnikom β-aktivirana kinaza 1 (TAK1) spada v razred protein kinaz in ima pomembno vlogo pri uravnavanju signalnih poti, povezanih z vnetnimi procesi in celičnim preživetjem. Prekomerno aktivna TAK1 poganja pozitivno povratno zanko nastanka vnetnih mediatorjev, zato je privlačna tarča za razvoj novih protivnetnih učinkovin. Takinib je trenutno najvidnejši predstavnik do sedaj znanih zaviralcev TAK1, ki do tarče izkazuje visoko afiniteto in zadovoljivo selektivnost. Kot klasičen zaviralec ima tudi nekaj splošnih pomanjkljivosti, ki lahko omejijo uporabnost molekule v in vivo okolju, kot so vzdrževanje višjih koncentracij učinkovine na mestu delovanja, delovanje po modelu zasedenosti in v stehiometričnem razmerju glede na tarčo. Z obetavnim pristopom tehnologije himernih razgrajevalcev ali molekul PROTAC (ang. proteolysis targeting chimeras) se tem pomanjkljivostim lahko izognemo. Gre za uporabo bifunkcionalnih molekul, sestavljenih iz liganda za tarčni protein, distančnika in liganda za izbrano ligazo E3, ki za svoje delovanje izkoriščajo ubikvitin proteasomski sistem za tarčno razgradnjo patološkega proteina – v našem primeru je to TAK1. Na osnovi derivata takiniba smo v okviru magistrske naloge pripravili tri molekule PROTAC, ki se med seboj razlikujejo po ligandu za izbrano ligazo E3 – to je bodisi ligand za CRBN, VHL ali IAP. Končne spojine smo biokemijsko vrednotili s pomočjo encimskih testov, njihovo sposobnost tarčne proteasomske razgradnje TAK1 pa smo ovrednotili na izbrani celični liniji MDA-MB-231 z metodo prenosa western. Ugotovili smo, da nekatere izmed njih izkazujejo dobro afiniteto do TAK1 in uspešno sprožijo njeno razgradnjo. Inflammatory diseases are a wide group of acutely and chronically conditioned diseases. We have acess to a wide range of drugs, which we can use to stop or at least control inflammation. However, these drugs exhibit certain side effects that makes their long-term use questionable. This is why the research of protein kinases, newer targets that are involved in intracellular inflammatory pathways and synthesis of inflammatory mediators, is gaining more recognition. By inhibiting these targets we could limit the body's inflammatory response in an alternative way and target the affected part of the body more specifically. Transforming growth factor β-activated kinase 1 (TAK1) belongs to the class of protein kinases and plays an important role in regulating signaling pathways related to inflammatory processes and cell survival. Overactive TAK1 drives a positive feedback loop of inflammatory mediator production, making it an attractive target for the development of new anti-inflammatory agents. Takinib is currently the most prominent representative of TAK1 inhibitors known to date, showing favorable affinity and selectivity within the kinome. As a classical inhibitor, it also has some general disadvantages, such as the need to maintain high concentrations of the active substance at the site of action in vivo, relying on the occupancy driven model and in a stoichiometric ratio to the target. These shortcomings can be avoided with proteolysis targeting chimeras (PROTACs). This approach involves the use of bifunctional molecules consisting of a ligand for the target protein, a linker and a ligand for the selected E3 ligase which exploit the ubiquitin proteasome system for the targeted degradation of the pathological protein which in our case is TAK1. As part of the master's thesis, we prepared three PROTACs based on the takinib derivative, which differ from each other in terms of the ligand for the selected E3 ligase – these are either the ligand for CRBN, VHL or IAP. The final compounds were evaluated biochemically with enzymatic assays and on the selected cell line MDA-MB-231. We found that some of the prepared molecules showed a good affinity for TAK1 and successfully induced its degradation.

Sistemska skleroza (SSc) je sistemska avtoimunska revmatska bolezen, katere patogeneza ni popolnoma razjasnjena, tudi obvladovanje glavnih kliničnih znakov, kot sta vnetje in fibroza, s farmakoterapijo pogosto ni učinkovito. Z namenom boljšega razumevanja patogeneze in odkrivanja novih terapevtskih učinkovin smo proučevali oksidativni stres in migracijo monocitov pri bolnikih s SSc ter učinke adiponektina in resolvina D1 na vnetje in fibrozo. Metodološki pristop je zajemal delo na vzorcih bolnikov s SSc in zdravih oseb, celičnih kulturah in živalskem modelu. Pri bolnikih s SSc smo izmerili povišan indeks oksidativnega stresa (na osnovi povišanih serumskih reaktivnih kisikovih metabolitov in/ali antioksidantne kapacitete), več prelomov DNA v levkocitih ter oslabljene popravljalne mehanizme poškodb DNA. Na površini monocitov bolnikov s SSc smo dokazali povečano izražanje adhezijske molekule CD62L, katere izražanje je bilo spodbujeno s komponentami seruma bolnikov. Spremembe v oksidativnem stanju in migratornem fenotipu monocitov so pri bolnikih s SSc sovpadale s pljučno prizadetostjo, prisotnostjo avtoprotiteles in uporabo farmakoterapije. Na skeletnomišičnih celicah in skeletnomišičnih fibroblastih smo pokazali, da se adiponektin vpleta v vnetje in mišično regeneracijo z aktivacijo znotrajceličnih signalnih poti ter s spodbujanjem izločanja interlevkina-6 in sfingozin-1-fosfata, kot možne posledice povečane ceramidazne aktivnosti adiponektinskih receptorjev. AdipoRon, analog adiponektina, primerljivih učinkov ni izkazal. Resolvin D1 je na mišjem modelu peritonealne fibroze znižal delež polimorfonuklearnih celic v sistemskem krvnem obtoku, medtem ko vpliva na lokalno vnetje in fibrozo v trebušni steni ni imel. Negativno je vplival na izražanje hemerina, regulatorja vnetnih procesov, ki se veže na isti receptor kot resolvin E1. V doktorski disertaciji smo pokazali, kako obsežnejše stanje oksidativnega stresa in večji potencial migracije monocitov pri SSc sovpada s klinično sliko bolezni ter tako pripomogli k boljšemu razumevanju patogeneze. Obenem smo dokazali, da ima adiponektin učinke na mišično vnetje in regeneracijo, resolvin D1 pa potencial obvladovanja vnetja v fibrotičnih boleznih tudi z regulacijo izražanja ligandov resolvinskih receptorjev. Systemic sclerosis (SSc) is a systemic autoimmune rheumatic disease whose pathogenesis remains poorly understood, and pharmacotherapy for the major clinical manifestations - inflammation and fibrosis - is often ineffective. To better understand the pathogenesis and find new therapeutic options, we investigated oxidative stress and monocyte migration in SSc and effects of adiponectin and resolvin D1 on inflammation and fibrosis. Our methodological approach included work with samples from SSc patients, cell cultures, and animal models. SSc patients exhibited a higher oxidative stress index (based on increased serum reactive oxygen metabolites and/or antioxidant capacity), more DNA breaks in leukocytes, and impaired DNA damage repair mechanisms. We found increased surface expression of the adhesion molecule CD62L on SSc monocytes and showed that CD62L expression is stimulated by SSc serum components. Both the altered oxidative status and monocyte phenotype were consistent with lung involvement, autoantibodies, and pharmacotherapy in SSc patients. In skeletal muscle cells and skeletal muscle fibroblasts, adiponectin acted on inflammation and muscle regeneration by activating intracellular signalling pathways and stimulating secretion of interleukin-6 and sphingosine-1-phosphate, possibly due to increased ceramidase activity of adiponectin receptors. AdipoRon, an adiponectin analogue, did not show comparable effects. In a mouse model of peritoneal fibrosis, resolvin D1 decreased the proportion of polymorphonuclear cells in the systemic circulation, whereas locally in the abdominal wall it had no effect on inflammation and fibrosis. It also downregulated the expression of chemerin, an important regulator of inflammation that binds to the same receptor as resolvin E1. In our work, we show that oxidative stress and increased migratory potential of monocytes in SSc correspond to the clinical picture of the disease. We show that adiponectin has effects on muscle inflammation and regeneration and that resolvin D1 has the potential to promote resolution of inflammation by regulating the expression of other resolvin receptor ligands.

High-intensity exercise can elicit acute changes in the biochemical and physiological processes in the body of an athlete, including increased oxidative stress and inflammation. The purpose of this study was to explore the effect of acute moderate-intensity exercise on total antioxidant capacity (TAC) and serum levels of anti-inflammatory adiponectin (APN), and inflammatory markers in competitive cyclists. Ten male cyclists (age 15–26 years, body mass index 19.4–24.7 kg/m$^2$) participated in this study. Each subject performed the maximal oxygen uptake test (VO$_{2peak}$) and completed a 10-min cycling exercise at a workload of 50% of the peak of VO$_{2peak}$. Blood samples were collected on three different occasions: after an overnight fasting and at the exercise workloads of 50% and 100% VO$_{2peak}$. We measured APN, TAC, inflammatory markers as well as assessed nutrient and energy intake for each participant. Baseline concentration of serum APN (10.92µg/mL) significantly increased at 50% and at 100% VO$_{2peak}$. In addition, TAC also increased after acute exercise (0.079 vs 0.093 nmol/µL). The concentration of APN at 50% VO$_{2peak}$ positively correlated with the CRP (r = 0.640, p = 0.046) and negatively correlated with TNF-α (r = −0.696, p = 0.025). This test showed that short (10min) and medium-intensity (50% VO$_{2peak}$) exercise activity in trained athletes evoked beneficial antioxidant and anti-inflammatory responses. Importantly, this response correlates with the increase in APN levels thereby showing that highly trained individuals have beneficial responses originating from adipose tissue. Our observations show that a short training at moderate activity can be an important preservative strategy during the recovery training period.

