Les cellules immunitaires proviennent d'un ensemble commun de cellules souches hématopoïétiques qui se différencient hiérarchiquement en lignées myéloïdes et lymphoïdes. Ce processus est étroitement régulé par un réseau entrelacé de facteurs de transcription et de régulateurs épigénétiques, qui contrôlent l'activation et la répression des gènes impliqués. Les travaux récents sur la reprogrammation cellulaire ont montré que certaines protéines peuvent reprogrammer des cellules différenciées, comme le facteur de transcription C/EBPa qui peut induire la trans-differenciation de cellules B en macrophages. De plus, une courte induction de Cebpa suivie de l’expression des quatre facteurs de transcription Oct4-Sox2-Klf4-cMyc permet une reprogrammation extrêmement rapide en cellules pluripotentes. Afin de déchiffrer le réseau de régulation moléculaire contrôlant la spécification et la reprogrammation des cellules immunitaires, j’ai combiné différentes méthodes à haut débit pour analyser l’expression des gènes et leur régulation épigénétique, et ce au court de la reprogrammation des cellules B. J’ai découvert des interactions entre différents facteurs de transcription, au niveau des régions régulatrices de gènes des différents programmes génétiques impliqués (lymphoide, myeloide et pluripotence), et j’ai identifié des facteurs régulant l’état de la chromatine également impliqués dans la reprogrammation (notamment Lsd1, Hdac1, Brd4 et Tet2). Enfin, J’ai intégré ces données dans un modèle dynamique du réseau moléculaire régulant la spécification des cellules B et des macrophages à partir de progéniteurs multipotents. J’ai utilisé à la fois des méthodes analytiques (analyse des états stables) et des simulations (simulations logiques asynchrones, chaînes de Markov à temps continu) pour étudier in silico la différenciation et la reprogrammation cellulaire. Ces analyses ont révélés des régulations transcriptionelles encore inconnues, que nous avons pu confirmer expérimentalement. Nous avons ainsi obtenu une meilleure compréhension des circuits de régulation contrôlant le destin cellulaire. / Immune cells arise from a common set of hematopoietic stem cells, which differentiate hierarchically into the myeloid and lymphoid lineages. This process is tightly regulated by an intertwined network of transcription and epigenetic factors, which control both the activation and repression of gene programs, to ensure cell commitment. However, recent work on cellular reprogramminghas shown that the ectopic expression of some specific factors can enforce the trans-differentiation of committed cells. The transcription factor C/EBPa can induce the reprogramming of B-cells into macrophages. Furthermore, a pulse of Cebpa expression in B cells followed by the expression of the four transcription factors Oct4-Sox2-Klf4-cMyc leads to an extremely fast and efficient reprogramming into induced pluripotent stem cells. Despite the many data we have on the molecular mechanisms by which specific genes are regulated, we are still lacking a global understanding of the interplay between these factors and how theycontrol cell fate. In order to decipher the molecular regulatory network controlling immune cell specification and their reprogramming, I have combined a variety of high-throughput methods to measure changes in gene expression and epigenetic regulation during B cells reprogramming. I have revealed the interplay between different transcription factors at enhancers regulating genes of the different programs (B cells, macrophages and pluripotent cells) and identified epigenetic regulators forming complexes and controlling enhancers activities (such as Lsd1, Hdac1, Brd4 and Tet2) and consequently regulating cell fate. Finally, I integrated these data together with published data, in a computational model of the regulatory network controlling the specification of B-cells and macrophages from multipotent progenitors. I used both analytic tools (stable states analysis) and simulations (logical asynchronous simulations, continuous time Markov chains) to study in silico differentiation and reprogramming.These analyses have revealed previouslyunknown transcriptional regulations, which weconfirmed experimentally, and allowed us to get abetter understanding of the regulatory circuitscontrolling cell fate commitment.

