No description available.

Autophagy, as the name suggests, is a cellular process of self-eating in which cytoplasmic debris is engulfed by a double membrane vesicle dubbed the autophagosome and is ultimately degraded and recycled by proteases in the lysosome. The process is initiated by a group of core ATG proteins, including a multi-pass transmembrane protein called ATG9A. Although ATG9A has been shown to be essential for both stress induced and basal autophagy, its mechanism and interaction network remain largely illusive. Our current study employs BioID proteomics to identify a network of interactors, including regulators of membrane fusion and vesicle trafficking, such as TRAPP, EARP, GARP, exocyst, AP-1 and AP-4 complexes, as well as members of the ULK1 autophagy kinase complex. Further investigations confirm that two components of the ULK1 complex, ATG13 and ATG101, directly interact with ATG9A. Using CRISPR, we show that deletion of ATG13 or ATG101 disrupts ATG9A trafficking and causes an accumulation of ATG9A at p62/SQSTM1-positive ubiquitin clusters. Lentivirus reconstitution and split-mVenus approaches using an ULK1 binding deficient mutant of ATG13 reveal that ATG9A interacts with ATG13 and ATG101 in an ULK1-independent manner. Together, these data reveal ATG9A interactions in vesicle trafficking and autophagy pathways, including a role for an ULK1- independent ATG13 complex in regulating ATG9A.

14-3-3 proteins are among a family of phospho-binding proteins that are known to regulate many essential cellular mechanisms. By binding to sites of phosphorylation, 14-3-3s are integrated into multiple signaling pathways that govern critical processes, such as apoptosis, cell cycle progression, autophagy, glucose metabolism, and cell motility. These processes are crucial for tumorigenesis and 14-3-3 proteins are known to play a central role in facilitating cancer progression. In this study, my colleagues and I discover two novel 14-3-3 interacting proteins, ATG9A and PTOV1, that are both vital to essential cellular functions and describe various mechanisms that these two proteins regulate. ATG9A is a multi-pass transmembrane lipid scramblase that is found primarily as a homotrimer in the ER or small ATG9A vesicles. It is essential in the cellular recycling process called autophagy and is believed to act at the earliest stages of autophagy by providing the seed for the growth of the double membrane vesicle called an autophagosome. Previous work in our lab demonstrated that upon hypoxic stress, AMPK, the master nutrient-sensing kinase, phosphorylates S761 on the C-terminus of ATG9A. This triggers the binding of 14-3-3ζ to contribute to ATG9A function in hypoxia induced autophagy. Despite this revelation, the exact function of ATG9A is still poorly understood, especially in unstimulated conditions where autophagy functions at a basal level and AMPK is inactive. In this study, we sought to understand ATG9A function more broadly by identifying novel interactors of ATG9A and the role ATG9A plays in basal autophagy. To do this, we employed BioID mass spectrometry and various biochemical approaches to identify LRBA as a bona fide ATG9A interactor and autophagy regulator. Furthermore, using deuterium labeling and quantitative whole proteome mass spectrometry, and various other biochemical techniques, we show that ATG9A regulates the basal degradation of p62 and is recruited to sites of basal autophagy by active poly-ubiquitination to initiate basal autophagy. PTOV1 is an oncogenic protein that is poorly understood. Our current understanding of PTOV1 is limited to a few studies, which demonstrate that PTOV1 is highly expressed in primary prostate tumor samples and is correlated with metastasis, drug resistance, and poor clinical outcomes. In this study, we identify a mechanism by which SGK2, a poorly understood kinase, phosphorylates PTOV1 at S36 to trigger 14-3-3 binding at that site to increase PTOV1 stability in the cytosol and increase c-Jun expression. Upon SGK2 inhibition, 14-3-3 releases PTOV1 and PTOV1 is shuttled into the nucleus where HUWE1, an E3 ubiquitin ligase, ubiquitinates PTOV1 and initiates PTOV1 degradation by the proteasome. This is the first detailed mechanism of regulation identified for the poorly understood oncogene, PTOV1, and sheds light on potential therapeutic targets for cancer treatments.

