As the survival rate increases, the importance of quality of life post-cancer is increasing. This, in conjunction with genetic screening, has increase the number of breast reconstructions 36%. The most common complication causing revision of reconstructive surgery is the formation of a dense scar capsule around the silicone implant called capsular contracture. Nearly all patients will experience this complication, though with different degrees of response, ranging from moderate scarring to major disfigurement and pain at the implant site. Presently, there is no way to predict the degree of contraction capsule formation that individual patients will suffer prospectively, nor is there clinical approach to preventing this complication. Patient information and tissue was collected in a uniform manner to address these lingering problems. Clinical data was used to construct a predictive model which can accurately predict capsular contracture severity in breast reconstruction patients. Histological analysis demonstrated differences in structure and cell composition between different capsule severities. Of particular note, a new region was described which could serve as the communication interface between innate immune cells and fibroblasts. RNA-seq analysis identified 1029 significantly dysregulated genes in severe capsules. Pathway enrichment was then performed which highlights IL4/13 signaling, extracellular matrix organization, antigen presentation, and interferon signaling as importantly dysregulated pathways. These RNA results were also compared to various clinical and histological measurements to evaluate novel correlations. PVT-1, a long non-coding RNA associated with cancer, was strongly correlated to capsules formed after cancer removal. This suggests cancerous transformations of cell types that remain after the tumor is removed. Furthermore, transgelin and caspase 7 correlated to myofibroblasts density, suggesting an abnormal fibroblasts that are resistant to cell death and may have enhanced contractile abilities. Capsule formation is a complex process however, with well controlled clinical models quantitative differences can be found. These results serve as stepping stone for the field to move beyond retrospective clinical trials and pursue treatments and preventative measures. / Ph. D. / As the survival rate increases, the importance of quality of life post-cancer is increasing. This, in conjunction with genetic screening, has increase the number of breast reconstructions 36%. The most common complication causing revision of reconstructive surgery is the formation of a dense scar capsule around the silicone implant called capsular contracture. Nearly all patients will experience this complication, though with different degrees of response, ranging from moderate scarring to major disfigurement and pain at the implant site. Presently, there is no way to predict the degree of contraction capsule formation that individual patients will suffer prospectively, nor is there clinical approach to preventing this complication. Patient information and tissue was collected in a uniform manner to address these lingering problems. Clinical data was used to construct a predictive model which can accurately predict capsular contracture severity in breast reconstruction patients. Histological analysis demonstrated differences in structure and cell composition between different capsule severities. Of particular note, a new region was described which could serve as the communication interface between innate immune cells and fibroblasts. RNA-seq analysis identified 1029 significantly dysregulated genes in severe capsules. Pathway enrichment was then performed which highlights IL4/13 signaling, extracellular matrix organization, antigen presentation, and interferon signaling as importantly dysregulated pathways. These RNA results were also compared to various clinical and histological measurements to evaluate novel correlations. PVT-1, a long non-coding RNA associated with cancer, was strongly correlated to capsules formed after cancer removal. This suggests cancerous transformations of cell types that remain after the tumor is removed. Furthermore, transgelin and caspase 7 correlated to myofibroblasts density, suggesting an abnormal fibroblasts that are resistant to cell death and may have enhanced contractile abilities. Capsule formation is a complex process however, with well controlled clinical models quantitative differences can be found. These results serve as stepping stone for the field to move beyond retrospective clinical trials and pursue treatments and preventative measures.