Neonatalna hipoksično-ishemična encefalopatija (HIE) je posledica obporodnega pomanjkanja kisika v osrednjem živčevju ter je eden najbolj pogostih vzrokov za trajno nevrološko okvaro otrok. Pri nekaterih otrocih se kasneje razvije cerebralna paraliza (CP). Pri HIE pride do povečanega nastanka reaktivnih kisikovih spojin in aktivacije vnetnih procesov. Genetska variabilnost v teh poteh bi lahko vplivala na nastanek CP po HIE. V raziskavi smo se osredotočili na pogoste polimorfizme v ključnih genih, ki sodelujejo pri antioksidativni obrambi in pri vnetnem odgovoru po HIE. Vključili smo 90 otrok z zmerno in hudo HIE. S kompetitivnim alelno-specifičnim PCR smo določili polimorfizme v izbranih genih za antioksidativne encime (SOD2 rs4880, CAT rs1001179, GPX1 rs1050450) in provnetne dejavnike (NLRP3 rs35829419, CARD8 rs2043211, IL1B rs1143623, IL1B rs16944, IL1B rs1071671, TNF rs1800629). Pri otrocih, ki so bili zdravljeni s TH in so po rojstvu opravili MR slikanje glave, je bila stopnja možganske okvare opredeljena po klasifikaciji Rutheford. Povezanost polimorfizmov z razvojem CP po HIE in z izidom na MR smo analizirali z uporabo standardnih statističnih testov. Povezave med izbranimi polimorfizmi in nastankom CP nismo dokazali. V skupini otrok, ki ni bila zdravljena s TH, je bil polimorfen alel NLRP3 rs35829419 nominalno značilno povezan z večjim tveganjem za nastanek CP (p = 0,034). V skupini, zdravljeni s TH, pa preučevani polimorfizmi niso bili povezani s pojavom CP. V skupini, zdravljeni s TH, je bil polimorfni alel CARD8 rs2043211 nominalno značilno povezan z normalno/blago možgansko poškodbo na MR glave (p = 0,040), IL1B rs1143623 C alel pa je bil nominalno povezan z zmerno/hudo možgansko poškodbo na MR (p = 0,040). Hipoteze, da so polimorfizmi v izbranih genih povezani s pojavom CP po HIE, nismo potrdili. Vpliv polimorfizmov na izid po HIE je bil do sedaj slabo poznan, zato rezultati predstavljajo izhodišče za nadaljnja raziskovanja na tem področju z večjim številom preiskovancev. Neonatal hypoxic-ischemic encephalopathy (HIE) results from peripartum or intrapartum brain tissue hypoxia and ischemia and is a common cause of long-term neurologic sequelae, including cerebral palsy (CP). Hypoxic-ischemic (HI) insult results in oxidative stress and activation of inflammatory processes. Genetic variability in these pathways could influence the development of CP after HIE. The aim of our study was to evaluate the association of the selected single nucleotide polymorphisms (SNPs) in the genes involved in response to oxidative stress and inflammation with the development of CP after HIE. Ninety children with moderate and severe HIE were included in the study. Genotyping using competitive allele-specific PCR was performed for polymorphisms in genes encoding antioxidant enzymes (SOD2 rs4880, CAT rs1001179, GPX1 rs1050450) and proinflammatory factors (NLRP3 rs35829419, CARD8 rs2043211, IL1B rs1143623, IL1B rs16944, IL1B rs1071676, TNF rs1800629) that could contribute to the development of CP after HIE. In children treated with TH where MRI was performed, it was classified according to the Rutheford classification. Standard statistical tests were used to evaluate the association of selected SNPs with the development of CP. No association between the selected SNPs and the development of CP was found. Polymorphic NLRP3 rs35829419 allele was nominally significantly associated with the development of CP in children who were not treated with TH (p = 0.034). None of the SNPs were associated with CP in children treated with TH. Among children treated with TH polymorphic allele CARD8 rs2043211 was nominally significantly associated with normal/mild MRI overall findings (p = 0.040) and IL1B rs1143623 C allele was nominally significantly associated with moderate/severe MRI overall findings (p = 0.040). The association of genetic variability with the outcome after HIE has so far not been well established, therefore the results of our study represent foundations for future research in larger studies.

Celice mikroglije so glialne celice centralnega živčnega sistema (CŽS), podobne makrofagom, ki vzdržujejo homeostazo možganov in so ene izmed najpomembnejših prirojenih celic imunskega sistema v CŽS. Pod vplivom patoloških in imunoloških dražljajev pride do polarizacije mikroglije v pro-vnetni fenotip M1, ki spodbuja procese nevrovnetja ali proti-vnetni fenotip M2, ki izkazuje zaščitno delovanje v možganih. Cisteinski katepsini, ki jih sprošča polarizirana mikroglija M1, so vse bolj prepoznani kot pomembni vnetni dejavniki, ki sprožijo signalne poti, kar vodi v poslabšanje nevrovnetja. Med njimi je cisteinska peptidaza katepsin X, ki je že bila prepoznana kot patogeni dejavnik nevrodegeneracije, povzročene z vnetjem. V okviru magistrske naloge smo želeli ovrednotiti vlogo katepsina X pri polarizaciji celic mikroglije. Najprej smo postavili celični model polarizirane mikroglije z uporabo celične linije BV2, kjer smo pokazali vpliv stimulacije z lipopolisaharidom (LPS), interferonom-γ (IFN-γ), interlevkinom-4 (IL-4) in IL-13 na usmeritev polarizacije mikroglije. Pri tem sta LPS in IFN-γ povzročila polarizacijo mikroglije v fenotip M1, kar smo pokazali s povišano ravnjo pro-vnetnih dejavnikov. Po drugi strani sta citokina IL-4 in IL-13 spodbudila polarizacijo v smeri fenotipa M2, kar smo potrdili z označevalci proti-vnetnega stanja. Nadalje smo opredelili izražanje, aktivnosti in vezikularno lokalizacijo katepsina X v stimuliranih celicah BV2. Stimulacija z LPS in IFN-γ je povišala znotrajcelično izražanje katepsina X, kar smo pokazali s pomočjo prenosa western, in hkrati znižala njegovo aktivnost zaradi sproščanja zrele oblike katepsina X iz celic mikroglije ter zmanjšala njegovo znotrajcelično lokalizacijo v lizosomih, ki smo jo določili s konfokalno mikroskopijo. Pri tem IL-4 in IL-13 na izražanje, aktivnost in lokalizacijo katepsina X nista imela vpliva. V zadnjem delu magistrske naloge smo vrednotili še vpliv ireverzibilnega zaviralca katepsina X, AMS36, na polarizacijo mikroglije. Pokazali smo, da je AMS36 z zaviranjem katepsina X po stimulaciji z LPS in IFN-γ preprečil polarizacijo mikroglije v fenotip M1, kar se je odražalo v upadu ravni pro-vnetnega dejavnika NO, medtem ko je pri stimulaciji celic BV2 z IL-4 in IL-13 spodbudil polarizacijo mikroglije v fenotip M2, kar smo pokazali s povečano aktivnostjo arginaze-1 in izražanjem označevalca CD206, ki sta značilna za polarizirano mikroglijo M2. Pridobljeni rezultati nakazujejo, da ima katepsin X pomembno vlogo pri polarizaciji mikroglije v pro-vnetni fenotip M1 oziroma proti-vnetni fenotip M2 in predstavlja potencialno terapevtsko tarčo za preprečevanje in zdravljenje nevrodegenerativnih bolezni, povezanih s prekomernim vnetjem. Microglia cells are glial cells of the central nervous system (CNS), similar to macrophages, which maintain brain homeostasis and are among the most important cells of the innate immune system in the CNS. Under the influence of pathological and immunological stimuli, the polarization of microglia occurs, either into the pro-inflammatory phenotype M1, which promotes the processes of neuroinflammation, or the anti-inflammatory phenotype M2, which exhibits a protective function in the brain. Cysteine cathepsins released by M1 polarized microglia are increasingly recognized as important inflammatory factors that trigger signaling pathways, which in turn lead to aggravation of neuroinflammation. Among them is the cysteine peptidase cathepsin X, which has already been recognized as a pathogenic factor in inflammation-induced neurodegeneration. As part of the master's thesis, we evaluated the role of cathepsin X in the polarization of microglia cells. First, we established a cellular model of polarized microglia using the BV2 cell line, where we demonstrated the effect of stimulation with lipopolysaccharide (LPS), interferon-γ (IFN-γ), interleukin-4 (IL-4) and IL-13 on the course of polarization. Here, LPS and IFN-γ induced polarization of microglia to the M1 phenotype, which we demonstrated with increased levels of pro-inflammatory factors. On the other hand, the cytokines IL-4 and IL-13 induced polarization towards the M2 phenotype, which was confirmed by markers of the anti-inflammatory state. We further characterized the expression, activity, and vesicular localization of cathepsin X in stimulated BV2 cells. Stimulation with LPS and IFN-γ increased the intracellular expression of cathepsin X, which was demonstrated by western blotting, and at the same time decreased its activity due to the release of the mature form of cathepsin X from microglial cells and reduced its intracellular localization in lysosomes, which was determined by confocal microscopy. IL-4 and IL-13 had no effect on cathepsin X expression pattern and localization. In the last part of the master's thesis, we also evaluated the influence of the irreversible cathepsin X inhibitor, AMS36, on the polarization of microglia. We showed that AMS36 prevented the polarization of microglia into the M1 phenotype by inhibiting cathepsin X after stimulation with LPS and IFN-γ, as is also indicated by the reduction of the pro-inflammatory factor NO, whereas stimulation of BV2 cells with IL-4 and IL- 13 in the presence of AMS36 enhanced polarization of microglia into the M2 phenotype, which was demonstrated by increased arginase-1 activity and the expression of the marker CD206, which are characteristic of M2 polarized microglia. The obtained results suggest that cathepsin X plays an important role in the polarization of microglia into the pro-inflammatory M1 and the anti-inflammatory M2 phenotype as such, it represents a potential therapeutic target for the prevention and treatment of neurodegenerative diseases associated with excessive inflammation.

