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Koronavirus, ki povzroča akutni respiratorni sindrom (SARS-CoV-2), se je decembra 2019 pojavil v kitajskem mestu Wuhan. Bolezen, ki jo virus povzroča, je svetovna zdravstvena organizacija poimenovala COVID-19. Virus se je hitro razširil po vsem svetu in močno spremenil naše vsakdanje življenje. Pandemija je močno pospešila razvoj novih diagnostičnih metod, saj je zgodnje odkritje okužbe s SARS CoV-2 bistvenega pomena za uspešen nadzor nad širjenjem virusa. Verižna reakcija s polimerazo v realnem času s predhodno reverzno transkripcijo trenutno velja za zlati standard v diagnostiki SARS-CoV-2. Vzorec izbire je že od začetka pandemije bris nosnega dela žrela. Zaradi pomanjkanja materialov ter neprijetnega odvzema brisa nosnega dela žrela se je pojavil interes iskanja alternativnih kužnin, kot je npr. slina. V okviru magistrskega dela smo primerjali testiranja brisov nosnega dela žrela in sline 624 preiskovancev, odvzetih med septembrom 2021 in februarjem 2022, za odkrivanje različic delta in omikron SARS-CoV-2. Ocenili smo razlike med virusnim bremenom v vzorcih, pridobljenih v posameznem valu okužbe. Poleg tega smo ugotavljali razlike v učinkovitosti metod molekularne diagnostike pri uporabi obeh vrst vzorcev, pri tem pa, v primerjavi s standardno, ovrednotili tudi novo molekularno diagnostično platformo. Dokazali smo, da povzroča genetska različica delta težji potek bolezni v primerjavi z različico omikron. Izkazalo se je, da imajo bolniki, okuženi z različico delta, višje virusno breme v obeh testiranih vzorcih. Hkrati je raziskava ovrgla hipotezo, da bi slina lahko nadomestila bris nosnega dela žrela kot kužnino izbire. Dokazali smo statistično značilno razliko v inhibiciji pomnoževanja nukleinskih kislin v vzorcih sline v primerjavi z vzorci brisov nosnega dela žrela. Pri ovrednotenju nove molekularne diagnostične platforme pa smo zaključili, da nova platforma ni primerna za odkrivanje genetske različice omikron. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible and virulent human-infecting coronavirus that emerged in late December 2019 in Wuhan, China, causing a respiratory disease called coronavirus disease 2019 (COVID-19). SARS-CoV-2 has massively impacted global public health and caused widespread disruption to daily life. The pandemic has greatly accelerated the development of new diagnostic methods, as early detection of SARS-CoV-2 infection is essential for successful outbreak control. Real-time polymerase chain reaction with prior reverse transcription is currently considered the gold standard in SARS-CoV-2 diagnostics. Since the beginning of the pandemic nasopharyngeal swab has been the sample of choice. Due to the lack of materials and the inconvenience of nasopharyngeal swab collection, there has been a great interest in finding an alternative sample, such as saliva. We compared the usefulness of nasopharyngeal swab and saliva samples of 624 participants, collected between September 2021 and February 2022, for the detection of Delta and Omicron SARS-CoV-2 variants. The study aimed to assess if any differences among participants from both waves could be observed and if any difference in molecular diagnostic performance could be observed among the two sample types. We also evaluated a new molecular diagnostic platform. We demonstrated that the Delta variant of SARS-CoV-2 caused a more serious progress of an infection compared to the Omicron variant. Participants infected with the Delta variant were shown to have a higher viral load in both viral specimens. At the same time the research refuted the notion that a saliva sample could substitute for nasopharyngeal swab, since nasopharyngeal swab was shown to provide a better viral specimen. In saliva samples, we demonstrated a higher percentage of nucleic acid amplification inhibition, compared to nasopharyngeal swab, with the difference being statistically significant. During the evaluation of the new molecular diagnostic platform, we concluded that the new platform is not suitable for detecting the Omicron genetic variant.