Biotecnologias reprodutivas como a produção in vitro de embriões e a transferência de núcleo apresentam grande potencial de aplicação na medicina veterinária seja para a correção de infertilidades, para o aumento na eficiência da produção animal ou mesmo para um melhor entendimento sobre os mecanismos envolvidos no desenvolvimento embrionário inicial. Porém, manipulações in vitro de gametas ou embriões levam a alterações na regulação epigenética, podendo causar altas taxas de anormalidades no desenvolvimento e no nascimento de indivíduos derivados. A geração de um modelo de indução da pluripotência in vitro, ou seja, a geração de células iPS (do inglês induced pluripotent stem cells) possibilitou estudar o processo de reprogramação in vitro de maneira robusta e precisa. Os genes OCT4 e SOX2 são fundamentais no processo de aquisição e manutenção da pluripotência celular, e recentemente foi reportado que a ação destes dois fatores exerce grande influência sobre a regulação de alguns genes imprinted, em especial, no locus H19/IGF2, sabidamente importantes para o desenvolvimento normal do embrião e de sua placenta. Este estudo propõe a geração de um modelo experimental in vitro onde os fatores em questão sejam estudados, juntos ou em combinação, quanto à sua influência na regulação do imprinting genômico. Para tal, três linhagens de fibroblastos fetais bovinos (bFF1, bFF2 e bFF3) foram transduzidas com vetores lentivirais contendo cDNAs de OCT4 ou SOX2 humanos. Os fibroblastos foram analisados através de citometria e as células positivas foram separadas e recuperadas (sorted). Os fibroblastos expressando OCT4, SOX2, ambos (OCT4 + SOX2), nenhum (controle) juntamente com um controle recuperado (não sorted) não transgênico (total de cinco tratamentos) foram investigados quanto à expressão de genes relacionados à pluripotência e expressão de genes imprinted, bem como a manutenção dos padrões de metilação do DNA no locus H19/IGF2. Além disso, estas células foram submetidas à reprogramação in vitro e produção de células iPS. A indução à pluripotência foi realizada através da transdução dos fibroblastos com o vetor policistrônico contendo o cDNAs murino ou humano dos fatores de transcrição OCT4, SOX2, c-MYC e KLF4 (OSMK, vetor STEMCCA). Os resultados da análise de fluorescência por citometria de fluxo foram, em média, de 40,4% para OCT4, 6,1% para SOX2 e 0,63% para OCT4 + SOX2. A bFF1 foi a única linhagem a apresentar uma recuperação pós-sorting, o que possibilitou sua utilização para a indução da pluripotência. De maneira interessante, as células que não passaram pela citometria geraram colónias de células iPS, enquanto que os demais grupos não. A quantificação de transcritos por qRT-PCR mostrou que a expressão de OCT4 e de SOX2 estava aumentada nos respectivos grupos, a expressão do gene H19 mostrou-se aumentada no grupo controle que passou pelo procedimento de sorting e a expressão do gene imprinted IGF2R não variou entre os grupos. Já a análise preliminar da manutenção do padrão de metilação de DNA na DMR do locus H19/IGF2 mostrou que o grupo controle sorted apresentou uma leve diferença no padrão de metilação quando comparada aos outros grupos. Neste estudo, portanto, o procedimento de separação e recuperação celular por citometria de fluxo celular, aliado ao elevado número de repiques celulares durante o cultivo prolongado pode ter levado a um efeito prejudicial sobre a eficiência de reprogramação in vitro / Reproductive biotechniques such as in vitro embryo production and somatic cell nuclear transfer may greatly contribute for fertility improvements, to enhance animal production or else to contribute to a better understanding of the underlying mechanism involved during initial embryonic development. However, in vitro manipulation of gametes or embryos may lead to possible disruptions on epigenetic regulation, causing high developmental abnormalities and decreased healthy calves born at term. The generation of induced pluripotency models (induced pluripotent stem cells, or iPS) made it possible to study the process of in vitro reprogramming in a more solid and precise manner. OCT4 and SOX2 are fundamental genes for the acquisition and maintenance process of cellular pluripotency. Recently, it has been reported that both factors may have a huge influence on the regulation of some imprinted genes, specially at locus H19/IGF2, known to be important for the normal development of embryo and placenta. Therefore, this study aimed to generate an in vitro experimental model where the above transcription factors will be studied together or separately regarding their influence on genomic imprinting regulation. For that, three bovine fetal fibroblasts