CD133, a pentaspan glycoprotein, has long been known to represent aggressive, stem-like populations across various human malignancies. While its expression correlates with numerous clinical outcomes including disease progression, metastasis, recurrence, and poor overall survival in numerous cancers, little is currently known about its function. In the brain cancer glioblastoma (GBM), CD133-expressing cells have previously been shown to initiate tumours, evade therapy and interestingly, self-renew, a key property of cancer stem cells. With an implied signalling role in driving self-renewal, we aim to elucidate the role of CD133 in glioblastoma. To understand the role of CD133, we aim to study its protein-protein interactions using the proximity-dependent labelling technique known as miniTurboID. By tagging proteins of interest with a promiscuous biotin ligase at both protein termini, potential interactors can be biotinylated and identified by subsequent mass spectrometry. While miniTurboID has traditionally been performed by synthetic transgenes expressing the tagged proteins of interest in commercial cell lines, overexpression may not recapitulate its native function. Thus, using CRISPR technology, we aim to insert the miniTurboID ligase at both the N- and C-terminus of CD133 in patient-derived human GBM lines. Although little is currently known about CD133 function, development of targeted therapies has presented a promising strategy in pre-clinical studies. In the Singh Lab, we previously developed a chimeric antigen receptor T-cell, or CAR-T, comprised of a T-cell expressing a synthetic receptor capable of recognizing a tumor-associated antigen and activating cytolytic-killing directed towards the target cell. Currently, CAR-T therapies are autologous, or patient-derived, in nature which may host a myriad of concerns including patient-specific qualitative and quantitative T-cell dysfunction, inconsistent generation of CAR products, and availability to rapidly progressing patients. To circumvent this concern, “off-the-shelf”, donor-derived or allogeneic CAR-T products may be generated for use in GBM patients. However, in addition to CAR integration, allogeneic products must be additionally modified to eradicate expression of the endogenous TCR, as this would induce a phenomenon known as graft versus host disease, in which healthy tissues are targeted. Thus, in this thesis, we show gene editing potential in human GBMs to perform an endogenous genomic knock-in of miniTurboID. With the identification of interacting proteins, defining the subsequent functionality of CD133 may elucidate oncogenic cellular programs, and highlight common nodes of interaction within divergent cell signaling pathways. To develop an allogeneic CAR-T product, we designed a two-step approach in which the CAR sequence was integrated into the TCR gene for simultaneous knock-out. We later show early pre-clinical efficacy in comparison to traditional autologous CAR-T in our patient-derived models of human GBM. Thus, by using CD133 as a centralizing concept in this thesis, we ultimately hope to develop our biological understanding of CD133, while testing the therapeutic development of a donor-derived CAR-T therapy. / Thesis / Master of Science (MSc) / Glioblastoma (GBM) is one of the most common malignant brain tumors in adults. Despite an aggressive therapy regimen, almost all patients relapse 7-9 months post-diagnosis. Therapy failure and poor patient outcome may be attributed to a small population of cells known as glioblastoma stem cells, or GSCs, that are able to escape therapy and seed disease recurrence. GSCs are most notably identified by the cell surface protein CD133, which has previously been shown to associate with pro-tumor properties including treatment resistance, tumor growth, maintenance, progression and metastasis. While expression of CD133 in cancer has been heavily characterized, little is currently known about its function. One such avenue to understand its mechanism of action in cancer, and more particularly GBM, is to define its interactions with other proteins. Protein-protein interactions play a pivotal part as the backbone of signalling pathways that drive tumor development and growth. Therefore, defining and mapping the CD133 interaction network may help us understand how this protein governs regulation of GSCs, and ultimately, GBM progression. While the biology of CD133 has yet to be elucidated, targeting CD133 on GSCs has presented a promising therapeutic strategy for patients with GBM. Previously in the Singh Lab, we developed an engineered T-cell therapy, known as a CAR-T, that can recognize CD133 to induce tumor cell death. While this showed success in our animal models of human GBM, other considerations must be addressed on its path to clinical development. As of current, CAR-T therapies are generated from T-cells taken from cancer patients. This hosts a myriad of concerns including the quality of patient T-cells, the time and cost to manufacture, and its availability for patients with rapidly progressing disease. To circumvent this issue, donor-derived CAR-T cells can be genetically engineered for safe usage in GBM patients as a readily available, “off-the-shelf” therapy. To define the function of CD133, we have attempted to use a technique known as BioID, which tags the protein of interest with a smaller biotin ligase. This biotin ligase can subsequently tag proteins that come within the vicinity of CD133, that may later be identified by sequencing as potential interactors. As current use of BioID may not reliably mimic the interaction of CD133, we sought to genetically engineer human GBM lines with the BioID protein to more closely resemble tumor-relevant behaviours of CD133. To develop a donor-derived CAR-T therapy, we similarly used genetic engineering of T-cells to ensure specific targeting of tumor cells with CD133, while sparing healthy tissues. By using CD133 as a centralizing concept in this thesis, we ultimately hope to develop our biological understanding of CD133, while testing the therapeutic development of a donor-derived CAR-T therapy.