Ozadje: Akutna bolezen presadka zoper gostitelja (aGvHD) je stanje, pri katerem celice T darovalca, v odzivu na aloantigene prejemnika, povzročijo disregulacijo citokinske kaskade in delujejo citotoksično na prejemnikova tkiva. Predstavlja enega od najpogostejših, v določenih primerih tudi življenje ogrožajočih zapletov po alogenski presaditvi krvotvornih matičnih celic. Kronične vnetne črevesne bolezni (KVČB) pa so ponavljajoča se vseživljenjska patološka stanja, pri katerih aktivirane imunske celice in vnetni citokini povzročajo poškodbe črevesnega tkiva in nenormalen sistemski imunski odziv. Izsledki številnih raziskav so pokazali veliko stopnjo učinkovitosti in varnosti mezenhimskih/ stromalnih matičnih celic (MSC) pri zdravljenju tkivnih poškodb in uravnavanju imunskega odziva, predvsem zaradi njihovih imunomodulatornih, pa tudi multipotentnih regenerativnih lastnosti. Pri tem je zlasti pomembna njihova sposobnost tarčnega lociranja poškodovanih tkiv, kjer nato proizvajajo protivnetne parakrine dejavnike. Namen našega dela je bil, s pomočjo sistematičnega pregleda objavljenih raziskav in meta-analize njihovih rezultatov, potrditi varnost in učinkovitost zdravljenja z MSC. Metode: Izvedli smo sistematični pregled literature v prosto dostopnih spletnih podatkovnih zbirkah. Iz pridobljenih zadetkov smo, glede na predhodno določene kriterije, izbrali študije s kontrolnimi skupinami, serije primerov ter poročila posameznih primerov. V izbrane študije sta bili vključeni tako odrasla kot pediatrična populacija bolnikov. Meta-analize smo izvedli s pomočjo programske opreme Review Manager 5.4 ter rezultate predstavili v obliki drevesnih diagramov. Rezultati: V meta-analizo rezultatov kliničnih študij s kontrolno skupino smo v primeru aGvHD vključili 4, v primeru KVČB pa 5 študij. Uspelo nam je dokazati statistično značilno zmanjšanje smrtnosti bolnikov, ki so prejemali MSC za zdravljenje aGvHD, medtem ko primerjave drugih izbranih parametrov niso pokazale statistično značilnih razlik. Prav tako nismo uspeli dokazati statistično značilnih razlik pri primerjavi različnih parametrov v primeru zdravljenja KVČB z MSC. Pomembno je tudi dejstvo, da zaradi heterogenosti raziskav nismo mogli vključiti vseh v meta-analizo vsakega posameznega parametra. Sklep: Terapije z MSC nedvomno lahko pomagajo pri zdravljenju aGvHD in KVČB. Pri tem pa treba izpostaviti dejstvo, da stanje bolnikov pomembno vpliva na določitev odmerkov celičnega zdravila in način njihove aplikacije, kar je še posebej opazno v primeru KVČB. Background: Acute graft-versus-host disease (aGvHD) is a condition in which donor T cells, in response to recipient alloantigens, cause dysregulation of the cytokine cascade and act cytotoxicly on recipient tissues. It represents one of the most common, in certain cases even life-threatening complications after allogeneic hematopoietic stem cell transplantation. Chronic inflammatory bowel diseases (IBD) are recurrent lifelong pathological conditions in which activated immune cells and inflammatory cytokines cause intestinal tissue damage and an abnormal systemic immune response. The results of many studies have shown a high level of efficiency and safety of mesenchymal/stromal stem cells (MSC) in the treatment of tissue damage and regulation of the immune response, mainly due to their immunomodulatory as well as multipotent regenerative properties. Particularly important here is their ability to target damaged tissues, where they then produce anti-inflammatory paracrine factors. The aim of our work was to confirm the safety and effectiveness of MSC treatment through a systematic review of published research and a meta-analysis of their results. Methods: We conducted a systematic review of the literature in accessible online databases. From the obtained results, according to previously defined criteria, we selected studies with control groups, case series, and individual case reports. Both adult and pediatric patient populations were included in the selected studies. Meta-analyses were performed using the software Review Manager 5.4, and the results were presented in the form of tree diagrams. Results: In the meta-analysis of the results of clinical studies with a control group, we included 4 studies in the case of aGvHD and 5 studies in the case of IBD. We were able to demonstrate a statistically significant reduction in mortality in patients who received MSCs for the treatment of aGvHD, while comparisons of other selected parameters did not show statistically significant differences. We also failed to demonstrate statistically significant differences when comparing various parameters in the case of IBD treatment with MSCs. Also important is the fact that, due to the heterogeneity of the research, we could not include all of them in the meta-analysis of each individual parameter. Conclusion: MSC therapies can undoubtedly help in the treatment of aGvHD and IBD. However, it is important to highlight the fact that the condition of the patients has a significant impact on the determination of the doses of the cellular drug and the way in which they are administered, which is particularly noticeable in the case of IBD.

