FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / A Escala de AvaliaÃÃo da Capacidade para Cuidar de CrianÃas Expostas ao HIV (EACCC-HIV) à um instrumento que estima o cuidado de mÃes Ãs crianÃas nascidas na vigÃncia do HIV. Objetivou-se avaliar a capacidade de mÃes para cuidar de crianÃas expostas ao HIV mediante a EACCC-HIV e verificar a associaÃÃo entre as dimensÃes da escala e as caracterÃsticas maternas. Estudo transversal desenvolvido em 2010 em Fortaleza-CE. Participaram 62 cuidadoras (mÃes) HIV+ com 64 filhos (dois gemelares) nascidos expostos ao HIV menores de 1 ano. Apreciaram-se as caracterÃsticas das mÃes e das crianÃas; as estratÃgias para reduÃÃo da transmissÃo vertical do HIV; Apgar familiar e a EACCC-HIV. A escala possui 52 itens e cinco fatores que sÃo utilizados para determinadas idades entre zero e 1 ano: Fator 1: capacidade para administrar o AZT xarope (crianÃas atà 42 dias de vida); Fator 2: capacidade para preparar e administrar o leite em pà (crianÃas atà 1 ano); Fator 3: capacidade para preparar e administrar alimentaÃÃo complementar (crianÃas > de 4 meses); Fator 4: capacidade para administrar a profilaxia com sulfametoxazol e trimetoprim (crianÃas > 42 dias); Fator 5: capacidade para garantir a adesÃo ao acompanhamento clÃnico e vacinaÃÃo (todas as crianÃas). As respostas sÃo mediadas por fator ou pela somatÃria de todos os itens, indicando-se o grau de cuidado desenvolvido pela mÃe. Para anÃlise utilizou-se o STATA 11.0, empregando-se nÃvel de significÃncia de 5%. A idade materna oscilou entre 18 e 42 anos, 33,9% com aids, 61,3% integrantes das classes D e E. Das crianÃas uma tinha aids (1,6%), 98,4% iniciaram o AZT nas primeiras horas de vida, 3,1% foram amamentadas, 61,3% tiveram consumo inadequado de leite artificial e 36,2% consumo inadequado de alimentaÃÃo complementar. O Apgar familiar indicou 34,4% severamente funcional. Enquanto o fator 1 da EACCC-HIV avaliou 11 crianÃas, das quais 72,7% recebiam cuidados considerados adequados, o fator 2 avaliou 64 crianÃas e indicou que 86,0% das mÃes possuÃam alta capacidade de cuidar. Pelo fator 3, o cuidado concentrou-se entre moderado (44,4%) e alto (50%). O fator 4 estimou o cuidado oferecido para 51 crianÃas, indicando que 76,5% tiveram alta capacidade para o cuidado, e o fator 5 avaliou respostas das 62 mÃes sobre as 64 crianÃas. Destas, 95,3% possuÃam alta capacidade para adesÃo ao acompanhamento clÃnico e vacinaÃÃo. Pela avaliaÃÃo global, 29,7% dos cuidados foram considerados como adequados (alta capacidade para o cuidado). A associaÃÃo de variÃveis indicou significÃncia entre Apgar da famÃlia e capacidade para administrar o leite em pà (fator 2); paridade e capacidade para administrar a profilaxia com sulfametoxazol e trimetropim; paridade e escolaridade e capacidade para garantir adesÃo ao acompanhamento clÃnico e vacinaÃÃo; e estÃdio evolutivo e tempo de diagnÃstico com avaliaÃÃo global da escala. Com a EACCC-HIV favoreceu-se avaliar o cuidado materno dispensado Ãs crianÃas e realizar intervenÃÃes em prol da saÃde infantil para a manutenÃÃo da qualidade de vida na vigÃncia da exposiÃÃo ao HIV ou para aquelas infectadas pelo vÃrus. / A Escala de AvaliaÃÃo da Capacidade para Cuidar de CrianÃas Expostas ao HIV (EACCC-HIV) à um instrumento que estima o cuidado de mÃes Ãs crianÃas nascidas na vigÃncia do HIV. Objetivou-se avaliar a capacidade de mÃes para cuidar de crianÃas expostas ao HIV mediante a EACCC-HIV e verificar a associaÃÃo entre as dimensÃes da escala e as caracterÃsticas maternas. Estudo transversal desenvolvido em 2010 em Fortaleza-CE. Participaram 62 cuidadoras (mÃes) HIV+ com 64 filhos (dois gemelares) nascidos expostos ao HIV menores de 1 ano. Apreciaram-se as caracterÃsticas das mÃes e das crianÃas; as estratÃgias para reduÃÃo da transmissÃo vertical do HIV; Apgar familiar e a EACCC-HIV. A escala possui 52 itens e cinco fatores que sÃo utilizados para determinadas idades entre zero e 1 ano: Fator 1: capacidade para administrar o AZT xarope (crianÃas atà 42 dias de vida); Fator 2: capacidade para preparar e administrar o leite em pà (crianÃas atà 1 ano); Fator 3: capacidade para preparar e administrar alimentaÃÃo complementar (crianÃas > de 4 meses); Fator 4: capacidade para administrar a profilaxia com sulfametoxazol e trimetoprim (crianÃas > 42 dias); Fator 5: capacidade para garantir a adesÃo ao acompanhamento clÃnico e vacinaÃÃo (todas as crianÃas). As respostas sÃo