The human GI tract has evolved to simultaneously absorb nutrients and be the frontline in host defence. These seemingly mutually exclusive goals are achieved by a single cell thick epithelial barrier, and a complex resident immune system which lives in symbiosis with the intestinal microflora and is also able to rapidly respond to invading pathogens. An immunological balance is therefore required to permit tolerance to the normal intestinal microflora, but also prevent the dissemination of pathogenic micro-organisms to the rest of the host. Inappropriate immune responses in genetically susceptible individuals are the hallmark of human inflammatory bowel disease (IBD) and are thus targeting effector immune cells and their cytokines remains the mainstay of treatment. However despite vigorous efforts to delineate the genetic contribution to IBD disease susceptibility using large multinational cohorts, the majority of disease heritability remains unknown. Epigenetics describes heritable changes in chromatin that are not conferred by DNA sequence. These incorporate changes to histones, chromatin structure and DNA methylation, which confer changes to gene transcription and thus gene expression and cellular function. Methylbinding proteins (MBD) have the ability to bind to methylated DNA and recruit large chromatin remodeling complexes that underpin a variety of epigenetic modifications. Methyl- CpG-binding domain protein 2 (MBD2) is one such MBD that is required for appropriate innate (dendritic cell) and adaptive (T cell) immune function, though its role has not been investigated in the GI tract. We hypothesized that alterations in chromatin are central to the reprogramming of normal gene expression that occurs in disease states. By defining the phenotype of immune cells in the absence of MBDs we hope to understand the mechanisms of chromatin-dysregulation that lead to immune-mediated diseases such as IBD. We therefore aimed to assess the role of MBD2 in colon immune cells in the steady state and in murine models of GI tract inflammation, thereafter identifying the culprit cell types and genes responsible for any observed changes. We envisaged that investigating heritable, epigenetic changes in gene expression that are inherently more amenable to environmental manipulation than our DNA code, may provide novel insight to a poorly understood mechanism of disease predisposition. In addition identifying the cellular and gene targets of Mbd2 mediated changes to immune homeostasis that may provide exciting and novel approaches to therapeutic modulation of pathological inflammatory responses. In chapter 3 we assessed the expression of Mbd2/MBD2 in the murine/human GI tract. Consistent with existing mouse data, levels of Mbd2 mRNA increased between anatomical divisions of small (duodenum, ileum, terminal ileum) and large intestine (caecum, colon, rectum). In addition MBD2 mRNA was greater in the rectum versus ileum, with active IBD associated with lower rectal MBD2 mRNA compared to quiescent IBD controls. Thus we sought to understand the role of Mbd2 in the colon, where mRNA levels were the highest in the GI tract and where appropriate immune function is central to prevent damaging inflammation. To address these aims required the development of existing methods of cell surface marker expression analysis using flow cytometry techniques to simultaneously identify multiple innate and adaptive immune populations. Using naïve Mbd2 deficient mice (Mbd2-/-) we observed CD11b+ CD103+ DCs were significantly reduced in number in Mbd2 deficiency. To understand the role of Mbd2 in colonic inflammation we employed a mouse model of chemical (DSS) and infectious (T. gondii) colitis comparing Mbd2-/- and littermate controls (WT). Mbd2-/- were extremely sensitive to DSS and T. gondii mediated colonic inflammation, characterized by increased symptom score, weight loss and histological score of tissue inflammation (DSS) and increased antibody specific cytokine responses (T. gondii) in Mbd2 deficient animals. Flow cytometry analysis of colon LP cells in both infectious and chemical colitis revealed significant accumulation of monocytes and neutrophils in Mbd2-/-. Indeed monocytes and neutrophils were the principal myeloid sources of IL-1b and TNF in DSS colitis and the number of IL-1b/TNF+ monocytes/neutrophils was significantly greater in Mbd2-/-. Lastly we employed our colon LP isolation techniques to analyse immune populations in active and quiescent IBD and healthy controls, using endoscopically acquired biopsy samples. Analysis revealed that as in murine colitis, active human IBD is characterized by the accumulation of CD14High monocyte-like cells, with an associated increased ratio of macrophage:monocyte-like cells. In Chapter 4 we sought to understand the cellular sources of Mbd2 that may explain the predisposition of Mbd2-/- to colitis. Firstly we restricted Mbd2 deficiency to haematopoietic cells using grafting Mbd2-/- bone marrow (BM) into lethally irradiated WT mice. These animals treated with DSS displayed increased weight loss, symptom score, neutrophil accumulation and histopathology score compared to mice irradiated and grafted with WT BM. Given the accumulation of monocytes in Mbd2-/- DSS treated mice, and existing literature supporting a pathogenic