HIV/AIDS annually kills millions of people worldwide. Those claimed by the disease are quickly replaced by those newly infected. Three times as many new infections occur globally, as patients who are likely to have access to antiretroviral therapy. We need a HIV vaccine. However, the better HIV protein to target for this vaccine in unknown. Structural proteins such as Group specific antigen (Gag) and Envelope (Env) were thought likely candidates due to viral structural proteins usually being highly conserved and constrained in their ability to mutate to escape T cell attack. To establish if Env-specific T cells could control viraemia, 2 large vaccine trials were conducted with 66 pigtail macaques participating. Also, 2 reversion trials involving 4 pigtail macaques were undertaken. Env-specific T cell epitopes were mapped in both SHIV (simian/human immunodeficiency virus) and SIV (simian immunodeficiency virus)-infected macaques using IFNγ intracellular cytokine staining and flow cytometry.

Le développement de la phase SIDA (Syndrome de l'ImmunoDéficience Acquise) chez les patients infectés par le virus VIH-1 (Virus de l'Immunodéficience Humaine) se caractérise par une diminution progressive du nombre de cellules T CD4. Orla majorité des cellules constituant cette déplétion sont non-infectées et appelées "bystander". En 2006, notre équipe a montré que le contact entre les cellules infectées exprimant les glycoprotéines de l’enveloppe (Env) et les cellules noninfectées exprimant les récepteurs CD4 et CXCR4 déclenche au sein de ces dernières la voie d’autophagie, ce qui mène à une mort cellulaire par apoptose. L'autophagie est un processus impliqué dans la dégradation de matériel cytoplasmiqueaprès sa séquestration au sein de vacuoles dans lesquelles il sera dégradé puis recyclé. Ce processus peut être hautement sélectif par l'action de protéines réceptrices tels que p62 ou NBR1.L’objectif de mon projet de thèse vise à comprendre comment l'autophagie induite par Env mène les cellules T CD4 bystander à leur mort par apoptose. Une précédente étude menée par notre équipe a démontré que les changements induits par Env au sein de ces cellules T CD4 non infectées comprenaient la production d’espèces oxygénées réactives (ROS) menant à un état de stress oxydatif. Nous montré que le stress oxydatif induit par Env est impliqué dans la mort des cellules T CD4 bystander par apoptose. Nous avons également observé que l’autophagie doit être dégradative pourmener ces cellules T CD4 à leur mort par apoptose. De plus, nous avons observé une dégradation des protéines peroxysomales par l’autophagie induite par Env dans le même modèle. Les peroxysomes sont des organelles essentielles de la cellule qui sont en partie responsables de la détoxification des ROS dans la cellule. Leur nombre est régulé par une dégradation sélective autophagique que l’on appelle la pexophagie.Dès lors, nous étudions l’hypothèse de l'induction par Env d’une dégradation sélective des systèmes antioxydants de la cellule par autophagie dans les cellules T CD4 bystander. Les peroxysomes étant des organites responsables de la réponse au stress oxydatif, leur dégradation sélective pourrait empêcher la cellule de faire face au stress oxydatif qu’elle subit et la mener vers une mort cellulaire par apoptose. En conclusion, nous montrons que l’autophagie induite par Env dégrade un système antioxydant important qui est un facteur clé nécessaire à la survie des cellules T CD4 bystander pour réduire le stress oxydatif induit par Env. / The development of AIDS (Acquired ImmunoDeficiency Syndrome) in HIV-1 (Human Immunodeficiency Virus)-infected patients is characterized by a progressive decrease in the number of CD4 