Glioblastom �GBM� kljub številnim uveljavljenim diagnostičnim in prognostičnim biološkim označevalcem in novim načinom zdravljenja še vedno velja za eno izmed najagresivnejših oblik raka s slabo napovedjo. Med glavne razloge za to uvrščamo visoko infiltrativno sposobnost tovrstnih rakavih celic, ki lahko migrirajo v še neprizadete dele možganov, poleg tega pa lahko kirurško odstranimo le majhen delež okoliškega tkiva. Z zdravljenjem običajno ne dosežemo migrirajočih celic, zato se tumor pogosto ponovno pojavi. Za boljšo uspešnost zdravljenja je ključnega pomena čim hitrejša postavitev diagnoze, zato predstavlja odkrivanje novih bioloških označevalcev, ki bi jih lahko pravočasno določali v enostavno dostopnih bioloških vzorcih bolnikov z GBM pomemben cilj trenutnih raziskav. Z njihovo pomočjo bi morda lahko bolezen odkrili v dovolj zgodnji fazi, predstavljali pa bi tudi nove potencialne tarče za zdravljenje te zahrbtne bolezni. Želja po razvoju učinkovitejših načinov zdravljenja, s katerimi bi lahko zdravili GBM na celovitejši način in s tem morebiti delovali tudi na invazivne celice, je pripeljala do množice novih pristopov, kot so npr. imunsko zdravljenje, uporaba mezenhimskih matičnih celic MMC, ciljanje signalnih celičnih poti in nenazadnje tudi vplivanje na izražanje genov preko uravnavanja količine miRNA v obolelih celicah. Molekule miRNA imajo kratko, enovijačno strukturo in igrajo pomembno vlogo v posttranskripcijski regulaciji. Skupaj z majhnimi interferenčnimi molekulami RNA siRNA namreč sestavljajo regulacijski biološki sistem, ki nadzira začetek in trajanje izražanja posameznih genov v celici. MiRNA sodelujejo pri diferenciaciji in proliferaciji celic, uravnavajo njihovo morfogenezo, delovanje endokrinega sistema, metabolizma, apoptozo in druge pomembne procese v celici. Tako zmanjšana kot povišana ekspresija miRNA sta lahko povezani s številnimi boleznimi, med drugim tudi z rakom, zato predstavljajo pomembno skupino tako potencialnih bioloških označevalcev kot morda tudi terapevtskih tarč in samih terapevtikov. V naši raziskavi smo z digitalnim profiliranjem s pomočjo tehnologije nCounter® analizirali plazemske koncentracije 734 človeških in s človekom povezanih virusnih miRNA. Omenjena tehnologija temelji na uporabi zmesi lovilnih in poročevalskih sond za določen nabor zrelih miRNA. Na ta način lahko kvantitativno digitalno določimo profil izražanja posamezne miRNA, in sicer na podlagi fluorescenčnih kod, ki so značilne zanjo. Tehnologija nCounter® se je v primeru analize nizkih koncentracij miRNA izkazala za bistveno občutljivejšo od splošno uporabljane metode PCR v realnem času. V raziskavo smo vključili 16 zdravih posameznikov in 16 bolnikov z GBM. Prvim smo odvzeli vzorce krvi trikrat znotraj enega meseca, drugim pa na dan operacije tumorja, dan po njej in en teden kasneje. Pri tem smo identificirali 19 miRNA s statistično značilno različnimi plazemskimi nivoji pri GB, v primerjavi s skupino zdravih posameznikov, pri čemer so bile razlike med ravnmi pri bolnikih v primerjavi z zdravimi prostovoljci najmanj 2‐kratne. Poleg teh smo odkrili še 24 miRNA, ki so bile statistično značilno povezane s preživetjem bolnikov po operativnem posegu. S prekrivanjem skupine miRNA, ki so potencialne kandidatke za biološke označevalce z diagnostično vrednostjo s skupino tistih, povezanih s preživetjem, smo opredelili 10 takih, ki bi lahko imele tako diagnostičen kot tudi prognostičen pomen. To so hsa‐miR‐1260a, hsa‐miR‐302c‐3p, hsa‐miR‐484, hsa‐miR‐493‐3p, hsa‐miR‐514a‐3p, hsa‐miR‐592, hsa‐miR‐124‐3p, hsa‐miR‐145‐5p, hsa‐miR‐30a‐5p in hsa‐miR‐483‐5p. Dve izmed njih, in sicer hsa‐miR‐592 in hsa‐miR‐514a‐3p, ki ju do sedaj še niso povezali z GBM, predstavljata novi kandidatni molekuli za selektivno biološko označevanje bolezni. Poleg tega smo odkrili tudi 11 virusnih miRNA s statistično