Uvod: Problem slepote kot posledice glavkoma se povečuje zaradi staranja prebivalstva. Nastanek in napredovanje primarnega glavkoma z odprtim zakotjem (POAG) sta povezana z oksidativnim stresom, vnetjem in apoptozo ganglijskih celic mrežnice. Različni dejavniki lahko povzročijo večje nastajanje reaktivnih kisikovih spojin in sproščanje mediatorjev imunsko-vnetnega odziva. Matriksne metaloproteinaze (MMP) so encimi, ki sodelujejo pri preoblikovanju izvenceličnega matriksa v trabekulumu in ciliarniku ter na ta način uravnavajo očesni tlak (IOP). Edini dokazano učinkovit način zdravljenja glavkoma je znižanje IOP. Z zdravili pogosto ne dosežemo zadostnega znižanja IOP, pri nekaterih bolnikih pa kljub nizkemu IOP bolezen napreduje. Latanoprost je prvi prostaglandinski analog, ki ga evropske glavkomske smernice, skupaj s selektivno lasersko trabekuloplastiko, priporočajo kot prvi izbor za zdravljenja glavkoma. Zaenkrat ne poznamo kliničnih ali bioloških označevalcev, s katerimi bi lahko napovedali tveganje za nastanek POAG, njegov potek ter odgovor na določeno vrsto zdravljenja. Hipoteze: Genetska variabilnost lahko vpliva na potek POAG. Farmakogenetska variabilnost napoveduje odgovor na zdravljenje z latanoprostom in s selektivno lasersko trabekuloplastiko (SLT) pri bolnikih s POAG in očesno hipertenzijo (OHT). Z ugotavljanjem genetskih označevalcev lahko napovemo, katero zdravljenje bo pri posameznem bolniku učinkovitejše in povezano z manj neželenimi učinki. Metode: V raziskavo smo vključili 307 bolnikov s POAG ali z OHT, starejših od 40 let in 339 kontrol. V retrospektivnem delu smo pri bolnikih in kontrolah analizirali polimorfizme posameznih nukleotidov (SNP) v genih, ki so vključeni v obrambo pred oksidativnim stresom (GSTM1, GSTT1, GSTP1, SOD2, CAT in GPX1), vnetne poti (TNF, IL1B in IL6) ter delovanje encimov MMP (MMP2, MMP3, MMP9, MMP14). V prospektivni del raziskave smo vključili nezdravljene bolnike, ki smo jih naključno razvrstili za zdravljenje z latanoprostom ali s SLT ter ovrednotili odgovor na zdravljenje po 1,5 meseca. Pri vseh bolnikih smo analizirali SNP v genih, ki so vključeni v antioksidativne in vnetne poti ter za encime MMP. Pri bolnikih zdravljenih z latanoprostom smo analizirali tudi SNP v genih, ki sodelujejo pri farmakodinamiki in farmakokinetiki latanoprosta (CES1,CES1P1, BCHE , SLCO2A1, PTGFR). Introduction: The problem of blindness because of glaucoma is increasing due to the aging population. The onset and progression of primary open-angle glaucoma (POAG) is associated with oxidative stress, inflammation, and apoptosis of retinal ganglion cells. Various factors cause increased formation of reactive oxygen species and release of mediators involved in the immune-inflammatory response. Matrix metalloproteinases (MMP) are enzymes involved in the formation of the extracellular matrix in the trabecular meshwork and ciliary body and thus regulate intraocular pressure (IOP). The only proven effective treatment is reduction of IOP. Medication often does not achieve sufficient reduction of IOP, and in some patients the disease progresses despite low IOP. The first prostaglandin analogue latanoprost and selective laser trabeculoplasty (SLT), have been both recommended as the first-choice treatment for POAG by the European Glaucoma Society Guidelines. Currently, there are no clinical or biological markers that would help to predict the risk of POAG, its course or the response to a particular type of treatment. Hypotheses: Genetic variability may influence the course of POAG. Pharmacogenetic variability predicts response to latanoprost and selective laser trabeculoplasty in patients with POAG and ocular hypertension (OHT). By identifying genetic markers, we can predict which treatment will be more effective and associated with fewer side effects in an individual patient. Methods: A total of 307 patients with POAG or OHT older than 40 years and 339 controls were included in the study. In the retrospective part, we analyzed common single nucleotide polymorphisms (SNPs) in the genes involved in the pathways of oxidative stress (GSTM1, GSTT1, GSTP1, SOD2, CAT and GPX1), inflammation (TNF, IL1B and IL6) and in the activity of MMP (MMP2, MMP9, MMP14). The prospective part of the study included treatment-naive patients randomized to treatment with latanoprost or SLT, and evaluated the treatment response after 1.5 months. In these patients we also analyzed SNPs in the genes involved in the oxidative and inflammatory pathways, and in MMP genes. In addition, in the latanoprost treatment group, we analyzed SNPs involved in the pharmacodynamics and pharmacokinetics of latanoprost (CES1, CES1P1, BCHE , SLCO2A1, PTGFR). Results: Homozygotes for GSTM1 gene deletion had a lower risk of developing OHT, while homozygotes for MMP3 rs3025058 had a higher risk of developing OHT. There were more subjects with a higher degree of glaucoma impairment among carriers of at least one GPX1 polymorphic allele, while among carriers of at least one MMP3 polymorphic allele there were more subjects with a lower degree of impairment. The SOD2 rs4880, CAT rs1001179, and TNF 1800629 polymorphisms were associated with an absolute decrease in IOP, whereas carriers of at least one copy of GSTT1 had a lower relative decrease in IOP after latanoprost treatment. IL6 polymorphism was associated with a smaller absolute and relative decrease in IOP after SLT treatment. The clinical-pharmacogenetic model better predicted the effectiveness of IOP reduction (30% and more) in SLT treatment compared to the clinical model, whereas in latanoprost treatment both models had similar predictive value. Conclusion: SNPs in genes involved in oxidative and inflammatory pathways may be potential biological markers for predicting the risk and progression of POAG, as well as the response to latanoprost and SLT treatment. A clinical-pharmacogenetic prediction model could be helpful in selecting a more effective treatment.

Konjska astma je pogosta kronična bolezen dihal, ki pomembno vpliva na zdravje in dobrobit konj. Trenutne strategije zdravljenja konjske astme temeljijo na lajšanju simptomov in ne zdravijo bolezni. Lahko imajo tudi znatne stranske učinke. Cilj naše raziskave je bil oceniti lokalne in sistemske učinke intrabronhialne administracije avtolognih mezenhimskih matičnih celic, pridobljenih iz maščobnega tkiva (Adipose derived mesenchymal stem cells, AD-MSC) na potek konjske astme. Konje smo naključno razdelili v dve skupini in jih zdravili z AD-MSC ali deksametazonom. Za ugotavljanje terapevtskih učinkov smo natančno opredelili klinično izražanje bolezni pri posameznem konju in opravili meritve pljučnih funkcij in endoskopski pregled dihalnih poti. Ob endoskopiji smo opravili tudi bronhoalveolarno lavažo, izpirke pa citološko pregledali in izmerili vrednosti izražanja sporočilne RNK določenih vnetnih citokinov in koncentracijo njihovih beljakovin. Po končani obravnavi smo konje spremljali še eno leto in primerjali dolgoročno stabilnost kliničnih znakov med obema skupinama. Kljub temu, da zdravljenje z AD-MSC ni povzročilo takšnega izboljšanja kliničnih znakov astme kot zdravljenje kontrolne skupine, smo zaznali znatno izboljšanje pri izražanju in koncentraciji posameznih vnetnih spremenljivk. V primerjavi s kontrolnim zdravljenjem, pa je imelo zdravljenje z AD-MSC pozitiven dolgoročni učinek na stabilnost klinične slike. Na podlagi rezultatov te raziskave zaključujemo, da ima intrabronhialno zdravljenje z AD-MSC omejen kratkoročen učinek in pozitiven dolgoročen učinek na simptome kronične astme pri konjih. Equine asthma is a common chronic disease of the respiratory tract that significantly affects horses' health and well-being. Current therapeutic strategies are based on symptomatic treatment and do not alter the disease's long-term progression, and their side effects are worrying. Our study aimed to evaluate the local and systemic effects of intrabronchial administration of autologous adipose-derived mesenchymal stem cells (AD-MSC) on severe equine asthma. Horses were randomly divided into two groups and treated with AD-MSC or dexamethasone. To determine the therapeutic effects of intrabronchial treatment with AD-MSC, we precisely defined clinical signs, and performed lung function tests and endoscopic examination of the airways in horses included in the study. Bronchoalveolar lavage was performed, and the samples were cytologically examined, and the mRNA expression values of specific inflammatory cytokines and the concentration of their proteins were measured. After treatment, the horses were monitored for another year, and the long-term stability of clinical signs between the two groups was compared. Although non-inferiority of AD-MSC treatment was not confirmed, improvement in certain inflammatory variables was observed after cell therapy. However, intrabronchial treatment with AD-MSC had a positive long-term effect on clinical stability of horses with severe asthma. Results of the study indicate that intrabronchial treatment with AD-MSC has a limited short-term anti-inflammatory effect and a positive long-term clinical effect in asthmatic horses.