Bacterial persistence is considered as one of the primary reason for antibiotic tolerance besides genetically acquired antibiotic resistance. Persisters are the subpopulation of a clonal bacterial population, which can survive environmental extremes and become invulnerable to stresses due to limited metabolic activities and physiological functions. Cognate toxin and antitoxin (TA) pairs, which are transcribed simultaneously from the same or different operons within the bacterial chromosomes or plasmids, play an important role for bacterial survival during stressful growth environments. Pseudomonas aeruginosa PAO1 is one of the most versatile microorganisms in the environment. Despite its ubiquitous presence, no studies have shown the differential expression pattern of its toxin-antitoxins, and persistence related genes. The purpose of the following study is to analyze differential expression of P. aeruginosa PAO1 type II toxin-antitoxins and persistence related genes under different growth conditions and to show how their stoichiometric ratio changes during different growth conditions. Differential expression analysis indicated that the toxins and antitoxin pairs behave differently under different growth conditions. In addition, the genes related to persistence presented relatively consistent differential expression pattern under different growth environment. / Master of Science / Bacterial persistence is one of the main reason for antibiotic tolerance and recurrent infections. Toxin-antitoxin molecules play an important role during bacterial persistence. Change in the expression of toxin, antitoxins, and persistence related genes and the ratio of the toxin to antitoxin mRNA molecules are important for bacterial survival in stressful environments. Pseudomonas aeruginosa PAO1 is one of most ubiquitous bacteria and responsible for recurrent infection in patients with weaker and compromised immunity. This mRNA sequence (RNA-Seq) analysis study of P. aeruginosa PAO1 showed different expression levels of toxin, antitoxin, and persistence related genes in various stressful growth conditions. This expression also showed the different ratios of the toxin to antitoxin mRNA molecules under different stress conditions. These implicate the different hypothetical roles of these toxin and antitoxin molecules in different growth conditions.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13609号 / 論医博第2319号 / 新制||医||1073(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 村川 泰裕, 教授 竹内 理, 教授 永井 純正 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

This study explores the use of coding-region, forensically-relevant single nucleotide polymorphisms (SNPs) from RNA sequencing data. SNPs present distinct advantages over short tandem repeat (STR) typing, particularly in niche scenarios, such as in samples with low-quantity DNA templates or in degraded samples with substantially fragmented DNA. While RNA is susceptible to rapid ex-vivo degradation, mRNA has demonstrated unexpected stability in dried body fluid stains, contingent upon the storage conditions. This paper presents a pipeline designed to identify forensically relevant coding region single nucleotide polymorphisms (cSNPs) from RNA-seq data. The forensically relevant cSNPs utilized in this study were sourced from a previously published paper that identified a panel of 35 body fluid-specific cSNPs. Our pipeline demonstrated effectiveness in identifying forensically relevant cSNPs across various tissue categories. However, the final analysis raises concerns about the overall specificity of this panel of cSNPs and issues with cross-reactivity for different body fluids. Overall, this study contributes to the advancement of forensic genetics by providing a robust and standardized pipeline for identifying cSNPs from RNA-seq data. While further evaluation and optimization are necessary, the demonstrated efficacy of this pipeline holds promise for enhancing genetic profiling in forensic contexts.

Zebrafish thrombocytes exhibit characteristics of human platelets and megakaryocytes, making them valuable for studying megakaryopoiesis and thrombopoiesis. Using single-cell RNA sequencing, we analyzed gene expression in young and mature zebrafish thrombocytes. We identified 394 protein-coding genes unique to young thrombocytes, many corresponding with human orthologs, suggesting shared regulatory mechanisms in zebrafish and humans. We hypothesized knocking down these 394 genes should identify the novel regulatory genes that control thrombocyte maturation. To address this, we used the piggyback knockdown method to knock down these genes to study their biological functions in zebrafish thrombopoiesis. We first found the knockdown of nfe2, nfe2l1a, and nfe2l3 reduced both young and mature thrombocyte counts, confirming their role in thrombopoiesis. A comprehensive knockdown screening of the uniquely expressed genes in young thrombocytes identified 7 candidate genes associated with thrombopoiesis. We selected the spi1b gene for further mutant characterization, which revealed its critical role in young thrombocyte development, with homozygous mutations leading to embryonic lethality. Considering megakaryocyte properties in thrombocytes, we studied the potential for polyploidization in zebrafish thrombocytes. The inhibition of AURKA led to the development of polyploid thrombocytes resembling mammalian megakaryocytes, suggesting the retention of genetic programs for megakaryocyte development in zebrafish thrombocytes and providing insights into the evolutionary basis of thrombopoiesis. Thus, our study reveals critical gene expression patterns and regulatory factors in zebrafish thrombocyte development, offering insights into conserved mechanisms relevant to developmental biology and research in thrombosis and hemostasis disorder.

