Submitted by Adriele Dandara Levorato Vinche (adrielelevorato@yahoo.com.br) on 2018-03-13T14:11:39Z No. of bitstreams: 1 Tese de Doutorado Adriele Dandara Levorato Vinche.pdf: 2200417 bytes, checksum: 23e69a47a9fa5126001d659e0803e67a (MD5) / Approved for entry into archive by Luciana Pizzani null (luciana@btu.unesp.br) on 2018-03-13T18:45:09Z (GMT) No. of bitstreams: 1 vinche_adl_dr_bot.pdf: 2200417 bytes, checksum: 23e69a47a9fa5126001d659e0803e67a (MD5) / Made available in DSpace on 2018-03-13T18:45:09Z (GMT). No. of bitstreams: 1 vinche_adl_dr_bot.pdf: 2200417 bytes, checksum: 23e69a47a9fa5126001d659e0803e67a (MD5) Previous issue date: 2018-02-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A paracoccidioidomicose (PCM) é micose granulomatosa sistêmica causada por fungos termodimórficos do gênero Paracoccidioides. O presente estudo teve como objetivos avaliar a atividade da liriodenina, extraída de Annona macroprophyllata Donn. Sm, sobre isolados clínicos e cepas padrão de fungos do gênero Paracoccidioides e outras espécies que causam micoses sistêmicas e, através de um estudo-piloto realizado em modelo murino, avaliar a absorção da liriodenina e possíveis efeitos indesejáveis. A concentração inibitória mínima (CIM) e concentração fungicida mínima (CFM) da liriodenina foram determinadas pelo método de microdiluição. As alterações celulares causadas pela liriodenina em P. brasiliensis / cepa padrão Pb18 foram avaliadas pela microscopia eletrônica de transmissão (MET) e de varredura (MEV). Em estudo-piloto, quatro camundongos isogênicos albinos, da linhagem BALB/c, foram utilizados para avaliar a absorção e os efeitos indesejáveis da liriodenina. Os animais, sem infecção, foram divididos em dois grupos: grupo 1, com dois camundongos que receberam a dose de 0,75 mg.kg-1 e grupo 2, com dois camundongos que receberam a dose de 1,50 mg.kg-1, em única tomada. Após seis e 12 horas após a administração, os animais foram sacrificados e amostras de sangue foram coletadas para a determinação dos níveis séricos. Os intestinos foram coletados para exame histológico. O teste de sensibilidade in vitro revelou que a liriodenina possui atividade sobre parte dos fungos do gênero Paracoccidioides, com valores de CIM entre 31,2 e 250 μg.mL-1 e sobre a cepa padrão de Histoplasma capsulatum (CIM de 1,95 μg.mL-1). No entanto, menor atividade foi observada em leveduras da maioria das espécies de Candida ensaiadas, com CIM de 125 a 250 μg.mL-1, e maior atividade sobre as cepas padrões de Cryptococcus neoformans e Cryptococcus gattii (CIM de 62,5 μg.mL-1), enquanto a cepa de Aspergillus fumigatus revelou-se resistente à maior concentração de liriodenina. Deve-se ressaltar a atividade da liriodenina sobre Candida krusei, que é intrinsecamente resistente ao fluconazol e, ao contrário, a ausência de atividade sobre Candida tropicalis. A liriodenina revelou ter atividade fungicida sobre todas as cepas padrão e isolados clínicos que foram inibidos in vitro. A MET revelou alterações citoplasmáticas e danos na parede celular de P.brasiliensis. O estudo-piloto revelou que, após 12 horas da administração da liriodenina, os animais que receberam a dose de 1,50 mg.kg-1 apresentavam gases intestinais e distensão abdominal, achados não observados nos que receberam 0,75 mg.kg-1. Os exames histológicos não evidenciaram alterações de intestino com nenhuma das doses empregadas. Níveis séricos de liriodenina foram detectados em ambos os grupos de animais. No entanto, os animais que receberam 0,75 mg.kg-1 de liriodenina apresentaram aumento da concentração com o passar do tempo, enquanto achado oposto foi observado nos que receberam 1,50 mg.kg-1. Os testes de sensibilidade in vitro indicaram que a liriodenina é uma alternativa promissora no tratamento de várias micoses sistêmicas. O estudo-piloto revelou que a liriodenina é absorvida após administração por gavagem, embora a dose de 1,50 mg.kg-1 tenha induzido alterações funcionais, caracterizadas por formação exagerada de gases e conseqüente distensão abdominal, que talvez possam explicar a diminuição de sua absorção. Os resultados do presente estudo estimulam a realização da avaliação