Natural products have, historically, played an important role in drug discovery. Nevertheless, drug resistance, pathogen evolution, and global climate change threaten human health and nearly all current anti-infective treatments on the market today. It is undeniable that new drug discovery efforts are needed with increasing urgency. Bolstered by a rich history of discovering treatments in the world around us, natural products chemists continue to look to the environment with increasing understanding and emerging technologies that allow efficient, effective isolation of new chemical entities. This thesis will describe one such endeavor. Focusing on fungal natural products, herein is described the isolation and structure elucidation of new, bio-active natural products. Further, the development and implementation of a large fungal screening program will be discussed, the results of which stand to advance microbial drug discovery in the Baker lab for years to come.

Mon travail a été d'étudier le rôle de l'expression de FZD9, un récepteur Wnt, dans l'hématopoïèse normale et maligne. Frizzled 9 (FZD9) est un composant clé de la voie de signalisation Wnt, une voie connue pour jouer un rôle important dans l'hématopoïèse normale et maligne. La méthylation d'un site CpG proximal de site d'initiation de la transcription du gène FZD9 est un facteur de mauvais pronostic dans les LAMs et sa déméthylation est un facteur prédictif de réponse aux thérapies épigénétiques. Sa fonction dans l'hématopoïèse n'est cependant pas connue. Au cours de ma thèse, j'ai démontré que le promoteur du gène FZD9 est dans un état non-permissif dans les cellules leucémiques. L'induction de la différenciation des cellules leucémiques restaure l'état permissif du promoteur, le recrutement du facteur E2F et de l'histone H3 acétyle permettant l'expression de FZD9. L'expression de FZD9 expression est également retrouvée au cours de la différenciation myéloïde d'une lignée IPS Dans les cellules de la lignée THP1 différenciées en monocytes, FZD9 est retrouvé dans un complexe comprenant LRP5/6 et Wnt5a. L'incubation des cellules différenciées par Wnt5a, un ligand de FZD9, déclenche la diminution de l'expression de -caténine et sa localisation nucléaire ainsi que l'expression des gènes cibles de la voie Wnt canonique (c-myc, Cyclin Dl and CD44). Nous n'avons détecté aucune augmentation des taux intracellulaires de calcium. Ces travaux suggèrent que la méthylation du promoteur de FZD9 dans les LAMs pourrait participer à la leucémogénèse en maintenant la voie b-caténine active / My goal was to study the importance of the expression of the FZD9, a Wnt receptor, in both normal and malignant hematopoiesis. Frizzled 9 (FZD9) is a key component of the Wnt signaling pathway, a pathway which has been shown to play a role in both normal and malignant hematopoiesis. Methylation of the CpG proximal to the transcription start site of the FZD9 gene is recognized as a prognostic factor in AML and its demethylation is a predictive factor for response to epigenetic therapy. Its function in hematopoiesis is however not known. The results show that the FZD9 promoter is in a non-permissive state in leukemic cells. Induction of myeloid differentiation in human myeloid leukemic cell fines restores FZD9 promoter permissiveness with recruitment of E2F and acetylated histone H3 and upregulation of FZD9 mRNA expression. FZD9 expression was progressively increased through the stages of myeloid differentiation in an IPS cell line. In differentiated monocytic THP1 cells, FZD9 was found in the LRPS/6 Wnt receptor complex. Incubation of differentiated cells with Wnt5a, a ligand of FZD9, triggered the decreased expression of - catenin and its nuclear localisation and the canonical Wnt-target genes (c-myc, Cyclin D1 and CD44). We detected no increase in calcium intracellular levels and thus activation of classical non-canonical pathway was not noted upon WntSa incubation. The results of my PhD suggest that the reported methylation of FZD9 promoter in AML and HR-MDS patients may participate in leukemogenesis by the maintenance of an activated Wnt canonical pathway.