A simplistic understanding of the central dogma falls short in correlating the number of genes in the genome to the number of proteins in the proteome. Post-transcriptional regulation, including alternative splicing and alternative polyadenylation contribute to the complexity of the proteome and is critical in understanding gene expression. In this dissertation, we aim to provide genome-wide detection and visualization of the transcript variants and quantify their significance in gene regulation. First, we propose a robust computational program to identify alternative splicing events from RNA-seq data, called AS-Quant. Our extensive experiments on simulated and real datasets demonstrate that AS-Quant can accurately quantify the splicing events among different biological conditions, as well as outperforming the other widely used baselines. The mammalian target of rapamycin (mTOR) pathway is crucial in energy metabolism and cell proliferation. We further interrogated the mTOR-activated transcriptome and found that hyperactivation of mTOR promotes transcriptome-wide exon skipping/exclusion, producing short isoform transcripts from genes. Among the RNA processing factors differentially regulated by mTOR signaling, we found that SRSF3 mechanistically facilitates exon skipping in the mTOR-activated transcriptome. This analysis reveals the role of mTOR in AS regulation and demonstrates that widespread AS is a multifaceted modulator of the mTOR-regulated functional proteome. Alternative Polyadenylation (APA) can occur either in the coding region or 3'-untranslated region (3'-UTR) of a transcript. 3'-UTR often serves as a binding platform for microRNAs and RNA-binding proteins. APA events in the 3'-UTR produce transcripts with shorter 3'-UTR, therefore provides a means to regulate gene expression at the post-transcriptional level and is known to promote translation. Current bioinformatics pipelines have limited capability in profiling 3'-UTR APA events due to incomplete annotations and a low-resolution analyzing power: widely available bioinformatics pipelines do not reference actionable polyadenylation (cleavage) sites but simulate 3'-UTR APA only using RNA-seq read coverage, causing false positive identifications. To overcome these limitations, we developed APA-Scan, a robust program that identifies 3'-UTR APA events and visualizes the RNA-seq short-read coverage with gene annotations. Additionally, we target to capture the novel APA events within the coding region boundary, specifically which occur in the introns of a transcript, referred to as Intronic PolyAdenylation (IPA). IPA is a key mechanism that can significantly alter a transcript's coding potential by truncating its translation region, thereby enhancing transcriptome and proteome diversity. This truncation can produce novel protein isoforms from the same gene with altered peptide sequences, which are linked to disease development, including cancer. To detect and quantify the de-novo IPA events, we developed a comprehensive computational pipeline for the precise identification and assessment of unannotated IPA events, named IPScan. IPScan has been benchmarked against other methods using simulated samples, data from various human and mouse cell lines, and TCGA breast cancer patient's data. Therefore, this dissertation aims to provide a comprehensive analysis to the researchers through extensive methodologies and experimental observations on the transcript variants and their functionalities.