da farmacocinética da liriodenina, de sua ação antifúngica em maior número de isolados de espécies de Candida spp, Cryptococcus spp, Histoplasma capsulatum e fungos do gênero Paracoccidioides e de sua eficácia em infecções experimentais em modelo murino. Por se tratar de composto antifúngico não pertencente às classes hoje utilizadas na terapia antifúngica, pode vir a contribuir com o tratamento de pacientes com infecções fúngicas resistentes. / Paracoccidioidomycosis (PCM) is systemic granulomatous mycosis caused by thermodymorphic fungi of the genus Paracoccidioides. The present study aimed to evaluate the activity of liriodenine, extracted from Annona macroprophyllata Donn. Sm on clinical isolates and standard strains of fungi of the genus Paracoccidioides and other species that cause systemic mycoses and, through a pilot study conducted in a murine model, to assess the absorption of liriodenine and possible undesirable effects. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (CFM) of liriodenine were determined by the microdilution method. Cellular alterations caused by liriodenin in P. brasiliensis / Pb18 standard strain were evaluated by transmission electron microscopy (SEM) and scanning electron microscopy (SEM). In a pilot study, four albino isogenic mice of the BALB / c strain were used to assess the absorption and undesirable effects of liriodenine. The animals, without infection, were divided into two groups: group 1, with two mice receiving the dose of 0.75 mg.kg-1 and group 2, with two mice receiving the dose of 1.50 mg.kg- 1 , in single outlet. After six and 12 hours after administration, the animals were sacrificed and blood samples were collected for the determination of serum levels. The intestines were collected for histological examination. The in vitro sensitivity test revealed that liriodenine has activity on part of fungi of the genus Paracoccidioides, with MIC values between 31.2 and 250 μg.mL-1 and on the standard Histoplasma capsulatum strain (MIC of 1.95 μg .mL-1 ). However, lower activity was observed in yeasts of most Candida species tested, with MIC of 125 to 250 μg.mL-1 , and greater activity on the standard strains of Cryptococcus neoformans and Cryptococcus gattii (MIC of 62.5 μg. mL-1 ), while the strain of Aspergillus fumigatus was resistant to the highest concentration of liriodenine. The activity of liriodenine on Candida krusei, which is intrinsically resistant to fluconazole and, on the contrary, the absence of activity on Candida tropicalis should be emphasized. Liriodenin reveale over all standard strains and clinical isolates that were inhibited in vitro. MET showed cytoplasmic alterations and damage to P. brasiliensis cell wall. The pilot study revealed that animals receiving the 1.50 mg.kg-1 dose had no intestinal gas and abdominal distension 12 hours after administration of liriodenine, which were not observed in those receiving 0.75 mg.kg-1 . Histological examinations showed no intestinal changes at any of the doses used. Serum levels of liriodenine were detected in both groups of animals. However, animals receiving 0.75 mg.kg-1 liriodenine showed increased concentration over time, while the opposite finding was observed in those receiving 1.50 mg.kg-1 . In vitro sensitivity tests indicated that liriodenine is a promising alternative in the treatment of various systemic mycoses. The pilot study revealed that liriodenine is absorbed after administration by gavage, although the 1.50 mg.kg-1 dose induced functional alterations, characterized by exaggerated gas formation and consequent abdominal distension, which may explain the decrease of its absorption. The results of the present study stimulate the evaluation of the pharmacokinetics of liriodenine, its antifungal action in a greater number of isolates of Candida species, Cryptococcus spp, Histoplasma capsulatum and fungi of the genus Paracoccidioides and their efficacy in experimental infections in murine model. Because it is an antifungal compound that does not belong to the classes currently used in antifungal therapy, it may contribute to the treatment of patients with resistant fungal infections.