značilno razliko med plazemskimi koncentracijami v obeh primerjanih skupinah. Znano je, da virusne miRNA vplivajo na celični cikel ter zavirajo imunske odzive in apoptozo. Njihovo delovanje se pri bolnikih z rakom prepleta s signali specifičnega tumorskega mikrookolja ter z odzivi imunskega in nevroendokrinega sistema. Za boljšo biološko interpretacijo smo rezultate obdelali z bioinformacijskimi orodji, in sicer na podlagi rudarjenja podatkov in z izrisom genskih mrež ter tako poskusili umestiti kandidatne miRNA v za GBM značilne signalne poti, oziroma razkriti njihovo povezavo z oslabljenimi imunskimi odzivi, ki vodijo do napredovanja bolezni. Najbolj nazorno smo uspeli prikazati njihovo vpetost v signalni poti p53 in Rb. Prisotnost GBM se torej nedvomno kaže v spremenjenih vzorcih plazemskih nivojev miRNA, vključno z virusnimi, kar predstavlja možnost za njihovo klinično uporabo v bodoče. Vsekakor bo potrebno vlogo miRNA, ki se na osnovi naših izsledkov ponujajo kot kandidatke za biološke označevalce, pri nastanku in poteku GBM dodatno raziskati in potrditi v še obsežnejših kliničnih študijah na neodvisnih skupinah bolnikov. Glioblastoma multiforme GBM is one of the most aggressive cancers with poor prognosis in spite of a plethora of well established diagnostic and prognostic biomarkers and treatment modalities. Poor treatment outcome is commonly linked to the fact that GBM cells infiltrate other sections of the brain. Besides, relatively small amount of surrounding tissue can be surgically removed, possibly leaving some tumor cells in the neighbourhood. Migrated tumor cells are often not affected by the treatment causing frequent tumor reocurrences. Early diagnosis is therefore an essential factor for imporvment of the treatment outcome. Current goal is a search for novel biomarkers, possibly easily accessible and detectable in blood of GBM patients that would enable early diagnosis, reliable prognosis and represent potential therapeutic targets, leading to more efficient therapeutic interventions. Development of effective terapies that would treat the disease in a more comprehensive way has led to many novel approaches such as immune therapies, messenhymal stem cell treatments, targeting molecules, involved in signaling pathways and gene expression manipulation through leveling of miRNA concentrations in affected cells. MiRNA molecules are short single‐stranded molecules playing an important role in posttranscriptional regulation. Together with siRNA molecules they form a biological system of RNA interference that contols the beginning and duration of gene expression in cells. MiRNAs are involved in differentiation and proliferation of cells, they balance morphogenesis, endocrine system, metabolism, apoptosis and other important cell processes. Decreased or increased miRNA expression can be correlated to various diseases including cancer, thus, they are foreseen as significant biomarkers or even therapeutics. Our research includes digital profiling of total 734 human and viral miRNAs in blood plasma samples using nCounter® technology. It uses mixture of capture and reporter probes for chosen set of mature miRNAs. Expression profiles can be digitally quantified with flourescent codes specific for each individual miRNA. Analysis of small miRNA concentrations using nCounter® technology prooved to be more sensitive compared to commonly used method RT‐qPCR. Our study included 16 healthy individuals and 16 patients with GBM. Samples of healthy individuals were taken three times within a period of one month. GBM patients were sampled on the day of the surgery, day after it and one week later. We identified 19 miRNAs with significantly different plasma levels in GBM patients, as compared to healthy individuals with the limit difference of at least 2‐fold. Additionally 24 miRNA