Дисертації з теми "Transcriptomic alterations"

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy due to its diagnosis at advanced stages, when the disease has already spread beyond the ovaries. EOC is generally sensitive to first line chemotherapy, and the vast majority of patients respond to platinum (Pt)-based therapy after debulking surgery. Unfortunately, more than 80% of Pt-responsive patients relapse with a disease that progressively becomes Pt-resistant. Based mainly on clinical evidence, the process by which disease relapses is still poorly understood. The aim is to identify biomarkers of sensitivity to chemotherapy and therapeutic targets in HGS-EOC by integrating transcriptomic data, coding and non-coding RNAs. The bioinformatic analysis was applied on microarray data and RNA-seq data, embracing different classes of patients (resistant, sensitive, partially sensitive and normal). Two complementary approaches have been adopted to identify biomarkers of therapy response in microarray data: i) a classic approach and ii) a network-based approach using micrographite. The results obtained with both procedures have then been used to reconstruct a regulatory circuit involved in therapy response. The final outcome is a regulatory cell signal pathway composed of genes and miRNAs mainly involved in the therapy response. Circuit has been validated using two external and independent cohorts by quantitative real-time PCR (qRT-PCR). However, in order to complete the characterization of network as prognostic factor we decided to consider in survival analysis defect of the Homologous Recombination (HR). Approaching in survival analysis, a signature of three genes (SDF2L1, PPP1R12A and PRKG1) found to be independent prognostic biomarkers, was able to predict, at the time of diagnosis, resistance to Pt-based chemotherapy. Also, a new approach has been evaluated in order to characterize new mechanisms of chemotherapy resistance in ovarian cancers. On microarray data, we tried to stratify patients for the immunotherapy, with recent improved understanding of the immune recognition and regulation of cancer cells. In addition, using RNA-seq data and somatic DNA mutations, we went deeper in immunogenicity of ovarian cancer trying to find new elements as therapy targets, neoantigens, not associated to this tumor till now. At last, in addition, the small amount of molecular differences between Pt-r and Pt-s patients suggested the presence of potential new transcripts involved in therapy response maybe due to aberrant splicing events. To investigate this hypothesis, we used a set of RNA-seq experiments, to identify new aberrant splicing such as circular RNAs. We reported 5 circRNAs differentially expressed between tumour resistance types, and a large number of class-specific circRNAs. In particular, circ_BARD1 showed a character as prognostic factor significative in OS and PFS, in multivariate analysis with residual tumour and age as covariates. The consistency of circular RNA expression, in conjunction with the regulatory circuit, may offer new candidates for cancer treatment and prognosis, revealing that the integration of coding and non-coding RNAs data may shed light on chemotherapy resistance mechanisms in ovarian cancer.

Orientador: Daisy Maria Fávero Salvadori
Resumo: A cardiotoxicidade induzida pela doxorrubicina (DOX), antraciclina isolada da actinobacteria Streptomyces peucetius e amplamente utilizada na terapia antineoplásica, corresponde a um dos mais importantes eventos patofisiológicos que limitam sua aplicação clínica. No entanto, não são completamente conhecidos todos os mecanismos envolvidos nessa toxicidade, o que diminui as possiblidades de intervenção e a redução dos efeitos colaterais para os pacientes sob tratamento. Uma das hipósteses é que os aldeídos gerados pela ação da DOX atuam sobre membranas mitocondriais, alterando o estado redox e formando adutos com proteínas, os quais prejudicam o correto funcionamento da organela. Atividades deletérias da DOX sobre outros componentes celulares, como, por exemplo, os ácidos ribonucleicos, são, também, possíveis mecanismos de toxicidade do antineoplásico. Várias estratégias têm sido utilizadas para minimizar os efeitos adversos da DOX. Uma delas, é a busca por compostos que possam proteger as células da ação citotóxica. Nesse sentido, a Alda-1, pertencente ao grupo das chaperonas e agonista da enzima aldeído desidrogenase mitocondrial (ALDH2), vem sendo testada com o objetivo de reduzir os efeitos adversos dos metabólitos e radicais gerados pelo antineoplásico. Para investigar outros possíveis mecanismos de ação da DOX e o efeito cardiprotetor da Alda-1, este estudo foi delineado utilizando duas abordagens distintas: experimentos in vivo, com ratos Wistar machos submetidos a trata... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The cardiotoxicity induced by doxorubicin (DOX), anthracycline isolated from the actinobacteria Streptomyces peucetius, and widely used as an antineoplastic drug, is one of the most important pathophysiological events that limit its clinical application. However, all the mechanisms involved in this toxicity are not fully understood. One hypothesis is that the aldehydes generated by DOX act on mitochondrial membranes, modifying the redox state and proting adducts with proteins. DOX activities on other cellular components, such as ribonucleic acids, are also possible mechanisms of toxicity. Several strategies have been used to reduce the DOX adverse effects. One of them is the identification of compounds that can protect cells against cytotoxic. Alda-1, which belongs to a group of chaperones and is an agonist of the mitochondrial aldehyde dehydrogenase (ALDH2), has been tested to reduce the adverse effects of metabolites and radicals generated by DOX. To investigate other possible DOX mechanisms of action, and the cardioprotective activity of Alda-1, this study was designed using two different approaches: in vivo, with male Wistar rats submitted to acute and chronic treatments; and, in vitro, in mice fibroblasts and cybrids with ND5 (gene that encodes the mitochondrial Complex I subunit) mitochondrial gene heteroplasmy. The expression profiling of genes related to beta oxidation pathways, Bax, Bcl-2, C1QBP, ALDH2 and miR34a microRNA (ALDH2 expression regulator), and the lipoper... (Complete abstract click electronic access below)