Ten percent of patients undergoing total hip arthroplasty (THA) require revision surgery. One of the reasons for THA are wear particles released from the implants that can activate the immune defense and cause osteolysis and failure of the joint implant. The discrepancies between reports on toxicity and immunogenicity of the implant materials led us to this study in which we compared toxicity and immunogenicity of well-defined nanoparticles from Al2O3, zirconia-toughened alumina (ZTA), and cobalt chrome (CoCr), a human THP-1 macrophage cell line, human PBMCs, and therefrom-derived primary macrophages. None of the tested materials decreased the viability of THP-1 macrophages nor human primary macrophages at the 24 h time point, indicating that at concentrations from 0.05 to 50 µm3/cell the tested materials are non-toxic. Forty-eight hours of treatment of THP-1 macrophages with 5 µm3/cell of CoCr and Al2O3 caused 8.3-fold and 4.6-fold increases in TNF-α excretion, respectively, which was not observed for ZTA. The comparison between THP-1 macrophages and human primary macrophages revealed that THP-1 macrophages show higher activation of cytokine expression in the presence of CoCr and Al2O3 particles than primary macrophages. Our results indicate that ZTA is a non-toxic implant material with no immunogenic effects in vitro., Slovenian Research Agency (ARRS)

Monoclonal antibodies (mAbs) have been extensively developed over the past few years, for the treatment of various inflammatory diseases. They are large molecules characterized by complex pharmacokinetic and pharmacodynamic properties. Therapeutic drug monitoring (TDM) is routinely implemented in the therapy with mAbs, to monitor patients’ treatment response and to further guide dose adjustments. Serum has been the matrix of choice in the TDM of mAbs and its sampling requires the visit of the patients to laboratories that are not always easily accessible. Therefore, dried blood spots (DBS) and various microsampling techniques have been suggested as an alternative. DBS is a sampling technique in which capillary blood is deposited on a special filter paper. It is a relatively simple procedure, and the patients can perform the home-sampling. The convenience it offers has enabled its use in the quantification of small-molecule drugs, whilst in the recent years, studies aimed to develop microsampling methods that will facilitate the TDM of mAbs. Nevertheless, hematocrit still remains an obstacle that hinders a more widespread implementation of DBS in clinical practice. The introduction of novel analytical techniques and contemporary microsampling devices can be considered the steppingstone to the attempts made addressing this issue.

Oxidative stress and neuroinflammation are involved in the pathogenesis and progression of glaucoma. Our aim was to evaluate the impact of selected single-nucleotide polymorphisms in inflammation and oxidative stress genes on the risk of glaucoma, the patients’ clinical characteristics and the glaucoma phenotype. In total, 307 patients with primary open-angle glaucoma or ocular hypertension were enrolled. The control group included 339 healthy Slovenian blood donors. DNA was isolated from peripheral blood. Genotyping was performed for SOD2 rs4880, CAT rs1001179, GPX1 rs1050450, GSTP1 rs1695, GSTM1 gene deletion, GSTT1 gene deletion, IL1B rs1143623, IL1B rs16944, IL6 rs1800795 and TNF rs1800629. We found a nominally significant association of GSTM1 gene deletion with decreased risk of ocular hypertension and a protective role of IL1B rs16944 and IL6 rs1800629 in the risk of glaucoma. The CT and TT genotypes of GPX1 rs1050450 were significantly associated with advanced disease, lower intraocular pressure and a larger vertical cup–disc ratio. In conclusion, genetic variability in IL1B and IL6 may be associated with glaucoma risk, while GPX and TNF may be associated with the glaucoma phenotype. In the future, improved knowledge of these pathways has the potential for new strategies and personalised treatment of glaucoma.

Glioblastoma is simultaneously the most common and most aggressive primary brain tumor in the central nervous system, with poor patient survival and scarce treatment options. Most primary glioblastomas reoccur and evolve radio- and chemoresistant properties which make them resistant to further treatments. Based on gene mutations and expression profiles, glioblastoma is relatively well classified; however, research shows that there is more to glioblastoma biology than that defined solely by its genetic component. Specifically, the overall malignancy of the tumor is also influenced by the dynamic communication to its immediate and distant environment, as important messengers to neighboring cells in the tumor microenvironment extracellular vesicles (EVs) have been identified. EVs and their cargo can modulate the immune microenvironment and other physiological processes, and can interact with the host immune system. They are involved in tumor cell survival and metabolism, tumor initiation, progression, and therapy resistance. However, on the other hand EVs are thought to become an effective treatment alternative, since they can cross the blood–brain barrier, are able of specific cell-targeting and can be loaded with various therapeutic molecules.

ObjectivesThe aim of this study was to quantitatively assess distinct immune cell subsets comprising inflammatory infiltrate in temporal artery biopsies (TABs) from patients with giant cell arteritis (GCA), and to link the obtained histopathological data with expression profiles of immune-related microRNAs (miRNAs).MethodsThe study included 68 formalin-fixed, paraffin-embedded TABs from treatment-naïve patients, including 30 histologically positive GCA and 16 negative GCA TABs, and 22 control non-GCA TABs. Quantitative assessment of histological parameters was performed using histopathological and immunohistochemical techniques. miRNA expression analysis was performed by quantitative real-time PCR.ResultsIntense transmural mononuclear inflammatory infiltrates in TAB-positive GCA arteries were predominantly composed of CD3+, CD4+ and CD8+ T lymphocytes, and CD68+ macrophages, accompanied by a strong nuclear overexpression of the nuclear factor of activated T cells, cytoplasmic 1 (NFATC) in the lymphocyte infiltrate fraction. Furthermore, TAB-positive GCA arteries were characterized by significant overexpression of nine pro-inflammatory miRNAs (miR-132-3p/-142-3p/-142-5p/-155-5p/-210-3p/-212-3p/-326/-342-5p/-511-5p) and a significant under-expression of six regulatory immune-related miRNAs (miR-30a-5p/-30b-5p/-30c-5p/-30d-5p/-30e-5p/-124-3p), whose expression levels significantly associated with most evaluated histopathological parameters. Notably, we revealed miR-132-3p/-142-3p/-142-5p/-155-5p/-212-3p/-511-5p as major promoters of arterial inflammation and miR-30a-5p/-30c-5p/-30d-5p as putative regulators of NFATC signaling in TAB-positive GCA arteries.ConclusionOverall, we demonstrated that an altered arterial tissue-specific pro-inflammatory miRNA signature favors enhanced T cell-driven inflammation and macrophage activity in TAB-positive GCA arteries. Moreover, dysregulation of several immune-related miRNAs seems to contribute crucially to GCA pathogenesis, through impairing their regulatory activity towards T cell-mediated immune responses driven by the calcineurin (CaN)/NFAT signaling pathway, indicating their therapeutic, diagnostic and prognostic potential.

Toll-like receptor 10 (TLR10) is the only member of the TLR family whose function and ligand have not been clearly described. Literature reports on its function are contradictory and suggest a possible immunomodulatory role that depends on the cell type, the pathogen, and the level of TLR10 expression. To investigate the regulatory role of TLR10 in A549 lung epithelial cells, we overexpressed TLR10 using CRISPRa technology and examined the differential expression of various genes involved in TLR signaling activated by different TLR ligands, namely dsRNA, LPS, and Pam3Cys. The expression of proinflammatory cytokines, such as IL1β, IFNβ, TNFα, IL8, CXCL10, and CCL20, decreased in the challenged cells overexpressing TLR10, whereas the expression of the anti-inflammatory cytokine IL10 and the antimicrobial peptide hβD-2 increased. For several of the regulated inflammatory markers, we were able to show the change in gene expression was translated to the protein level. It appears that TLR10 can function as an anti-inflammatory in A549 cells, depending on its expression level and that the mode of action may be virulence factor-specific. The potential suppression of inflammation by regulating expression of TLR10 in lung epithelial cells may allow the development of new approaches to balance an inflammatory response and prevent extensive tissue damage in respiratory diseases.

