Chromatin regulation is a major aspect of cancer development, progression, and treatment. Several small molecule inhibitors of chromatin regulators are currently used for treatment of certain hematological malignancies. However, there is still opportunity for many more patients to benefit from therapeutic approaches that target chromatin regulation, especially in the context of solid tumors. A critical unmet need is the identification of robust biomarkers that can guide the application of epigenetic inhibitors in a precise and personalized manner. In my dissertation, I aim to address this important knowledge gap by studying how perturbation of chromatin can target metabolic and lineage abnormalities in solid tumors for therapeutic benefit. To do this, I have focused on genetic and pharmacological perturbations of chromatin pathways in two cancer models: (1) lung adenocarcinoma (LUAD) with NRF2 activation and (2) neuroendocrine esophageal carcinoma (NEC). In the study on NRF2-active LUAD, we found that histone deacetylase (HDAC) inhibitors can be repurposed to reprogram the epigenomic and metabolic landscape, which leads to specific and potent anti-tumor effects in the context of NRF2 activation. Specifically, we employed a chromatin-focused genetic screen to identify dependencies on chromatin regulators. The screen revealed an NRF2-specific dependency on class I histone deacetylases. Experiments in mouse and human LUAD cell lines in vitro and in vivo indicated an NRF2-specific sensitivity to the class I HDAC inhibitor Romidepsin. Mechanistically, profiling of histone acetylation and gene expression upon Romidepsin treatment revealed a relative loss of histone H4 acetylation at promoters which was associated with reduced gene expression. Many downregulated genes were more essential for the survival of NRF2 hyperactive cancer cells, including genes involved in glutamine and serine metabolism, c-Myc and several of its targets involved in purine and pyrimidine synthesis. These transcriptional changes had corresponding effects on altering the metabolic pathways that NRF2-active cells selectively require for survival. In the study on neuroendocrine esophageal carcinoma (NEC), we identified a crucial role for epigenetic regulation of lineage fate through transcriptional control of the key epidermal transcription factor p63. This project originated from data from my collaborators that indicates a role for p63 in the suppression of basal-to-neuroendocrine identity transition in the developing esophagus. Consistently, I found that p63 is silenced in NEC through a non-genetic mechanism. Reintroducing p63 isoforms in a human NEC cell line showed that ΔNp63α was sufficient to restore squamous marker expression. An epigenetic drug screen assessing p63 gene expression and subsequent validation experiments revealed that inhibition of EZH2, a histone methyltransferase, induced expression of ΔNp63α and genes related to the squamous identity. Analysis of the chromatin state in the TP63 locus showed that EZH2 inhibition led to a loss histone H3 methylation and a gain of histone H3 acetylation and its reader BRD4. These results support the hypothesis that the squamous identity can be reactivated epigenetically in NEC through de-repression of ΔNp63α as a potential therapeutic strategy. Together, these studies contribute to our understanding of the transcriptional response to chromatin perturbation and show that this can be leveraged to modulate cell metabolism and identity, as well as to achieve therapeutic benefit in new contexts of cancer.

The purpose of this study was to determine the effect of prostaglandin (PG) inhibition on the sympathetic and pressor responses to isometric handgrip (HG) at 40% of maximal voluntary contraction (MVC) to exhaustion and postcontraction muscle ischemia (PC). To accomplish this heart rate (HR), arterial pressure (n=10) and plasma norepinephrine (NE) levels (n=8) were measured in 10 healthy male subjects during HG at 40% of MVC to exhaustion and during PC. The subjects were given a double-blind administration of either placebo (PLAC) or a single 100 mg dose of indomethacin (IND). The order of administration was counterbalanced and a one week drug washout period was provided between conditions. Mean arterial pressure increased 25±5 vs. 22±4 mmHg during the second minute of HG and 26±2 vs. 21±5 during the last minute of PC in PLAC vs. IND (P>.05), respectively. Heart rate was increased 21±4 vs. 17±3 bpm during the second minute of HG in PLAC vs. IND (P>.05), respectively and returned to control values during PC in both trials. Plasma NE increased 343189 vs. 289±89 pg/ml after HG and 67514132 vs. 6324132 pg/ml after PC (P>.05) in PLAC vs. IND, respectively. Therefore, PG inhibition does not alter sympathetic or arterial pressure responses during sustained isometric exercise in humans. This may suggest that 1) PGs not important in metaboreceptor stimulation of sympathetic or pressor responses to sustained isometric contractions in humans or 2) PGs may play only a small role in the regulation of these variables which may be masked by the effects of other stimuli. Index terms: prostaglandins, pressor reflex, muscle sympathetic nerve activity, static exercise / Ph. D.

