No description available.

No description available.

No description available.

No description available.

<p>RanBPM is a conserved putative scaffold protein of unknown function. Loss-of-function in <em>RanBPM</em> leads to pleiotropic phenotypes such as reduced locomotion, decreased size and larval lethality in the <em>Drosophila melanogaster</em>.</p> <p><em>dRanBPM</em> mutants have decreased branching and boutons at the neuromuscular junction, which may contribute to their locomotory defect. To investigate if dRanBPM is involved in controlling synaptic architecture at the neuromuscular junction, levels of two cytoskeletal proteins, Futsch and profilin, were assessed in <em>dRanBPM</em> mutants.</p> <p>Due to time constraints, immunoblots for Futsch were not fully optimized for protein measurement. Immunoblots for profilin, on the other hand, were successfully carried out. However, results from the reproduction of a blot demonstrating the negative regulation of <em>Drosophila</em> FMRP on profilin did not agree with that of the literature. In addition, results from an epistatic experiment demonstrated that profilin levels were not affected in FMRP deficient flies when compared to those with additional decrease in dRanBPM function.</p> <p>Targeted expression of <em>dRanBPM</em> to neurosecretory cells is able to rescue size and lethality of <em>dRanBPM</em> mutants, suggesting a common pathway through which both phenotypes operate is disrupted in these mutants. Activation of the insulin signaling pathway was indeed found to be downregulated in <em>dRanBPM</em> mutants. A longevity assay was alternatively carried out to demonstrate decreased insulin pathway activation in <em>dRanBPM</em> mutants. Unfortunately, due to inappropriate controls used for this experiment, no conclusive points can be made. Together, these findings contribute to the knowledge that RanBPM plays and to designing future experiments to test for RanBPM function.</p> / Master of Science (MSc)

Recent breakthroughs in high-throughput biotechnology have promoted the integration of multi-platform data to investigate signal transduction pathways within a cell. In order to model complicated dynamics and heterogeneity of biological pathways, sophisticated computational models are needed to address unique properties of both the biological hypothesis and the data. In this dissertation work, we have proposed and developed methods using Markov Chain Monte Carlo (MCMC) techniques to solve complex modeling problems in human cancer research by integrating multi-platform data. We focus on two research topics: 1) identification of transcriptional regulatory networks and 2) uncovering of aberrant intracellular signal transduction pathways. We propose a robust method, called GibbsOS, to identify condition specific gene regulatory patterns between transcription factors and their target genes. A Gibbs sampler is employed to sample target genes from the marginal function of outlier sum of regression t statistic. Numerical simulation has demonstrated significant performance improvement of GibbsOS over existing methods against noise and false positive connections in binding data. We have applied GibbsOS to breast cancer cell line datasets and identified condition specific regulatory rewiring in human breast cancer. We also propose a novel method, namely Gibbs sampler to Infer Signal Transduction (GIST), to detect aberrant pathways that are highly associated with biological phenotypes or clinical information. By converting predefined potential functions into a Gibbs distribution, GIST estimates edge directions by learning the distribution of linear signaling pathway structures. Through the sampling process, the algorithm is able to infer signal transduction directions which are jointly determined by both gene expression and network topology. We demonstrate the advantage of the proposed algorithms on simulation data with respect to different settings of noise level in gene expression and false-positive connections in protein-protein interaction (PPI) network. Another major contribution of the dissertation work is that we have improved traditional perspective towards understanding aberrant signal transductions by further investigating structural linkage of signaling pathways. We develop a method called Structural Organization to Uncover pathway Landscape (SOUL), which emphasizes on modularized pathways structures from reconstructed pathway landscape. GIST and SOUL provide a very unique angle to computationally model alternative pathways and pathway crosstalk. The proposed new methods can bring insight to drug discovery research by targeting nodal proteins that oversee multiple signaling pathways, rather than treating individual pathways separately. A complete pathway identification protocol, namely Infer Modularization of PAthway CrossTalk (IMPACT), is developed to bridge downstream regulatory networks with upstream signaling cascades. We have applied IMPACT to breast cancer treated patient datasets to investigate how estrogen receptor (ER) signaling pathways are related to drug resistance. The identified pathway proteins from patient datasets are well supported by breast cancer cell line models. We hypothesize from computational results that HSP90AA1 protein is an important nodal protein that oversees multiple signaling pathways to drive drug resistance. Cell viability analysis has supported our hypothesis by showing a significant decrease in viability of endocrine resistant cells compared with non-resistant cells when 17-AAG (a drug that inhibits HSP90AA1) is applied. We believe that this dissertation work not only offers novel computational tools towards understanding complicated biological problems, but more importantly, it provides a valuable paradigm where systems biology connects data with hypotheses using computational modeling. Initial success of using microarray datasets to study endocrine resistance in breast cancer has shed light on translating results from high throughput datasets to biological discoveries in complicated human disease studies. As the next generation biotechnology becomes more cost-effective, the power of the proposed methods to untangle complicated aberrant signaling rewiring and pathway crosstalk will be finally unleashed. / Ph. D.