O principal mecanismo de defesa de hospedeiros infectados pelo Paracoccidioides brasiliensis (Pb), fungo dimórfico que causa a mais importante micose sistêmica da América Latina, é a imunidade celular. Neste processo participam macrófagos ativados por IFN-<font face=\"Symbol\">g e a IL-10 parece ser a citocina que se contrapõe a esta ativação. Tanto na patologia humana como em modelos experimentais há fortes indicações de que a IL-10 age como supressora da imunidade celular causando efeitos deletérios aos hospedeiros; entretanto, estudos diretos sobre a função da IL-10 na paracoccidiodomicose (PCM) não tinham sido ainda realizados. Então o objetivo fundamental deste trabalho foi estudar a função da IL-10 nos mecanismos da imunidade inata e adaptativa contra o Pb utilizando como modelo experimental camundongos geneticamente deficientes de IL-10 (IL-10 nocaute, IL-10 KO) em comparação com seus controles normais (WT). Demonstramos in vitro que macrófagos peritoneais normais de camundongos IL-10 KO apresentam uma maior atividade fagocítica e fungicida que os macrófagos de camundongos WT e isto esteve associado à maior produção de IFN-<font face=\"Symbol\">g, TNF-<font face=\"Symbol\">&#945, óxido nítrico (NO) e da quimiocina MCP-1. Verificamos, entretanto, que a produção de IFN-<font face=\"Symbol\">g era realizada por células NKT contaminantes de macrófagos aderentes nos camundongos IL-10 KO, sugerindo uma possível participação destas células na ativação de macrófagos e de células T de camundongos IL-10 KO. Estudos in vivo revelaram que a partir da 2ª semana de infecção os camundongos IL-10 KO apresentaram uma resposta imune precoce em relação aos WT, pois os pulmões dos primeiros apresentavam carga fúngica significativamente menor, associada com aumento da maioria dos isótipos de anticorpos (IgM, IgG1, IgG2a, IgG2b) específicos contra o fungo. Observamos também a inibição total da produção das citocinas IL-4 e IL-5, mostrando a ação reguladora da IL-10 na síntese de citocinas Th2. Na 4ª semana pós-infecção a carga fúngica continuou menor nos animais IL-10 KO, mas não foram observadas as diferenças quanto à síntese de anticorpos entre as duas linhagens. A análise dos leucócitos infiltrantes nos pulmões revelou um aumento na frequência de linfócitos T CD4+ ativados nos camundongos IL-10 KO em relação aos WT. A partir da 8ª semana, a carga fúngica pulmonar dos camundongos IL-10 KO reduziu consideravelmente e não ocorreu disseminação para outros órgãos. Observou-se também, nesta linhagem, um aumento na resposta de hipersensibilidade do tipo tardio (HTT), confirmando o padrão de resposta desviado para um perfil Th1. A análise histológica dos órgãos dos camundongos IL-10 KO revelou ausência de granulomas e fungos no pulmão em contraste aos seus controles WT que apresentavam elevado número de granulomas e fungos neste órgão. A análise dos linfócitos infiltrantes nos pulmões dos camundongos IL-10 KO mostrou redução na frequência de células B, o que é coerente com a baixa síntese de anticorpos observada. Houve aumento marcante na freqüência de células T CD4+ e T CD8+ memória/efetora, caracterizando mais uma vez a eficiente resposta imune celular nesses animais associada à ausência do papel regulador negativo da IL-10. Em fase mais tardia (16 semanas pós-infecção) a regressão da infecção nos camundongos IL-10 KO continuou evidente pelo pequeno número de fungos recuperados do pulmão, acompanhada de baixa síntese de citocinas (IL-5 e IL-2) e de anticorpos anti- P. brasiliensis. Na 23ª semana de infecção além da baixa carga fúngica nos camundongos IL-10 KO, associada à baixa frequência de células responsáveis pela imunidade celular ocorreu também redução na freqüência de células Treg, indicando o papel regulador da IL-10 nesta subpopulação celular. A elevada sobrevida (90%) dos animais IL-10 KO foi coerente com a baixa carga fúngica e eficiente resposta imune no curso da infecção. Em conjunto, nosso trabalho demonstra o importante papel regulador da IL-10 na imunidade da PCM. / Cellular immunity is the main defense mechanism of hosts infected by the Paracoccidioide brasiliensis (Pb), a dimorphic fungus that causes the most important systemic mycosis in Latin America. IFN-<font face=\"Symbol\">g activated macrophages participate in this activity that is antagonized by IL-10 an anti-inflammatory cytokine. Both, in the human pathology and in experimental models, there are a number of evidences indicating that IL-10 acts as a suppressor of cellular immunity leading to deleterious effects to the hosts. However, direct studies aimed at investigating the function of IL-10 in the immunity to paracoccidioidomycosis (PCM) have not been performed. Thus, the fundamental objective of this work was to study the function of IL-10 in the mechanisms of innate and adaptive immunity against Pb using as experimental model IL-10 deficient mice (IL-10 Knockout, IL-10 KO) compared to their respective wild type (WT) controls. We demonstrated in vitro that normal peritoneal macrophages of IL-10 KO mice presented increased phagocytic and microbicidal activities than macrophages of WT mice and this was associated