Background: The Atlantic salmon, Salmo salar L., is a prominent member of the Salmonidae family, and has been the focus of intense research because of its environmental and economic significance as an iconic sporting species and its global importance as an aquaculture species. Furthermore, salmonids constitute ideal organisms for the study of evolution by gene duplication as they are pseudotetraploid descendants of a common ancestor whose genome was duplicated some 25 to 100 million years ago. Whole-genome duplication is considered a major evolutionary force capable of creating vast amounts of new genetic material for evolution to act upon, promoting speciation by acquisition of new traits. Recently, large-scale comparison of paralogous genes in Atlantic salmon suggested that asymmetrical selection was acting on a significant proportion of them. However, to elucidate the physiological consequences of gene and genome duplications, studies integrating molecular evolution and functional biology are crucial. To this end, sequence and molecular analyses were performed on duplicated Elovl5 fatty-acyl elongases of Atlantic salmon, as they are responsible for a rate-limiting reaction in the elongation process of long-chain polyunsaturated fatty acids (LC-PUFA), critical components of all vertebrates. The aim of the research presented here was to investigate the role of gene duplication as an evolutionary process capable of creating genetic novelty, and to identify the potential ecological and physiological implications. Results: Linkage analyses indicated that both fatty-acyl elongases segregated independently and located elovl5 duplicates on different linkage groups. Genetic mapping using microsatellites identified in each elovl5 locus assigned elovl5a and elovl5b to chromosomes ssa28 and ssa13, respectively. In silico sequence analysis and selection tests indicated that both salmon Elovl5 proteins were subject to purifying selection, in agreement with previous results showing indistinguishable substrate specificities. Gene expression and promoter analysis indicated that Elovl5 duplicates differed in response to dietary lipids and tissue expression profile. Lipid biosynthesis and metabolic gene expression profiling performed in Atlantic salmon SHK-1 cells, suggested that the control of lipid homeostasis in fish is similar to that described in higher vertebrates, and revealed the particular importance of Lxr and Srebp transcription factors (TFs) in the regulation of LC-PUFA biosynthetic enzymes. Sequence comparison of upstream promoter regions of elovl5 genes showed intense differences between duplicates. Promoter functional analysis by co-transfection and transcription factor transactivation showed that both elovl5 duplicates were upregulated by Srebp overexpression. However, elovl5b exhibited a higher response and its promoter contained a duplication of a region containing response elements for Srebp and NF-Y cofactors. Furthermore, these studies indicated an Lxr/Rxr dependant response of elovl5a, which was not observed in elovl5b. Analysis of the genomic sequences of elovl5 duplicates by comparison to various sequence databases showed an asymmetrical distribution of transposable elements (TEs) in both introns and promoter regions. Further comparison to introns of the single elovl5 gene in pike indicated much higher TE distribution in salmon genes compared to the pike. Conclusions: Although not conclusive, the most parsimonious origin for the salmon elovl5 duplicates is that they are derived from a WGD event. This conclusion is also supported by the close similarity of two elovl5 paralogs in the recently available rainbow trout genome. Regardless of their origin, Atlantic salmon elovl5 genes have been efficiently retained in the genome under strong functional constraints indicating a physiological requirement for both enzymes to be functionally active. In contrast, upstream promoter regions have strongly diverged from one another, indicating a relaxation of purifying selection following the duplication event. This divergence of cis-regulatory regions has resulted in regulatory diversification of the elovl5 duplicates and regulatory neofunctionalisation of elovl5a, which displayed a novel Lxr/Rxr-dependant response not described in sister or other vertebrate lineages. Promoter analysis indicated that the observed elovl5 differential response to dietary variation could be partly attributed to varying transcriptional regulation driven by lipid-modulated TFs. The distribution of TEs in elvol5 genes of Atlantic salmon shows a clear increase in TE mobilisation after the divergence of esocids and salmonids. This must have occurred after the elongase duplication and thus the salmonid WGD event and contributes to the observed regulatory divergence of elovl5 paralogs.