mediadas por fator ou pela somatÃria de todos os itens, indicando-se o grau de cuidado desenvolvido pela mÃe. Para anÃlise utilizou-se o STATA 11.0, empregando-se nÃvel de significÃncia de 5%. A idade materna oscilou entre 18 e 42 anos, 33,9% com aids, 61,3% integrantes das classes D e E. Das crianÃas uma tinha aids (1,6%), 98,4% iniciaram o AZT nas primeiras horas de vida, 3,1% foram amamentadas, 61,3% tiveram consumo inadequado de leite artificial e 36,2% consumo inadequado de alimentaÃÃo complementar. O Apgar familiar indicou 34,4% severamente funcional. Enquanto o fator 1 da EACCC-HIV avaliou 11 crianÃas, das quais 72,7% recebiam cuidados considerados adequados, o fator 2 avaliou 64 crianÃas e indicou que 86,0% das mÃes possuÃam alta capacidade de cuidar. Pelo fator 3, o cuidado concentrou-se entre moderado (44,4%) e alto (50%). O fator 4 estimou o cuidado oferecido para 51 crianÃas, indicando que 76,5% tiveram alta capacidade para o cuidado, e o fator 5 avaliou respostas das 62 mÃes sobre as 64 crianÃas. Destas, 95,3% possuÃam alta capacidade para adesÃo ao acompanhamento clÃnico e vacinaÃÃo. Pela avaliaÃÃo global, 29,7% dos cuidados foram considerados como adequados (alta capacidade para o cuidado). A associaÃÃo de variÃveis indicou significÃncia entre Apgar da famÃlia e capacidade para administrar o leite em pà (fator 2); paridade e capacidade para administrar a profilaxia com sulfametoxazol e trimetropim; paridade e escolaridade e capacidade para garantir adesÃo ao acompanhamento clÃnico e vacinaÃÃo; e estÃdio evolutivo e tempo de diagnÃstico com avaliaÃÃo global da escala. Com a EACCC-HIV favoreceu-se avaliar o cuidado materno dispensado Ãs crianÃas e realizar intervenÃÃes em prol da saÃde infantil para a manutenÃÃo da qualidade de vida na vigÃncia da exposiÃÃo ao HIV ou para aquelas infectadas pelo vÃrus. / The Assessment Scale of the Ability to take Care of Children Exposed to HIV (EACCC-HIV) estimates mothersâ care delivery to children born in conditions of HIV. The goal was to assess mothersâ ability to take care of children exposed to HIV through the EACCC-HIV and to verify the association between scale dimensions and maternal characteristics. This cross-sectional study was carried out in Fortaleza-CE in 2010. Participants were 62 HIV+ caregivers (mothers) with 64 children (two twins) exposed to HIV at birth and younger than one year. The mothers and childrenâs characteristics were evaluated; strategies to reduce vertical HIV transmission: Family Apgar and EACCC-HIV. The scale contains 52 items and five factors, used for certain ages between zero and 1 year: Factor 1: ability to administer AZT syrup (children up to 42 days of life); Factor 2: ability to prepare and administer powder milk (children up to 1 year); Factor 3: ability to prepare and administer complementary feeding (children > 4 months); Factor 4: ability to administer prophylaxis with sulfamethoxazole and trimethoprim (children > 42 days); Factor 5: ability to guarantee adherence to clinical monitoring and vaccination (all children). Answers are mediated by a factor or by the sum of all items, indicating the degree of care the mother develops. STATA 11.0 software was used for analysis, with significance set at 5%. Maternal age ranged between 18 and 42 years, 33.9% suffering from aids, 61.3% in lower socioeconomic classes. One of the children had aids (1.6%), 98.4% had starting AZT in the first hours of life, 3.1% was breastfed, 61.3% showed inadequate artificial milk consumption and 36.2% inadequate complementary feeding consumption. The Family Apgar indicated 34.4% severely functional. While factor 1 of the EACCC-HIV assessed 11 children, 72.7% of whom received adequate care, factor 2 assessed 64 children and indicated high ability for care delivery in 86.0% of the mothers. According to factor 3, care was concentrated between moderate (44.4%) and high (50%). Factor 4 estimated the care offered to 53 children, indicating high ability for care delivery in 76.5%, and factor 5 assessed the 62 mothersâ answers on the 64 children. In total, 95.3% showed high ability for adherence to clinical monitoring and vaccination. According to the global assessment, 29.7% of care was considered adequate (high ability for care delivery). The association between variables indicated significance between family Apgar and ability to administer powder milk (factor 2); parity and ability to administer prophylaxis with