Doutor

L’objectif principal de cette thèse est d’étudier les mécanismes liés au pouvoir pathogène de V. tapetis.Pour cela, nous avons développé 2 axes de recherche. Le premier axe vise à étudier la virulence de V. tapetis en répondant aux 2 problématiques suivantes : Quels sont les gènes impliqués dans la virulence de V. tapetis ? et Existe-t-il des marqueurs hôtes-spécifiques de la virulence de V. tapetis ? Le second axe de recherche concerne l’interaction hôte pathogène et répond aux 2 problématiques suivantes : Quels sont les gènes exprimés lors de l’infection chez l’hôte ? et Quelles sont les modulations au sein de l’animal associées au pH et à la température lors de l’infection ?Les principales découvertes de cette thèse sont : (i) La bactérie V. tapetis, dans le cadre de la MAB, induit une sous expression des gènes impliqués dans la réponseimmunitaire et une dérégulation des gènes impliqués dans la stabilisation et la synthèse des filaments d’actine (ii) Ce pathogène induit également une diminution de l’activité lysosomale sur les hémocytes exposés (iii) L’effet de V. tapetis sur le cytosquelette d’actine et sur la diminution de l’activité lysosomale est indépendante du système de sécrétion de type IV (T4SS) (iv) Le système de sécrétion de type IV (T4SS) est impliqué dans le développement de la MAB mais n’est pas essentiel pour induire cette affection(v) Dans le cadre de la MAB et de la perte des adhérences des hémocytes in vitro, V. tapetis est capable de moduler le pH des fluides extra-palléaux, respectivement dans les premiers jours et premières heures de l’infection (vi) Enfin, l’approche de « strains typing » basée sur la technique MALDI-TOF permet de discriminer les souches de V. tapetis en fonction de leur pouvoir pathogène vis à vis de la palourde japonaise
The main objective of this thesis is to study the mechanisms related to the pathogenicity of V. tapetis. For this purpose, we developed 2 research axes. The first one aimed at studying the virulence of V. tapetis by answering the following 2 issues: What are the genes involved in the virulence of V. tapetis? and Are there host-specific markers of the virulence of V. tapetis? The second research axis concerned pathogen-host interactions and addressed the following 2 issues: What are the genes expressed during infection in the host? and What are the modulations in the animal associated with pH and temperature during infection? The main findings of this thesis are: (i) V. tapetis, in the context of BRD, induces a down expression of genes involved in the immune response anda deregulation of genes involved in the stabilization and synthesis of actin filaments (ii) This pathogen also induces a decrease in lysosomal activity on exposed hemocytes (iii) The effect of V. tapetis on the actin cytoskeleton and on the decrease in lysosomal activity is independent of the type IV secretion system (T4SS) (iv) The type IV secretion system (T4SS) is involved in the development of BRD but is not essential to induce this disease (v) In the context of BRD and of the loss of hemocyte adhesions properties in vitro, V. tapetis is able to modulate the pH of extrapallial fluids, respectively in the first days and hours of infection (vi) Finally, the "strains typing" approach based on MALDITOF makes it possible to discriminate between V. tapetis strains according to their pathogenicity with regard to Manila clam

Doutoramento em Biologia
The genetic code is not universal. Alterations to its standard form have been discovered in both prokaryotes and eukaryotes and demolished the dogma of an immutable code. For instance, several Candida species translate the standard leucine CUG codon as serine. In the case of the human pathogen Candida albicans, a serine tRNA (tRNACAGSer) incorporates in vivo 97% of serine and 3% of leucine in proteins at CUG sites. Such ambiguity is flexible and the level of leucine incorporation increases significantly in response to environmental stress. To elucidate the function of such ambiguity and clarify whether the identity of the CUG codon could be reverted from serine back to leucine, we have developed a forced evolution strategy to increase leucine incorporation at CUGs and a fluorescent reporter system to monitor such incorporation in vivo. Leucine misincorporation increased from 3% up to nearly 100%, reverting CUG identity from serine back to leucine. Growth assays showed that increasing leucine incorporation produced impressive arrays of phenotypes of high adaptive potential. In particular, strains with high levels of leucine misincorporation exhibited novel phenotypes and high level of tolerance to antifungals. Whole genome re-sequencing revealed that increasing levels of leucine incorporation were associated with accumulation of single nucleotide polymorphisms (SNPs) and loss of heterozygozity (LOH) in the higher misincorporating strains. SNPs accumulated preferentially in genes involved in cell adhesion, filamentous growth and biofilm formation, indicating that C. albicans uses its natural CUG ambiguity to increase genetic diversity in pathogenesis and drug resistance related processes. The overall data provided evidence for unantecipated flexibility of the C. albicans genetic code and highlighted new roles of codon ambiguity on the evolution of genetic and phenotypic diversity.