levels were significantly correlated with patients’ survival. Moreover, the overlap between the group of candidate miRNAs for diagnostic biomarkers and those associated with survival, consisted of 10 miRNAs with both, diagnostic and prognostic potential. These are hsa‐miR‐1260a, hsa‐miR‐302c‐3p, hsa‐miR‐484, hsa‐miR‐493‐3p, hsa‐miR‐514a‐3p, hsa‐miR‐592, hsa‐miR‐124‐3p, hsa‐miR‐145‐5p, hsa‐miR‐30a‐5p and hsa‐miR‐483‐5p. Hsa‐miR‐592 and hsa‐miR‐514a‐3p have not been previously related to GBM and therefore represent novel candidates for selective biomarkers. Besides, 11 viral miRNAs were found to be differentially expressed in plasma of GBM patients. Viral miRNAs allow for viral persistence in the host by inhibiting the cell‐mediated immunity, apoptosis and cell‐cycle, thereby providing conditions favouring latency. These mechanisms may link the viral infection to tumorigenesis, where similar processes are deregulated. We analysed our results using bioinformatic tools for their better interpretation. This approach enabled us to link the revealed miRNAs to specific signalling pathways and to the impaired immune responses leading to progression of disease. The GBM burden is reflected in alteration of plasma miRNA patterns, including those of viral miRNAs and represents a new potential for future clinical applications. However, additional validation of these newely proposed biomarker candidate miRNAs in a larger clinical study using independent cohort of patients is needed.

Pandemija COVID-19 je zelo obremenila zdravstveni sistem. Čeprav se COVID-19 v večini primerov izraža v blagi obliki, lahko, še posebej pri pacientih s prisotnimi dejavniki tveganja, poteka v hujši obliki, ki je lahko življenje ogrožajoča. Namen naše naloge je ovrednotiti varnost in učinkovitost posameznih monoklonskih protiteles ali njihovih kombinacij pri zdravljenju ne-hospitaliziranih pacientov s COVID-19, ki imajo vsaj en dejavnik tveganja za težji potek bolezni. Metode: Izvedli smo sistematični pregled literature, in sicer z uporabo prosto dostopnih bibliografskih spletnih zbirk MEDLINE in Google Scholar. V magistrsko nalogo smo vključili raziskave, kjer so spremljali varnost in učinkovitost aplikacije monoklonskih protiteles bamlanivimaba, sotrovimaba in regdanvimaba ter varnost in učinkovitost njihovih kombinacij, in sicer bamlanivimaba in etesevimaba ter casirivimaba in imdevimaba. Magistrska naloga je sestavljena iz dveh delov. V prvem smo izvedli meta-analizo randomiziranih dvojno slepih kliničnih študij s pomočjo programa RevMan, v drugem pa smo ovrednotili varnost in učinkovitost aplikacij monoklonskih protiteles pacientom s COVID-19, ki so bili vključeni v serije primerov oz. v poročila posameznih primerov. Rezultati: S sistematičnim pregledom smo pridobili 9 dvojno slepih randomiziranih kliničnih raziskav s kontrolno skupino. V 5 od teh poročajo o varnosti in učinkovitosti monoterapije z monoklonskimi protitelesi, v 4 pa o varnosti in učinkovitosti njihovih kombinacij. Z meta-analizo smo dokazali, da aplikacija monoklonskega protitelesa v monoterapiji zmanjša število hospitalizacij, RR: 0,35 (95 % CI: 0,23 – 0,52). Tudi kombinacija monoklonskih protiteles je, v primerjavi s placebom, zmanjšala število hospitalizacij, RR: 0,31 in (95 % CI: 0,22 – 0,45). Z meta-analizo pa smo dokazali tudi zmanjšano število smrti ob uporabi kombinacije monoklonskih protiteles v primerjavi s placebom, RR = 0,14 (95 % CI: 0,04 – 0,45). Število in resnost neželenih učinkov pa sta bila v vseh študijah primerljiva med placebo (kontrolno) in eksperimentalno skupino. Sklep: Naši rezultati potrjujejo varnost in učinkovitost monoklonskih protiteles, uporabljenih pri pacientih s COVID-19, ki niso bili hospitalizirani in imajo prisotne dejavnike za težji potek bolezni. Background: COVID -19 has placed a massive burden on the health