1. Diferencialno izražanje genov in učinek filtriranja nizko izraženih genov pri analizi RNA sekvenciranjaŠpela Fabčič, 2025, magistrsko delo Opis: Uvod: Sekvenciranje RNA (analiza RNA-seq) se sooča z izzivi obdelave podatkov in razvojem metod za učinkovito shranjevanje, pridobivanje in obdelavo velikih količin podatkov z namenom, da se zmanjša število napak določevanja nukleotidnega zaporedja med drugim tudi na način z odstranitvijo odčitkov nižje kakovosti.
Metode: S pomočjo programskega orodja R smo iz treh različnih podatkovnih baz za tri različne bolezni (Crohnova bolezen, astma, miomi maternice) izvedli RNA-seq analizo v katero smo vključili filtriranje nizko izraženih z metodo CPM, kjer smo določili prag filtriranja nizko izraženih genov na podlagi statističnega izračuna minimuma, maksimuma in povprečja izražanja genov.
Rezultati: V okviru naloge smo ugotovili, da različni pragi filtriranja ne spremenijo števila nabora genov v končnih rezultatih analize RNA-seq. Pri vplivu postavitve različnih pragov filtriranja na nabor prvih 20 najbolj statistično značilno diferencialno izraženih genov je bila izrazita razlika med različno zastavljenimi pragi filtriranja samo v primeru Crohnove bolezni. Pri testiranju, ali se izločeni geni pri danem pragu filtriranja pojavijo kot statistično značilno diferencialno izraženi geni v analizi brez filtriranja, smo ugotovili, da se velik delež izločenih genov, še posebej pri nižjih pragovih filtriranja, še vedno pojavi kot statistično značilen v analizi brez filtriranja.
Razprava in sklep: Na podlagi rezultatov smo ugotovili, da postavitev različnih pragov filtriranja nima posebnega učinka na rezultate diferencialnega izražanja genov.
Ključne besede: genska ekspresija, astma, Crohnova bolezen, miomi maternice
Objavljeno v DKUM: 06.05.2025; Ogledov: 0; Prenosov: 0
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2. MFUM-BrTNBC-1, a newly established patient-derived triple-negative breast cancer cell line : molecular characterisation, genetic stability, and comprehensive comparison with commercial breast cancer cell linesKristijan Skok, Lidija Gradišnik, Helena Sabina Čelešnik, Marko Milojević, Uroš Potočnik, Gregor Jezernik, Mario Gorenjak, Monika Sobočan, Iztok Takač, Rajko Kavalar, Uroš Maver, 2022, izvirni znanstveni članek Opis: Triple-negative breast cancer (TNBC) is a breast cancer (BC) subtype that accounts for
approximately 15–20% of all BC cases. Cancer cell lines (CLs) provide an efficient way to model the
disease. We have recently isolated a patient-derived triple-negative BC CL MFUM-BrTNBC-1 and
performed a detailed morphological and molecular characterisation and a comprehensive comparison
with three commercial BC CLs (MCF-7, MDA-MB-231, MDA-MB-453). Light and fluorescence
microscopy were used for morphological studies; immunocytochemical staining for hormone receptor,
p53 and Ki67 status; RNA sequencing, qRT-PCR and STR analysis for molecular characterisation; and
biomedical image analysis for comparative phenotypical analysis. The patient tissue-derived MFUMBrTNBC-1 maintained the primary triple-negative receptor status. STR analysis showed a stable and
unique STR profile up to the 6th passage. MFUM-BrTNBC-1 expressed EMT transition markers and
displayed changes in several cancer-related pathways (MAPK, Wnt and PI3K signalling; nucleotide
excision repair; and SWI/SNF chromatin remodelling). Morphologically, MFUM-BrTNBC-1 differed
from the commercial TNBC CL MDA-MB-231. The advantages of MFUM-BrTNBC-1 are its isolation
from a primary tumour, rather than a metastatic site; good growth characteristics; phenotype identical
to primary tissue; complete records of origin; a unique identifier; complete, unique STR profile;
quantifiable morphological properties; and genetic stability up to (at least) the 6th passage.