T cells. The majority of dying cells are noninfected and called bystander CD4 T cells. In 2006, our team demonstrated that the contact between infected cells (expressing the envelope glycoproteins (Env)) and non-infected cells (expressing the CD4 and CXCR4 receptors) was responsible for enhancing the autophagic pathway which lead to their cellular death by apoptosis. The autophagicpathway is involved in the degradation of cytoplasmic material after its sequestration into vacuoles wherein it will be degraded and then recycled. This process can be highly selective through the involvement of receptor proteins such asp62 or NBR1.We aim at understanding how Env-mediated autophagy can lead to apoptosis in bystander CD4 T cells. A precedent workof our team showed that the changes induced by Env in bystander CD4 T cells included the production of reactive oxygen species (ROS) leading to an oxidative stress state. We showed that the oxidative stress induced by Env is involved in thecellular death by apoptosis of bystander CD4 T cells. We also show that the autophagic process involved has to be a degradative process to lead these CD4 T cells to their death by apoptosis. Moreover, we have observed that Env-mediatedautophagy was degrading peroxisomal proteins. Peroxisomes are essential organelles in the cell responsible partly for the detoxification of ROS in the cell. Their number is regulated through a selective autophagic degradation known aspexophagy.Therefore, we hypothesized that Env induced a selective degradation by autophagy of the cell antioxidant system in bystander CD4 T cells. Since peroxisomes are responsible for regulating the cellular response to an oxidative stress state, their selective degradation could prevent the cell from overcoming this event and eventually lead to its death by apoptosis. In conclusion, we are showing that Env-mediated autophagy degrades important antioxidant systems which are a key survival factor necessary to the bystander CD4 T cells to reduce the oxidative stress induced by Env.

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Jaagsiekte sheep retrovirus (JSRV) is a simple betaretrovirus that causes a contagious lung adenocarcinoma in sheep. One unique feature of JSRV is that its envelope (Env) glycoprotein functions as an active oncogene that induces oncogenic transformation in vitro and in vivo. While oncogenesis by JSRV Env protein has been extensively studied, the entry mechanism of JSRV has not been investigated. In this study, we showed that JSRV entry was specifically inhibited by lysosomotropic agents and bafilomycin Al (BafAl), indicating that JSRV is pH-dependent. Interestingly, oncoretroviral pseudotypes bearing JSRV Env protein were not inactivated by an acidic pH treatment, suggesting that additional factors besides low pH are involved in JSRV entry. Indeed, we found that JSRV entry was also blocked by dominant-negative mutants of dynamin and caveolin, raising the possibility that JSRV may use a dynamin-dependent, caveolae-associated pathway for entry. To determine a possible role of JSRV receptor, hyaluronidase 2 (Hyal2), in JSRV entry, we replaced the glycosylphosphatidyl-inositol (GPI) anchor of Hyal2 with the membrane-spanning domain and cytoplasmic tail of vesicular stomatitis virus G protein (VSV-G). We showed that although the transmembrane version of Hyal2 functioned efficiently as a JSRV receptor, JSRV entry mediated by the mutated Hyal2 was no longer inhibited by lysosomotropic agents and BafAl. Taken together, we conclude that JSRV entry is pH-dependent, but appears to use a non-classical pathway for entry. JSRV may provide an exciting novel model for a better understanding of retrovirus entry.