witch an elevated production of IFN-<font face=\"Symbol\">g, TNF-<font face=\"Symbol\">&#945, nitric oxide (NO) and the chemokine MCP-1. However, the production of IFN-<font face=\"Symbol\">g seen to be performed by NTK cells contaminating adherent macrophages, suggesting a possible participation of these cells in the activation of macrophages and T cells of IL-10 KO mice. In vivo studies revealed that at the second week of infection IL-10 KO mice presented an earlier immune response when compared to wild-type mice, since their lungs exhibited a significantly reduced fungal burden and an increased production of almost all antibodies isotypes (IgM, IgG1, IgGM, IgG2b). This effect was accompanied by a total absence of IL-4 and IL-5, showing a regulatory action of IL-10 in the synthesis of Th2 cytokines. Four weeks post-infection, the fungal load was still lower in the IL-10 KO mice but no differences in antibody synthesis was observed. However, the analysis of lung infiltrating leukocytes revealed an increased frequency of TCD4+ and TCD4+CD44 high lymphocytes in IL-10 KO mice, again demonstrating an early activation of cellular immunity in IL-10 KO mice. When compared with WT mice, the pulmonary fungal loads of IL-10 KO mice at week 8 of infection were drastically reduced and no dissemination to other organs were observed. The histopathological analysis revealed an absence of granulomas and fungi in the lungs of IL-10 KO in comparison with WT mice. The analysis of lung infiltrating leukocytes showed that IL-10 KO mice had a reduction in the frequency of B cells, in agreement with the reduced synthesis of immunoglobulins. An increased frequency of activated T CD4+ and a drastic increase of TCD4+ and T CD8+ effector/memory cells charactering once again an efficient immune response associated with IL-10 deficiency. In later stages, sixteen weeks after infection, a regressive infection of IL-10 KO mice was further characterized by low numbers of fungi in the lung, reduced synthesis of cytokines (IL-4 and IL-5) and anti- P. brasiliensis antibodies. By week 23 after infection, in addition to the characteristic reduction of fungal loads and reduced frequency of immune cells, we observed a decrease in the frequency of Treg cells, demonstrating the implication of IL-10 in the control of this T cell population. The elevated survival (90%) of IL-10 KO mice was in total agreement with the low fungal burdens and efficient immune response observed during infection. In conclusion, our work demonstrates for the first time that IL-10 plays a major role in the control of innate and adaptive immunity to Pb infection.

The sterol biosynthetic pathway is essential for growth of all eukaryotic cells and the main target of antifungal agents. The emergence of resistance to these antifungals in an already ill patient population indicates a need to develop drugs that have a broad spectrum of activity among pathogenic fungi and have minimal patient toxicity. Squalene synthase is the first committed step in the sterol pathway and has been studied intensively for development of antifungal agents. While the overall architecture of this enzyme is identical throughout eukaryotes, it was shown that plant and animal genes cannot complement a squalene synthase knockout mutation in yeast unless the carboxy-terminal domain is swapped for one of fungal origin. This implies that there is a component of the fungal carboxy-terminal domain that is responsible for the complementation phenotype and that is unique to the fungal kingdom of life. To determine the role of the carboxy-terminal domain of squalene synthase in the sterol pathway, we used the yeast Saccharomyces cerevisiae with a squalene synthase knockout mutation and expressed squalene synthases originating from fungi, plants, and animals. In contrast to previous observations, all enzymes tested could partially complement the knockout mutation when the genes were weakly expressed. When induced, non-fungal squalene synthases could not complement the knockout mutation and instead led to the accumulation of carboxysterol intermediates, suggesting an interaction between squalene synthase and the downstream sterol C4-decarboxylase. Overexpression of a sterol C4-decarboxylase from any kingdom of life both decreased the accumulation of carboxysterol intermediates and allowed non-fungal squalene synthases to complement the squalene synthase knockout mutation. Using chimeric squalene synthases from each kingdom of life, the motif in the C-terminal domain responsible for preventing this toxicity was mapped to a kingdom-specific 26-amino acid hinge motif adjacent to the catalytic domain. Furthermore, over-expression of the carboxy-terminal domain alone containing a hinge motif from fungi, not from animals or plants, led to growth inhibition of wild-type yeast. Since this hinge region is unique to and highly conserved within each kingdom of life, this data provides evidence for the development of an antifungal therapeutic as well as for tools to develop an understanding of triterpene catalytic activity and identify similar motifs in other biosynthetic pathways.