care system. COVID -19 progresses mildly in most cases, but can also be severe and life-threatening, especially in patients with existing risk factors. The aim of this master's thesis is to evaluate the safety and efficacy of single monoclonal antibodies or a combination of monoclonal antibodies in non-hospitalised patients who have at least one risk factor for severe COVID -19. Methods: A systematic review of the literature was performed using one of the free online bibliographic databases MEDLINE and Google Scholar. We selected the studies that reported the safety and efficacy of the following antibody monotherapy: bamlanivimab, sotrovimab and regdanvimab, or the following combination of antibodies: bamlanivimab and etesevimab, or casirivimab and imdevimab. The master thesis consists of two parts. In the first part, we performed a meta-analysis using RevMan with data from randomized, double-blind, placebo-controlled clinical trials. In the second part, we evaluated the safety and efficacy of using monoclonal antibodies in patients included in case reports or case series. Results: With a systematic review of the literature, we obtained 9 double-blinded randomized placebo-controlled clinical studies. 5 of them reported the safety and efficacy of the usage of single monoclonal antibodies and 4 reported the safety and efficacy of using a combination of monoclonal antibodies. The meta-analysis showed a reduction in the number of hospitalizations with the application of one monoclonald antibody, RR = 0.35, (95% CI 0.23-0.52). The meta-analysis also showed a reduction in hospitalizations with the use of combinations of monoclonal antibodies, RR: 0.31 (95% CI 0.22 - 0.45). Using meta-analysis, we also demonstrated a reduction in the number of deaths with the application of combination of monoclonal antibodies compared to placebo, RR = 0.14, (95% CI 0.04 - 0.45). The number and severity of adverse effects were comparable in all studies in the treatment and placebo groups. Conclusion: The obtained results confirm safety and efficacy of monoclonal antibodies in non-hospitalized COVID-19 patients, who have present risk factors for severe COVID-19.

Osteoartroza (OA) je kronična degenerativna bolezen, za katero je značilna obraba sklepnega hrustanca. Gre za heterogeno bolezen, s prevladujočimi vnetnimi in presnovnimi procesi, ki privedejo do razgradnje sklepnega hrustanca in spremenjene strukture kostnega tkiva. Napredovana bolezen najpogosteje prizadene kolčne in kolenske sklepe, hrbtenico ter sklepe dlani in stopal. Trenutno terapijo OA sestavljajo različni načini za blažitev bolečine in vnetja, v končni fazi bolezni pa kirurška zamenjava poškodovanega sklepa. Velike obete pri zdravljenju, in ne le zgolj lajšanju simptomov bolezni, zaradi svojih regenerativnih zmožnosti, ponujajo matične celice. Mezenhimske matične/stromalne celice (MSC, angl. mesenchymal stem/ stromal cells) so heterogena populacija multipotentnih celic, z veliko zmožnostjo samoobnove in diferenciacije v različne celične linije. Podvrsta MSC so nedavno odkrite človeške skeletne matične celice (hSSCs, angl. human skeletal stem cells), ki so ravno tako multipotentne, a za razliko od MSC tkivno specifične, saj imajo natančno določen imunofenotip (CD73+/CD146-/PDPN+/CD164+) in zmožnost preobrazbe v osteoblaste in hondrocite, tako v pogojih in vitro kot in vivo. Ker imajo pomembno vlogo pri razvoju, regeneraciji in homeostazi kostnega tkiva, veliko obetajo v zdravljenju degenerativnih sklepnih bolezni, med katere sodi tudi OA. Celične terapije z MSC, ki se trenutno uporabljajo v klinični praksi, namreč omejuje pomanjkanje specifičnih celičnih označevalcev, ki bi omogočili njihovo natančno identifikacijo. V okviru magistrske naloge smo proučevali, kako različna stopnja OA kolka vpliva na lastnosti kostnih MSC teh bolnikov, in