O código genético não é universal. Alterações à identidade de vários codões descobertas em procariotas e eucariotas invalidam a hipótese dum código genético universal e imutável. Por exemplo, várias espécies do género Candida traduzem o codão CUG de leucina como serina. Em Candida albicans, um único tRNA de serina (tRNACAGSer) incorpora in vivo 97% de serina e 3% de leucina nas proteínas em resposta a codões CUG presentes nos mRNAs deste fungo patogénico. Esta ambiguidade é flexível e a incorporação de leucina aumenta em condições de stress. De forma a elucidar a função desta ambiguidade e determinar se a identidade dos codões CUG podia ser revertida de serina para leucina, desenvolvemos uma estratégia de evolução forçada e uma proteína recombinante fluorescente cuja actividade depende da incorporação de leucina num codão CUG. Construímos estirpes que incorporam leucina nas proteínas em resposta a codões CUGs em níveis que variam entre 0,64% e 98,46%. Esta reversão de uma alteração ao código genético demostrou de modo inequívoco que o código é flexível e pode evoluir. Testes de crescimento em diferentes meios de cultivo revelaram uma série impressionante de fenótipos com elevado potencial adaptativo nas estirpes mais ambíguas, nomeadamente tolerância a antifúngicos. A sequenciação dos genomas das estirpes que construímos revelou que a ambiguidade do codão CUG resulta na acumulação de polimorfismos de nucleótido únicos (SNP) no genoma. Verificámos também perda de heterozigozidade (LOH) nos cromossomas 5 e R das estirpes que incorporam 80,84% e 98,46% de leucina em locais proteicos codificados por codões CUG. Os SNPs acumularam-se preferencialmente em genes envolvidos na adesão celular, no crescimento filamentoso e na formação de biofilmes, sugerindo que C. albicans utiliza a sua ambiguidade natural para aumentar a diversidade genética dos processos relacionados com a patogénese e resistência a drogas. Estes resultados evidenciam uma notável flexibilidade do código genético de C. albicans e revelam funções inesperadas da ambiguidade do código genético na evolução da diversidade genética e fenotípica.

Le cancer du pancréas est un problème majeur de santé publique. Le mauvais pronostic de l'adénocarcinome du pancréas est lié à un diagnostic tardif, une progression rapide, et à une mauvaise réponse aux traitements médicaux disponibles. Une caractérisation complète des altérations génétiques moléculaires sont aujourd'hui nécessaires pour permettre une détection plus précoce et identifier/élaborer de nouvelles thérapies ciblées dans le traitement de l'ADK du pancréas. En utilisant la technique d'hybridation génomique comparative, nous avons étudié les altérations du génome de 39 ADK. Plusieurs pertes récurrentes ont été observées, ainsi que des gains de matériel génétique. A partir de ces résultats, nous avons voulu aller plus loin en identifiant les gènes qui pourraient avoir des conséquences au niveau transcriptomique. A partir de données publiques, nous avons comparé les profils d'expression d'ADK (n=419) et de pancréas normal (n=105). Parmi les gènes trouvés amplifiés et/ou gagnés par aCGH, 170 (48%) étaient surexprimés dans les ADK par rapport au tissus normal. Les principales voies de signalisation impliquées touchaient la régulation du cycle cellulaire, les voies TP53 et TGFß. Parmi les gènes délétés en aCGH, 141 (41%) étaient sous exprimés dans les ADK du pancréas par rapport au tissus normal et étaient essentiellement liés au « métabolome » de la cellule pancréatique. Enfin, nous avons identifié une dizaine de gènes dont l'expression pourrait être liée à la survie des patients. Certains de ces gènes pourraient être des candidats à tester en tant que biomarqueurs pronostic ou cibles pour le développement de nouvelles thérapeutiques
Pancreatic adenocarcinoma (PDCA) is a major public health problem in France and worldwide. The inoperability and the poor prognosis of the PDCA are due to late diagnosis, rapid tumor progression (>80% of patients displayed metastases at diagnosis), early recurrences after resection, and poor response to available therapies. Innovative approaches and a comprehensive characterization of molecular genetic alterations are dearly needed to help develop techniques of early detection, identify new molecular targets and devise novel targeted-therapies (Hidalgo, 2010). Using high-resolution array-comparative genomic hybridization (aCGH), we studied the genome alterations of 39 fine-needle aspirations from PDCA. Recurrent losses were observed and comprised several known tumor suppressor genes. We identified frequent genetic gains. With this study, we decided to go one step further by identifying genes that might also be deregulated at the transcriptomic level. We started our analysis with a population of PDCA (n=419) versus normal pancreas (n = 105). Among the 352 genes found amplified and/or gained by aCGH, 170 (48%) were up regulated at the transcriptional level in PDCA compared to normal pancreatic tissues. Major pathways involved were cell cycle, TP53 and TGFß. Among the genes located in regions of losses, 141 (41%) were down regulated in PDCA compared to normal tissues. Furthermore, some genes were found related to a patients' survival With this study, we highlighted novels genes associated to PDCA oncogenesis. Some of those candidates should be further investigated as prognosis markers or as potential targets for new therapeutic approaches

Colorectal cancer (CRC) is one of the most frequent malignancies affecting western societies. Currently, the gold standard of CRC treatment is cetuximab, a monoclonal antibody, alone or in combination with chemotherapy. Not all patients positively respond to cetuximab: the analysis of KRAS mutational status at tumor site, a highly invasive analysis, is the only universally accepted genetic predictor for patient s response. However, some KRAS wild type patients, potential good responders, don t benefit from this therapy. To overcome these obstacles, research is focusing on the identification of new biomarkers detectable in circulating blood or other body fluids that can be used for diagnosis as well as for predicting the response to certain therapies. miRNAs, small RNA molecules involved in all aspects of cellular metabolism through regulation of gene expression, have been identified as new biomarkers for many diseases and cancers, including CRC. On the other hand, the scientific research is investigating on new molecules providing high