Angiogênese é o processo de formação de novos vasos sanguíneos a partir dos vasos existentes. É um processo vital, mas muitas doenças também dependem deste mecanismo para obter nutrientes e progredir. Estas \"doenças dependentes de angiogênese\" incluem cânceres, retinopatias e degeneração macular. Alguns inibidores da angiogênese foram desenvolvidos na última década, com o objetivo de auxiliar no manejo dessas doenças e melhorar a qualidade de vida dos pacientes. A maioria destes compostos funciona inibindo a ligação de VEGFA/VEGFR2, que também é um elemento importante para a sobrevivência de células endoteliais quiescentes; e isso pode explicar parcialmente eventos adversos observados em alguns ensaios clínicos. Nossa hipótese é que a melhoria das terapias anti-angiogênicas depende de uma compreensão melhor e mais ampla desse processo, especialmente quando relacionada à progressão das doenças. Utilizando RNA-Seq e um modelo animal bem aceito de angiogênese, o modelo murino de Retinopatia Induzida por Oxigênio, exploramos o transcritoma e identificamos 153 genes diferencialmente expressos durante a angiogênese. Uma extensiva validação de vários genes realizada por qRT-PCR e hibridização in-situ confirmou a superexpressão de Esm1 em células endoteliais de tecidos com angiogênese ativa. A análise de enriquecimento desta lista de genes confirmou a ligação da angiogênese com genes frequentemente mutados em tumores, consistente com a conhecida ligação entre câncer e angiogênese, e forneceu sugestões de fármacos já aprovados que podem ser reutilizados para controlar a angiogênese em circunstâncias patológicas. Finalmente, com base neste panorama amplo da angiogênese, fomos capazes de criar um biomarcador molecular com poder prognóstico para a predição da recidiva de câncer de mama, com aplicações clínicas promissoras. Em resumo, este trabalho revelou com sucesso genes relacionados à angiogênese e forneceu novas alternativas terapêuticas, incluindo potenciais fármacos para reposicionamento. Esse conjunto de genes diferencialmente expressos é também um recurso valioso para investigações futuras. / Angiogenesis is the process of formation of new blood vessels based on existing vessels. It is a vital process but many diseases also rely on this mechanism to get nourishment and progress. These so called angiogenesis-dependent diseases include cancers, retinopathies and macular degeneration. Some angiogenesis inhibitors were developed in the past decade, aiming to help the management of such diseases and improve patients quality of life. Most of these compounds work by inhibiting VEGFA/VEGFR2 binding, which is also a key element to the survival of quiescent endothelial cells; this may partly explain unanticipated adverse events observed in some clinical trials. We hypothesize that the improvement of anti-angiogenesis therapies hinges on a better and broader understanding of the process, especially when related to diseases\' progression. Using RNA-seq and a well accepted animal model of angiogenesis, the murine model of Oxygen Induced Retinopathy, we have explored the transcriptome landscape and identified 153 genes differentially expressed in angiogenesis. An extensive validation of several genes carried out by qRT-PCR and in-situ hybridization confirmed Esm1 overexpression in endothelial cells of tissues with active angiogenesis, providing confidence on the results obtained. Enrichment analysis of this gene list endorsed a narrow link of angiogenesis and frequently mutated genes in tumours, consistent with the known connection between cancer and angiogenesis, and provided suggestions of already approved drugs that may be repurposed to control angiogenesis under pathological circumstances. Finally, based on this comprehensive landscape of angiogenesis, we were able to create a prognostic molecular biomarker for prediction of breast cancer relapse, with promising clinical applications. In summary, this work successfully unveiled angiogenesis-related genes, providing novel therapeutic alternatives, including potential drugs for repositioning. The set of differentially expressed genes is also a valuable resource for further investigations.