Ključne besede: hormonal receptors, MFUM-BrTNBC-1, MCF-7, MDA-MB-231, MDA-MB-453
Objavljeno v DKUM: 10.04.2025; Ogledov: 0; Prenosov: 3
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3. Genska ontologija farmakogenomike bioloških zdravil pri imunsko pogojenih boleznih : magistrsko deloTino Kovačič, 2024, magistrsko delo Opis: V magistrskem delu smo z uporabo genske ontologije preučevali farmakogenomiko bioloških zdravil pri imunsko pogojenih boleznih, s poudarkom na atopijskem dermatitisu, astmi in multipli sklerozi. Namen raziskave je bil ugotoviti vpliv bioloških označevalcev na učinkovitost zdravljenja z biološkimi zdravili ter identificirati skupne in specifične molekularne poti teh bolezni. S sistematskim pregledom literature smo analizirali že objavljene članke, ki poročajo o genih povezanih z odzivom na zdravljenje z biološkimi zdravili pri vseh treh boleznih, nato pa smo izvedli analizo genske ontologije s pomočjo programske opreme Cytoscape.
Primerjali smo individualne analize za vsako bolezen posebej in izvedli komparativno analizo genske ontologije. Ugotovili smo, da so statistično najbolj pomembni pojmi za vse tri bolezni vključujejo: aktivacijo B celic (p vrednost = 1,16 × 10-17), diferenciacijo T celic (p vrednost = 2,15 × 10-33), diferenciacijo CD4+ alfa-beta T celic (p vrednost = 6,65 × 10-25) ter celično apoptozo (p vrednost = 5,28 × 10-18). Pri vseh posameznih analizah so se pojavili tudi statistično pomembni pojmi, kot so mikroglialna aktivnost, regulacija imunoglobulina E in vitamina D, ki posredno ali neposredno vplivajo na odziv na biološka zdravila. Diferenciacija Bergmannovih glialnih celic (p vrednost = 1,67 × 10-4) je bil statistično pomemben pojem, ki se je pojavil le pri analizi multiple skleroze.
Povzamemo lahko, da je v tej nalogi razkritih več GO izrazov, ki so vključeni v molekularne poti atopijskega dermatitisa, astme in multiple skleroze ter služijo kot pokazatelji odziva na zdravljenje z biološkimi zdravili za te bolezni.
Ključne besede: genska ontologija, biološka zdravila, biološki označevalci, atopijski dermatitis, astma, multipla skleroza
Objavljeno v DKUM: 16.01.2025; Ogledov: 0; Prenosov: 23
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4. Single-cell transcriptomic and targeted genomic profiling adjusted for inflammation and therapy bias reveal CRTAM and PLCB1 as novel hub genes for anti-tumor necrosis factor alpha therapy response in Crohn’s diseaseMario Gorenjak, Boris Gole, Larisa Goričan, Gregor Jezernik, Uršula Prosenc Zmrzljak, Cvetka Pernat Drobež, Pavel Skok, Uroš Potočnik, 2024, izvirni znanstveni članek Opis: The lack of reliable biomarkers in response to anti-TNFα biologicals hinders
personalized therapy for Crohn’s disease (CD) patients. The motivation behind our study is to shift
the paradigm of anti-TNFα biomarker discovery toward specific immune cell sub-populations using
single-cell RNA sequencing and an innovative approach designed to uncover PBMCs gene expression
signals, which may be masked due to the treatment or ongoing inflammation; Methods: The singlecell
RNA sequencing was performed on PBMC samples from CD patients either naïve to biological
therapy, in remission while on adalimumab, or while on ustekinumab but previously non-responsive
to adalimumab. Sieves for stringent downstream gene selection consisted of gene ontology and
independent cohort genomic profiling. Replication and meta-analyses were performed using publicly
available raw RNA sequencing files of sorted immune cells and an association analysis summary.