The purpose of this study was to determine the prevalence of Lyme Disease (LD) bacteria in adult Ixodes pacificus ticks collected from the mountains of San Bernardino County in Southern California. Seven hundred fifty four I. pacificus adults were collected from the Pacific Crest Trail and adjacent areas. The Polymerase Chain Reaction (PCR) was used to screen ticks for Borrelia burgdorferi infection by targeting two different DNA loci. Oligonucleotide primers targeting both the ospA and fla genes were used in the assay. Ticks were processed in pools of three, and genomic DNA from the ticks was extracted with a commercial mini-kit utilizing silica matrix spin-columns. All ticks tested negative for B. burgdorferi infection regardless of primer pair used. In addition, ticks were negative following examination by dark-field microscopy. This study confirms previous reports that the prevalence of LD in Southern California is quite low.

Chlamydia is the most common bacterial sexually transmitted infection in the United States and worldwide. It often goes unnoticed due to lack of symptoms and left untreated it can ascend the female genital tract to cause sequelae like pelvic inflammatory disease and irreversible tubal infertility. In reproductive-aged women, female sex hormones estrogen (E2) and progesterone (P4) concentrations fluctuate during the menstrual cycle and are influenced by hormonal contraceptives and hormone replacement therapy. E2 and P4 influence genital Chlamydia infection in women and mice, but these multifactorial interactions are not entirely mapped out. The complex interplay of E2 and P4 with Chlamydia and the host response demand further study to determine the effect of hormonal environment and host susceptibility to Chlamydia. E2 primarily signals through estrogen receptors (ER) ERα and ERβ. We used ERα or ERβ knockout (KO) mice to study the role of E2 and ERs in chlamydial progression and examined the host immune response at day 9 post-infection, when we expected the immune response to be the most robust. ERαKO, but not ERβKO mice had significant differences in the progression of Chlamydia and the host immune response. Future studies should test the immune response at additional timepoints, and a model should be utilized wherein ERα and ERβ are simultaneously silenced by chemical knockdown of ERβ in ERα knockout mice using ER agonist ICI 182, 680. 3 Mice are widely used in Chlamydia research, but due to its short estrus cycle, infection cannot be established naturally before infected cells are shed. To overcome this, mice are pretreated with depot medroxyprogesterone acetate (DMPA), an exogenous progesterone that halts the estrus cycle. However, a mouse model not reliant on DMPA pretreatment is needed because 1.) DMPA can affect the immune response and 2.) the hormonal environment in women is not static. Our model uses mice that are ovariectomized to stop the production of endogenous E2 and P4, then treated with physiologically relevant levels of E2 and P4 via implantation of a hormone-filled capsule. We observed that E2 protected mice from Chlamydia, making our model a good alternative for in vivo Chlamydia studies.

Tuberculosis (TB), a transmissible disease caused by Mycobacterium tuberculosis (Mtb), is a global health threat. To design an effective vaccine, we need to better understand how different elements of our immune system collaborate to fight against Mtb. CD4 T cells are crucial in protective immunity to Mtb because they produce cytokines including interferon-γ. In contrast, CD8 T cells are thought to play a modest role. Whether CD4 T cells act as “helper” cells to promote optimal CD8 T cell responses during TB is unknown. We argue CD8 T cells’ role are likely underestimated because CD8 T cell functions are compromised without CD4 T cells. Here, using two independent models, I show that CD4 T cell help promotes CD8 T cell effector functions and prevents CD8 T cell exhaustion. I demonstrate CD4 and CD8 T cells synergistically enhance the survival of infected mice. Purified helped, but not helpless, CD8 T cells effectively restrict intracellular Mtb growth. Thus, CD4 T cell help is indispensable for generating protective CD8 T cell responses. In addition, I investigate the mechanisms of CD4 T cell help. Signals from CD4 T cells, and signals relayed by antigen presenting cells collectively shape CD8 T cell responses. We infer that vaccines aimed for eliciting both CD4 and CD8 T cells, in which CD8 T cells are properly helped by CD4 T cells, are more likely to be successful.