specificity for the key players of the main cellular pathway affected in cancer. The main pathway involved in CRC is MAPK/ERK signaling pathway, which members are good targets for designing new specific inhibitors that could help to overcome the problems related to non-responsive patients to EGFR-targeted therapy. This thesis is focused on the relationship between the response to certain drugs and miRNA transcriptome changes in CRC human cellular models, based on KRAS mutational status. We profiled the expression of 667 miRNAs in 2 human CRC cell lines (Caco-2, KRAS wild type, and HCT-116, KRAS mutated), and 745 miRNAs in 3 CRC cell lines (Caco-2, HCT-116 and SW-620, another KRAS mutated cell line) after treatment with cetuximab and three specific inhibitors of MAPK pathway, respectively. Our aim was the identification of typical miRNA transcription profiles associated to cetuximab response, as well as the investigation on the global involvement of miRNAs within MAPK/ERK pathway. In the first analysis we identified substantially unique subsets of differentially expressed miRNAs in the sensitive cell line compared to the resistant one. Global network functional analysis on their targets suggested a role of these miRNAs in cancer related processes and identified hubs involved in EGFR internalization. In the second analysis we identified six differentially expressed miRNAs, that we have demonstrated to be involved in cell proliferation, migration, apoptosis, and to globally affect the regulation circuits centered on MAPK/ERK signaling. We evaluated the expression of the main candidate miRNAs identified in both studies in biopsies from CRC patients, previously categorized for their KRAS status: two miRNAs from the first study (miR-146b-3p and miR-486-5p) and four from the second (miR-92a-1*, miR-135b*, miR-372, miR-720) resulted highly expressed in biopsies from CRC patients than in normal controls. Moreover, the last four miRNAs are also overexpressed in CRC patients with mutated KRAS than in wild-type genotypes. The identification of miRNAs, which expression is linked to the efficacy of therapy, should help to predict the patients response to treatment and possibly lead to a better understanding of the molecular mechanisms of drug response. Our results contribute to deepen current knowledge on some features MAPK/ERK pathway, pinpointing new oncomiRs in CRC and allowing their translation into clinical practice and CRC therapy. Data shown in this thesis were published in 2010 and 2012 (Ragusa M, Majorana A, Statello L, et al. Specific alterations of microRNA transcriptome and global network structure in colorectal carcinoma after cetuximab treatment. Mol Cancer Ther. 2010 Dec; 9:3396-409; Ragusa M, Statello L, Maugeri M, et al. Specific alterations of the microRNA transcriptome and global network structure in colorectal cancer after treatment with MAPK/ERK inhibitors. J Mol Med (Berl). 2012 Jun 4).

Bien qu'une diminution conséquente de la pollution atmosphérique soit constatée depuis les années 1990, cette dernière demeure un problème de santé publique majeur, à l'origine de plus de 4,2 millions de décès prématurés par an dans le monde. À l'heure actuelle, l'attention des experts se concentre sur les particules ultrafines (PM0,1 ou PUF) en raison de leur capacité à transloquer dans la circulation systémique pour atteindre les organes périphériques où elles seront alors susceptibles d'avoir un impact néfaste. Néanmoins, les connaissances en termes de mécanismes cellulaires et moléculaires impliqués dans la toxicité de ces particules restent encore très parcellaires et demeurent, le plus souvent, centrées sur leur cible principale qu'est le poumon. Ainsi, ce projet de thèse avait pour objectifs principaux d'apporter des éléments novateurs sur la toxicocinétique (i.e., distribution/persistance) et la toxicodynamique (i.e., mécanismes physiopathologiques, voies de signalisation associées) de PUF prélevées en milieu urbain, d'une part, et les effets organo-spécifiques des PUF et l'utilisation des miARN circulants comme indicateurs d'exposition chronique et/ou cumulées aux PUF dans un modèle murin, d'autre part. Afin de répondre à ces interrogations, des souris Balb/cJRj ont été exposées durant 3 mois à différentes doses de PUF prélevées dans la zone urbaine de Lille, puis des analyses ont été réalisés au sein de différents organes-cibles richement vascularisés, et par conséquent directement exposés aux PUF lors de leur phase de translocation et de distribution systémique. Les résultats obtenus ont démontré que, dans l'ensemble des organes cibles, le potentiel oxydant intrinsèque des PUF induisait indéniablement la production d'espèces pro-oxydantes et l'activation de défenses antioxydantes en quantité suffisante pour rétablir un état d'homéostasie redox mais ne parvenant pas, cependant, à éviter l'apparition d'une réponse inflammatoire au niveau pulmonaire, cardiaque et cérébral. Des approches transcriptomiques réalisés au sein des poumons, organes cibles présentant les effets délétères les plus marqués, ont suggéré la dérégulation de nombreuses voies de signalisation en relation avec les réponses oxydante et inflammatoire, qui constituent les mécanismes centraux de toxicité des PUF mais aussi avec des mécanismes de toxicité plus originaux tels que la dysfonction mitochondriale, la transition épithélio-mésenchymateuse et le remodelage tissulaire, dont la modulation a également été validée d'un point de vue fonctionnel. Ces données prometteuses pourraient à terme contribuer à une meilleure prise de décision quant à la réduction des émissions des PUF de même qu'à la réactualisation des normes réglementaires actuellement en vigueur