Thesis advisor: Welkin E. Johnson / Endogenous retroviruses (ERVs) make up a significant portion of vertebrate genomes, and serve as a fossil record of past retroviral infections. Although most ERV genes acquire inactivating mutations over time, some loci retain open reading frames (ORFs) across one or more of the viral genes. The ERV-Fc family, for example, endogenized in multiple mammalian hosts 10 to 30 million years ago, yet many copies maintain intact ORFs corresponding to the env gene, including loci in humans (HERV-Fc1-env) and baboons (babERV-Fc2-env). We previously identified intact ERV-Fc-related env sequences in eight additional mammalian species: chimpanzee, bonobo, aardvark, grey mouse lemur, squirrel monkey, marmoset, dog, and panda. Here we present the results of our assays of expression of these full-length Env proteins. We found that most of the precursors were not cleaved to form the functional surface (SU) and transmembrane (TM) subunits, even when a canonical furin cleavage site was still intact. An exception was babERV-Fc2, in which reconstruction of the cleavage site led to cleavage into SU and TM subunits. Furthermore, removal of 22 residues from the C-terminus of the cytoplasmic tail of babERV-Fc2 enhanced syncytia formation and the ability of babERV-Fc2 pseudotyped virions to infect 293T cells, suggesting the presence of an R-peptide cleavage mechanism. A survey of a small panel of cells revealed that only human cell lines were infectable by babERV-Fc2 pseudotyped murine leukemia virus (MLV) particles, whereas cells of old world monkey, canine, feline and chicken origin were not susceptible to infection. Ectopic expression of native Env codon optimized babERV-Fc2 Env can also inhibit infection by reconstructed babERV-Fc2 pseudotyped virus, raising the possibility that the endogenous glycoprotein encoded in the baboon genome may function as a viral entry inhibitor. Our results suggest that exaptation of ERV Env proteins as antiviral defense genes involves a combination of selective pressures: selection to preserve the receptor-binding and receptor interference functions of Env, but also selection to eliminate the membrane fusion related functions. / Thesis (PhD) — Boston College, 2018. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

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Urbanization, the conversion of natural habitat into area fit for human exploitation, is the greatest contemporary threat to natural ecosystems. With urbanization only projected to increase in magnitude as human populations continue to grow it is becoming increasingly important to evaluate the potential negative impacts urbanization can have on vital ecosystem functions and services. One such important ecosystem service is pollination. Roughly 87.5% of flowering plants are animal pollinated, with pollination contributing over $175 billion dollars to the global economy each year. The fundamental ecologic and economic importance of pollination, coupled with the growing threat of urbanization, makes it important to better understand how pollination success may be impacted by urban development. Though, studies on the impact of urbanization on pollination success vary wildly in their findings, with some studies finding a negative impact of urbanization, while others find a negligible or even positive impact. This discordance in past studies is likely due to the fact that pollination studies typically focus on just one aspect of the pollination process, whether that be pollinator community, pollen removal/deposition, or fruit/seed production. Urbanization, however, may induce differential impacts across different stages of the pollination process. Focusing on just one pollination aspect may limit our understanding of the potential underlying mechanisms urbanization may impart on different stages of the pollination process. The goal of this study was to evaluate multiple aspects of pollination success across an urbanization gradient. Specifically, pollinator community (in terms of visitation rate and community composition), pollen removal/deposition, and fruit/seed production were surveyed across 12 common milkweed (Asclepias syriaca) populations, six of which were in natural habitats and six of which were in areas of moderate urbanization. By evaluating all pollination aspects together we can pinpoint which step(s) of the pollination process are impacted by urbanization, which will inform decisions on how to best conserve the integrity of pollination in anthropogenically disturbed environments. Overall, urbanization increased pollinator visitation rate (0.151 visits per minute per flower in urban populations, compared to 0.067 at natural populations), and altered pollinator composition, with more small bees and beetles present in more urbanized areas. Pollen removal also increased with urbanization (2.00 pollen removals per flower in urban populations, compared to 1.41 at natural populations), while pollen deposition, fruit production, and seed production were unchanged by urbanization. Thus, suggesting that the more abundant pollinators in more urbanized areas were effective at removing pollen, but ineffective at depositing pollen, resulting in no change in reproductive output (i.e., equal fruit and seed production). The results of this study suggest that increased urbanization can variably impact various steps in the pollination process, thus stressing the importance of studying multiple pollination aspects in tandem.