POVZETEK 1.8. Uvod Sladkorna bolezen tipa 1 (SB1) je ena najpogostejših kroničnih bolezni otroštva. Slabo urejena sladkorna bolezen vpliva na delovanje možganov. Z raziskavami so ugotovili, da imajo bolniki s SB1 in SB2 med akutno hiperglikemijo nižje dosežke pri govornem in celostnem IQ preizkusu. Vzrok kognitivnega upada pri bolnikih s SB1 ni znan, predpostavlja se, da bi bil lahko povezan z oksidativnim stresom in vnetjem. Prav tako še ni znano, katera področja možganov so najbolj prizadeta. 1.9. Cilji Cilji raziskave so bili: ? s fMRI opredeliti vpliv akutne hiperglikemije (20 mmol/l) na področja možganov, povezanih z načrtovanjem, prostorskim spominom in impulzivnostjo pri otrocih in mladostnikih s SBT1 ? z 1 H-MRS opredeliti vpliv 2-urne hiperglikemije na koncentracijo glukoze, mioinozitola, N- acetilaspartata, holina v področjih možganov, ki so vključene pri nadzornih funkcijah (talamus, frontalna možganska skorja, frontalna bela možganovina) pri otrocih in mladostnikih s SBT1. ? opredeliti vpliv 2-urne hiperglikemije na raven vnetnih mediatorjev (IL-6, fibrinogen) v periferni krvi pri otrocih in mladostnikih s SBT1 1.10. Metode V raziskavi je sodelovalo 20 otrok s SB1, starih med 11 in 19 let, brez kliničnih zapletov SB1, s podobno urejenostjo SB 1 v zadnjih 3 letih (HbA1c), normalno prehranjenih (ITM), nekadilcev, zdravljenih z intenzivirano inzulinsko shemo ali z inzulinsko črpalko, brez diabetične ketoacidoze ali težke hipoglikemije v zadnjem letu, s trajanjem SB1 5-10 let ter 20 zdravih prostovoljcev, skladnih po starosti. Otroci s SB1 so med evglikemično in hiperglikemično zanko opravili tako fMRI (Flankerjeva naloga, Londonski stolp, kapaciteta delovnega spomina) kot tudi H 1 -MRS preiskave. Vse MR preiskave so bile opravljene na 3T tomografu. Otrokom s SB1 smo na tešče in po 120-minutni hiperglikemiji odvzeli periferno kri za določitev kazalcev vnetja (IL-6,fibrinogen). Zdravi prostovoljci opravili enake preiskave fMRI in H 1 -MRS kot otroci s SB1, dvakrat zapored v istem dnevu, brez vpliva na vrednost krvnega sladkorja. 1.11. Hipoteze Pričakovali smo, da bomo opredelili: ? povečano delovanje možgan med izvajanjem naloge načrtovanja (Londonski stolp) med kratkotrajno hiperglikemijo v primerjavi z evglikemijo ? povečano delovanje možgan med izvajanjem naloge prostorskega spomina med kratkotrajno hiperglikemijo v primerjavi z evglikemijo ? večjo impulzivnost med kratkotrajno hiperglikemijo v primerjavi z evglikemijo ? spremembo koncentracije nevrotransmiterjev v določenih področjih možgan, povezanih z nadzornimi funkcijami, kar bi kazalo na spremembe v prenosu signala, ter funkciji celičnih membrane med kratkotrajno hiperglikemijo v primerjavi z evglikemijo povečanje koncentracije kazalcev vnetja v periferni krvi po kratkotrajni hiperglikemiji 1.12. Pomembnost Zaenkrat še ni objave fMRI med akutno hiperglikemijo, z izjemo abstrakta predstavljanega na ADA srečanju leta 2015, ko so pokazali, da je med akutno hiperglikemijo v stanju mirovanja povišana aktivacija možgan (fMRI resting state), zato bodo rezultati naše raziskave povsem izvirni. Ocena možnih škodljivih vplivov akutne hiperglikemije na kognicijo je pomembna za nadaljnje smernice zdravljenja sladkorne bolezni tipa 1, v smislu intenzivnosti inzulinske terapije in ciljev glede urejenosti. 1.13. Rezultati Med nalogo prostorskega spomina smo zaznali zmanjšano zmogljivost prostorskega delovnega spomina med akutno hiperglikemijo in pomembne razlike v aktivaciji regij zanimanja v različnih fazah prostorskega delovnega spomina (p = 0,014). Prostornina sive možganovine (p

Tehnološki izzivi na trg prinašajo vedno nove nanomateriale, ki so tako majhni, da zlahka vstopijo v naše telo. Nekateri med njimi poškodujejo pljučno bariero, nekateri pa povzročijo akutno ali celo kronično vnetje, ki se lahko razvije v še težje oblike bolezni. Da bi lahko ocenili, ali so določeni nanomateriali dolgoročno varni oz. nevarni za človeka in okolje, je potrebno take materiale testirati, za kar se ponavadi uporabljajo živalska testiranja in v njih izvaja dalj časa trajajoče študije. Slednje je drago in počasno, zato se razvijajo alternativne metode. Eno takih alternativnih metod testiranja proženja kroničnega vnetja so objavili Kokot in sodelavci (Advanced Materials 2020) in v okviru svoje študije pokazali na mehanizem ter možnost napovedi na omejenem izboru nanomaterialov iz kovinskih oksidov. Mi smo v pričujočem delu izbrali dodatne štiri nanomateriale, pomembne v različnih industrijskih panogah, za katere smo želeli preveriti možnost uporabe omenjenega alternativnega in vitro modela za analizo toksičnosti in napoved proženja vnetnih stanj. Tako smo poleg titanovega dioksida izbrali še barijev sulfat, cerijev dioksid, molibdenov trioksid in volframov oksid, kot nanodelce kovinskih oksidov, ki se pogosto uporabljajo v industriji zaradi svojih edinstvenih lastnosti. V in vivo študijah na miših in podganah so namreč raziskovalci pokazali, da večina omenjenih nanomaterialov razen barijevega sulfata povzroča vsaj akutno vnetje v pljučih. Barijev sulfat povzroči le rahel prehodni vnetni odziv, ki ne povzroča nepopravljivih učinkov na zdravje, cerijev dioksid pa npr. sproža ne le akutno temveč tudi kronično vnetje v pljučih. Na razvoj vnetnih stanj, ki jih potencialno prožijo nanodelci, vplivajo njihove najrazličnejše fizikalno-kemijske lastnosti, med katerimi pa je zelo težko izbrati tiste, ki prispevajo k proženju dolgoročnih zdravstvenih zapletov. Da bi ocenili slednje, smo na kombinaciji celičnih kultur izvedli detekcije različnih zgodnjih molekularnih dogodkov. Takšno alternativno in vitro testiranje je hitrejše, cenejše, lažje ponovljivo, etično manj problematično, obenem pa lahko zagotovi ustreznejše informacije. Zaradi številnih prednosti smo se tako odločili preveriti možnost napovedi vnetnih stanj za omenjene nanomateriale na osnovi mikroskopije ter ekspresij signalnih proteinov in vitro. Pokazali smo, da so rezultati pridobljeni z in vitro testiranjem skladni z rezultati in vivo študij, kar lahko predstavlja osnovo za nadaljnji razvoj alternativnih metod določevanja toksičnosti nanomaterialov. Technological challenges bring ever new nanomaterials to the market that are so small that they easily enter our body. Some of these nanomaterials damage the pulmonary barrier, and some cause acute or even chronic inflammation, which can develop into even more severe forms of the disease. In order to be able to establish whether certain nanomaterials are safe in the long run to humans and the environment, such materials need to be tested, for which animal testing is usually used and long-term studies are being performed. This kind of testing is expensive and slow, so alternative methods are being developed. One of these alternative methods of testing for chronic inflammatory triggering was published by Kokot et al. (Advanced Materials 2020). In their study, they demonstrated the mechanism and the possibility of prediction on a limited selection of nanomaterials from metal oxides. In the present work, we selected four additional nanomaterials relevant in various industries for which we wanted to test the possibility of using the mentioned alternative in vitro model for toxicity analysis and prediction of triggering inflammatory conditions. Thus, in addition to titanium dioxide, we also selected barium sulfate, cerium dioxide, molybdenum trioxide and tungsten oxide as nanoparticles of metal oxides, which are often used in industry due to their unique properties. In in vivo studies in mice and rats, researchers have shown that most of these nanomaterials, except barium sulfate, cause at least acute inflammation in the lungs. Barium sulphate causes only a slight transient inflammatory response that does not cause irreversible effects on health. On the other hand, cerium dioxide e.g. triggers not only acute but also chronic inflammation in the lungs. The development of inflammatory conditions potentially triggered by nanoparticles is influenced by their various physicochemical properties, among which it is very difficult to select those that contribute to the triggering of long-term health complications. To evaluate the latter, we performed detections of various early molecular events on a combination of cell cultures. Such alternative in vitro testing is faster, cheaper, easier to replicate, ethically less problematic, and at the same time can provide more relevant information. Due to many advantages, we decided to examine the possibility of predicting inflammatory conditions for these nanomaterials based on microscopy and expression of signal proteins in vitro. We have shown that the results obtained by in vitro testing are consistent with the results of in vivo studies, which may be the basis for the further development of alternative methods for determining the toxicity of nanomaterials.

Življenjski slog vpliva na delovanje prirojenega in pridobljenega imunskega sistema. Eden izmed dejavnikov, ki regulirajo imunski odziv, je prehrana. Z njo vplivamo na črevesno mikrofloro, ki predstavlja pomemben steber obrambe. Za protivnetni učinek moramo zaužiti dovolj kompleksnih ogljikovih hidratov, nenasičenih maščobnih kislin, beljakovin, vitaminov in mineralov. Ob okužbi je viden vpliv stresa, ki na nas deluje preko aktivacije HPA osi in simpatičnega živčnega sistema. Kratkoročni stres izboljša imunski odziv, nasprotno pa ga kronični oslabi. Tudi pomanjkanje spanca aktivira omenjeni poti in posledično se zviša koncentracija vnetnih citokinov v krvi. Urejen spalni cikel povzroči utišanje teh poti in deluje protivnetno. Podobno kot pri stresu ima lahko tudi fizična aktivnost dvojni učinek. Ob pretirani telesni dejavnosti nastane sindrom prekomernega treninga, pride do vnetij tkiv in povečane dovzetnosti za okužbe zgornjega dihalnega trakta. Po drugi strani se zmerna telesna aktivnost odraža v zmanjšani produkciji vnetnih citokinov, preprečevanju reaktivacije latentnih virusov in boljšem odzivu na cepiva. Tudi zmerno uživanje alkohola spodbudi imunski odziv na cepiva, nasprotno pa kronično pitje zmanjšuje število in aktivnost celic imunskega sistema. K razvoju bolezni doprinese še kajenje, ki negativno učinkuje na ustrezen imunski odziv. Lifestyle has a big impact on how our innate and adaptive immune system functions. One of the factors, that regulates the immune response, is diet. Diet affects the gut microflora, which is an important defense mechanism. To achieve an anti-inflammatory effect, we have to eat enough complex carbohydrates, unsaturated fatty acids, proteins, vitamins, and minerals. Along with the infection, we can also see the impact of stress, which works through the activation of the HPA axis and sympathetic nervous system. Short-term stress improves immune response, while chronic stress weakens it. Previously mentioned pathways can also be activated by sleep deprivation, resulting in increased amount of inflammatory cytokines. On the contrary, a good sleeping schedule reduces the inflammatory effect. Physical activity can have a double effect as well. Excessive physical activity leads to overtraining syndrome, tissue inflammation, and increased susceptibility to upper respiratory tract infections. In contrast, moderate exercise reduces inflammatory cytokines production, prevents reactivation of latent viruses, and enhances positive response to vaccines. This response is also enhanced by moderate alcohol consumption, whereas chronic alcoholism reduces the number and activity of immune cells. Furthermore, smoking negatively affects the immune response and increases the risk of diseases.