role in this model, we then investigated the role of Mbd2 in monocyte function. Colon monocytes sorted from Mbd2-/- and WT DSS treated mice displayed similar expression for many pro-inflammatory genes (Il6, Il1a, Il1b, Tnf), but demonstrated significantly dysregulated expression for some others (Regb, Lyz1, Ido1, C4a). To investigate this in a more refined model, we lethally irradiated WT mice and repopulated them with a WT:Mbd2-/- BM mix. This enabled the analysis of WT and Mbd2-/- haematopoietic cells in the same animal. Colon WT and Mbd2-/- monocyte recruitment and cytokine production in DSS treated mixed BM chimeras was equivalent between genotypes suggesting that Mbd2 deficiency in monocytes alone did not explain the increased susceptibility of Mbd2-/- to DSS colitis. We then restricted Mbd2 deficiency to CD11c expressing cells, given the known role for Mbd2 in their function, and for CD11c+ cells in DSS, using a CD11cCreMbd2Fl/Fl system. DSS treated mice with Mbd2 deficient CD11c+ cells demonstrated increased weight loss, symptoms score, histolopathology score, monocyte and neutrophil colon accumulation compared to controls. To further explore the role of Mbd2 in colon CD11c+ cells, macrophage and DCs from DSS treated WT and Mbd2-/- mice were purified and their gene expression analysed. Mbd2-/- versus WT macrophages demonstrated significantly altered expression of both pro- (Il1a, C6, Ido1, Trem2) and antiinflammatory (Tgfbi, Retnla) pathways that we hypothesized was a method for attempted host control of excessive colon damage in Mbd2-/- mice. DC gene expression analysis was hampered by small sample size, but demonstrated a large number of small expression changes, including IL-12/IL-23 (Jak2) and autophagy (Lrrk2) pathways. Lastly levels of costimualtory molecules (CD40/CD80) were increased in Mbd2-/- but not CD11cΔMbd2 colon LP DCs/macrophages suggesting that non-CD11c+ cellular sources of Mbd2 were required to produce increased activation phenotype in these cells. Finally in Chapter 5 we explored the role for Mbd2 in non-haematopoietic cells, namely the colonic epithelium. Here we first developed a novel method for identifying and purifying these cells using flow cytometry. Mbd2 deficient colonic epithelium demonstrated increased expression of activation markers MHC II and LY6A/E in the steady state and in DSS / T. muris mediated colonic inflammation. Indeed FACS purified colon epithelial cells from naive and DSS treated, Mbd2-/- and WT mice revealed conserved dysregulated gene expression independent of inflammation: Both naïve and inflamed Mbd2 deficient epithelium displayed significantly increased expression of genes responsible for antigen processing/presentation (MHC I, MHC II, immunoproteasome) and decreased expression of genes involved in cell-cell adhesion (Cldn1, Cldn4). Lastly we investigated whether the observed differences in Mbd2-/- cell types conferred alterations in the makeup of the intestinal microflora. Interestingly independent of co-housing of Mbd2-/- and WT animals, Mbd2 deficiency consistently predicted the microbial composition, with increased levels of Clostridales and decreased levels of Parabacteroides bacteria. Collectively we have identified CD11c+ cells, monocytes and colon epithelial cells as key cell types for Mbd2 mediated changes in gene expression that affect mucosal immune responses. These data thus identify Mbd2 gene targets within these cell types as exciting new areas for investigation and therapeutic modulation to limit damaging GI tract inflammation.

Paediatric high grade glioma (HGG), including diffuse intrinsic pontine glioma (DIPG) are highly aggressive tumours with no effective cures. Lack of understanding of the molecular biology of these tumours, in part due to lack of well-characterised pre-clinical models, is a great challenge in the development of novel therapies. Analysis of paired cell culture/biopsy samples in this study revealed that paediatric HGG short-term cell cultures retain many of the tumour characteristics in vivo. Using a genome-wide approach, copy number, gene and miRNA expression, and methylation changes were characterised in 17 paediatric HGG-derived short-term cell cultures including 3 from DIPG. The majority of the genomic changes were unique from those arising in adult HGG. Approximately 65% (11/17) of paediatric HGG short-term cell cultures had balanced genetic profiles resembling normal karyotypes. The most frequent copy number gain and loss were detected at 14q11.2 (94%) and 8p11.23-p11.22 (59%), respectively. H3F3A (K27M) mutation was present in 2/17 (12%) cases and concurrent loss of CDKN2A and BRAFV600E in 1/17 (6%) case. Genes involved in reelin/PI3K signaling (DAB1), RTK signaling (PTPRE), and arginine biosynthesis (ASS1 and ASL) were frequently deregulated by methylation in these tumours. The anti-growth and anti-migratory properties of DAB1 and PTPRE were demonstrated in vitro. Preliminary investigations validated the therapeutic potential of ADI-PEG20 (arginine depletion), and PI-103 (PI3K/mTOR inhibition) in a subset of paediatric HGG short-term cell cultures. This study has identified novel genetic and epigenetic changes in paediatric HGG that may, following further validation, be translated into potential biomarkers and/or therapeutic targets.