Although there has been a significant reduction in air pollution since the 1990s, it remains a major public health problem, responsible for over 4.2 million premature deaths worldwide every year. At present, experts' attention is focused on ultrafine particles (PM0.1 or UFP) because of their ability to translocate into the systemic circulation and reach peripheral organs, where they are likely to have a harmful impact. Nevertheless, the knowledge of the cellular and molecular mechanisms involved in the toxicity of these particles is still very patchy, and most often remains focused on their main target, the lung. Thus, the main objectives of this thesis project were to provide innovative insights into the toxicokinetics (i.e., distribution/persistence) and toxicodynamics (i.e., pathophysiological mechanisms, associated cell signaling pathways) of UFP collected in urban environments, on the one hand, and the organospecific effects of UFP and the use of circulating miRNA as indicators of chronic and/or cumulative exposure to UFP in a mouse model, on the other hand. To answer these questions, Balb/cJRj mice were exposed for 3 months to various doses of UFP collected in the urban area of Lille, then analyzed in various target organs richly vascularized, and therefore directly exposed to UFP during their translocation and systemic distribution phase. The results showed that, in all target organs, the intrinsic oxidative potential of UFP undeniably induced the production of oxidative oxygen species and the activation of antioxidant defenses in sufficient quantities to restore a state of redox homeostasis, but were unable to prevent the onset of an inflammatory response in the lungs, heart and brain. Transcriptomic approaches carried out in the lungs, the target organ with the most marked deleterious effects, have suggested the deregulation of numerous signaling pathways in relation to oxidative and inflammatory responses, which constitute the central mechanisms of UFP toxicity, but also with more original toxicity mechanisms such as mitochondrial dysfunction, epithelial-mesenchymal transition and tissue remodeling, whose modulation has also been validated from a functional point of view. These promising data could ultimately contribute to better decision-making on the reduction of UFP emissions, as well as to the updating of current regulatory standards

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality worldwide and is a risk factor for lung cancer development. COPD encompasses both emphysema and chronic bronchitis, the pathogenesis of which are unclear. In this dissertation, I leveraged genome-wide gene-expression studies of emphysema and lung cancer to investigate pathogenesis and carcinogenesis in COPD. Tobacco smoke is the primary cause of emphysema. The most severe form is also associated with alpha1-antitrypsin deficiency (AATD) resulting from a mutation. In this study, I leveraged multiple lung samples from patients with emphysema, with or without AATD. While genes involved in tissue repair decreased with emphysema severity, the unfolded protein response (UPR) was uniquely changed in AATD lungs. AATD may play multiple roles in emphysema and UPR activation suggests AAT replacement therapy may be insufficient to treat this form of emphysema. Emphysema is a progressive disease, and the mean linear intercept (Lm) can serve as a surrogate of progression. I evaluated whether Lm increases in non-diseased lungs may represent similar processes to those occurring in emphysema, and could offer insight into early stages of disease or homeostasis. Genes involved in tissue repair increased with Lm in controls but decreased in disease. Tissue repair processes may be active in even the non-insulted lung, suggesting their activity is necessary for lung homeostasis and their deficiency may drive emphysema progression. Finally, COPD patients are at increased lung cancer risk, and transcriptomic changes common to both diseases could explain this risk. In both COPD and lung cancer, I discovered that H3K27Me3 regulated genes are repressed, and that the methyltransferase responsible for H3K27me3, EZH2, is induced. H3K27Me3, an oncogenic histone modification, may drive carcinogenesis and pathogenesis in COPD. Though usual and AATD emphysema share transcriptomic signatures associated with tissue repair, which may be active in the normal homeostatic lung, the UPR changes in AATD emphysema only; successful therapeutic strategies in emphysema will need to account for this difference. In COPD, H3K27Me3 may play a role in both pathogenesis and carcinogenesis, making it an attractive target for therapeutic interventions, but one that would need further augmentation in AATD.
2019-11-01T00:00:00Z

Epigenetic modifications remain dynamic in most somatic cells to enable flexible gene activity. Simultaneously, this creates a cellular environment prone to “epigenetic mistakes” and this is evident in most diseases, including cancers. It is well established that cancer initiation and progression is caused by a complex series of genetic and epigenetic changes encompassing altered patterns of DNA methylation, histone modifications and the physical chromatin structure. The consequence of these collective changes is an abnormal gene expression signature that can act as a catalyst for disease. Prostate cancer is a highly heterogeneous disease and owing to this, only a few highly penetrant genomic drivers have been associated with the disease. Epigenetic studies can provide additional insight into disease onset and progression, with DNA methylation being one of the most studied epigenetic alterations in prostate cancer. The transcriptomic and DNA methylation profiles of ‘local’ and ‘metastatic’ cells are typically integrated and compared to benign or ‘normal’ states. However, the metastasis of primary tumours is the main cause of prostate cancer related deaths, and it is important to identify gene expression and epigenetic signatures that may drive this process. To address this, the overall aim of this thesis was to identify and validate divergent transcriptomic events that may explain the biology of metastasis by drawing comparisons between cells obtained from local tumours and those of metastasised cancer. A cell line model of prostate cancer that represents ‘localised’ prostate cancer (22Rv1 cell line) and two prostate cancer ‘metastasis’ cell lines (LNCaP, lymph node; PC3, bone) was utilised, in combination with whole transcriptomic data and genome-wide DNA methylation analysis to identify candidate genes and test the impact of DNA methylation inhibitors. Putative drivers of metastasis were identified in three parts, firstly by differential gene expression; secondly, by differential alternative splicing and thirdly, by differential promoter DNA methylation. Using whole transcriptomic RNA sequencing (RNA-seq), genes of interest exhibited at least two fold difference between 22Rv1 cells and either of LNCaP or PC3 cell lines, with the genes that were commonly identified in both pairwise comparisons subject to clustering and pathway analyses. This revealed a list of 629 robust candidates, including GUCY1A2 and KISS1R, which displayed higher levels of expression in LNCaP and PC3 metastatic cell lines compared