La liaison de l’insuline à son récepteur permet le recrutement de protéines adaptatrices, ce qui conduit notamment à l’activation de la voie mitogénique des MAPK et des voies impliquées dans le métabolisme du glucose. Deux voies complémentaires contribuent au recrutement du transporteur de glucose, GLUT4, à la membrane ;la voie de la PI3-kinase qui implique la formation du messager secondaire PtdIns(3,4,5)P3 conduisant à l’activation de la PKB et la voie de la petite protéine G, TC10, qui implique les protéines APS, CAP et c-Cbl. <p><p>La phosphatidylinositol 5-phosphatase 2 (SHIP2) contrôle négativement la voie des MAPK par interaction avec des acteurs de la cascade et la voie de la PI3-kinase en hydrolysant le PtIns(3,4,5)P3 en PtIns(3,4)P2. De plus, il a été montré dans notre laboratoire que SHIP2 peut interagir directement avec la protéine CAP ainsi que co-immunoprécipiter avec le récepteur de l’insuline et c-Cbl, participant ainsi à un complexe multiprotéique formé des protéines CAP et c-Cbl et du récepteur. Au cours de ce travail, nous avons tenté de mieux comprendre l’implication moléculaire de SHIP2 dans la cascade TC10. Comme APS est la première protéine de la cascade à être recrutée au récepteur suite à une stimulation par l’insuline et qu’elle peut interagir directement avec le récepteur et les protéines CAP et c-Cbl, nous avons étudié dans un premier temps le lien potentiel entre APS et SHIP2.<p><p>Nous avons montré que SHIP2 interagit de manière directe avec la protéine adaptatrice APS tant dans un système de sur-expression (CHO-IR) que dans un système endogène (3T3-L1). Bien qu’une stimulation par l’insuline ne semble pas modifier cette interaction, elle induit néanmoins le recrutement d’une fraction des protéines APS et SHIP2 du cytoplasme vers la membrane plasmique. L’étude des domaines d’interaction a montré que la région centrale de SHIP2 qui comprend le domaine catalytique est nécessaire pour cette association. <p><p>Nous avons ensuite montré que cette association entre APS et SHIP2 peut moduler certaines de leurs propriétés biochimiques. D’une part, bien que la sur-expression de SHIP2 n’influence pas le recrutement d’APS au récepteur, SHIP2 diminue, indépendamment de son activité enzymatique, la phosphorylation sur tyrosine d’APS induite par l’insuline et l’interaction entre APS et c-Cbl, qui sont deux étapes cruciales dans la cascade TC10. Ainsi, SHIP2 pourrait non seulement influencer la cascade de l’insuline par son activité enzymatique, mais également par interaction avec des acteurs de la cascade. D’autre part, APS augmente l’activité 5-phosphatase de SHIP2 dans un test in vitro. Elle pourrait ainsi, outre son rôle positif dans la cascade, participer au rétrocontrôle négatif de la voie de signalisation à l’insuline. Finalement, nous avons déterminé comment ces deux protéines influencent les cascades de l’insuline. Alors qu’APS n’influence pas l’activation de la PKB, ni le taux de PtdIns(3,4,5)P3, la sur-expression d’APS et de SHIP2 induit une inhibition plus forte de l’activation de la PKB comparée à celle provoquée par SHIP2 seul. De plus, une sur-expression de SHIP2 abolit l’augmentation induite par APS de la phosphorylation des MAPK. Cette activation des MAPK par APS semble dépendre de sa liaison au récepteur car les domaines PH et essentiellement SH2 sont indispensables pour cet effet positif.<p><p>En conclusion, nous avons mis en évidence l’existence d’une association entre APS et SHIP2. Cette interaction modifie certaines de leurs propriétés biochimiques et fournit un nouveau mécanisme d’action pour ces protéines dans le contrôle négatif de la voie de signalisation à l’insuline. <p><p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

The cellular regulation of acidification and intracellular ph (pHi) was studied in the integument of Rana pipiens, a model renal epithelium. Acidification was enhanced by : (1) chronic metabolic acidosis, (2) high salinity adaptation, and (3) ibuprofen treatment.