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Made available in DSpace on 2016-12-08T16:24:14Z (GMT). No. of bitstreams: 1 PGCA13MA113.pdf: 865063 bytes, checksum: 41861f94a8b447b6d17dbc06ba954319 (MD5) Previous issue date: 2013-08-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Bovine leukemia virus (BLV) is a member of the family Retroviridae, genus Deltaretrovirus and it is an important agent, being primarily responsible for economic losses in dairy herds, causing losses of around 10% in production. The infection of BLV, can manifest itself in two ways, benign one, which reaches approximately 30% of the animals infected with concomitant persistent lymphocytosis and malignant condition, often fatal, affecting approximately 5% of animals with tumor appearance lymphoid (lymphosarcomas).Currently, several studies have been made about of BLV genotypes, finding at least seven genotypes, in samples of different parts of the world. The aim of this study was the molecular characterization of samples of BLV from seropositive dairy cattle in Santa Catarina State. Were collected 454 blood samples of dairy cattle of the 31 properties and four buffaloes, being initially performed serology using agar gel immunodiffusion test (IDGA). Also collected tumor samples with different locations: lymph node, heart, intestine and muscle near the coxal, from three cattle. After serology 191 samples were found to be seropositive then submitted to DNA extraction and in 62 samples were performed the polymerase chain reaction (PCR) for amplification of a 440 bp fragment of the env gene, as well as the tumor samples. Nineteen samples extracted from blood and tumors samples were submitted to restriction fragment lenght polymorphism analysis (RFLP) by digestion of the PCR fragment for five restriction endonucleases, BamHI, HaeIII, Tru9I, TaqI and MwoI. The results obtained in serology demonstrated 42% seropositive animals (191/454) and 68% positives properties 7 (21/31), buffaloes no positive and tumor samples were positive in its entirety in PCR. In RFLP analysis identified five different profiles corresponding to five genotypes circulating in the State. Among the genotypes found in this study, the highest prevalence was observed genotype X (47.4%). This study allows us to know some the viral genotypes present in cattle in the Santa Catarina state and these results useful for future epidemiological studies, as well as identify the existence of new variants and their current prevalence / O vírus da Leucose Bovina (BLV) membro da família Retroviridae, gênero Deltaretrovirus, é um agente importante em bovinos, sendo responsável por perdas econômicas principalmente em rebanhos leiteiros, podendo causar prejuízos em torno de 10% na produção. A infecção pelo BLV, pode manifestar-se de duas formas, uma benigna, que atinge aproximadamente 30% dos animais infectados, cursando com linfocitose persistente, e a forma maligna, muitas vezes fatal, que atinge aproximadamente 5% dos animais, com surgimento de tumores linfóides (linfossarcomas). Atualmente, vários estudos têm sido realizados a cerca dos genótipos do BLV, encontrando-se pelo menos sete genótipos, em amostras de diferentes locais do mundo. O presente estudo teve como objetivo, a genotipagem do BLV, em rebanhos leiteiros do estado de Santa Catarina. Foram coletadas 454 amostras de sangue de bovinos leiteiros de 31 propriedades e quatro búfalos sendo realizada inicialmente a sorologia pelo teste de imunodifusão em gel de ágar (IDGA). Coletaram-se também amostras de tumores com diferentes localizações: linfonodo, coração, intestino e musculatura próximo ao coxal, de três bovinos. Após a sorologia 191 amostras foram constatadas como soropositivas sendo submetidas então à extração de DNA e em 62 amostras realizou-se a reação da polimerase em cadeia (PCR), visando a amplificação de um fragmento 440 pb do gene env, assim como nas amostras de tumores. Dezenove amostras extraídas de sangue e dos tumores foram submetidas à análise de polimorfismo dos fragmentos de restrição (RFLP), através da digestão do fragmento da PCR por 5 cinco endonucleases de restrição, BamHI, HaeIII, Tru9I, TaqI e MwoI. Os resultados obtidos na sorologia demonstraram 42% dos soros dos bovinos analisados positivos (191/454), 68% de propriedades positivas (21/31), nenhum soro bubalino positivo e as amostras de tumores foram positivas em sua totalidade na PCR. Nas análises por RFLP identificamos cinco perfis diferentes correspondendo a cinco genótipos circulantes no Estado. Dentre os genótipos encontrados neste trabalho, o mais prevalente foi o genótipo X (47,4%). Este estudo permitiu conhecer alguns dos genótipos virais presentes em bovinos no Estado de Santa Catarina sendo estes resultados úteis para futuros estudos epidemiológicos, assim como identificar a existência de novas variantes circulantes e sua prevalência