Quorum sensing molecules have become a recent focus of study to learn if and how they can be used, both on their own and in conjecture with current antimicrobial methods, as a means of bacterial control. One such quorum sensing molecule is the sesquiterpene alcohol, Farnesol, which is synthesized and released by the fungus, Candida albicans. In most in-vivo cases, our laboratory has shown that Alcaligenes faecalis overtakes C. albicans, preventing its growth. However, as a way to counteract this inhibitory effect, Farnesol may be one way that Candida has found to fight back. In this study, we focused on the inhibitory properties of Farnesol for growth and motility of A. faecalis, as well as, the molecule’s ability to prevent Alcaligenes from creating biofilms and/or degrading them once they have already been established. Our experiments show evidence that Farnesol is able to inhibit both the growth and motility of A. faecalis, and determination of the specific concentrations of Farnesol needed to see the largest effects on A. faecalis biofilms. Our hope is that in future studies, we will be able to add varying concentrations of the Farnesol to known and widely used antibiotics in order to increase the effectiveness of antibiotics against bacterial strains, both in the Alcaligenes genus and in other genus, that have previously been considered “antibiotic resistant”.

In 2008 the Nobel Prize in Physiology or Medicine was awarded to the co-discoverers of the Human Immunodeficiency Virus Type 1 (HIV-1), the causative agent of Acquired Immunodeficiency Syndrome (AIDS). This award acknowledged the enormous worldwide impact of the HIV-1/AIDS pandemic and the importance of research aimed at halting its spread. Since the syndrome was first recognized, 25 million people have succumbed to AIDS and over 33 million are currently infected with HIV-1 (www.unaids.org). The most effective strategy for ending the pandemic is the creation of a prophylactic vaccine. Yet, to date, all efforts at HIV-1 vaccine design have met with very limited success. The consistent failures of vaccine candidates stem in large part from the unprecedented diversity of HIV-1. Among the novel theories of vaccine design put forward to address this diversity is the targeted vaccine approach. This proposal is based on the finding that mucosal transmission of HIV-1, the most prevalent form, occurs across a selective bottleneck such that typically only a single (or a few) variants of the viral swarm present in a donor are passed to the recipient. While the mechanisms controlling the selection are largely unknown, the targeted vaccine approach postulates that once they are identified, we can utilize this understanding to design vaccines specifically targeted to the characteristics shared by the rare, mucosally transmissible HIV-1 variants. The studies described in this work were conducted to improve our understanding of the factors influencing viral variant selection during mother-to-child-transmission of HIV-1, a route of mucosal transmission which has globally become the leading cause of child infection. A unique panel was generated, consisting of nearly 300 HIV-1 envelope genes cloned from infected mother-infant pairs. Extensive characterization of the genotypes, phenotypes and phylogeny of these clones was then done to identify attributes differentiating early infant from maternal variants. Low genetic diversity of HIV-1 envelope variants was detected in early infant samples, suggesting a bottleneck and active selection of variants for transmission. Transmitted variants did not differ from non-transmitted variants in CD4 and CCR5 use. Infant isolates replicated poorly in macrophages; a cell subtype hypothesized to be important in the establishment of infection. The sensitivity of infant envelope variants to neutralization by a panel of monoclonal antibodies, heterologous and autologous plasmas and HIV-1 entry inhibitors varied. Most intriguingly, envelopes cloned from infants infected during delivery exhibited a faster entry phenotype than maternal isolates. Together, these findings provide further insight into viral variant selection during mother-to-child transmission. Identification of properties shared by mucosally transmitted viral variants may allow them to be selectively targeted, resulting in improved methods for preventing HIV-1 transmission.

Mycobacterium tuberculosis isolates from multiple drug resistant or extensively drug resistant patients show a particular set of mutations in drug targets conferring resistance. However, the selection of drug-resistant strains in vitro yields an alternative set of mutations, thought to result from the cost-benefit associated with drug resistance. Mutations allowing for survival under antibiotic may not be beneficial when presented with the host environment or with a drug-free environment. These fitness effects drive the natural evolution of this bacterium. Using recombineering a large cohort of mutations was generated within two drug targets, inhA and gyrA, to study in vitro the variability of mutations allowable under either isoniazid or ofloxacin, respectively. As a proof of concept this process was carried out in Mycobacterium smegmatis. Analysis of survivors allowed for identification of novel mutations and substitutions, as well as showing mutations previously found only in clinical isolates can be present in laboratory isolates.