sicer na nivojih njihove sposobnosti osteogeneze ter imunofenotipa novih označevalcev hSSC v pogojih in vitro. Najprej smo z metodo kvantitativne verižne reakcije s polimerazo v realnem času (qPCR) izvedli preliminarno študijo na vzorcih cDNA, pridobljenih iz kostnih MSC 32 preiskovancev, s katero smo preverili, ali med bolniki z zgodnjo in pozno obliko OA ter skupino kontrolnih oseb brez OA morda obstajajo razlike v izražanju genov za nove označevalce hSSC. Na podlagi teh rezultatov smo nadaljevali s študijo na izbranih vzorcih kostnih MSC 12 preiskovancev, pri čemer smo ovrednotili njihovo sposobnost osteogeneze ter s pomočjo pretočne citometrije določili njihove imunske fenotipe za označevalce hSSC. Med skupinami bolnikov z različnima stopnjama OA kolka nismo dokazali statistično značilnih razlik v sposobnosti osteogeneze in imunofenotipu novih označevalcev hSSC njihovih kostnih MSC. Čeprav je naša raziskava prva, ki je določila lastnosti kostnih MSC na nivoju nedavno odkritih hSSC pri bolnikih z različnima stopnjama OA, pa ostajamo do rezultatov kritični in dopuščamo možnost, da bi lahko z vključitvijo večjega števila preiskovancev dobili drugačne rezultate. Osteoarthritis (OA) is a chronic degenerative disease with typical loss of joint cartilage. It is a heterogeneous disease accompanied by an inflammatory and catabolic processes that lead to decomposition of joint cartilage and changes in tissue structure. It most commonly affects hip and knee joints, spine and joints of palms and feet. The therapy of OA currently involves pain relieving and anti – inflammatory medications. When disease progresses the only treatment option is surgical replacement of the joint with the artificial prosthesis. Significant breakthrough in the treatment options are mesenchymal stem/ stromal cells (MSCs) since they possess regenerative abilities and would therefore be capable of disease-modifying not only of relieving symptoms of OA. MSCs are heterogeneous population of multipotent cells with regenerative properties and capability of multilineage differentiation. Recently discovered sub-population of MSCs are human skeletal stem cells (hSSCs), also multipotent but in a contrast to MSC, hSSC are tissue specific, with defined immunophenotype CD73+/CD146-/PDPN+/CD164-, capable of in vitro and in vivo differentiation into cartilage and bone. hSSC play a significant role in bone forming and bone remodelling process and are therefore promising treatment option for degenerative diseases such as OA. The clinical use of the current cell therapies is limited by the lack of reliable cell surface markers, which hinders their proper identification and isolation. In this thesis, we researched the influence that different stages of hip OA might have on bone-derived MSC features such as in vitro osteogenic differentiation and immunophenotype for new hSSC markers. First, we performed a gene expression analysis on cDNA samples of bone-derived MSCs from 32 donors, where we searched for potential differences in gene expression of new molecular markers for hSSC in patients with early and late OA, and patients without OA. We then chose 12 samples of bone-derived MSCs for further in vitro analyses of osteogenic differentiation using Alizarin red S staining. On the same set of samples, we then performed flow cytometry and determined the immunophenotype for new hSSC markers. Results showed that there are no significant differences in the osteogenic potential and immunophenotype for new hSSC markers in bone-derived MSCs between patient groups with different stages of hip OA. This is the first research study determining the recently identified hSSC-like features of bone-derived MSCs, in patients with different stages of hip OA. Due to small group of donors, further studies are suggested to confirm the results of our study.