cell lines (bFF1, bFF2 and bFF3) were transduced with lentiviral vectors containing human OCT4 or SOX2 cDNAs. The fibroblasts were analyzed trough cell cytometry and positive cells were sorted. Fibroblasts expressing OCT4, SOX2, both (OCT4+SOX2), none (control) together with a non-sorted and non-transgenic control (five treatments) were investigated regarding pluripotency and imprinted gene expression, as well maintenance of DNA methylation patterns at H19/IGF2 locus. Further, these cells were also submitted to in vitro induced reprogramming and production of iPS cell colonies. Induction into pluripotency was realized by transducing fibroblasts with polycistronic excisable vector containing the murine or human cDNA of OCT4, SOX2, c-MYC and KLF4 transcription factors (OSMK, STEMCCA vector). The results of fluorescence analysis by flow cytometry were, on average, 40.4% for OCT4, 6.1% for SOX2 and 0,63% for OCT4+SOX2 groups. bFF1 was the only lineage presenting a post-sorting recovery that enabled its use for pluripotency induction. Interestingly, non-sorted cells generated biPS colonies whereas sorted cells (control non transgenic, OCT4, SOX2 and OCT4+SOX2 expressing cells) did not generate biPS cells. The transcript quantification by qRT-PCR showed that OCT4 and SOX2 expression were increased in the respective groups, the expression of H19 gene was increased in the control sorted group and IGF2R expression was not different between groups. Preliminary results of imprinting pattern methylation at H19/IGF2 locus showed that sorted group was slightly different from others. In this study, therefore, analysis and sorting procedure by flow citometry, together with an extended period in culture may have lead to a detrimental effect on in vitro reprogramming efficiency

Au début de ma thèse, je me suis intéressé aux processus qui sous-tendent la communication cellulaire et plus spécifiquement les interactions cellule-cellule. Pourquoi une cellule établit-elle un contact spécifique avec une autre cellule ? Comment les cellules répondent-elles à cette interaction et quels en sont les effets ? J'ai utilisé comme modèle d'étude l'interaction entre les cellules souches/stromales mésenchymateuses (CSM) et des lignées de cancer du sein. L'objectif de mon travail a été d'analyser les mécanismes de ces interactions entre CSM et cellules cancéreuses et d'en évaluer les effets sur les fonctions des cellules cancéreuses. En effet, des mécanismes de recrutement des CSM aux sites tumoraux ont été décrits avec des effets sur la progression tumorale, ce qui ouvre par ailleurs des perspectives pour de nouvelles approches thérapeutiques. J'ai tout d'abord développé un système expérimental de microscopie confocale en temps réel pour observer le type d'interaction qui est produit entre les CSM humaines et les cellules de carcinomes mammaires MDA-MB-231 et MCF7. J'ai constaté la formation dynamique de structures tubulaires entre les deux types cellulaires et, de façon surprenante, le passage des mitochondries des CSM vers les cellules cancéreuses. En un deuxième temps, j'ai utilisé un système d'invasion dans une matrice 3D de collagène, que nous avons adapté à la coculture, afin d'observer les effets de l'interaction des MDA-MB-231 avec les CSM. En accord avec la littérature, nous avons constaté une augmentation du pouvoir invasif des cellules cancéreuses, effet qui pouvait être lié au transfert des mitochondries provenant des CSM. Pour répondre à cette question, j'ai mis au point un protocole pour transférer spécifiquement des mitochondries, isolées à partir de cellules, vers d'autres cellules. Ce protocole, exploité dans ce manuscrit pour le transfert de mitochondries de CSM vers les cellules cancéreuses MDA-MB-231, peut être transposé à d'autres types cellulaires et fait l'objet d'une demande de brevet. Nos données indiquent que l'acquisition de mitochondries de CSM par les cellules cancéreuses modifie leurs propriétés fonctionnelles et augmente leur capacité de prolifération et d'invasion. Concernant leur activité métabolique, on observe une augmentation de leur respiration mitochondriale et de leur production d'ATP. Nos données préliminaires suggèrent aussi une augmentation de l'expression transcriptionnelle d'enzymes impliquées dans la synthèse des lipides et l'oxydation des acides gras. Ces données, générées grâce au protocole de transfert artificiel de mitochondries mis au point, montrent pour la première fois que les mitochondries des CSM peuvent majorer certaines propriétés cellulaires liées à la progression tumorale, comme la prolifération et l'invasion, et contribuer à une reprogrammation métabolique des cellules cancéreuses. Elles s'intègrent au rôle proposé