Deux phénomènes liés au TFIIH ont été étudiés : l'influence des mutations spécifiques dans la sous-unité XPD de TFIIH sur la réponse transcriptionnelle de certains gènes après l'irradiation UV, et l'interaction entre le TFIIH et la transcription des gènes de classe III. Une analyse détaillée de la dynamique du transcriptome a été effectuée pour la réponse des cellules humaines mutantes XP-D/CS à l'UV. Il a été démontré que la dysrégulation sélective observée de l’expression des gènes était liée à l'incapacité pour la ré-initiation transcriptionnelle et à l'hétérochromatinisation suivante, où l'histonedésacétylase SIRT1 a été identifiée comme le principal facteur. Son inhibition a permis de recouvrer l'expression normale d'un nombre substantiel des gènes affectés. Une étude de la participation pangénomique du coeur de TFIIH dans latranscription a découvert son association avec les gènes actifs de classe III. Cette association a été démontrée être indépendante de Pol II. Le coeur de TFIIH a été montré participer directement à la transcription effectuée in vitro par Pol III. / In this work, two TFIIH-related phenomena were investigated : the influence of specific mutations in TFIIH XPD subunits on the transcriptional response of different genes on UV irradiation and the interaction between TFIIH and transcription of class III genes. For the first time the detailed investigation of transcriptome dynamics was carried out for the response of XP-D/CS mutant human cells to UV-irradiation. The transcription regulation nature of the observed selective gene expression dysregulation was clearly observed. Its relation to failure of transcription re-initiation and consequentheterochromatisation was demonstrated. SIRT1 histone deacetylase was identified as the main driver of the repressive chromatin establishment on the certain genes upon UV. Inhibition of SIRT1 was found to recover normal expression of substantial number of affected genes. SIRT1 mediated mechanism was shown to be XP-D/CS specific. A potential link between this longevity related protein and progeria features of XP-D/CS mutants was hypothesised. Genome-wide study of the involvement of the core TFIIH in transcription revealed its association with active class III genes, not described previously. This association was demonstrated to be Pol II-independent. The core TFIIH was shown to be directly involved in Pol III mediated transcription in vitro.

Le nombre de gènes codant pour des protéines chez l’'homme, le vers rond et la mouche des fruits est du même ordre de grandeur. Cette absence de correspondance entre le nombre de gènes d’un eucaryote et sa complexité phénotypique s’explique en partie par le caractère alternatif de l’épissage.L'épissage alternatif augmente considérablement le répertoire fonctionnel de protéines codées par un nombre limité de gènes. Ce mécanisme, très actif lors du développement embryonnaire, participe au devenir cellulaire. De nombreux troubles génétiques, hérités ou acquis (en particulier certains cancers), se caractérisent par une altération de son fonctionnement.Les technologies de séquençage à haut débit de l'ARN donnent accès a une information plus riche sur le mécanisme de l’épissage. Cependant, si la lecture à haut débit des séquences d’ARN est plus rapide et moins coûteuse, les données qui en sont issues sont complexes et nécessitent le développement d’outils algorithmiques pour leur interprétation. En particulier, la reconstruction des transcrits alternatifs requiert une étape de déconvolution non triviale.Dans ce contexte, cette thèse participe à l'étude des événements d'épissage et des transcrits alternatifs sur des données de séquençage à haut débit de l'ARN.Nous proposons de nouvelles méthodes pour reconstruire et quantifier les transcrits alternatifs de façon plus efficace et précise. Nos contributions méthodologiques impliquent des techniques de régression parcimonieuse, basées sur l'optimisation convexe et sur des algorithmes de flots. Nous étudions également une procédure pour détecter des anomalies d'épissage dans un contexte de diagnostic clinique. Nous suggérons un protocole expérimental facilement opérant et développons de nouveaux modèles statistiques et algorithmes pour quantifier des événements d’épissage et mesurer leur degré d'anormalité chez le patient. / The number of protein-coding genes in a human, a nematodeand a fruit fly are roughly equal.The paradoxical miscorrelation between the number of genesin an organism's genome and its phenotypic complexityfinds an explanation in the alternative natureof splicing in higher organisms.Alternative splicing largely increases the functionaldiversity of proteins encoded by a limitednumber of genes.It is known to be involved incell fate decisionand embryonic development,but also appears to be dysregulatedin inherited and acquired human genetic disorders,in particular in cancers.High-throughput RNA sequencing technologiesallow us to measure and question splicingat an unprecedented resolution.However, while the cost of sequencing RNA decreasesand throughput increases,many computational challenges arise from the discrete and local nature of the data.In particular, the task of inferring alternative transcripts requires a non-trivial deconvolution procedure.In this thesis, we contribute to deciphering alternative transcript expressions andalternative splicing events fromhigh-throughput RNA sequencing data.We propose new methods to accurately and efficientlydetect and quantify alternative transcripts.Our methodological contributionslargely rely on sparse regression techniquesand takes advantage ofnetwork flow optimization techniques.Besides, we investigate means to query splicing abnormalitiesfor clinical diagnosis purposes.We suggest an experimental protocolthat can be easily implemented in routine clinical practice,and present new statistical models and algorithmsto quantify splicing events and measure how abnormal these eventsmight be in patient data compared to wild-type situations.