to the localized 22Rv1 cancer cells. Additionally, global alternative splicing analysis revealed that QSOX1 showed intron retention concomitant with overall higher levels of gene expression in the LNCaP and PC3 metastatic cell lines compared to the localized 22Rv1 cancer cells. DNA methylation information was generated by Illumina Infinium MethylationEPIC Beadchip array. A total of 16,866 differentially methylated regions (DMRs) were identified between 22Rv1 and PC3 comprising of 218,341 total CpG dinucleotides. These DMRs were filtered based on FDR<0.25, and mean beta fold change >0.25, and to include regions with promoter methylation patterns that tightly correlated with gene expression patterns. These stringent criteria identified 147 regions of interest spanning 2,050 CpG sites, including two DMRs overlapping the PXMP4 and SLC36A4 gene promoters. Interestingly, the DNA methylation profiles of these promoters was inverse, with PXMP4 displaying DNA hypermethylation and SLC36A4 showing DNA hypomethylation in PC3 compared to 22Rv1 cells. As predicted, this DNA methylation pattern correlated with expression patterns; PXMP4 was repressed while SLC36A4 was expressed in the metastasized PC3 compared to localized 22Rv1 cancer cells. To investigate whether these genes were regulated by DNA methylation, genes of interest were tested for responsiveness to the DNA methyltransferase inhibitor, 5-aza-2’- deoxycytidine. Drug treatment caused a significant upregulation of PXMP4 in the metastatic cell lines while no effect was seen in 22Rv1 cells. No significant change in expression was noted for SLC36A4, GUCY1A2 and KISS1R. This suggests that repression of PXMP4 in metastasis could possibly be due to the modulation of DNA methylation. Overall, this study provides insights into the genome-wide transcriptomic and methylation differences identifying a number of promising candidate genes ideal for further functional investigation in the process of prostate cancer metastasis.

The human bronchial epithelium is composed of multiple, discrete cell types that cooperate to perform mucociliary clearance. While previous studies have shown that cigarette smoke can alter bronchial epithelial gene expression, the underlying effects of this exposure on specific cell types are not well understood. In this thesis, single-cell RNA sequencing was used to profile bronchial epithelial cells from six current smokers and six never smokers. Thirteen cell clusters were identified that were defined by expression of unique combinations of nineteen distinct gene sets. This clustering revealed that smoke exposure induced expression of a toxin metabolism program that specifically associated with ciliated cells. Extensive airway remodeling was also observed, in which smoking was associated with loss of club cells as well as goblet cell expansion and hyperplasia. Additionally, a previously uncharacterized CEACAM5+ KRT8+ epithelial subpopulation was identified in the airways of smokers. While it has been shown that most smoking-associated gene expression alterations can be reversed upon smoking cessation, a subset of these alterations persists in former smokers. The basal layer of the bronchial epithelium is comprised of a multipotent progenitor subpopulation. When abnormalities persist in the bronchial epithelium despite normal tissue turnover, the source of these abnormalities may be traced to this progenitor population and its program of differentiation. Therefore, basal cells were procured from three current smokers and three never smokers, differentiated in vitro, and profiled by RNA sequencing at eight time points spanning the differentiation procedure. Twenty-seven unique sets of co-expressed genes associated with differentiation were identified and functionally characterized, a subset of which were abnormally expressed in smoker cells. Robust expression of genes involved with the unfolded protein response was specifically detected in smoker basal cells. Additionally, a smoking-associated delay in the onset of expression of genes involved with ciliogenesis was observed. These data therefore indicate that smoking has long-term consequences on the differentiated state of the airway epithelium. Collectively, the observations outlined in this thesis demonstrate that smoking drives a complex landscape of alterations that affects the function and composition of the human bronchial epithelium.
2020-10-24T00:00:00Z

碩士
國立中央大學
系統生物與生物資訊研究所
100
Antrodia cinnamomea, an endemic fungus of Taiwan, is well known as a herbal medicine to remedy various illnesses, including liver cancer, a major cause of death in Asia. However, the early mechanism of its anti-cancer effect was unclear. In this study, we explored the anti-cancer mechanism induced by Antrodia cinnamomea fruiting body ethanol crude extract (AcFBE) on human hepatocellular carcinoma cell (SK-HEP-1) through in vitro assay in conjunction with miRNA and mRNA transcriptome profiling using next generation sequencing (NGS). Results indicated that 500μg /ml of AcFBE can induce apoptotic cell death on SK-HEP-1 within 2 hours, as characterized by cell viability assay, cell morphology alteration, cell membrane extroversion, caspases activation, and DNA fragmentation. The sequence data revealed 354 known miRNAs from miRNA libraries. To our surprise, 85.4% of known miRNAs in SK-KEP-1 were decreased, while only 57.3% of known miRNAs were found decreased for normal mouse liver cells (BNL CL.2). The AcFBE-affected miRNAs expression profiles showed dramatic down-regulation in SK-HEP-1 cells, with 95.2% (141/148) and 84.1% (122/145) affected after 2 hr and 4 hr AcFBE treatment, respectively, suggesting that AcFBE caused a global inhibition of miRNA level. This result was supported by western blotting showing decreases of miRNA biogenesis proteins, Dicer and Drosha, together with an increase of XRN2 involved in miRNA degradation. Transcriptome analysis showed 2077 (58.3%) and 1491 (40.1%) up-regulated genes in 2-hr and 4-hr libraries, respectively, based on 2 fold cutoff. We then analyzed 365 genes most significantly regulated by AcFBE using GO term analysis on transcriptome data, indicating a strong association with apoptosis. Overall, our study suggests that AcFBE is able to globally reduce SK-HEP-1 miRNA expression and thereafter affect gene expression regulation, causing cancer cell death by apoptosis. This is the first report implicating miRNA in the anti-cancer effects of A. cinnamomea fruiting body.