Many virus infections induce a transient state of immune suppression in the infected host. Virus-induced T cell suppression can be caused by T cell activation-induced cell death (AICD), dendritic cell (DC) apoptosis, DC dysfunction, and/or the enhanced expression of immune-suppressive cytokines. It has been previously demonstrated that naïve bystander CD8 T cells derived from hosts experiencing an acute virus-specific T cell response underwent AICD when polyclonally activated by anti-CD3 in vitro (Zarozinski et al., 2000). Susceptibility of naïve bystander T cells to AICD could prevent the development of a new T cell response during an ongoing immune response, and thus render infected hosts immune suppressed. Although immune suppression could result in an enhanced susceptibility to superinfections, virus-infected individuals are more commonly resistant to superinfecting pathogens. Because of these seemingly contradictory conditions, we sought to investigate how acute viral infections impact naïve bystander CD8 T cells in vivo. More specifically, we asked whether bystander CD8 T cells are susceptible to immune suppression or whether they can contribute to the resistance to superinfections. In order to address this, we examined the responses of bystander CD8 T cells activated with cognate antigen during acute viral infections in vivo. We generated several in vivomodels using P14 (LCMV glycoprotein-specific), HY (male antigen-specific), and OT-I (ovalbumin-specific) transgenic CD8 T cells, which we defined as bystander during acute infections with lymphocytic choriomeningitis virus (LCMV), Pichinde virus (PV), vaccinia virus (VV), and murine cytomegalovirus (MCMV). Consistent with the enhanced susceptibility to cell death noted in vitro, we found that bystander CD8 T cells activated with cognate antigen in vivo during acute viral infections underwent markedly reduced proliferation. Virus-induced transient T cell suppression in vivo was not exclusively mediated by Fas-FasL- or TNF-induced AICD or due to an enhanced susceptibility to apoptosis. Instead, immune suppression in vivowas associated with a delayed onset of division, which we found not to be due to a defect in antigen presentation, but rather due to a T cell intrinsic defect. Despite the suppressed proliferation of TCR-stimulated bystander CD8 T cells in vivo, we found an enhancement of the effector functions exerted by bystander CD8 T cells activated during acute viral infections. During acute viral infections or after stimulation with type 1 IFN (IFN-αβ) inducers, some bystander CD8 T cells were sensitized to immediately exert effector functions such as IFN-γ production and degranulation upon stimulation with high affinity cognate antigen. Sensitization of naïve CD8 T cells required self-MHC I and indirect effects of IFN-αβ, while IL-12, IL-18, and IFN-γ were not individually required. IL-15 was not required for the rapid expression of IFN-γ, but was required for up-regulation of granzyme B (GrzB). P14 and OT-I CD8 T cells, which are capable of homeostatic proliferation, could be sensitized by poly(I:C), but HY CD8 T cells, which are poor at homeostatic proliferation, could not, suggesting that the requirement for MHC I may be to present low affinity cryptically cross-reactive self antigens. Sensitized naive CD8 T cells up-regulated the t-box transcription factor Eomesodermin (Eomes), which can regulate these rapid effector functions. In conclusion, we demonstrate in this thesis that acute viral infections impact naïve bystander CD8 T cells such that their response to cognate antigen is altered. Prior to cognate antigen engagement, bystander CD8 T cells up-regulated Eomes, CD122, and GrzB. Following cognate antigen engagement, bystander CD8 T cells rapidly degranulated and expressed the effector cytokine IFN-γ. The ability of bystander CD8 T cells to rapidly exert effector functions may contribute to the resistance of virus-infected individuals to superinfections. Despite these rapid effector functions, the proliferation of TCR-stimulated bystander CD8 T cells was markedly inhibited. This reduced proliferation was found not to be a defect in antigen presentation, but was a T cell intrinsic defect in initiating division. Thus, bystander CD8 T cells were also susceptible to virus-induced immune suppression. It is also likely that virus-specific CD8 T cells that are not activated until later in the response, so-called latecomer CD8 T cells, may also be susceptible to immune enhancement and suppression. Thus, latecomer CD8 T cells would be able to rapidly exert effector functions at the expense of proliferation. Taken together, we propose that during an immune response, due to spatial and temporal gradients of antigen and inflammation, it is likely that a combination of heterogeneous T cells with different signal strengths and sequences of exposure from cytokines and peptide-MHC constitute the total T cell response to pathogens.