RAS est une GTPase qui transduit les signaux extracellulaires envers des voies de signalisation intracellulaires, en liant ses effecteurs. RAS peut activer la voie Hippo qui inhibe la croissance cellulaire et qui est souvent dérégulée dans le cancer. Les protéines RASSF suppresseurs de tumeurs relient RAS à la voie Hippo. L’expression exogène de KRASG12V avec RASSF1 ou RASSF5 conduit à l'activation de la voie Hippo, bien que KRAS et RASSF1 ne s’associent pas directement. Ce projet de maîtrise vise à identifier les protéines impliquées dans l'activation de la voie Hippo par RAS. Nous avons effectué plusieurs expériences BioID, une technique qui permet d’identifier les interacteurs proximaux d’une protéine d’intérêt, dans des lignées cellulaires U2OS stables et inductibles exprimant les protéines KRASG12V, RASSF1 ou RASSF5 seules ou coexprimées, permettant de comparer les conditions où la voie Hippo inactive ou active. Nous avons élucidé l'interactome d'un mutant de KRAS avec affinité accrue envers RASSF5 et affinité réduite envers RAF, permettant d’étudier les voies activées en aval de RASSF5, avec une activation réduite de la voie MAPK. Nos données montrent que RASSF1 et RASSF5 relâchent les kinases Hippo MST1 et MST2 lorsque la voie Hippo est active, conformément aux données in vitro démontrant un rôle inhibiteur de l'interaction RASSF/MST. De plus, nous avons démontré que KRAS est un interacteur proximal des protéines VAMP3 et SNAP23. Comprendre comment l'oncoprotéine RAS active des effecteurs et des voies de signalisation moins étudiés, en particulier ceux qui ont des fonctions suppressives de tumeurs a des implications importantes pour le développement de nouvelles thérapies ciblées pour les cancers induits par RAS. / RAS is a small GTPase that transduces signals from membrane-bound receptors to intracellular pathways, by signaling to downstream effector proteins. RAS can activate the Hippo pathway, a growth-suppressive pathway that is often dysregulated in cancer. The tumor suppressor RASSF proteins link RAS to Hippo signaling. Co-expression of KRASG12V with either RASSF1 or RASSF5 leads to Hippo pathway activation, despite KRAS and RASSF1 not being direct binding partners. This M.Sc. project aims to identify proteins that are involved in RAS-mediated activation of the Hippo pathway. We performed BioID, a proteomic technique which is used to identify the proximal interactors of a protein of interest, in stable and inducible U2OS osteosarcoma lines expressing KRASG12V, RASSF1, or RASSF5 proteins alone, as well as in lines co-expressing both KRASG12V and the RASSF proteins, allowing for a comparison between inactive and active Hippo pathway interactomes. Furthermore, we mapped the interactome of a double mutant of KRAS that displays increased affinity for RASSF5 and decreased affinity for the effector RAF, allowing us to study KRAS signaling downstream of RASSF5, with decreased activation of the MAPK pathway. Our BioID data shows that RASSF1 and RASSF5 disengage the Hippo kinases MST1 and MST2 when the Hippo pathway is active, in line with the inhibitory role of the RASSF/MST interaction observed in vitro. Furthermore, we show that KRAS is a proximal interactor of the SNARE proteins VAMP3 and SNAP23. Understanding how the oncoprotein RAS signals to less studied effectors and pathways, particularly those with tumor suppressive functions has significant implications for understanding oncogenesis, and for development of new targeted therapies for RAS-driven cancers.