The development of drug resistance during treatment is possibly the most important factor hampering the success of cancer therapy. In order to survive in the presence of chemotherapeutic drugs cells must quickly adapt to their altered environment. This may involve a collective stress response of interacting cells, whose mechanism is not yet clear. In the course of this work we interrogated the conceptual framework used to describe cancer and examined different aspects of drug resistance. While the main focus was on the role of ABC transporters in the rapid acquisition of drug resistance following a short period of drug treatment, the long-term adaptation to continuous drug treatment was also studied. As a tangent to this subject, the possible role of endocytosis in the process of adaptation to continuous presence of drug and subsequent resistance was also assessed. Cancer cell populations inexorably develop resistance to therapeutic treatment. In addition to selection of genetic variants, resistance may arise through two possible non-genetic mechanisms, (1) Darwinian selection of cells occupying (non-genetic) resistant microstates, or (2) Lamarckian instruction, in which cells adopt a resistant (treatment) induced phenotype. To examine the relative contribution of these two mechanisms we studied the population dynamics of leukemic cells (HL60 cell line) following treatment with the mitotic inhibitor vincristine. Single-cell analysis and mathematical modelling of state transition kinetics demonstrated that the appearance of multi-drug resistance phenotype within 24h was overwhelmingly the result of instruction. Transcriptome dynamics pointed towards a genome-wide state transition into a stress response state. Resistance induction correlated with Wnt pathway upregulation and was suppressed by beta-catenin knockdown, revealing a new opportunity for early therapeutic intervention against the development of drug resistance. By addressing the adaptation of the cell culture to prolonged drug treatment we observed that the survivor cells mounted a cellular response that neutralised the cytotoxic stress. That response involved the stabilisation of a transcriptome state that confers drug resistance. Our results suggested that the positive correlation between Wnt signalling and ABC transporters expression is important not only for the short-term survival but also for the enduring MDR phenotype. As we explored population heterogeneity we realised that the dead cells might also help the rest of the population to survive. Thus, our results support the need for examining the role of each population fraction, and ultimately each individual cell, in the overall story of cancer adaptation towards multidrug resistance. Subsequently we examined the differential endocytic behaviour between drug-sensitive and drug-resistant cells. By combining confocal time-lapse microscopy with flow cytometry we demonstrated that fluid-phase endocytosis was reduced in the resistant cells. The differences in the endocytic pathway only became noticeable after MDR1 expression has become constitutive, suggesting another protective role of the ABC transporters. All the results obtained support the idea that acquired drug resistance is not simply the passive selection of pre-existing mutants but can be accelerated by active adaptation. Cancer treatment is a double-edge sword: while the weakest cells die, the survivors cope cell-autonomously with the therapeutic perturbation.