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Les maladies dégénératives de la rétine sont une des causes principales de cécité. Parmi différentes stratégies thérapeutiques actuellement étudiées, notre équipe s’intéresse au potentiel régénératif de la rétine. Une source cellulaire d'intérêt sont les cellules de Müller, principal type de cellules gliales de la rétine, capables de se réactiver en cas de dégénérescence et d’adopter certaines caractéristiques de cellules souches. Elles entrent alors dans un état appelé gliose réactive. Tandis que chez certaines espèces comme le poisson, elles permettent la régénération de la rétine, elles ont des capacités régénératives très limitées et inefficaces chez les mammifères. Une meilleure connaissance des mécanismes moléculaires régissant la gliose réactive des cellules de Müller est donc essentielle si l’on veut identifier des cibles thérapeutiques capables de stimuler le potentiel de régénération de ces cellules. Dans ce contexte, le but de mon projet de thèse a été d’étudier le rôle du co-facteur de transcription YAP dans la réactivation des cellules de Müller. Cette protéine est l’effecteur de la voie de signalisation Hippo, connue pour son implication dans la régulation des cellules souches et la régénération de certains organes.Dans un premier temps, nous avons réalisé une analyse transcriptomique qui a montré que la voie Hippo/YAP est une des principales voies dérégulées dans un modèle de dégénérescence rétinienne chez la souris. Nous avons ensuite montré que la protéine YAP est spécifiquement exprimée dans les cellules de Müller et que son expression et son activité transcriptionnelle sont augmentées au cours de la dégénérescence lorsque les cellules de Müller deviennent réactives. Ces données suggèrent pour la première fois un lien entre YAP et la gliose réactive dans la rétine. Par conséquent, dans un second temps, mon projet de thèse a consisté en l’étude fonctionnelle de YAP dans les cellules de Müller. Dans ce but, nous avons généré par croisements chez la souris un modèle inductible de délétion du gène Yap spécifiquement dans ces cellules. Ce modèle a permis de montrer qu’en absence de Yap en conditions physiologiques, plusieurs gènes spécifiques des cellules de Müller sont dérégulés, suggérant un dysfonctionnement de ces cellules. L’étude phénotypique a permis de révéler que ces dérégulations moléculaires conduisent à un vieillissement prématuré des cellules de Müller et à une baisse de la vision chez les souris âgées. Ces données suggèrent que YAP est requis pour le fonctionnement normal des cellules gliales de Müller. Nous avons ensuite examiné l’impact de la perte de Yap dans les cellules de Müller en conditions de dégénérescence des photorécepteurs. Une analyse transcriptomique a permis de montrer que différents aspects de la réponse moléculaire des cellules de Müller réactives sont affectés. Parmi les processus biologiques dérégulés, nous nous sommes intéressés à la régulation de la prolifération cellulaire. Nous avons montré que YAP est nécessaire à l’augmentation de l’expression de gènes associés à la réentrée dans le cycle cellulaire de la glie de Müller. Par ailleurs, nos résultats suggèrent que des composants de la voie de signalisation EGFR, connue pour son rôle central dans la réactivation des cellules de Müller, sont régulés par YAP.Dans l’ensemble, ces résultats révèlent l’importance de YAP (i) dans le fonctionnement des cellules de Müller en conditions physiologiques pour maintenir l’homéostasie rétinienne, et (ii) dans la régulation des processus de réactivation de ces cellules en conditions dégénératives. De plus, ces données permettent de proposer un modèle selon lequel YAP serait impliqué dans le contrôle de la réentrée des cellules de Müller dans le cycle cellulaire via une interaction avec la voie de signalisation EGFR. Ce travail a donc contribué à approfondir nos connaissances du réseau de signalisation impliqué dans la réactivation des cellules de Müller de la rétine des mammifères. / Retinal dystrophies are one of the main causes of blindness. Among the different therapeutic strategies currently studied, our team is interested in the regenerative potential of endogenous retinal cells. A cellular source of interest are Müller cells, which are the main type of glial cells in the retina. These cells are able to reactivate in case of retinal degeneration and adopt various characteristics of stem cells. They enter a state called reactive gliosis. While in some species such as the fish, they allow the complete regeneration of the retina, they have very limited and ineffective regenerative capacities in mammals. Increasing our knowledge of the complex molecular response of Müller cells to retinal degeneration is thus essential for the development of promising new therapeutic strategies. In this context, the aim of my thesis project was to study the role of the co-transcription factor YAP in Müller cells reactivation. This protein is the main effector of the Hippo signaling pathway which is a crucial player in the field of stem cell biology and regeneration.As a first step, we performed a transcriptomic analysis, which revealed that the Hippo/YAP pathway is one of the main signaling deregulated in a mouse model of photoreceptor degeneration. In particular, we found that YAP is specifically expressed in Müller cells and strongly upregulated upon retinal degeneration, when these cells are reactivated. We thus uncovered for the first time a link between the Hippo/YAP pathway and reactive gliosis in the retina. Consequently, the second part of my thesis project was to undertake a functional study of YAP in Müller cells. For this purpose, we generated, by crossing, a mouse model allowing for Yap conditional knockout specifically in these cells. This model allowed us to show that Yap deletion leads to deregulation of several Müller cell specific genes. A phenotypic analysis revealed that these molecular deregulations lead to premature aging of Müller cells and visual defects in old mice. These results suggest that YAP is required for normal function of Müller glial cells. We then studied the impact of Yap deletion in Müller cells under degenerative conditions. A transcriptomic analysis revealed that various aspects of the molecular response of reactive Müller cells are affected in the absence of Yap. Among the deregulated biological processes, we focussed in particular in the regulation of cell proliferation. We found that YAP is required to trigger cell cycle gene upregulation that occurs in Müller glial cells following photoreceptor cell death. Furthermore, our results suggest that some components of the EGFR signaling pathway, which is known for its central role in the reactivation of Müller cells in pathological conditions, are regulated by YAP in Müller cells.Taken together, these results highlight the importance of YAP (i) in Müller cell function under physiological conditions to maintain retinal homeostasis, and (ii) in the regulation of Müller cell reactivation process under degenerative conditions. Moreover, these data allow us to propose a model in which YAP would be involved in the control of Müller glia cell cycle re-entry through its interaction with the EGFR signaling pathway. Therefore, this work has contributed to increase our knowledge of the signaling network involved in the reactivation of Müller cells in the mammalian retina.