Background. Inborn errors of immunity (IEI) are rare diseases, which makes diagnosis a challenge. A better description of the initial presenting manifestations should improve awareness and avoid diagnostic delay. Although increased infection susceptibility is a well-known initial IEI manifestation, less is known about the frequency of other presenting manifestations. Objective. We sought to analyze age-related initial presenting manifestations of IEI including different IEI disease cohorts. Methods. We analyzed data on 16,486 patients of the European Society for Immunodeficiencies Registry. Patients with autoinflammatory diseases were excluded because of the limited number registered. Results. Overall, 68% of patients initially presented with infections only, 9% with immune dysregulation only, and 9% with a combination of both. Syndromic features were the presenting feature in 12%, 4% had laboratory abnormalities only, 1.5% were diagnosed because of family history only, and 0.8% presented with malignancy. Two-third of patients with IEI presented before the age of 6 years, but a quarter of patients developed initial symptoms only as adults. Immune dysregulation was most frequently recognized as an initial IEI manifestation between age 6 and 25 years, with male predominance until age 10 years, shifting to female predominance after age 40 years. Infections were most prevalent as a first manifestation in patients presenting after age 30 years. Conclusions. An exclusive focus on infection-centered warning signs would have missed around 25% of patients with IEI who initially present with other manifestations.

Inflammation and oxidative stress are recognized as important contributors of brain injury in newborns due to a perinatal hypoxic-ischemic (HI) insult. Genetic variability in these pathways could influence the response to HI and the outcome of brain injury. The aim of our study was to evaluate the impact of common single-nucleotide polymorphisms in the genes involved in inflammation and response to oxidative stress on brain injury in newborns after perinatal HI insult based on the severity and pattern of magnetic resonance imaging (MRI) findings. The DNA of 44 subjects was isolated from buccal swabs. Genotyping was performed for NLRP3 rs35829419, CARD8 rs2043211, IL1B rs16944, IL1B rs1143623, IL1B rs1071676, TNF rs1800629, CAT rs1001179, SOD2 rs4880, and GPX1 rs1050450. Polymorphism in CARD8 was found to be protective against HI brain injury detected by MRI overall findings. Polymorphisms in IL1B were associated with posterior limb of internal capsule, basal ganglia, and white matter brain patterns determined by MRI. Our results suggest a possible association between genetic variability in inflammation- and antioxidant-related pathways and the severity of brain injury after HI insult in newborns.

Sladkorna bolezen tipa 2 je metabolična in vnetna bolezen, katere pojavnost v zadnjih letih izrazito narašča. Zanjo je poleg hiperglikemije značilen tudi pojav dislipidemije. Slednja v telesu povečuje vnetno stanje ter oksidativni stres. Na to kako buren bo vnetni odziv, vpliva tudi izražanje provnetnih genov, na katero pa vplivajo genetske spremembe v regulatornih ali kodirajočih področjih genov. Vnetje in oksidativni stres sta dejavnika, ki pospešujeta krajšanje telomerov, kompleksov deoksiribonukleinske kisline in proteinov, ki se nahajajo na koncih kromosomov ter so sestavljeni iz dolgih tandemskih ponovitev deoksiribonukleinske kisline. Njihova vloga je zaščita genoma pred poškodbami. Namen magistrskega dela je bil ugotoviti, ali je lipidni status povezan s pospešenim krajšanjem dolžine telomerov pri bolnikih s sladkorno boleznijo tipa 2 ter ali na dolžino telomerov pri teh bolnikih vpliva genetska variabilnost provnetnih genov IL6, TNF in IL1B. V raziskavo smo vključili 150 bolnikov starih med 41 in 75 let s sladkorno boleznijo tipa 2 ter z zelo veliko srčno žilno ogroženostjo, ki je bila ocenjena glede na smernice Evropskega kardiološkega združenja za preventivo srčno-žilnih bolezni iz leta 2016 ter slovenske prilagoditve iz leta 2012. Bolnikom smo določili, ali imajo povišane/normalne vrednosti celokupnega holesterola, povišane/normalne vrednosti holesterola lipoproteinov nizke gostote ter triacilglicerolov in znižane/normalne vrednosti holesterola lipoproteinov visoke gostote. Za posameznega bolnika smo z modificirano Cawthonovo metodo verižne reakcije s polimerazo v realnem času določili relativne dolžine telomerov ter s kompetitivno alelno specifično verižno reakcijo s polimerazo določili polimorfizme genov IL1B (rs16944), IL6 (rs1800795) in TNF (rs1800629). S statistično analizo podatkov nismo uspeli dokazati povezave med dolžino telomerov ter lipidnim statusom pri bolnikih s sladkorno boleznijo tipa 2. Analizo smo tudi prilagodili na starost, spol, zdravljenje s statini ter trajanje sladkorne bolezni, t. j. dejavniki, ki bi lahko dodatno vplivali na dolžino telomerov, vendar povezave tudi v tem primeru nismo odkrili. Z dolžino telomerov prav tako nismo povezali polimorfizmov provnetnih genov IL6, TNF in IL1B pri omenjenih bolnikih. Povezave ni bilo prisotne tudi po prilagoditvi na starost, spol, zdravljenje s statini ter prisotnost povišanih oz. normalnih vrednosti celokupnega holesterola. Za dokončno pojasnitev zastavljenega raziskovalnega problema bi bile potrebne nadaljnje raziskave, predvsem na večjem številu bolnikov. Type 2 diabetes is an inflammatory and metabolic disease, with significant increase in incidence over the past years. Besides hyperglycemia, type 2 diabetes is also characterised with dyslipidemia, which increases inflammatory state and oxidative stress of the body. Inflammatory response is dependent on expression of proinflammatory genes, while their expression is influenced by genetic changes in regulatory and coding parts of genes. Inflammation and oxidative stress are two factors, associated with accelerated shortening of telomeres. Telomeres are deoxyribonucleic acid–protein complexes located at the end of chromosomes with typical long tandem repeats of deoxyribonucleic acid. Their primal role is protection of the genome from deoxyribonucleic acid damage. The goal of this master’s thesis was evaluation of the association of lipid state and accelerated shortening of telomere length of type 2 diabetes patients and evaluation of association of telomere length and genetic variability of inflammatory genes IL6, TNF, IL1B in these patients. We included 150 patients aged between 41 and 75 with type 2 diabetes and very high cardiovascular risk, as evaluated according to the 2016 European Society of Cardiology Guidelines on cardiovascular disease prevention and 2012 Slovenian adjustments. We determined, whether patients have elevated/normal levels of total cholesterol, elevated/normal levels of low-density lipoprotein cholesterol and triacylglycerols or decreased/normal levels of high-density lipoprotein cholesterol. For each patient, we had used modified Cawthon real-time polymerase chain reaction method to determine relative telomere length and competitive specific allele polymerase chain reaction, to determine gene polymorphism variants IL1B (rs16944), IL6 (rs1800795) and TNF (rs1800629). Using statistical analysis, we could not have proven the association between telomere length and lipid state of type 2 diabetes patients. We have adjusted the analysis regarding age, sex, statin treatment and the duration of diabetes, e.g. factors, that could additionally influence telomere length, but found no association. In our cohort of patients, the telomere length was not associated with polymorphisms of inflammatory genes IL6, TNF and IL1B. There was no association even after adjustment of age, sex, statin treatment and having elevated/normal values of total cholesterol. To get final elucidation of the scientific questions posted in this study, further research is required, preferably on larger group of patients.

Several plant polyphenols have been shown to reduce osteoarthritis symptoms due to their antioxidant, anti-inflammatory and immunomodulatory properties. We investigated the effects of two different polyphenolic extracts (Belinal, Pycnogenol) and two different polyphenols (resveratrol, quercetin) on the chondrogenic potential of bone-derived mesenchymal stem/stromal cells (MSCs) from healthy donors and patients with osteoarthritis. Our main aim was to determine whether Belinal, a commercially available polyphenolic extract from silver fir (Abies alba L.) branches, has comparable chondrogenic potential with the other tested extract and the polyphenols under inflammatory and non-inflammatory conditions. In our study, Belinal promoted significantly greater chondrogenesis compared to the untreated (p = 0.0289) and resveratrol-treated (p = 0.0468) MSCs from patients with hip osteoarthritis under non-inflammatory conditions. Under inflammatory conditions, chondrogenesis was significantly enhanced for MSCs treated with Belinal compared to the control (p = 0.0483). The other extract and the polyphenols did not show any significant effects on chondrogenesis under non-inflammatory or inflammatory conditions. None of the tested extracts and polyphenols showed significant effects on chondrogenesis in healthy donors, under either non-inflammatory or inflammatory conditions. Our data show that Belinal can boost the chondrogenesis of MSCs derived from patients with osteoarthritis, under both non-inflammatory and inflammatory conditions.