Ozadje: Eksosomi so membranski vezikli, ki jih celice izločajo v zunajcelični prostor. Sodelujejo pri številnih fizioloških in patoloških procesih, kot so: imunski odziv, obnavljanje tkiv, vnetje in rak. Udeleženi so tudi v medcelični komunikaciji, saj prenašajo številne signalne molekule: proteine, lipide in nukleinske kisline (DNA, mRNA, mikro RNA). Tovor, ki ga eksosomi prenašajo, odraža sestavo celic iz katerih izvirajo, zato so pomemben vir raznolikih diagnostičnih označevalcev. Vse več kliničnih raziskav se ukvarja predvsem s terapevtsko rabo eksosomov matičnih celic, ki imajo številne prednosti v primerjavi s samimi celicami. Metode: Sistematični pregled literature smo izvedli v bibliografskih spletnih bazah, Clinicaltrials.gov, Cochrane library ter MEDLINE. Za analizo smo izbrali tiste raziskave, v okviru katerih so proučevali normalne in spremenjene človeške eksosome za terapevtske ter diagnostične namene. Pri slednjih smo se omejili na rakava obolenja prostate in pljuč. Zaradi velikega števila še potekajočih študij smo izvedli analizo globalnih trendov izvajanja omenjenih raziskav. Izbrane parametre kot so število preiskovancev, vrsta uporabljenih eksosomov, itd., smo prikazali v obliki grafov. Značilnosti vseh analiziranih raziskav pa smo predstavili v preglednicah. Rezultati: S sistematičnim pregledom smo pridobili skupno 53 raziskav, od katerih jih je 30 obravnavalo terapevtsko, 23 pa diagnostično rabo človeških eksosomov. V terapevtske namene največkrat uporabljajo eksosome iz mezenhimskih/ stromalnih matičnih celic, s katerimi eksperimentalno tretirajo predvsem bolezni dihal, neoplazme, nevrološke in kožne bolezni. Večina analiziranih kliničnih preskušanj je bila odprtih, nerandomiziranih in v fazi I kliničnih raziskav. V povprečju so te raziskave trajale 1,9 let. V primeru diagnostične rabe eksosomov pa smo zasledili največ kohortnih študij. Veliko je takih, ki validirajo teste na podlagi eksosomskih označevalcev. Kot nove diagnostične označevalce pa raziskujejo predvsem eksosomske mikro RNA. Tovrstne raziskave trajajo v povprečju 3 leta. Sklep: Področje uporabe eksosomov je novo in nakazuje njihov velik potencial za terapevtsko in diagnostično uporabo, pri čemer je slednja pravzaprav že postala realnost. Background: Exosomes are membrane vesicles which cells secrete into the extracellular space. They are involved in many physiological processes, like immune response and tissue regeneration, as well as pathological processes, e.g. inflammation and cancer. They are also involved in intercellular communication, as they carry many signaling molecules, such as proteins, lipids and nucleic acids (DNA, mRNA, microRNA). Due to their load, which reflects the composition of the originating cell, they are an important source of various diagnostic markers. More and more clinical research is conducted in the therapeutic use of exosomes, which have many advantages especially compared to stem cells from which they originate. Methods: Systematic review of literature has been performed using the following online bibliographic databases: Clinicaltrials.gov, Cochrane library and MEDLINE. We have selected those clinical research studies in which human exosomes or altered human exosomes were used for therapeutic or diagnostic purposes. In the latter case, we limited ourselves to prostate and lung cancers. Due to a large number of ongoing studies, we performed the analysis of their global trends. Selected parameters, such as number of participating subjects, types of exosomes used, etc., were presented graphically. The selected properties of all studies were presented in tables. Results: Following stringent inclusion/ exclusion criteria, 53 studies were selected for analysis. Thirty of them were dealing with therapeutic and 23 with diagnostic use of human exosomes. In the first group the most commonly used exosomes in clinics were derived from mesenchymal stem cells, and were mostly investigated in treatment of respiratory diseases, neoplasms, and neurological and skin diseases. Most clinical trials were open-label, non-randomised, and phase I. Therefore, the average length of a clinical trial in this group is 1,9 years. Most of the analysed diagnostic studies were cohort studies. There were also many validating diagnostic tests based on exosomal markers. In terms of new diagnostic markers, microRNAs were those being mainly investigated. On average, this type of research takes 3 years. Conclusion: The field of exosomes is rather new, and holds a great potential for their therapeutic and diagnostic use.