碩士
國立中興大學
生命科學系所
103
The purpose of this thesis is to systemically investigate molecular modulations when euryhaline brackish medaka, Oryzias dancena under cold stress. Fresh water (FW)- and seawater (SW)-acclimated brackish medaka were transferred from 28°C to 18°C. After cold exposure to 18°C for a week, brain, gill and liver were sampled. Next generation sequencing was applied for de novo assembly and transcriptome analyses. The assembled transcripts were further used to KEGG annotation. The numbers of genes in KEGG mapping results were mainly categorized into signal transduction group in all three organs under low temperature. In brain, no specific gene groups or signaling pathways were up-regulated under low temperature comparing with other organs. Expression of some specific genes in pathways belong to environmental information processing were activated in the gill. Up-regulations of gene groups in the liver were mainly found among the pathway of metabolic groups especially in carbohydrate metabolism. Nevertheless, gill is the only organ found with largest numbers of up-regulated gene under low temperature. After cold transfer, the following experiments were performed. In the gill of SW-acclimated brackish medaka, the mRNA abundance of anti-apoptosis factor, baculoviral inhibitor of apoptosis repeat-containing 5 (birc5) and protein level of cleaved-caspase-3 increased. The osmoregulatory protein, Na+, K+-ATPase (NKA) protein and activity declined and ATP-sensitive inward rectifier potassium channel 1 (kcnj1) mRNA abundance increased. On the other hand, decreased mRNA abundance of tight junction and related gene crumbs homolog 3 (crb3) mRNA abundance with increased Na+, K+, 2Cl--cotransporter (NKCC) and kcnj1 were found in the gill of FW-acclimated brackish medaka. In this thesis, large numbers of complete coding sequences and pathways classified of mRNA were found to advantage next experimental usage. To combine pathway speculate and physiological change, different adaptation to 18°C of brackish medaka were found: To compare 18°C and 28°C medaka, kcnj1 mRNA abundant were upregulated at both SW18°C and FW18°C, but apoptosis protein were only upregulated at SW18°C. Coupling with mRNA abundant relating to tight junction were downregulated and NKCC mRNA abundant were upregulated at FW18°C. In conclusion, cell permeability of gill and different reaction of ionocyte to cold stress were critical issues in euryhaline fish.

Tese de doutoramento do Programa Interuniversitário de Doutoramento em Envelhecimento e Doenças Crónicas, apresentada à Faculdade de Medicina da Universidade de Coimbra
Head and neck squamous cell carcinoma (HNSCC) is an emergent health problem worldwide. These tumors present heterogeneity at phenotypic, aetiological, biological and clinical level. In developed countries, smoking and alcohol are implicated in the increase of HNSCC cases, and human papillomavirus is an important risk factor, especially in the rise of oropharyngeal tumors without smoke and alcohol habits. A significant percentage of HNSCC patients develop loco-regional and distant recurrences. Even with progresses in surgery, radiation and chemotherapy, approximately half of all patients die of the disease. Risk stratification for HNSCC is essencial in order to decrease mortality and improve quality of life of the patients. The great HNSCC heterogeneity makes difficult to understand the molecular carcinogenesis process as well as to develop early detection and therapeutic strategies for these tumors. Nowadays, the majority of genome-wide molecular profiling studies of HNSCC are limited to single approaches, which hampers the identification of accurate and robust biomarkers of early diagnosis and prognosis. Indeed, there is a lack of proven biomarkers for predicting clinical outcomes and response to treatment. The present work aimed to perform a molecular characterization of HNSCC in order to predict recurrence/metastasis development and signaling pathways associated to targeted therapy and resistance to conventional drugs through the identification of different molecular groups with apparently different survival profiles using genomic, epigenetic and transcriptomic approaches. We analyzed the same HNSCC patients through different molecular technologies, being the identified biomarkers and molecular signatures validated with TCGA (The Cancer Genome Atlas) data. First, we performed a direct genetic and epigenetic characterization of HNSCC patients, using specific Multiplex Ligation-dependent Probe Amplification (MLPA) and Methylation-Specific MLPA (MS-MLPA) probe panels. We reported different genetic signatures related to tumor stage and anatomic site as well as tobacco use. Additionally, specific genomic and epigenetic signatures associated to patients' risk of recurrence/metastasis development after treatment of primary tumor and also to survival were identified. The genetic analysis of non-tumor samples (from surgical margins) revealed some imbalances similar to those identified in the tumor samples, which reinforce the importance of molecularly analyze the high-risk patients even before the visible morphological changes and also the suspicious lesions in order to early diagnose these tumors and their recurrences. Secondly, we moved forward to a high-throughput genomic and transcriptomic approaches and we identified molecular signatures with capability to predict the recurrent/metastatic disease development and clinical outcome. In these studies using either direct probe panels or genome-wide approaches we identified the most common chromosomal regions with imbalances and altered genes. As expected, whole-genome techniques revealed new chromosomal regions and genes that seem to have a role in HNSCC development and behavior. Overall, through these comprehensive genomic, epigenetic and transcriptomic characterization we identified biomarkers and molecular signatures of prognosis and survival, which open the door for personalized medicine in HNSCC patients. Finally, we applied these genomic and epigenetic technologies to perform a molecular characterization of four paradigmatic HNSCC patients in order to prove the benefit of these molecular knowledgement to the clinical management of the HNSCC patients. Several chromosomal regions and genes related to radiation and/or chemotherapy resistance and to patients' prognosis and survival were identified, which could help and guide the type or intensify of treatment modalities. Moreover, molecular characterization of commercial HNSCC cell lines and primary cell cultures established from these patients was conducted, which revealed the ploidy and the complex structural chromosomal rearrangements of HNSCC tumors. This comprehensive characterization enables cell models for further studies both in radiation and pharmacogenomics fields, as well as to understand the molecular mechanisms of HNSCC development and progression. With this work we performed a robust molecular characterization of HNSCC, using different omic approaches in the same tumor samples, which allowed the identification of new prognostic biomarkers and molecular signatures with potential to be translated to clinical practice.