par la communauté scientifique pour les CSM dans le microenvironnement tumoral. La technique de transfert artificiel de mitochondries nous permettra de répondre à d'autres questions restées ouvertes, comme le rôle possible des mitochondries des CSM dans les résistances développées par les tumeurs vis-à-vis des agents anti-cancéreux. Le protocole de transfert de mitochondries développé au laboratoire constitue une technique de choix et offre de nombreux avantages comparativement à d'autres techniques comme la micro-injection et la génération des hybrides cytoplasmiques. Sa mise en œuvre est en effet simple et reproductible et permet de traiter une grande quantité de cellules. Cette méthode permet d'envisager de nombreuses perspectives et applications dans le domaine de la reprogrammation métabolique, comme par exemple de restaurer les capacités d'une cellule dysfonctionnelle par le transfert de mitochondries issues d'une cellule saine et « métaboliquement active ». / At the beginning of my thesis, I was interested in the process involved in cell communication, more specifically in cell-to-cell interactions. Why does a cell specifically establish contacts with another one, how do cells respond to these interactions and what are the effects? As a model to answer these questions, I studied the interactions between MSCs and two breast cancer cell lines. The study of the communications between MSCs and tumor cells is an alternative to explore and understand tumor progression. MSC recruitment to the tumor is shown to favor the progression of the disease. The mechanisms of this dialogue are multiple and are the object of a great number of studies that aim at finding new therapeutic approaches. The objective of this work was to analyze the interactions between MSCs and cancer cells and evaluate the potential effects of this communication in tumor progression. First, I developed an experimental system of real time confocal microscopy in order to observe the interaction produced between MSCs and the breast carcinoma MDA-MB-231 and MCF-7 cells. I noticed the dynamic formation of tubular structures between the two different cell types and, surprisingly, the passage of mitochondria from MSCs to the cancer cells. Second, we used a 3D system of cell invasion in a collagen matrix, which we adapted for the coculture, in order to observe the effects of the interactions between the MDA-MB-231 and MSCs. In agreement with the literature, we observed an increase in the migratory potential of the cancer cells, an effect that could be linked to the transfer of mitochondria from MSCs to the cancer cells. To answer this question, I set up a protocol to specifically transfer to the cancer cells mitochondria isolated from the MSCs and test directly the functional consequences for the cancer cells. This protocol can be used to transfer mitochondria, not only from MSCs but also from other cells. This method is currently submitted to a patent process. Our results show that the transfer of MSC mitochondria to the cancer cells modifies cancer cells functional properties and increase their invasive and proliferative capacities. Concerning the metabolic activity, we noticed an increase in mitochondrial respiration and ATP production. We also observed an increase in the transcription level of enzymes related to the lipid synthesis and fatty acid oxidation. The results generated with this new protocol of mitochondria transfer show, for the first time, that mitochondria originating from MSCs can improve cellular capacities linked to the tumor progression. The role proposed by the scientific community for the interactions of MSCs with the tumor cells fits with the data generated in our work. Several questions remain open. In particular, could the transfer of mitochondria from MSCs to the cancer cells contribute to the acquisition of resistance to anti-cancer agents observed in patients? The protocol of transfer of mitochondria that we developed in the laboratory is a technique of choice and offers many advantages over other techniques such as microinjection and cytoplasmic hybrids; its implementation is simple and reproducible and can target large numbers of cells. This method opens numerous perspectives and potential applications such as the study of metabolic reprogramming. Thus, we could consider restoring the activity of dysfunctional cells by transferring mitochondria from “metabolically active” or healthy cells. In the long term, one of the applications could be transferring healthy or genetically modified mitochondria to zygotes carrying mitochondrial DNA mutations, in order to treat pathologies like infertility, neuro-degenerative diseases, cancer and premature aging.