Systematic characterization of microbes in several tumors including colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) has revealed the presence of multiple species of intracellular bacteria within tumors. However, there is limited knowledge on how these bacteria colonize tumors, how they survive inside host cells, how they modulate host cell phenotypes, and if their elimination should complement cancer therapy. This is, in part, due to the lack of representative animal models, challenges in co-culture of host epithelial cells and bacteria, and limited resolution of available analytical techniques to study host-microbial interactions. I have addressed these challenges by harnessing multiple technologies from microbiology, genetic engineering, tissue engineering, and microfluidics, in order to investigate the role of an emerging oncomicrobe, Fusobacterium nucleatum, in the tumor microenvironment (TME). F. nucleatum is a Gram-negative, anaerobic bacterium that is normally found within the oral cavity. However, its selective enrichment in CRC and PDAC tumors is correlated with poor clinical outcomes. My work along with collaborators in the Verbridge, Slade, and Lu labs at Virginia Tech has revealed a multifactorial impact of F. nucleatum in influencing cancer progression. First, in CRC, we discovered that F. nucleatum infection of host cancer cells induced robust secretion of select cytokines that increased cancer cell migration, impacted cell seeding, and enhanced immune cell recruitment. In PDAC, we uncovered additional cytokines that were secreted from both normal and cancerous pancreatic cell lines upon infection with F. nucleatum that increased cancer cell proliferation and migration via paracrine and autocrine signaling, notably in the absence of immune cell participation. In order to examine the contribution of a hypoxic TME on infection dynamics, we used a multi-omics approach that combined RNA-seq and ChIP-seq of H3K27ac to determine epigenomic and transcriptomic alterations sustained within hypoxic CRC cells upon infection with F. nucleatum. Our findings revealed that F. nucleatum can subvert host cell recognition in hypoxia and can modulate the expression of multiple cancer-related genes to drive malignant transformation. Insights gained from this research will pave the way for future studies on the impact of the tumor microbiome in cancer and will identify novel targets for therapy and clinical intervention to control bacteria-induced exacerbation of cancer. / Doctor of Philosophy / Colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) are the second and third leading causes of cancer death in the United States, respectively. Recent systematic characterization of various tumor types revealed the presence of distinct bacteria within tumors. However, there is limited knowledge on how these bacteria colonize tumors, how they survive inside host cells, how they modulate host cell phenotypes, and if their elimination should complement cancer therapy. This is, in part, due to the lack of representative animal models, challenges in developing host cell-microbe co-culture models, and limited resolution of available analytical techniques to study host-microbial interactions. I have addressed these challenges by harnessing multiple technologies from microbiology, genetic engineering, tissue engineering, and microfluidics, in order to investigate the role of an emerging cancer-associated microbe, Fusobacterium nucleatum, in the tumor microenvironment (TME). F. nucleatum is a microbe commonly found within the oral cavity. However, clinical studies revealed that selective enrichment of F. nucleatum in CRC and PDAC tumors significantly correlated with poor prognosis. My work along with collaborators in the Verbridge, Slade, and Lu labs at Virginia Tech has revealed a multifactorial impact of F. nucleatum in influencing cancer progression. First, in CRC, we discovered that F. nucleatum invasion of host cancer cells induced the secretion of select proteins called cytokines that cells use to signal and communicate with each other. These cytokines directly stimulated the cell migration of host cancer cells which is usually associated with increased cancer aggressiveness. In PDAC, F. nucleatum infection induced the secretion of additional cytokines from both cancer cells and normal cells that, in addition to cell migration, impacted the proliferation of cancer cells, another feature of aggressive cancers. F. nucleatum usually thrives in a low oxygen environment that is prevalent in cancer tissue and hence, we examined how a low oxygen environment can influence infection dynamics using sequencing technologies that probe the genomic constitution within cells. Our findings revealed that F. nucleatum can escape recognition in low oxygen environments and can modulate the expression of multiple cancer-related programs within the cell to drive cancer progression. Insights gained from this research will pave the way for future studies on the impact of the tumor-associated microbes in cancer and will identify novel targets for therapy and clinical intervention to control bacteria-induced exacerbation of cancer.

No description available.

Afin de protéger durablement les écosystèmes face aux rejets planifiés ou accidentels de radionucléides dans l’environnement, il est essentiel d’évaluer l’impact de l’exposition des organismes aux radiations ionisantes sur le long terme, à l’échelle de plusieurs générations. Dans ce contexte, ce travail de doctorat vise à améliorer la caractérisation des processus moléculaires et la prédiction des effets transgénérationnels lors d’une exposition aux radiations gamma. Une approche expérimentale concerne l’étude des modifications épigénétiques radio-induites, c’est-à-dire des modifications des mécanismes régulant l’activité des gènes sans modification de la séquence d’ADN elle-même et de leur transmission au fil des générations. Cette étude a permis de mettre en évidence que certaines modifications de la méthylation de l’ADN, l’un des mécanismes épigénétiques les plus étudiés, peuvent être transmises par la lignée germinale aux les générations non-exposées (génération F3) suite à une exposition parentale (génération F0) externe aux radiations gamma (6,5 µGy.h-1 et 41,3 mGy.h-1) pendant 25 jours. Dans une seconde approche, un modèle mécaniste DEBtox (Budget Energétique Dynamique appliqué à la toxicologie) est modifié pour permettre l’analyse des effets des radiations gamma sur la croissance et la reproduction de D. magna à l’échelle de plusieurs générations. Pour ce faire, on utilise des compartiments de dommage, dont le niveau peut être hérité d’une génération à la suivante. Le modèle est ajusté aux données avec des méthodes d’inférence bayésienne afin d’estimer les paramètres tout en tenant compte des incertitudes qui leur sont associées. / In order to durably protect ecosystems facing planned or accidental releases of radionuclides, the long-term impact of organism exposure to ionizing radiation must be studied on a multigenerational scale. The aim of this PhD is to improve the characterization of molecular processes and the prediction of transgenerational effects during a gamma irradiation. First, an experimental approach investigated on radio-induced modifications of epigenetic processes, i.e. changes in mechanisms that regulate gene expression without changing DNA sequence itself and on the transmission of these modifications to subsequent generations. Significant changes in DNA methylation, a well-studied epigenetic mechanism, detected in generation F3 clearly showed that epigenetic modifications could be transmitted to unexposed generations, in response to the exposure of a parental generation (F0) to external gamma radiation (6.5 µGy.h-1 et 41.3 mGy.h-1) for 25 days. Second, a mechanistic modelling approach used a modified version of the DEBtox model (Dynamic Energy Budget model applied to toxicology) in order to analyze effects of gamma radiation on D. magna growth and reproduction over several generations. To that end, damage compartments, with damage levels that were transmitted from one generation to the next, were included. The model was fitted to data using Bayesian inference methods, in order to estimate the parameters while considering their associated uncertainty.

No description available.