Machine learning, Mendelian randomization, and oligogenic risk score methods were deployed to
validate DEGs highly relevant to anti-TNFα therapy response; Results: This study found PLCB1 in
CD4+ T cells and CRTAM in double-negative T cells, which met the stringent statistical thresholds
throughout the analyses. An additional assessment proved causal inference of both genes in response
to anti-TNFα therapy; Conclusions: This study, jointly with an innovative design, uncovered
novel candidate genes in the anti-TNFα response landscape of CD, potentially obscured by therapy
or inflammation.
Ključne besede: inflammatory bowel diseases, Crohn’s disease, tumor necrosis factor alpha, adalimumab, single-cell gene expression analysis
Objavljeno v DKUM: 10.12.2024; Ogledov: 0; Prenosov: 7
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5. Discovery of novel biomarkers with extended non-coding RNA interactor networks from genetic and protein biomarkersGregor Jezernik, Damjan Glavač, Pavel Skok, Martina Krušič, Uroš Potočnik, Mario Gorenjak, 2024, izvirni znanstveni članek Opis: Curated online interaction databases and gene ontology tools have streamlined the analysis of highly complex gene/protein networks. However, understanding of disease pathogenesis has gradually shifted from a protein-based core to complex interactive networks where non-coding RNA (ncRNA) is thought to play an essential role. As current gene ontology is based predominantly on protein-level information, there is a growing need to analyze networks with ncRNA. In this study, we propose a gene ontology workflow integrating ncRNA using the NPInter V5.0 database. To validate the proposed workflow, we analyzed our previously published curated biomarker datasets for hidden disease susceptibility processes and pharmacogenomics. Our results show a novel involvement of melanogenesis in psoriasis response to biological drugs in general. Hyperpigmentation has been previously observed in psoriasis following treatment with currently indicated biological drugs, thus calling attention to melanogenesis research as a response biomarker in psoriasis. Moreover, our proposed workflow highlights the need to critically evaluate computed ncRNA interactions within databases and a demand for gene ontology analysis of large miRNA blocks.
Ključne besede: gene ontology, non-coding RNA, disease pathogenesis
Objavljeno v DKUM: 06.12.2024; Ogledov: 0; Prenosov: 7
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6. Meta-analytic comparison of global RNA transcriptomes of acute and chronic myeloid leukemia cells reveals novel gene candidates governing myeloid malignanciesStaša Jurgec, Gregor Jezernik, Mario Gorenjak, Tomaž Büdefeld, Uroš Potočnik, 2022, izvirni znanstveni članek Opis: Despite advances in the understanding of genetic risk factors and molecular mechanisms underlying acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), clinical outcomes of current therapies in terms of disease relapse and mortality rate pose a great economic and social burden. To overcome this, the identification of new molecular prognostic biomarkers and pharmacological targets is crucial. Recent studies have suggested that AML and CML may share common pathogenic mechanisms and cellular substrates. To this end, in the present study, global transcriptome profiles of AML and CML at the molecular and cellular level were directly compared using a combination of meta-analysis and modern statistics, and novel candidate genes and specific biological processes associated with the pathogenesis of AML and CML were characterized. Our study significantly improves our current understanding of myeloid leukemia and will help develop new therapeutic targets and biomarkers for disease progression, management and treatment response.
Ključne besede: AML, CML, meta-analysis, lincRNA, spliceosome
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7. Identification of novel loci involved in adalimumab response in Crohn’s disease patients using integration of genome profiling and isoform-level immune-cell deconvoluted transcriptome profiling of colon tissueMario Gorenjak, Gregor Jezernik, Martina Krušič, Pavel Skok, Uroš Potočnik, 2022, izvirni znanstveni članek Opis: Crohn’s disease is a consequence of dysregulated inflammatory response to the host’s microbiota. Although anti-TNF treatment improves the quality of the patient’s life, a large proportion of patients lose response to the treatment. The past decade of research has led to a continuum of studies showcasing the heterogeneity of anti-TNF response; thus, the aim of the present study was to dissect transcriptome-wide findings to transcript isoform specific levels and combine the analyses with refined information of immune cell landscapes in colon tissue, and subsequently select promising candidates using gene ontology and genomic integration. We enrolled Slovenian Crohn’s disease patients who were naïve with respect to adalimumab treatment. We performed colon tissue RNA sequencing and peripheral blood mononuclear cell DNA genotyping with a subsequent contemporary integrative approach to combine immune cell deconvoluted isoform transcript specific transcriptome analysis, gene ontology layering and genomic data. We identified nine genes (MACF1, CTSE, HDLBP, HSPA9, HLA-DMB, TAP2, LGMN, ANAPC11, ACP5) with 15 transcripts and 16 variants involved in the adalimumab response. Our study identified loci, some of which were previously shown to contribute to inflammatory bowel disease susceptibility, as novel loci involved in adalimumab response in Crohn’s disease patients.