Calcium (Ca2+) plays an important role as a second messenger, transmitting the message of arrival of stimuli such as hormones and neurotransmitters to the intracellular system that carries out the cellular response to the stimulus. The universality of Ca2+ as an intracellular messenger depends on its enormous versatility. This versatility is exploited to control processes as diverse as fertilization, proliferation, development, learning and memory, contraction and secretion, and must be accomplished within the context of Ca2+ being highly toxic. Ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs) are Ca2+ -release channels located on intracellular membranes of the endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR) that perform essential functions as key targets of hormone/neurotransmitter action to initiate intracellular Ca2+ signals. The purpose of this project was to study the role of RyR2 in Ca2+ signaling in the NG115-401L neuronal cell line. siRNA transfection methods were employed to knockdown RyR2 expression levels in NG115-401L cells. We used reverse transcription and real-time PCR to evaluate RyR2 gene expression in transfected/untransfected cells. We also evaluated cytosolic Ca2+ changes induced by RyR activators or regulators, using fura-2 AM as the Ca2+ indicator. Successful RyR2 gene knockdown allowed us to carry out some initial experiments to characterize the specific roles played by the RyR2 receptor isoform. We examined cell responses to FK-506 under the condition of RyR2 knockdown, finding that RyR2 appears to confer selectivity to this response. Finally, the effects of siRNA transfection and FK-506 treatment on NG115-401L cell growth were evaluated. These experimental results may contribute to future studies of RyR2, and help develop novel treatments for RyR2-base d dysfunctional diseases.

Skin is the largest organ in the body that protects the organism from environmental, chemical and physical traumas of each passing day. The protective skin epidermal permeability barrier (EPB) is formed within the exterior layers of the epidermis, which are regularly sloughed off and repopulated by movement of inner cells. The epidermal permeability barrier is established during in utero development and maintained through lifetime. Impaired epidermal barrier formation is one of the major features of several dermatoses such as psoriasis and atopic dermatitis. Chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein 2 (Ctip2), also known as Bcl11b, is a C���H��� zinc finger protein expressed in many organs and tissues. It has been shown to regulate the development of thymocyte, tooth and corticospinal motor neurons. Ctip2 is highly expressed in mouse epidermis during skin organogenesis and in adulthood. It is crucial for epidermal homeostasis and protective barrier formation in developing mouse embryos. Germline (Ctip2- null mice) and selective ablation of Ctip2 in mouse epidermis (Ctip2[superscript ep-/-] mice) leads to increased transepidermal water loss (TEWL), impaired epidermal proliferation and terminal differentiation as well as altered lipid distribution during embryogenesis. Sphingolipids account for ~50% of total skin lipids by weight and are crucial components of epidermal barrier. We have recently identified Ctip2 as a key regulator of skin lipid metabolism. Germline deletion of Ctip2 in mouse embryos leads to altered lipid composition in the developing mouse epidermis by modulating the expression levels of key enzymes involved in lipid metabolism (bio-synthesis and catabolism). We also demonstrated that Ctip2 is recruited to the promoter regions of several genes involved in the ceramide and sphingomyelin biosynthesis pathways and could directly regulate their expression. Thus, we have identified Ctip2 as a key regulator of several lipid metabolizing genes and hence epidermal sphingolipid biosynthesis during skin development. To study the role of Ctip2 in adult skin homeostasis, we have utilized Ctip2[superscript ep-/-] mouse model in which Ctip2 is selectively deleted in epidermal keratinocytes. We showed that keratinocytic ablation of Ctip2 leads to atopic dermatitis (AD)-like skin inflammation, characterized by alopecia, pruritus and scaling, as well as high infiltration of T lymphocytes and immune cells. We have also observed increased expression of Th2-type cytokines and chemokines in the mutant skin, as well as systemic immune responses that share similarity with human AD patients. Furthermore, we discovered that thymic stromal lymphopoietin (TSLP) expression is significantly upregulated in the mutant epidermis as early as postnatal day 1 and Ctip2 was recruited to the promoter region of the TSLP gene in mouse epidermal keratinocytes. The results suggest that upregulation of TSLP expression in the Ctip2[superscript ep-/-] epidermis could be due to a derepression of gene transcription in absence of Ctip2. Thus, our data demonstrated a cell-autonomous role of Ctip2 in barrier maintenance and epidermal homeostasis in adult skin, as well as a non-cell autonomous role of keratinocytic Ctip2 in suppressing skin inflammatory responses by regulating the expression of Th2-type cytokines in adult mouse skin. Present results establish an initiating role of epidermal TSLP in AD pathogenesis via a novel repressive regulatory mechanism mediated by Ctip2 in mouse epidermal keratinocytes. Altogether, our study indicates that Ctip2 could be involved in a diverse range of biological events in skin including barrier formation, maintenance and epidermal homeostasis. Ctip2 appears to be a master regulator in skin barrier functions by directly regulating the transcription of a subset of genes involved in lipid metabolism and inflammatory responses. / Graduation date: 2013