Angiogênese é o processo de formação de novos vasos sanguíneos a partir dos vasos existentes. É um processo vital, mas muitas doenças também dependem deste mecanismo para obter nutrientes e progredir. Estas \"doenças dependentes de angiogênese\" incluem cânceres, retinopatias e degeneração macular. Alguns inibidores da angiogênese foram desenvolvidos na última década, com o objetivo de auxiliar no manejo dessas doenças e melhorar a qualidade de vida dos pacientes. A maioria destes compostos funciona inibindo a ligação de VEGFA/VEGFR2, que também é um elemento importante para a sobrevivência de células endoteliais quiescentes; e isso pode explicar parcialmente eventos adversos observados em alguns ensaios clínicos. Nossa hipótese é que a melhoria das terapias anti-angiogênicas depende de uma compreensão melhor e mais ampla desse processo, especialmente quando relacionada à progressão das doenças. Utilizando RNA-Seq e um modelo animal bem aceito de angiogênese, o modelo murino de Retinopatia Induzida por Oxigênio, exploramos o transcritoma e identificamos 153 genes diferencialmente expressos durante a angiogênese. Uma extensiva validação de vários genes realizada por qRT-PCR e hibridização in-situ confirmou a superexpressão de Esm1 em células endoteliais de tecidos com angiogênese ativa. A análise de enriquecimento desta lista de genes confirmou a ligação da angiogênese com genes frequentemente mutados em tumores, consistente com a conhecida ligação entre câncer e angiogênese, e forneceu sugestões de fármacos já aprovados que podem ser reutilizados para controlar a angiogênese em circunstâncias patológicas. Finalmente, com base neste panorama amplo da angiogênese, fomos capazes de criar um biomarcador molecular com poder prognóstico para a predição da recidiva de câncer de mama, com aplicações clínicas promissoras. Em resumo, este trabalho revelou com sucesso genes relacionados à angiogênese e forneceu novas alternativas terapêuticas, incluindo potenciais fármacos para reposicionamento. Esse conjunto de genes diferencialmente expressos é também um recurso valioso para investigações futuras. / Angiogenesis is the process of formation of new blood vessels based on existing vessels. It is a vital process but many diseases also rely on this mechanism to get nourishment and progress. These so called angiogenesis-dependent diseases include cancers, retinopathies and macular degeneration. Some angiogenesis inhibitors were developed in the past decade, aiming to help the management of such diseases and improve patients quality of life. Most of these compounds work by inhibiting VEGFA/VEGFR2 binding, which is also a key element to the survival of quiescent endothelial cells; this may partly explain unanticipated adverse events observed in some clinical trials. We hypothesize that the improvement of anti-angiogenesis therapies hinges on a better and broader understanding of the process, especially when related to diseases\' progression. Using RNA-seq and a well accepted animal model of angiogenesis, the murine model of Oxygen Induced Retinopathy, we have explored the transcriptome landscape and identified 153 genes differentially expressed in angiogenesis. An extensive validation of several genes carried out by qRT-PCR and in-situ hybridization confirmed Esm1 overexpression in endothelial cells of tissues with active angiogenesis, providing confidence on the results obtained. Enrichment analysis of this gene list endorsed a narrow link of angiogenesis and frequently mutated genes in tumours, consistent with the known connection between cancer and angiogenesis, and provided suggestions of already approved drugs that may be repurposed to control angiogenesis under pathological circumstances. Finally, based on this comprehensive landscape of angiogenesis, we were able to create a prognostic molecular biomarker for prediction of breast cancer relapse, with promising clinical applications. In summary, this work successfully unveiled angiogenesis-related genes, providing novel therapeutic alternatives, including potential drugs for repositioning. The set of differentially expressed genes is also a valuable resource for further investigations.