Ključne besede: Crohn’s disease, adalimumab, transcriptome, isoforms, deconvolution
Objavljeno v DKUM: 05.12.2024; Ogledov: 0; Prenosov: 4
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8. Isoform-level transcriptome analysis of peripheral blood mononuclear cells from breast cancer patients identifies a disease-associated RASGEF1A isoformHelena Sabina Čelešnik, Mario Gorenjak, Martina Krušič, Bojana Crnobrnja, Monika Sobočan, Iztok Takač, Darja Arko, Uroš Potočnik, 2024, izvirni znanstveni članek Opis: Breast cancer (BC) comprises multiple subtypes with distinct molecular features, which differ in their interplay with host immunity, prognosis, and treatment. Non-invasive blood analyses can provide valuable insights into systemic immunity during cancer. The aim of this study was to analyze the expression of transcriptional isoforms in peripheral blood mononuclear cells (PBMCs) from BC patients and healthy women to identify potential BC immune biomarkers. Methods: RNA sequencing and isoform-level bioinformatics were performed on PBMCs from 12 triple-negative and 13 luminal A patients. Isoform expression validation by qRT-PCR and clinicopathological correlations were performed in a larger cohort (156 BC patients and 32 healthy women). Results: Transcriptional analyses showed a significant (p < 0.001) decrease in the ENST00000374459 RASGEF1A isoform in PBMCs of BC compared to healthy subjects, indicating disease-related expression changes. The decrease was associated with higher ctDNA and Ki-67 values. Conclusions: The levels of the RASGEF1A transcriptional isoform ENST00000374459 may have the potential to distinguish between BC and healthy subjects. The downregulation of ENST00000374459 in breast cancer is associated with higher proliferation and ctDNA shedding. Specialized bioinformatics analyses such as isoform analyses hold significant promise in the detection of biomarkers, since standard RNA sequencing analyses may overlook specific transcriptional changes that may be disease-associated and biologically important.
Ključne besede: breast cancer, peripheral blood, isoform-level RNA-seq, RASGEF1A ENST00000374459, ctDNA, Ki-67
Objavljeno v DKUM: 26.11.2024; Ogledov: 0; Prenosov: 5
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9. Izražanje izoform gena DDB2 v mononuklearnih celicah periferne krvi pri različnih podtipih raka dojke : diplomsko delo univerzitetnega študijskega programa I. stopnjeKarin Rajh, 2024, diplomsko delo Opis: Rak dojke (RD) je najpogosteje diagnosticiran rak pri ženskah in je glavni vzrok smrti zaradi raka pri ženskah po vsem svetu. Gre za heterogeno bolezen, ki se razlikuje na molekularni, histološki in klinični ravni. Ločimo več podtipov RD, ki jih delimo na podlagi histopatoloških in molekularnih značilnosti. Podtipa trojno negativni RD in HER2-pozitivni RD (TNRD in HER2+) sta na splošno bolj imunogena in povezana z boljšo odzivnostjo na imunoterapijo kot luminalna podtipa (luminalni A in B).