En dehors du cadre pathologique, la notion de temps est la base essentielle dans le processus de vieillissement de l’organisme et des systèmes associés. Ces derniers vont progressivement présenter un déclin de leur(s) fonction(s) où de nombreux mécanismes complexes vont intervenir à différents niveaux. Parmi les premiers constats proposés, le vieillissement est la conséquence d’un processus inéluctable, d’une succession d’agressions au niveau cellulaires qui pourraient être réparées voire évitées, ouvrant ainsi la voie à de futures études qui permettront à terme de proposer une explication détaillée, complète et claire de ce processus. Pour exemple, les travaux que nous avons menés tentent d’apporter un élément de réponse afin d’établir un lien entre sénescence et vieillissement, avec pour base de générer par reprogrammation cellulaire des cellules hiPSCs à partir de cellules issues de biopsies de patients jeunes et âgées, sénescentes ou prolifératives. Outre la caractérisation de leur état pluripotent comparable à celui des cellules souches embryonnaires, nous avons également mis en évidence après une différenciation spécifique en fibroblastes, que les caractéristiques cellulaires de ces fibroblastes présentaient un effacement des marques du vieillissement, signe d’une plasticité cellulaire possible au cours du vieillissement. A présent, étendre une telle étude au modèle tissulaire cutané par la mise en place de protocoles de différenciation dans le lignage épidermique permettra à l’avenir de mieux comprendre pour mieux appréhender les pathologiques associées au vieillissement, et ainsi pouvoir offrir aux patients une médecine appropriée et concrète. / Beside the pathological context, time is the essential basis in the aging process of the body and associated systems. These will gradually introduce a decline in function(s) where many complex mechanisms will take part at different levels. Among proposed findings, aging is the result of an inevitable process, a succession of cellular stress that could be prevented or repaired, opening the way for future studies that will eventually offer an explanation detailed, clear and complete which occur. For example, our work provide a response element to establish a link between senescence and aging, based on the generation of hiPSCs by cellular reprogramming of cells from biopsies of young, older, senescent and proliferating cells patients. Further characterization of their pluripotent state comparable to that of human embryonic stem cells, we have also showed by a specific differentiation into fibroblasts, that cellular characteristics of those fibroblasts had erased aging features. Next step, is to extend such study in cutaneous tissue model by the introduction of differentiation protocols in the epidermal lineage which will able us to better understand aging-associated diseases, and thus bring the ability to propose an appropriate and cocnrete medicine to aged patients.

La transition fleur-fruit, appelée nouaison, est déclenchée par la pollinisation des fleurs et ce processus est essentiel pour cycle reproducteur des plantes, la formation des semences et le rendement de production. Les mécanismes moléculaires contrôlant cette importante transition développementale ont été peu explorés. Les marques histones et la méthylation de l'ADN sont les deux principaux modes de régulation épigénétique, mais à ce jour, leurs contributions respectives à la reprogrammation transcriptionnelle qui est associée au programme d’initiation des fruits charnus n’ont pas fait l’objet d’aucune étude sur aucune espèce de plante. Afin d’explorer l’importance dans la transition fleur-fruit de ces deux types de régulation épigénétique, des approches de transcriptomique "genome-wide", de ChIP-seq se et de séquençage bisulfite d'ADN ont été mises en place chez la tomate, une espèce économique majeure et un modèle d’étude pour les fruits charnus. Les résultats révèlent une corrélation étroite entre le repositionnement des marques histones et les changements observés de l'expression génique globale. L’étude montre aussi que les marques H3K9ac et H3K4me3 agissent en synergie pour activer la transcription génique, alors que la marque H3K27me3 a un effet répressif. A l’inverse, il n’y a pas de corrélation entre les variations de la méthylation de la cytosine et l’évolution des profils transcriptomiques. Il ressort donc que ce sont les changements au niveau des marques histones plutôt que de la méthylation de l'ADN qui constituent le moteur principal de la reprogrammation génétique associée au processus de transition fleur-fruit chez la tomate. En concordance avec cette idée, le niveau d'expression des gènes associés à l’initiation du fruit, tels que ceux liés au métabolisme hormonal, à la division cellulaire ou au développement embryonnaire, est corrélé avec les modifications des marques H3K9ac ou H3K4me3, mais pas avec la méthylation de l'ADN. En outre, l'étude comparative des profils transcriptomiques associés à la formation du fruit dépendant et indépendant de la pollinisation révèle l'intervention complexe de multiples voies de signalisation hormonales. Au total, notre étude présente un nouvel aperçu du contrôle de la reprogrammation génétique nécessaire à l’initiation du développement du fruit et révèle le rôle important du contrôle épigénétique dans ce processus de transition développementale. Dans le même temps, l’étude identifie un groupe de gènes impliqués dans la régulation épigénétique qui offrent des cibles potentielles pour les programmes d’amélioration de la nouaison