Made available in DSpace on 2014-12-17T14:16:30Z (GMT). No. of bitstreams: 1 TherezaMMP_DISSERT.pdf: 3247575 bytes, checksum: b56626eecb3cf571b5b30d6c0ab92d04 (MD5) Previous issue date: 2013-03-22 / Bioidentical hormones are defined as compounds that have exactly the same chemical and molecular structure as hormones that are produced in the human body. It is believed that the use of hormones may be safer and more effective than the non-bioidentical hormones, because binding to receptors in the organism would be similar to the endogenous hormone. Bioidentical estrogens have been used in menopausal women, as an alternative to traditional hormone replacement therapy. Thermal data of these hormones are scarce in literature. Thermal analysis comprises a group of techniques that allows evaluating the physical-chemistry properties of a drug, while the drug is subjected to a controlled temperature programming. The thermal techniques are used in pharmaceutical studies for characterization of drugs, purity determination, polymorphism identification, compatibility and evaluation of stability. This study aims to characterize the bioidentical hormones estradiol and estriol through thermal techniques TG/DTG, DTA, DSC, DSC-photovisual. By the TG curves analysis was possible to calculated kinetic parameters for the samples. The kinetic data showed that there is good correlation in the different models used. For both estradiol and estriol, was found zero order reaction, which enabled the construction of the vapor pressure curves. Data from DTA and DSC curves of melting point and purity are the same of literature, showed relation with DSC-photovisual results. The analysis DTA curves showed the fusion event had the best linearity for both hormones. In the evaluation of possible degradation products, the analysis of the infrared shows no degradation products in the solid state / Horm?nios bioid?nticos s?o compostos que t?m exatamente a mesma estrutura qu?mica e molecular dos horm?nios end?genos humanos. Acredita-se que a utiliza??o desses horm?nios pode ser mais segura e eficaz que os horm?nios n?o-bioid?nticos, pois a liga??o aos receptores no organismo se daria de forma semelhante aos horm?nios end?genos. Estrog?nios bioid?nticos v?m sendo utilizado, em mulheres na menopausa, como uma alternativa ? terapia de reposi??o hormonal tradicional. Dados t?rmicos desses horm?nios s?o escassos na literatura. A an?lise t?rmica ? um conjunto de t?cnicas que possibilita medir as propriedades f?sico-qu?micas de uma subst?ncia em fun??o da temperatura. As t?cnicas t?rmicas v?m sendo utilizadas na ?rea farmac?utica em diversas aplica??es, como na caracteriza??o de f?rmacos, determina??o do grau de pureza, identifica??o de polimorfismo, estudos de estabilidade e compatibilidade. Este trabalho tem como objetivo a caracteriza??o dos horm?nios bioid?nticos estradiol e estriol atrav?s das t?cnicas t?rmicas TG/DTG, DTA, DSC, DSC-fotovisual. A partir da an?lise das curvas TG, foi poss?vel calcular os par?metros cin?ticos para as amostras. Os dados cin?ticos mostraram boa correla??o entre os diferentes modelos empregados. Tanto para o estradiol como para o estriol, foi encontrada ordem zero de rea??o, o que possibilitou a constru??o das curvas de press?o de vapor. Dados das curvas DSC e DTA sobre ponto de fus?o e pureza s?o condizentes com a literatura, sendo poss?vel correlacionar estes resultados com o DSC-fotovisual. As an?lises das curvas DTA mostraram o evento de fus?o como o de melhor linearidade para os dois horm?nios. Na avalia??o dos poss?veis produtos de degrada??o, a an?lise do infravermelho mostra que n?o houve produtos de degrada??o no estado s?lido

No description available.