Endometrial adenocarcinoma (EAC) is the most frequently diagnosed gynecological cancer of the female genital tract in the Western world. Research studies in EC is difficult to conduct on human tumor samples due to the complex nature of tumor arousal and genetic heterogeneousness in the human population. Therefore, inbred animal models can be promising tools to use in EC research due to similar histopathology and pathogenesis as humans. Studies performed on MAP2K1 and CCND1 has shown that their altered expression play a crucial role in carcinogenesis. CCND1 has been demonstrated to have oncogenic properties when overexpressed in human neoplasias. The aim of this study is to investigate gene expression levels of MAP2K1 and CCND1 in BDII rat model of endometrial adenocarcinoma cells. Quantitative real-time PCR was used to analyze expression levels of MAP2K1 and CCND1 genes in BDII/Han rat model of endometrial cancer cells using TaqMan approach. The differences in gene expression levels of MAP2K1 and CCND1 between pathologically EAC malignant and nonmalignant cells showed an upregulation of MAP2K1 and CCND1 in EAC malignant cells. The analyzed data presented observable mean differences between MAP2K1 and CCND1 in several endometrial cell lines that were examined. Although no statistical significance was reached, an alteration in gene expression levels in malignant and nonmalignant endometrial cells could be observed. Furthermore, this present study shows observable upregulation of MAP2K1 and CCND1 in endometrial carcinoma cells vs. nonmalignant endometrium cells and encourages further investigation of the role of CCND1 and MAP2K genes in endometrial carcinogenesis.

Our laboratory is primarily interested in novel pharmacological intervention of cell proliferation and survival pathways expressed in various types of cancer. These cyto-protective pathways can be activated in response to growth factor stimulation, toxic insult and radiation. In our studies, we utilized novel drug combinations with and without radiation to enhance breast & prostate tumor cell death both in vitro and in vivo. Previous studies from our group have shown that UCN-01 and MEK1/2 inhibitors interact to cause tumor cell death in transformed cell lines in vitro. We extended this observation to an in vivo animal model system using the estrogen dependent breast cell carcinoma line MCF-7 and the estrogen independent breast cell carcinoma line MDA-MB-231. This drug combination was shown to profoundly reduce tumor cell proliferation in vivo and also exhibited the ability to significantly reduce ex-vivo tumor cell colony formation 30 days after cessation of the combination drug treatment. In addition, tumor cell death coincided with decreased ERK112 phosphorylation, reduced immunoreactivity of Ki67 and CD31. Overall, these studies demonstrate that UCN-01 and MEK112 inhibitors have the potential to suppress mammary tumor growth in vivo which is independent of p53 status, estrogen dependency, caspase-3 levels or oncogenic K-RAS expression. In our LnCap prostate carcinoma cell studies we demonstrated the impact of hCG and lovastatin in combination with ionizing radiation to radiosensitize and enhance tumor cell lethality. This enhancement was attributed to the hCG-induced activation of ERBB1 via a GPCR, MEK112 and metalloprotease dependent paracrine mechanism which was further enhanced by radiation. This enhanced cell killing effect was shown to involve prolonged activation of PARP1 which could be suppressed by inhibition of ERBB1, MEKl , PI3 kinase or PARP1. Therefore, the combination of hCG, lovastatin and radiation may represent a novel approach to kill prostate cancer cells and potential new therapy.