des fruits, un processus majeur affectant le rendement de production / The flower-to-fruit transition, so-called fruit setting, is triggered by flower pollination and this process is essential for plant reproductive success, seed formation and crop yield. The underlying molecular mechanisms controlling this developmental transition remain unclear. Histone marking and DNA methylation are the main epigenetic modes for genetic reprogramming, however, their respective contribution to the fruit set-associated transcriptomic reprogramming is also unknown. To address the contribution of the two types of epigenetic regulation to fruit set, genome-wide transcriptomic profiling, ChIP-sequencing and DNA bisulfite sequencing were applied to tomato, a major economic crop and a model system for fleshy fruit. The study emphasizes the tight correlation between histone repositioning and gene expression changes revealing that H3K9ac and H3K4me3 histone marks synergistically promote gene transcription, whereas H3K27me3 marking has a repressive effect. We concluded that changes in histone marks rather than in DNA methylation are the main drivers of genetic reprogramming associated with the fruit set transition in tomato, and H3K9ac and H3K4me3 marking is the primary players in this control mechanism. Consistently, the expression level of fruit set-associated genes such as those related to hormone metabolism, cell division, and embryo development correlated with changes in H3K9ac or H3K4me3 marking, but not with DNA methylation. In addition, comparative study of transcriptomic profiling between pollination-dependent and -independent fruit set, uncovered the complex intervention of multiple hormone signaling pathways involved in the flower-to-fruit transition. Auxin appears as the central hormone triggering the extensive transcriptomic reprogramming associated with the initiation of early fruit growth. Altogether, the study provides new insight into the control of gene reprogramming underlying fruit the shift from flower to fruit and uncovers a set of genes encoding modifiers of epigenetic marks which may provide new targets for breeding programs aiming to improve fruit setting, a major process impacting crop yield.

L'adénocarcinome canalaire pancréatique (ADKp) possède une architecture compacte, où les cellules tumorales forment des glandes emprisonnées dans un bouclier fibrotique, composé à 50% de collagènes. Ce bouclier empêche la vascularisation, limite l'apport en nutriments et oxygène. Beaucoup de cellules meurent, mais certaines survivent, en reprogrammant en particulier leur métabolisme. Ula plus étudiée est l'utilisation constitutive de la glycolyse, indépendamment de la présence d'oxygène (Effet Warburg). Nous montrons que la population hypoxique de l'ADKp dépend aussi de la dégradation de la glutamine, et que l'activité concomitante de la glycolyse et de la glutaminolyse entraîne la réactivation de la biosynthèse des hexosamines. Ces composés participent à la prolifération tumorale en stabilisant les transporteurs au glucose, ou des oncogènes. L'activité glycolytique intense des cellules hypoxiques permet la synthèse de lactate qui sert de ressource nutritive aux cellules oxygénées adjacentes aux cellules hypoxiques. Nous montrons que certaines cellules oxygénées sont capables de survivre au stress nutritif en dégradant le collagène (écophagie), en utilisant la proline qu'il contient. Les cellules tumorales captent et dégradent les fragments de collagènes pour survivre. Des traçages isotopiques de collagène marqué permettent d'appuyer que la proline internalisée provient du collagène matriciel. Cette proline est transformée en glutamate et fournit le cycle de Krebs pour favoriser la survie tumorale. Ces travaux montrent l'importance de l'étude de la reprogrammation métabolique dans l'ADKp, et le rôle de l'hypoxie ou du collagène dans la progression tumorale. / Pancreatic ductal adenocarcinoma (PDAC) has a compact architecture wherein the tumor cells are organized in glands and trapped in a fibrotic shield (stroma) made of up to 50% of collagen. This shield prevents blood supply, limits nutrients and oxygen intake. Many cells die, but some survive, and proliferate particularly by reprogramming their metabolism. The most studied metabolic reprogramming remains tumor cells addiction to glucose and the constitutive use of glycolysis, regardless of the presence of oxygen (Warburg effect). We show that the hypoxic population of PDAC also depends on glutamine degradation, and the concomitant activity of both glycolysis and glutaminolysis reactivates the hexosamine biosynthetic pathway. These compounds contribute to tumor proliferation by stabilizing glucose transporters, or oncogenes. The intense glycolytic activity of hypoxic cells allows the synthesis of lactate. Excreted in the microenvironment, it serves as a nutritive resource to oxygenic cells adjacent to the hypoxic population and enables their proliferation. We show that some oxygenated cells are also able to survive under nutrient stress by degrading collagen (ecophagy) and use proline it contains. Tumor cells intake and degrade collagen fragments to survive. Isotopic tracer experiments using labeled collagen support the idea that proline comes from the extracellular collagen. This proline is degraded and converted into glutamate, fueling the Krebs cycle for anaplerosis and promotes tumor survival. These studies therefore show the importance to study the metabolic reprogramming of PDAC, and the role of hypoxia or collagen matrix in tumor progression.