Le virus de l’immunodéficience humaine de type 1 (VIH-1) est l’agent étiologique du SIDA, un rétrovirus complexe encodant les protéines accessoires : Nef, Vif, Vpr et Vpu. La fonction principale de ces protéines est de moduler l’environnement cellulaire afin de promouvoir la réplication virale. Les travaux présentés dans cette thèse portent sur la protéine virale Vpr, une protéine bien connue pour son activité d’arrêt du cycle cellulaire en phase G2/M dans les cellules en division et pour l’avantage réplicatif qu’elle confère au virus durant l’infection de cellules myéloïdes. Les évènements sous-jacents à ces deux activités restent pour l’heure mal compris. Le but des travaux regroupés dans cet ouvrage est d’identifier de nouveaux facteurs cellulaires pouvant éventuellement expliquer les activités de Vpr précédemment décrites. Pour ce faire, nous avons utilisé une approche d’identification des partenaires de proximité par biotinylation, appelée BioID. L’avantage du BioID est de permettre un marquage in cellulo des protéines à proximité de la protéine d’intérêt. La mise en place et la caractérisation de cette approche font l’objet de la première section de cette thèse. En utilisant cette approche, nous avons défini un réseau de 352 partenaires cellulaires de la protéine Vpr. Parmi ces partenaires de Vpr, plusieurs sont organisés sous forme de complexes ou réseaux protéiques incluant notamment le complexe promoteur de l’anaphase/cyclosome (APC/C) et les centrosomes. Étant donné que le complexe APC/C est l’un des principaux régulateurs du cycle cellulaire, nous avons décidé d’analyser sa relation avec Vpr. Nous avons découvert que Vpr formait un complexe non seulement avec APC1, une sous-unité essentielle du complexe APC/C, mais aussi avec les coactivateurs (CDH1 et CDC20) de ce complexe. Nous avons par la suite démontré que Vpr induisait la dégradation d’APC1 et que celle-ci pouvait être prévenue par une double-mutation N28S-G41N de Vpr. Cette dégradation d’APC1 ne semblerait pas être reliée aux activités précédemment décrites de Vpr. Ces travaux font l’objet de la seconde section de cette thèse. Enfin, dans une troisième section, des travaux effectués en collaboration et analysant la relation entre les centrosomes et Vpr sont présentés. Cette thèse identifie 200 nouveaux partenaires de Vpr, ouvrant la porte à l’exploration de nouvelles cibles et activités de Vpr. Elle décrit également une nouvelle cible de Vpr : le complexe APC/C. Globalement nos résultats contribuent à une meilleure compréhension de la façon dont le VIH-1 manipule l’environnement cellulaire de l’hôte à travers la protéine virale Vpr. / Human immunodeficiency virus (HIV-1) is the AIDS causal agent. This complex retrovirus encodes several accessory proteins; namely Nef, Vif, Vpr and Vpu; whose functions are to manipulate the cellular host environment in order to favor HIV-1 viral replication. This thesis focused on Vpr whose main activities are to induce a cell cycle arrest in the G2/M phase in dividing cells and to provide a replicative advantage to HIV-1 during infection of myeloid cells such as macrophages. The cellular mechanisms underlying these two activities are up to now misunderstood. The main goal of the work presented in this thesis is to identify new cellular factors that could potentially explain the previously described Vpr activities. To do so, we used the proximity labelling approach called BioID. The main strength of BioID is to tag in cellulo partners of the protein of interest. The development as well as optimization of the BioID approach is presented in the thesis first section. Using BioID, we defined a network containing 352 cellular partners in close proximity with the viral protein Vpr. Amongst these cellular partners, several were organized into protein complexes or networks such as the anaphase promoting complex/cyclosome (APC/C) or the centrosome. Given that APC/C is a cell cycle master-regulator, we analyzed the interplay governing Vpr and APC/C interactions. We first demonstrated that Vpr could form a complex containing the scaffolding subunit APC1. APC/C coactivators, namely CDH1 and CDC20, could also be found in association with Vpr. We next showed that Vpr was inducing APC1 degradation and that Vpr residues N28 and G41 were essential to this activity. Surprisingly, the APC1-Vpr interplay does not relate to previously described Vpr activities. This work is presented in the second section of this thesis. Lastly, in the third section, a work done in collaboration analyzed the interplay between Vpr and the centrosomes. In this thesis we identified 200 new potential partners of Vpr, opening the doors to discover novel Vpr targets and activities. This thesis also defined APC/C as new Vpr target. Taken together our results allow a better understanding on how HIV-1 modulates the cellular environment by using the viral accessory protein Vpr.