No description available.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-07-12T15:39:14Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação Juan Luiz Coelho da Silva.pdf: 2693101 bytes, checksum: b946d507d9f21698d6349e8ecf91e259 (MD5) / Made available in DSpace on 2017-07-12T15:39:14Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação Juan Luiz Coelho da Silva.pdf: 2693101 bytes, checksum: b946d507d9f21698d6349e8ecf91e259 (MD5) Previous issue date: 2016-03-11 / FACEPE / Algumas evidências destacam mutações no gene ASXL1 como um evento importante na evolução clínica de pacientes com neoplasias hematológicas, particularmente em leucemias mieloides agudas e síndrome mielodisplásicas. Contudo, seu impacto prognóstico em neoplasias mieloproliferativas (NMP) ainda é pouco explorado. Aqui, nós caracterizamos 208 pacientes com NMP cromossomo Filadélfia (Ph) negativo (policitemia vera, PV; trombocitemia essencial, TE; mielofibrose primária, MFP), de acordo com mutações no gene ASXL1, e correlacionamos esses achados com características clinico-laboratoriais desses pacientes. A pesquisa das mutações foi realizada por sequenciamento sanger, em que polimorfismos germinativos e mutações sinonímias foram excluídas das análises. Mutações no ASXL1 foram detectadas em 22/208 pacientes (10%), das quais quatro foram observadas em pacientes com PV (4/54; 7%), onze em pacientes com TE (11/123; 9%) e sete com MFP (7/31; 22%). As características clínicas e laboratoriais foram similares entre pacientes com ASXL1 mutado e não mutado. Quando as entidades foram avaliadas individualmente (PV, TE e MFP), observou-se associação entre mutações no ASXL1 e idade mais avançada em pacientes com TE (P = 0,049) e desenvolvimento de esplenomegalia em pacientes com MFP (P = 0,026). Com uma mediana de seguimento de 5,1 anos (IC95%: 4,5 a 7,3 anos), 136 pacientes (65%) desenvolveram algum tipo de manifestação clínica, sendo o desenvolvimento de complicações vasculares o mais frequente (n=54; 26%), seguido por esplenomegalia (n=47; 22%), eventos hemorrágicos (n=30; 14%) e trombose (n=21; 10%). Mutações no gene ASXL1 não foram associadas com o desenvolvimento das referidas manifestações. Dentro deste seguimento, apenas dois pacientes evoluíram para síndrome mielodisplásica e um para leucemia mieloide aguda, todos sem mutações no gene ASXL1. / Accumulating evidences report mutation in ASXL1 as an important predictor to clinical outcomes of patients with hematological malignancies, particularly acute myeloid leukemia and myelodysplastic syndrome. However, the prognostic impact in myeloproliferative neoplasm (MPN) remains underexplored. Here, we evaluated clinical and laboratory features of 208 Philadelphia negative MPN patients (polycythemia vera, PV; essential thrombocythemia, ET; primary myelofibrosis, PMF), according to mutations in ASXL1. Screening for ASXL1 mutations were performedby Sanger sequencing. Germline variations were excluded. ASXL1 mutations were detected in 22/208 patients (10%), of which four in PV patients (4/54-7%), 11 in ET patients (11/123-9%) and seven in PMF (7/31-22%). Baseline features were similar between ASXL1-mutated and non-mutated patients. Evaluated individually (PV, ET, PMF), we observed that ET patients harboring ASXL1 mutations were older (P = 0,049) than ASXL1 non-mutated patients. Similarly, PMF patients presented higher frequency of splenomegaly in ASXL1mutated group (P = 0,026). No other features were associated with ASXL1mutations. The median follow-up was 5,1 years (CI95%: 4,5-7,3 years). One hundred and thirty six patients (65%) developed some of the clinical common manifestations, which the most frequent was vascular complications (n=54; 26%), followed by splenomegaly (n=47; 22%), bleeding (n=30;14%) and thrombosis (n=21;10%). ASXL1 mutations were not associated with development of such events. In our cohort, only two patients have evolved for myelodysplastic syndrome and one for acute myeloid leukemia, all of them without mutations in ASXL1.

<div><div><div><p>Transcriptomic regulation of human liver is a tightly controlled and highly dynamic process. Genetic and environmental exposures to this process play pivotal roles in the development of multiple liver disorders. Despite accumulating knowledge have gained through large-scale genomics studies in the developed adult livers, the contributing factors to the interindividual variability in the pediatric livers remain largely uninvestigated. In the first two chapters of the present study, we addressed this question through an integrative analysis of both genetic variations and transcriptome-wide RNA expression profiles in a pediatric human liver cohort with different developmental stages ranging from embryonic to adulthood. Our systematic analysis revealed a transcriptome-wide transition from stem-cell-like to liver-specific profiles during the course of human liver development. Moreover, for the first time, we observed different genetic control of hepatic gene expression in different developmental stages. Motivated by the critical roles of genetics variations and development in regulating hepatic gene expression, we constructed robust predictive models to impute the virtual liver gene expression using easily available genotype and demographic information. Our model is promising in improving both PK/PD modeling and disease diagnosis for pediatric patients. In the last two chapters of the study, we analyzed the genomics data in a more liver disease- related context. Specifically, in the third chapter, we identified Macrophage migration inhibitory factor (MIF) and its related pathways as potential targets underlying human liver fibrosis through an integrative omics analysis. In the last chapter, utilizing the largest-to-date publicly available GWAS summary data, we dissected the causal relationships among three important and clinically related metabolic diseases: non-alcoholic fatty liver disease (NAFLD), type 2 diabetes (T2D), and obesity. Our analysis suggested new subtypes and provided insights into the precision treatment or prevention for the three complex diseases. Taken together, through integrative analysis of multiple levels of genomics information, we improved the current understanding of human liver development, the pathogenesis of liver disorders, and provided implications to precision medicine.</p></div></div></div>