Throughout society’s continual war against cancer, we have attempted pharmacological intervention only to find that tumors develop modes of resistance. It is well known that genetics play an integral role in cancer. Technological advances have greatly improved our ability to study cancer biochemistry beyond the genome by measuring changes in the expression and activity of RNA, proteins, and lipids in experimental models and human patients. As our techniques and technology to perform cancer research progresses, it is becoming more evident that cancer cells develop stress tolerance mechanisms at multiple levels within the central dogma, including altering mRNA expression, enzyme concentrations, and functional activity of cellular proteins and lipids. In the first chapter, I review previous discoveries demonstrating the importance of metabolic reprogramming in cancer cells and how shifts in metabolic pathways contribute to cancer progression and therapeutic challenges. I discuss how mass spectrometry is a multifunctional research tool that can be used to identify global shifts in gene expression, identify oncogenic roles of specific metabolites and corresponding metabolic pathways, conduct enzyme activity assays, and understand the effects of drugs on cell signaling and metabolic flux through specific pathways. While metabolic reprogramming is a complex and multifaceted concept, the following chapters focus on two specific stress tolerance pathways of lipid and protein metabolism we have shown to significantly promote cancer cell evolution, proliferation, and drug resistance in models of human pancreatic and colon cancer. I describe novel mass spectrometry-based lipidomics and proteomics methods we developed to measure and determine the biological impact of these pathways in each model. I discuss the contributions we have made toward increasing general knowledge of metabolic reprogramming networks in cancer and how they may be targeted in more specific and effective manners to sensitize cancers to therapeutic drugs. Specifically, the second chapter entails our study of a pro-survival lipid metabolic pathway driven by the sphingolipid modifying enzyme sphingosine kinase in a panel of differentially reprogrammed pancreatic cancer subclones. The third chapter describes our novel kinetic proteomics approach to identify how the cellular degradation system autophagy is used to selectively remodel the proteome of colon tumor cells in a xenograft mouse model of colon cancer. Lastly, I discuss how these and other projects completed during my graduate work lay a foundation for ongoing research to further our fundamental understanding of cancer metabolism and treatment development.

Here we describe some of the crucial steps to generate induced pluripotent stemcells (iPSCs) usingmRNA transfection. Our approach uses a V. virus-derived capping enzyme instead of a cap-analog, ensuring 100% proper cap orientation for in vitro transcribedmRNA. V. virus\'' 2′-O-Methyltransferase enzymecreates a cap1 structure found in higher eukaryotes and has higher translation efficiency compared to other methods. Use of the polymeric transfection reagent polyethylenimine proved superior to other transfection methods. The mRNA created via this method did not trigger an intracellular immune response via human IFN-gamma (hIFN-γ) or alpha (hIFN-α) release, thus circumventing the use of suppressors. Resulting mRNA and protein were expressed at high levels for over 48 h, thus obviating daily transfections. Using this method, we demonstrated swift activation of pluripotency associated genes in human fibroblasts. Low oxygen conditions further facilitated colony formation. Differentiation into different germ layers was confirmed via teratoma assay. Reprogramming with non-synthetic mRNA holds great promise for safe generation of iPSCs of human origin. Using the protocols described herein we hope to make this method more accessible to other groups as a fast, inexpensive, and non-viral reprogramming approach.