V magistrskem delu najprej predstavimo zgradbo in delovanje nuklearnega transkripcijskega faktorja kappa B (NF-κB), ki je izražen v skoraj vseh vrstah celic. Le-ta je ob odsotnosti specifičnih stimulov latenten in se v citoplazmi nahaja v kompleksu z inhibitorjem NF-κB (IκB). Aktivira se šele pod vplivom vezave različnih stimulov na receptorje na celici, nakar začne prehajati v jedro celice, kjer se veže na DNA. To vodi do povečanega izražanja specifičnih genov. V normalnem fiziološkem stanju NF-κB inducira gene, ki imajo pomembno vlogo v procesih vnetja in tiste, ki so pomembni pri razvoju in normalnem delovanju številnih tkiv, kot so kosti, koža, mlečne žleze in centralni živčni sistem. Nepravilno delovanje signalne poti NF-κB pa lahko vodi v vnetne in avtoimune bolezni, nastanek in širjenje raka ter v rezistenco rakavih celic na kemoterapevtike. V nalogi opišemo dve najbolj znani signalni poti, preko katerih se aktivira NF-κB – klasično (kanonično) signalno pot, ki je odgovorna za kontrolo prirojene imunosti in vnetja, ter alternativno (nekanonično) signalno pot, ki ima ključno vlogo pri kontroli razvoja, organizacije in funkcije sekundarnih limfatičnih organov ter v promociji zorenja in preživetja limfocitov B. Nadalje obravnavamo matematični model, ki omogoča podrobno analizo kinetike pomembnih molekul v kanonični signalni poti: kinaz IκB (IKK), NF-κB, proteinov A20, IκBα, njihovih kompleksov in transkriptov. Ugotovimo, da se ta model v veliki meri sklada z izmerjenimi vrednostmi in z rezultati drugih modelov ter, da ob različnih pogojih in vrednostih parametrov lahko reproducira različno dinamiko sistema – oscilirajočo ali dvofazno. Identificiramo tiste parametre in njihove vrednosti, ki posledično pomembno vplivajo na časovno dinamiko izražanja genov. In the master's thesis we first present the structure and function of the nuclear transcription factor kappa B (NF-κB), which is expressed in almost all cell types. In the absence of specific stimuli, it is latent in the cytoplasm and is found in a complex with the NF-κB inhibitor (IκB). It is activated by the binding of different stimuli to the cell receptors, which induces their transport into the cell nucleus. There it binds to DNA, which leads to increased expression of specific genes. Under the normal physiological conditions, NF-κB induces genes that play an important role in the inflammation processes and those that are important in the development and normal functioning of many tissues, such as bones, skin, mammary glands, and the central nervous system. Malfunctioning of the NF-κB signaling pathway may, however, lead to inflammatory and autoimmune diseases, the emergence and spread of cancer, and the resistance of cancer cells to chemotherapeutic agents. Two of the best known signaling pathways via which NF-κB is activated are described here – the classical (canonical) pathway that is responsible for the control of innate immunity and inflammation, and the alternative (noncanonical) pathway, which plays a key role in the development control as well as in the organization and function of secondary lymphatic organs and in promoting the maturation and survival of B lymphocytes. We further discuss and study a mathematical model that allows a detailed analysis of the kinetics of important molecules in the canonical signaling pathway: kinases of IκB (IKK), NF-κB, proteins A20, IκBα, their complexes and transcripts. We find that this model is largely consistent with the measured values and the results of other models. Moreover, that under different conditions and parameter values, it can reproduce different system dynamics - oscillatory or biphasic. We identify those parameters and their values that have a significant effect on the dynamics of gene expression.

Exosomes, a type of nanovesicle, are distinct cellular entities specifically capable of carrying various cargos between cells. It has been hypothesized that exosomes, as an enriched source of biomolecules, may serve as biomarkers for various diseases. This review introduces general aspects of exosomes, presents the challenges in exosome research, discusses the potential of exosomes as biomarkers, and describes the contribution of microfluidic technology to enable their isolation and analysis for diagnostic and disease monitoring. Additionally, clinical applications of exosomes for diagnostic purposes are also summarized.

Background Inflammation and oxidative stress are recognized as important contributors to Parkinson’s disease pathogenesis. As such, genetic variability in these pathways could have a role in susceptibility for the disease as well as in the treatment outcome. Dopaminergic treatment is effective in management of motor symptoms, but poses a risk for motor and non-motor adverse events. Our aim was to evaluate the impact of selected single-nucleotide polymorphisms in genes involved in inflammation and oxidative stress on Parkinson’s disease susceptibility and the occurrence of adverse events of dopaminergic treatment. Methods In total, 224 patients were enrolled, and their demographic and clinical data on the disease course were collected. Furthermore, a control group of 146 healthy Slovenian blood donors were included for Parkinson’s disease’ risk evaluation. Peripheral blood was obtained for DNA isolation. Genotyping was performed for NLRP3 rs35829419, CARD8 rs2043211, IL1β rs16944, IL1β rs1143623, IL6 rs1800795, CAT rs1001179, CAT rs10836235, SOD2 rs4880, NOS1 rs2293054, NOS1 rs2682826, TNF-α rs1800629, and GPX1 rs1050450. Logistic regression was used for analysis of possible associations. Results We observed a nominally significant association of the IL1β rs1143623 C allele with the risk for Parkinson’s disease (OR = 0.59; 95%CI = 0.38–0.92, p = 0.021). CAT rs1001179 A allele was significantly associated with peripheral edema (OR = 0.32; 95%CI = 0.15–0.68; p = 0.003). Other associations observed were only nominally significant after adjustments: NOS1 rs2682826 A allele and excessive daytime sleepiness and sleep attacks (OR = 1.75; 95%CI = 1.00–3.06, p = 0.048), SOD2 rs4880 T allele and nausea/vomiting (OR = 0.49, 95%CI = 0.25–0.94; p = 0.031), IL1β rs1143623 C allele and orthostatic hypotension (OR = 0.57, 95%CI = 0.32–1.00, p = 0.050), and NOS1 rs2682826 A allele and impulse control disorders (OR = 2.59; 95%CI = 1.09–6.19; p = 0.032). We did not find any associations between selected polymorphisms and motor adverse events. Conclusions Apart from some nominally significant associations, one significant association between CAT genetic variability and peripheral edema was observed as well. Therefore, the results of our study suggest some links between genetic variability in inflammation- and oxidative stress-related pathways and non-motor adverse events of dopaminergic treatment. However, the investigated polymorphisms do not play a major role in the occurrence of the disease and the adverse events of dopaminergic treatment.

Izhodišča in namen: Protin je bolezen, ki vpliva na sklepe in se izraža z vnetjem sklepov zaradi nepravilne prehrane. Pacienti večinoma uživajo hrano z veliko vsebnostjo purinov, večina jih pa tudi pije alkohol, predvsem pivo. Bolezen je kronična, vendar pacient ob upoštevanju diete nima večjih težav. Bolezen lahko vpliva na pacienta tudi psihično, zaradi velikih sprememb v življenju. Z raziskavo želimo ugotoviti, pri katerih življenjskih aktivnostih imajo pacienti največ težav in kako si lahko sami pomagajo pri tem. Raziskovalne metode: Uporabljena je bila metoda pregleda literature in deskriptivna metoda dela. Izvedena je bila študija primera, kjer smo uporabili delno strukturiran intervju, v katerega je bil vključen pacient z diagnosticiranim protinom. Študija primera temelji na konceptualnemu modelu Virginije Henderson in 14-ih osnovnih življenjskih aktivnostih. Rezultati: S pomočjo študije primera smo ugotovili, da ima pacient sedem negovalnih diagnoz. Bolečina najbolj ovira starostnika pri opravljanju življenjskih aktivnostih. Razprava in sklep: Pri starostniku z diagnozo protin je najpomembnejše lajšanje bolečine. Pri tem si največ lahko pomaga sam z upoštevanjem diete in jemanjem zdravil, ki mu jih je predpisal zdravnik. Kot zelo pomembno je potrebno izpostaviti tudi podporo družine in prijateljev, ki so ključni dejavniki pri soočanju z boleznijo. Introduction and purpose: Gout is a disease that affects joints, resulting in joint inflammation and is caused by inappropriate nutrition. The majority of patients with gout eat foods with high levels of purines and consume alcoholic beverages, especially beer. Gout is a chronic disease, patients can reduce symptoms by following an appropriate diet. The disease can lead to psychological stress, since patients have to change their lifestyle significantly in order to treat the disease. The purpose of this research is to examine which patients' activities of daily living are affected the most by the disease and how patients can cope with it. Research methodology: The research problem in this paper was defined and described by reviewing professional literature. In empirical part, a descriptive research method was used. For the purposes of a case study a semi structured interview with a gout patient was conducted. The case study is based on the Virginia Henderson's conceptual model and its 14 activities of daily living. Results: The case study showed that the patient has seven nursing diagnoses. Pain prevents the elderly from performing the activities of daily living. Discussion and conclusion: The case study showed that pain relief plays the most important role for the examined patient. The patient himself can contribute the most to pain relief by following his diet and taking medications prescribed by his doctor. Another crucial aspect in dealing with the disease is support of family and friends.