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21663号 / 医博第4469号 / 新制||医||1035(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 篠原 隆司, 教授 万代 昌紀, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22727号 / 医博第4645号 / 新制||医||1045(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 濵﨑 洋子, 教授 羽賀 博典, 教授 伊藤 貴浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22880号 / 医博第4674号 / 新制||医||1047(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 篠原 隆司, 教授 近藤 玄, 教授 万代 昌紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Les Eléments Transposables (ET) sont des parasites du génome caractérisés par leur capacité à se répliquer plus rapidement que les autres éléments génétiques du génome. La régulation par la voie des piARN joue un rôle essentiel pour limiter l’expansion des ET dans les lignées germinales des animaux.La première question posée est comment le génome répond face à une nouvelle invasion par un ET. Dans ce but, nous avons introduit le transposon de Classe II mariner (sous-famille mos1) chez D. melanogaster, qui ne contient naturellement pas l’élément. Nous avons montré, qu’après son amplification autonome dans le génome, l’élément avait atteint un équilibre en termes de nombre de copies, depuis qu’une régulation de novo par les piARN avait été acquise.Deuxièmement, nous avons étudié la mobilisation de l’élément mariner au cours du processus de colonisation des régions géographiques tempérées. A partir d’un large panel de populations naturelles nous avons trouvé que l’activité moyenne de mariner était remarquablement augmentée dans les populations non-Africaines en comparaison aux populations Africaines. Ces variations peuvent s’expliquer par un fort polymorphisme d’expression (transcriptionnel et traductionnel) des gènes de la voie des piARN.Le troisième chapitre soutient que la forte activité des ET dans la lignée germinale mâle est un phénomène global chez les drosophiles. Par ailleurs, le contenu en ET chez les espèces sœurs (D. melanogaster et D. simulans) a fortement divergé et, cela a affecté la réponse associée à la production des piARN. Chez D. melanogaster, de nombreux « burst » de transposition ont eut lieu récemment. Ces familles d’ET sont activement réprimées par les piARN dans l’ovaire et donc, se retrouvent massivement surexprimés dans les testicules. Chez D. simulans, nous pensons que la réponse par les piARN résulte principalement d’une régulation passée qui semble être la relique d’anciennes invasions d’ET.La voie des piARN est supposé être « garante de l’intégrité du génome » de par son rôle actif dans la défense contre les ET. Cependant, si la sélection naturelle purge les génomes de ces parasites délétères, il semble que les mécanismes de régulation de l’hôte contribuent au maintien de l’homéostasie du génome en limitant leur expansion, et quelque part en favoriser le maintien sur long terme. Ainsi, une autre interprétation pourrait être que la voie des piARN est « garante de la diversification du génome » de par son rôle à faciliter l’accumulation des ET. / Transposable Elements (TEs) are genomic parasites characterized by their ability to replicate faster than any other genetic element in the genomes. The piRNA mediated silencing is of central importance to limit TE expansion in the germline of animal species. The present dissertation explores the relationship between TEs and piRNAs alongside their evolutionary dynamics.The first question raised here was to understand how the genome responds to a new TE invasion. For that purpose, we injected a mariner Class II transposon into D. melanogaster genome that does not naturally contain the element. We found that, after its self-replication into the genome, the element have reached a copy number equilibrium since a de novo piRNA mediated regulation have been acquired.Second, we studied the mariner rewiring activity during the colonization of geographical temperate regions. From a large sampling of D. simulans natural populations, we found the mean activity of mariner to be strikingly higher in non-African populations compared to the African ones. These findings support the idea that selection acting on piRNA effector proteins has been of central importance to explain TE lineages diversification during colonization process.The third chapter provides evidences to propose that, the strong TE activity in testes, is a general phenomenon in Drosophila. We also observed that TE landscape divergence between the two sister species, have affected the genomic response mediated by the piRNAs. As a response of their recent bursts of transposition, TEs overexpressed in testes are preferentially silenced by piRNAs in D. melanogaster ovaries. By contrast, we assumed the D. simulans piRNA response to be the relic of a past regulation that still persists mostly against inactive TEs.The piRNA silencing in the germline, is assumed to be the “vanguard of genome” defense and integrity due to its active role against TEs. However, while natural selection purifies the genome from its deleterious parasites, it seems that the host regulation contributes to genome homeostasis by limiting their expansion, and somehow, favors their longterm maintenance. Thus, another interpretation would have been that piRNA silencing is the “vanguard of genome” diversification due to its active role in facilitating TE accumulation