O carcinoma epidermóide da cabeça e pescoço (CECP) é um problema emergente de saúde em todo o mundo. Estes tumores são heterogéneos a nível fenótipico, etiológico, biológico e clínico. Nos países desenvolvidos, o tabaco e o álcool estão implicados no aumento do número de casos de CECP e o papiloma vírus humano é um fator de risco importante para o aumento dos tumores da orofaringe não relacionados com hábitos tabágicos e de álcool. Uma percentagem significativa de doentes com CECP desenvolve recidivas loco-regionais e à distância. Mesmo com os progressos na cirurgia, radioterapia e quimioterapia, cerca de metade de todos os doentes morre devido ao CECP. A estratificação do risco de CECP é essencial de forma a contribuir para a diminuição da mortalidade e melhoria da qualidade de vida destes doentes. A heterogeneidade do CECP dificulta por um lado a compreensão dos processos moleculares da carcinogénese e por outro lado o desenvolvimento de estratégias de deteção precoce e de terapêutica. Atualmente, a maioria dos estudos moleculares de grande escala são restritos, o que dificulta a identificação robusta e precisa de biomarcadores de diagnóstico e prognóstico. De facto, há falta de biomarcadores para predizer o desenlace clínico e resposta ao tratamento. O presente trabalho teve como objetivo caraterizar molecularmente o CECP de forma a prever o desenvolvimento de recidivas/metástases e a identificação de vias de sinalização associadas a terapias alvo e resistência às terapias convencionais, através da identificação de diferentes grupos moleculares com diferentes sobrevivências, usando abordagens de genómica, epigenética e transcriptómica. Neste estudo, analisámos os mesmos doentes com CECP usando diferentes tecnologias moleculares, tendo validado os biomarcadores e assinaturas moleculares identificados usando dados do portal TCGA (The Cancer Genome Atlas). Em primeiro lugar, realizámos uma caraterização genética e epigenética do CECP direcionada, utilizando painéis de sondas específicos de Multiplex Ligation-dependent Probe Amplification (MLPA) e Methylation-Specific MLPA (MS-MLPA). Identificaram-se diferentes assinaturas genéticas relacionadas com o estadio do tumor e as localizações anatómicas, bem como com o consumo de tabaco. Adicionalmente, uma assinatura genética e epigenética associada ao risco dos doentes desenvolverem recidivas/metástases após o tratamento do tumor primário e também associada à sobrevivência, foi identificada. A análise genética das amostras não tumorais (provenientes das margens cirúrgicas) revelou alguns desequilíbrios similares aos identificados nas amostras tumorais, o que reforça a importância de analisar molecularmente os doentes de elevado risco mesmo antes de qualquer alteração morfológica visível e também das lesões suspeitas, de forma a diagnosticar precocemente estes tumores e as suas recidivas. Na segunda parte do estudo utilizámos abordagens genómicas e transcriptómicas de larga escala e, identificámos assinaturas moleculares capazes de prever o desenvolvimento de recidivas/metástases e evolução clínica dos doentes. Estes estudos, usando quer painéis de sondas direcionados quer abordagens de todo o genoma, permitiram identificar as regiões cromossómicas e genes mais comummente alterados. As técnicas de análise de todo o genoma revelaram novas regiões cromossómicas e genes que parecem desempenhar um papel no desenvolvimento e evolução clínica do CECP. No geral, através desta caraterização genómica, epigenética e trasncriptómica, identificámos biomarcadores e assinaturas moleculares de prognóstico e sobrevivência, o que abre novas portas para a medicina personalizada no CECP. Finalmente, utilizámos estas tecnologias de genómica e epigenética para caraterizar quatro doentes paradigmáticos com CECP de forma a provar o benefício deste conhecimento molecular na conduta clínica. Várias regiões cromossómicas e genes relacionados com a resistência à radiação, quimioterapia, prognóstico e sobrevivência foram identificados, o que poderia ajudar na escolha do tipo e intensidade das modalidades de tratamento. Adicionalmente, foi realizada a caraterização molecular de linhas comerciais de CECP e de culturas primárias estabelecidas a partir destes doentes de CECP, o que revelou a ploidia e rearranjos estruturais complexos destes tumores, garantindo modelos celulares para futuros estudos no campo da radiação e farmacogenómica e ainda para uma melhor compreensão dos mecanismos moleculares de desenvolvimento e progressão do CECP. Este trabalho permitiu, de uma forma robusta, caracterizar molecularmente o CECP, usando diferentes abordagens ómicas nas mesmas amostras tumorais, ajudando assim a identificar novos biomarcadores de prognóstico e assinaturas moleculares com potencial translação à clínica.