Lignin polymers provide natural rigidity to plant cell walls by forming complex molecular networks with polysaccharides such as cellulose and hemicellulose. This evolved strategy equips plants with recalcitrance to biological and chemical degradation. While naturally beneficial, recalcitrance complicates the use of inedible plant materials as feedstocks for biofuel production. Genetically modifying lignin biosynthesis is an effective way to generate varieties of bioenergy crops with reduced recalcitrance, but certain lignin-modified plants display undesirable phenotypes and/or unexplained effects on lignin composition, suggesting that the process and regulation of lignin biosynthesis is not fully understood. Given the intrinsic complexities of metabolic pathways in plants and the technical hurdles in understanding them purely with experimental methods, the objective of this dissertation is to develop novel computational tools combining static, constraint-based, and dynamic, kinetics-based modeling approaches for a systematic analysis of lignin biosynthesis in wild-type and genetically engineered plants. Pathway models are constructed and analyzed, yielding insights that are difficult to obtain with traditional molecular and biochemical approaches and allowing the formulation of new, testable hypotheses with respect to pathway regulation. These model-based insights, once they are verified experimentally, will form a solid foundation for the rational design of genetic modification strategies towards the generation of lignin-modified crops with reduced recalcitrance. More generically, the methods developed in this dissertation are likely to have wide applicability in similar studies of complex, ill-characterized pathways where regulation occurring at the metabolic level is not entirely known.

Current feed formulations within the aquaculture industry have tended to rely on high dietary lipid thus offsetting relatively expensive protein as a source of energy. In this way, protein can be ‘spared’ for synthesis of new tissue and the high lipid content can also fulfil both fish and consumer essential fatty acid (EFA) requirements. However, the main disadvantage of feeding high lipid levels to farmed fish is a surplus of fat deposition in the flesh and other important tissues, which can detrimentally impact on quality characteristics central to the human consumer. However, based on previous work in other animal models, it is entirely feasible that supplementation of the diet with bioactive fatty acids such as conjugated linoleic acid (CLA) and tetradecylthioacetic acid (TTA) may mitigate the deleterious effects of feeding farmed fish high fat diets by reducing fat deposition in particular. The general objective of this research work was to test the hypothesis that CLA and/or TTA could augment growth, reduce fat deposition and enhance fatty acid composition via incorporation of these bioactive fatty acids, and increase n-3 highly unsaturated fatty acid (HUFA) levels in the flesh of commercially important fish species such as Atlantic salmon (Salmo salar), Atlantic cod (Gadus morhua L.) and rainbow trout (Oncorhynchus mykiss). This project also considered the influence of CLA and TTA on enzymes and transcription factors thought to be pivotal in lipid metabolism and fatty acid oxidation in particular. A subsidiary aim of this research work was to investigate the immunological impact of dietary CLA and TTA administration in these fish. The results of this project have revealed that the hypothesis was only partly proved. There was no effect in growth or biometry after either CLA or TTA supplementation in any of the fish species investigated. Additionally, there were few physiologically significant effects on fat levels on fish as a result of TTA or CLA administration. However, there were a number of effects on fatty acid metabolism including inhibition of steroyl coenzyme desaturase (SCD) in cod and trout in particular and also enhancement of hepatic n-3 HUFA levels in trout. Importantly, it was determined that both TTA and CLA could be incorporated into the flesh thus providing a vehicle through which these bioactive fatty acids can be delivered to the consumer. There were also a number of beneficial effects on activity and gene expression of a number of enzymes and transcription factors thought to be fundamental to the modulation of fatty acid oxidation in particular. However, the effects on gene transcription and biochemistry had little impact at the whole body level. This research work also showed that there were no detrimental effects on immune status after supplementation with dietary CLA or TTA. Conclusively, this thesis has contributed to the overall understanding of the influence of dietary CLA and TTA in farmed fish.

No description available.