Kronične rane predstavljajo velik socialno-ekonomski problem v zahodnem svetu. Eno najpomembnejših vrst kroničnih ran predstavlja kronična venozna razjeda, ki je odgovorna za okoli 80% vseh kroničnih razjed. Nova histo-patološka spoznanja so pokazala, de je lokalno, kronično vnetje v veliki meri odgovorno za patološke procese, ki vodijo do nastanka razjede kot tudi do upočasnjenega procesa celjenja. V tej študiji je predstavljen nov možen pristop k terapiji kronične venozne razjede, ki temelji na lokalnem dajanju potentne protivnetne zdravilne učinkovine, pentoksifilin (PTX). V ta namen so bile razvite ne-toksične polisaharidne večslojne medicinske obloge iz hitozana in alginata, ki zraven nadzorovanega sproščanja zdravilne učinkovine, že same pozitivno vplivajo na potek celjenja kroničnih ran. V okviru razvoja in optimizacije postopkov priprave omenjenih medicinskih oblog so bili pripravljeni in okarakterizirani najprej 2D modelni večslojni nosilni materiali, ter nato še obložena 3D alginatna koprena. S pomočjo različnih analiznih metod, smo dokazali uspešno pripravo večslojnih medicinskih oblog z dodanim PTX. Z analizo in vitro sproščanja je bilo pokazano, da tako pripravljene medicinske obloge omogočajo nadzorovano, dvo-stopenjsko sproščanje vodotopnega PTX. S pomočjo študij na humanih kožnih celicah smo pokazali biokompatibilnost večslojnih matric z dodatkom PTX. Za konec smo na osnovi eksperimenta na humanih monocitih potrdili tudi, da so pripravljene večslojne medicinske obloge preko delovanja PTX, uspešno zavrle sproščanje TNF-alfa, ki je ključen vnetni mediator pri kroničnih venoznih razjedah. Chronic wounds represent a huge socio-economical problem in the western sociality. One of the most common types of chronic wounds, is the chronic venous ulceration, which accounts for 80% of all chronic wounds. New histo-pathological insights have revealed that chronic local inflammation, which is believed to be preferentially caused by by chronic venous insufficiency, is one of the major causes for the development and poor healing of chronic wounds. In this study we represent a new approach for the treatment of chronic venous ulcers, which is based on the local application of a potent anti-inflammatory drug, pentoxifyllin (PTX). TO achieve controlled release of PTX, and hence provide an efficient and safe local treatment, a non-toxic, multi-layered polysaccharide matrix based on alginate and chitosan was developed. Besides the controlled drug release, such composition has a well-known positive effect on wound healing on its own. As part of their development, 2D model multilayer matrices, as well as functionalized 3D alginate fleece was prepared and characterized. With the help of different analytical methods, it was possible to demonstrate the successful preparation of multilayer matrices with the added PTX. By analysing the in vitro release, it has been shown that the prepared matrices allow a controlled release of the incorporated substance. Further analysis of the drug release profiles has shown, that a two-step release mechanism of the substance was achieved. The biocompatibility of multilayer matrixces with the added PTX was demonstrated through cellular studies on human skin derived fibroblasts. Additional cellular testing using human monocytes confirmed our hypothesis, that the controlled release of PTX is able to inhibit the secretion of TNF-alpha.

We explored the assembly of intestinal microbiota in healthy male participants during the randomized crossover design of run-in (5 day) and experimental phases (21-day normoxic bed rest (NBR), hypoxic bed rest (HBR) and hypoxic ambulation (HAmb) (hypoxic ~4000 m simulated altitude)) in a strictly controlled laboratory environment, with balanced fluid and dietary intakes, controlled circadian rhythm, microbial ambiental burden and 24/7 medical surveillance. Intestinal transit spanning Bristol Stool Scale, defecation rates, zonulin, α1-antitrypsin, eosinophil derived neurotoxin, bile acids, reducing sugars, short chain fatty acids, total soluble organic carbon, water content, diet composition and food intake, intestinal electrical conductivity, sterol and polyphenol content and diversity, indole, aromaticity and spectral characteristics of dissolved organic matter, along with nuclear magnetic resonance metabolomics and trace metal makeup of intestinal environment were assessed. Furthermore (i) abundance, structure and diversity of intestinal butyrate producing microbial community sequencing butyrate pathway genes but and buk, (ii) structure and diversity of microbial community sequencing bacterial and archaeal 16S rRNA and (iii) taxonomic and functional description of bacteria, archaea and fungi using shot-gun metagenomics were investigated. Inactivity negatively affected fecal consistency and in combination with hypoxia aggravated the state of gut inflammation (p < 0.05). On the other hand many of the microbial parameters such as butyrate producing microbial community, the general bacterial and archaeal microbial community were shown to lag behind the changes in human physiology and intestinal environment, since significant changes in bacterial community were delayed until week four in HBR only, where members of the genus Bacteroides and proteins involved in iron acquisition and metabolism, cell wall, capsule, virulence and mucin degradation were enriched. This suggest a time-dependent and complex interplay between the host physiology (including apparent constipation), immunity (inflammation), controlled diet, intestinal environment variables and microbiome physiology during the acute cessation of exercise. Finally, yo-yo dieting and active/inactive lifestyle, along with its effects, seems not to be a human peculiarity but rather a common evolutionary adaptation of mammals to survive food shortage and seasons rotation. Preiskovali smo sestavo črevesne mikrobiote pri zdravih moških preiskovancih v navzkrižno zasnovanem poskusu s 5 dnevnim prilagajanjem na kontrolirano okolje in tremi 21-dnevnimi izvedbami: (i) horizontalnega mirovanja v normoksičnih pogojih (NBR), (ii) horizontalnega mirovanja v hipoksičnih pogojih (HBR) in (iii) gibanja v hipoksičnih pogojih (HAmb) (hipoksično ~ 4000 m simulirane nadmorske višine), v strogo nadzorovanem laboratorijskem okolju, z uravnoteženimi vnosi tekočine in prehrane, kontroliranim cirkadianim ritmom, minimaliziranim mikrobnim vnosom in 24-urnim zdravstvenim nadzorom. Preučevali smo parametre črevesnega okolja: zaprtost črevesja, zonulin, α1-antitripsin, nevrotoksin eozinofilcev, žolčne kisline, reducirajoče sladkorje, kratko-verižne maščobne kisline, vsebnost vode, črevesno električno prevodnost, sterole, polifenole, indol, spektralne lastnosti raztopljenih organskih snovi, celokupne črevesne metabolite in elemente v sledovih. Črevesno mikrobioto smo opredelili glede na: (i) preučevanje številčnosti, strukture in raznovrstnosti mikrobnih skupin, ki proizvajajo masleno kislino, (ii) preučevanje strukture in raznovrstnosti bakterijske in arhejske mikrobiote na osnovi 16S rRNA genov, in (iii) taksonomski ter funkcionalni opis bakterij, arhej in gliv na osnovi sekvenciranja celokupne mikrobne DNA. Telesna neaktivnost je povzročila zaprtje črevesja in v kombinaciji s hipoksijo še poslabšala vnetje v črevesju. Po drugi strani je mikrobiota oz. njen odziv zaostajal za spremembami v človeški fiziologiji in v črevesnem okolju, saj so se bistvene spremembe v bakterijski mikrobioti zgodile v zadnjem tednu poskusa v fiziološko najbolj prizadeti skupini (HBR), kjer je prišlo do obogatitve bakterij rodu Bacteroides in genov vključenih v privzem železa, ter genov za celično steno, kapsulo, virulentnost in razgradnjo mukoznega sloja gostiteljevega črevesja. Slednje kaže na časovno odvisen in zapleten medsebojni vpliv med gostiteljsko fiziologijo (vključno z očitnim zaprtjem), imunskim odgovorom (vnetje črevesja), parametri črevesnega okolja in lastnostmi mikrobiote ob nadzorovanem prehranskem vnosu in med akutnim prenehanjem fizične aktivnosti. Drastične spremembe v prehrani (angl. yo-yo dieting) in nihanje med aktivnim ter neaktivnim življenjskim slogom, ki smo jim priča v vsakdanjem življenju, skupaj s pripadajočimi učinki, niso človeška posebnost, temveč skupna evolucijska prilagoditev sesalcev, razvita kot posledica preživetja zaradi pomanjkanja hrane in prilagajanja na spremembe v letnih časih.

Nekatere rane, definirane kot kronične rane, se v daljšem časovnem obdobju ne zacelijo. Njihovo celjenje je ustavljeno v vnetni fazi, na kar vpliva več dejavnikov, na primer slaba cirkulacija krvi, visoka starost, težave s premikanjem, razne bolezni, ki zavirajo delovanjo imunskega sistema. V kroničnih ranah se veliko bolj kot v akutnih izražajo vnetni citokini, ki poskušajo odpraviti intenzivno vnetje v rani. Med temi citokini so: interlevkini (IL), interferoni in tumor zavirajoči faktor (TNF). Vrednotenje celjenja rane je bilo do sedaj omejeno bodisi na presojo zdravnika ali pa na merjenje kvalitativnih podatkov o celjenju, medtem ko so poskusi določanja poteka celjenja na kvantitativen, napovedno veliko boljši, način, še v povojih. V diplomskem delu smo se zato lotili vrednotenja celjenja prav na tej osnovi, torej poiskati kvantitativno merljive količine, ki jih lahko povežemo s celjenjem. Osredotočili smo na analizo izražanja vnetnih in imunskih genov: IL4, IL5, IL8, IL10 in CSF2 v kronični diabetični razjedi, kot modelni rani. Rezultati so pokazali, da se vsi geni najbolj izražajo na robovih rane in vedno manj proti sredini. To nakazuje na smer celjenja rane, in sicer od njenega roba proti sredini, kar je v skladu tudi s siceršnjimi opazovanju celjenja. Rezultati so zelo vzpodbudni in so osnova za nadaljnje preiskovanje omenjenega pristopa kot možnega modernega protokola za oceno celjenja ran. Some wounds, defined as chronic wounds, do not heal through long period of time. Their healing is inhibited in the inflammatory phase and influenced by several factors, such as poor blood circulation, high age, difficulty moving and various diseases that suppress the immune system. In chronic wounds the inflammatory cytokines are much more expressed than in acute wounds. Among these cytokines are interleukins (IL), interferons and tumor necrosis factor (TNF). Evaluation of wound healing has so far been limited either to the physician's judgment, or the measurement of qualitative data about healing, while attempts to determine a healing process in a quantitative, predictive much better way, are still in infncy. In this thesis, therefore, we have undertaken the evaluation of healing on this basis, to find quantitatively measurable quantities. We focused on the analysis of the expression of inflammatory and immune genes: IL4, IL5, IL8, IL10 and CSF2 in chronic diabetic ulcer as a model wound. The results showed that are all genes most expressed at the edges of the wound and less expressed towards the center. This indicates the direction of the wound healing from its edges to the center of the wound, which is in line with other observations of wound healing. The results are very encouraging and are the basis for further investigation of approach as a possible modern protocol for evaluation of wound healing.

Cilj rada bio je prikazati osnovne karakteristike tendinitisa Ahilove tetive, simptome i uzroke. Takodjer prikazani su osnovni principi fizioterapije.