Analize krvi omogočajo vpogled v sistemski imunski odziv na tumorske celice. Odvzem krvi je lahko dostopna metoda pridobivanja bioloških vzorcev, ki lahko pripomore k zgodnjemu odkrivanju RD. V Centru za humano genetiko in farmakogenomiko Medicinske fakultete v Mariboru so z metodo RNA-seq opravili sekvenciranje transkriptoma imunskih celic iz periferne krvi (mononuklearnih celic periferne krvi, PBMC) pri 25 bolnicah z RD, od tega 13 s podtipom luminalni A in 12 s podtipom TNRD. Z bioinformatsko analizo transkripcijskih izoform so identificirali izoformo ENST00000378603 gena DDB2 kot potencialno pomembno, saj je pokazala značilno diferencialno izražanje med tema dvema podtipoma RD. V nasprotju z omenjeno izoformo pa so se druge izoforme gena DDB2, na primer izoforma ENST00000256996, enako izražale pri podtipih TNRD in luminalni A.
Namen te diplomske naloge je bila validacija ter razširitev teh ugotovitev z analizo izoform gena DDB2 z metodo RT-qPCR na večjem številu vzorcev, ki so vključevali 182 vzorcev bolnic z RD vseh podtipov in 37 vzorcev zdravih oseb. Da bi lahko ovrednotili izražanje izoform gena DDB2 z metodo RT-qPCR, je bilo najprej potrebno optimizirati reakcijske pogoje s specifičnimi oligonukleotidnimi začetniki za obe izoformi. Po optimizaciji reakcijskih pogojev smo z RT-qPCR izmerili izražanje izoforme ENST00000256996 pri bolnicah z RD in zdravih osebah. V nasprotju pa je bila optimizacija reakcijskih pogojev za izoformo ENST00000378603 neuspešna.
Ugotovili smo, da se izoforma ENST00000256996 statistično značilno bolj izraža v vzorcih zdravih oseb kot v vzorcih pacientk z RD, kar je v skladu z našo hipotezo, saj je produkt gena DDB2 udeležen pri popravljanju poškodb DNA, njegova odsotnost pa je povezana s kopičenjem DNA-napak in kancerogenezo. Rezultati so potrdili tudi, da se analizirana izoforma enako izraža v podtipih RD TNRD in luminalni A.
Ključne besede: Rak dojke, TNRD, PBMC, DDB2, transkripcijske izoforme, biooznačevalci
Objavljeno v DKUM: 20.09.2024; Ogledov: 0; Prenosov: 41
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10. Optimizacija in vitro stimulacije limfocitov s fitohemaglutininom (PHA) in lipopolisaharidi (LPS) : diplomsko delo univerzitetnega študijskega programa I. stopnjePatricija Počivavšek, 2024, diplomsko delo Opis: Stimulacija limfocitov s fitohemaglutininom(PHA) in lipopolisaharidi(LPS) je ključna metoda za preučevanje imunskih odzivov v eksperimentalnih pogojih. Optimizacija koncentracij omenjenih imunostimulatorjev je bistvena za zagotavljanje fiziološko relevantnih in natančnih rezultatov. V diplomski nalogi smo preučevali odziv limfocitov na različne koncentracije PHA in LPS, ki inducirajo 70% maksimalnega odziva. Pri tem smo prav tako želeli ugotoviti, če najvišje koncentracije PHA in LPS povzročijo celično smrt. Limfocite smo izolirali iz mononuklearnih celic periferne krvi(PBMC) zdravih darovalcev in jih gojili v prisotnosti različnih koncentracij PHA in LPS. Proliferacijo celic smo merili s pomočjo barvila alamarBlue in določili celično viabilnost z uporabo pretočne citometrije. Rezultati so pokazali, da je PHA pri vmesni koncentraciji 1-2,5 μg/mL učinkovit stimulator za doseg 70% maksimalnega odziva limfocitov, hkrati pa najvišje koncentracije PHA povzročajo celično smrt. Nasprotno je bil LPS neuspešen zaradi velike variabilnosti med donorji. Naše ugotovitve prispevajo k boljši standardizaciji eksperimentalnih protokolov pri imunoloških raziskavah.
Ključne besede: limfociti, stimulacija, pretočna citometrija, PHA, LPS, optimizacija
Objavljeno v DKUM: 19.09.2024; Ogledov: 0; Prenosov: 29
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