Robustness is a ubiquitous property of biological systems, however, underlying mechanisms that help reinforce the optimal phenotypes despite environmental or physiological perturbations are poorly understood. C. elegans development consists of four larval stages (L1-L4) and well-characterized invariant cell lineages, within which the heterochronic pathway controls the order and timing of cell-fates. Environmental or physiological stress signals can slow or temporarily halt larval stage progression; remarkably, however, temporal cell-fate progression remains unaffected. We show that two widely conserved signaling pathways, insulin and TGF- β, that regulate C. elegans larval stage progression in response to starvation and crowding, respectively, also regulate a rewiring of the heterochronic pathway so that cell-fates remain temporally anchored to appropriate larval stages. This rewiring is mediated by the nuclear hormone receptor DAF-12, and it involves a shift from the reliance on let-7-family microRNAs to the reliance on LIN-46 for proper downregulation of the transcription factor, Hunchback-like-1 (HBL-1), which promotes L2 cell-fates and opposes L3 cell-fates. LIN-46 (which is a homolog of bacterial molybdopterin molybdenum transferase (moeA) and human gephyrin) post-translationally inhibits HBL-1 activity. LIN-46 expression is repressed by the RNA-binding protein LIN-28 at the early stages to permit HBL-1 activity and hence the proper execution of L2 cell-fates. Our results indicate that robustness mechanisms of temporal cell-fate progression in C. elegans involves 1) coordinated regulation of temporal cell-fates and larval stage progression and 2) collaboration between translational regulation exerted by microRNAs and post-translational regulation exerted by LIN-46 to coordinate HBL-1 downregulation with stage progression.

Cancer immunotherapy has gained attention in recent years for its successes in potentiating immune responses that can elicit tumor control. In particular, adoptive cell therapy (ACT), which involves the autologous/allogeneic transplant of ex vivo-cultivated tumor-specific T lymphocytes, can mediate potent tumor recognition and killing; however, durable clinical responses are often difficult to obtain in solid tumors. Solid tumors and their unique microenvironments have the capacity to evade and suppress antitumor immune responses and represent significant hurdles for effective ACT. Recently, we have discovered that chemical inhibition of histone deacetylases via MS-275 (Entinostat) during ACT can subvert tumor resistance to foster potent, broad-spectrum antitumor immunity. Overall, the work described supports the efficacy of ACT in the treatment of immunosuppressive, solid tumors; however, consistency in durable clinical outcomes can only be achieved through the concurrent therapeutic targeting of tumor resistance mechanisms. This thesis uses pre-clinical models to describe how tumor resistance to ACT can manifest, and demonstrates that concurrent MS-275 delivery drives extensive immunomodulation to promote sustained tumor clearance. This includes: 1) The polarization of tumor-infiltrating myeloid cells into cytotoxic effectors with the ability to reject immune escape variants 2) The inflammatory remodeling of the tumor microenvironment to potentiate epitope spreading against secondary tumor antigens 3) The transcriptional reprogramming of adoptively transferred T cells to overcome tumor-burden-dependent exhaustion We expect that the results will help facilitate the development of next-generation ACT platforms that will feature strategies for multi-mechanistic perturbation of tumor resistance. / Thesis / Doctor of Philosophy (PhD) / The host immune system has the ability to recognize and destroy tumor cells. Therapeutic platforms that leverage antitumor immune cells, specifically T cells, have shown potency in the elimination of cancer. In the clinic, cancer immunotherapies have demonstrated early success against hematological malignancies; however, are unreliable in the treatment of solid tumors. Solid tumors utilize intrinsic and adapted mechanisms of resistance to mitigate the effectiveness of cancer immunotherapy. This thesis pursues research questions aimed at understanding how tumors resist immunotherapy, what mechanisms are utilized, and how to overcome these obstacles. We anticipate that these results will contribute to the development and incorporation of strategies to subvert tumor resistance and potentiate T cells against solid tumors.