sulfamethoxazole and trimethropim; parity and education level and ability to guarantee adherence to clinical monitoring and vaccination: and staging and diagnosis time with global assessment of the scale. Through the EACCC-HIV, maternal care for the children could be assessed and interventions could be made to enhance child health, with a view to maintaining quality of life in cases of exposure to or contamination by HIV. / The Assessment Scale of the Ability to take Care of Children Exposed to HIV (EACCC-HIV) estimates mothersâ care delivery to children born in conditions of HIV. The goal was to assess mothersâ ability to take care of children exposed to HIV through the EACCC-HIV and to verify the association between scale dimensions and maternal characteristics. This cross-sectional study was carried out in Fortaleza-CE in 2010. Participants were 62 HIV+ caregivers (mothers) with 64 children (two twins) exposed to HIV at birth and younger than one year. The mothers and childrenâs characteristics were evaluated; strategies to reduce vertical HIV transmission: Family Apgar and EACCC-HIV. The scale contains 52 items and five factors, used for certain ages between zero and 1 year: Factor 1: ability to administer AZT syrup (children up to 42 days of life); Factor 2: ability to prepare and administer powder milk (children up to 1 year); Factor 3: ability to prepare and administer complementary feeding (children > 4 months); Factor 4: ability to administer prophylaxis with sulfamethoxazole and trimethoprim (children > 42 days); Factor 5: ability to guarantee adherence to clinical monitoring and vaccination (all children). Answers are mediated by a factor or by the sum of all items, indicating the degree of care the mother develops. STATA 11.0 software was used for analysis, with significance set at 5%. Maternal age ranged between 18 and 42 years, 33.9% suffering from aids, 61.3% in lower socioeconomic classes. One of the children had aids (1.6%), 98.4% had starting AZT in the first hours of life, 3.1% was breastfed, 61.3% showed inadequate artificial milk consumption and 36.2% inadequate complementary feeding consumption. The Family Apgar indicated 34.4% severely functional. While factor 1 of the EACCC-HIV assessed 11 children, 72.7% of whom received adequate care, factor 2 assessed 64 children and indicated high ability for care delivery in 86.0% of the mothers. According to factor 3, care was concentrated between moderate (44.4%) and high (50%). Factor 4 estimated the care offered to 53 children, indicating high ability for care delivery in 76.5%, and factor 5 assessed the 62 mothersâ answers on the 64 children. In total, 95.3% showed high ability for adherence to clinical monitoring and vaccination. According to the global assessment, 29.7% of care was considered adequate (high ability for care delivery). The association between variables indicated significance between family Apgar and ability to administer powder milk (factor 2); parity and ability to administer prophylaxis with sulfamethoxazole and trimethropim; parity and education level and ability to guarantee adherence to clinical monitoring and vaccination: and staging and diagnosis time with global assessment of the scale. Through the EACCC-HIV, maternal care for the children could be assessed and interventions could be made to enhance child health, with a view to maintaining quality of life in cases of exposure to or contamination by HIV.

The innate immune system is a multicomponent response governed by intricate mechanisms of induction, regulation and resolution to elicit antimicrobial defenses. In recent years, the complexity of eukaryotic transcriptomes has become the subject of intense scrutiny and curiosity. It has been established, that RNA polymerase II (RNAPII) transcribes hundreds to thousands of long noncoding RNAs (lncRNAs), often in a stimulus and cell-type specific manner. However, the functional significance of these transcripts has been particularly controversial. While the number of identified lncRNAs is growing, our understanding of how lncRNAs themselves regulate other genes is quite limited. In chapter 2, a novel lncRNA is identified, more specifically, a natural antisense transcript, that mediates the transcription of the pro-inflammatory cytokine IL-1α. Through loss-of-function studies, I report the necessity of this transcript in mediating IL-1α mRNA expression by affecting RNAPII binding to the IL-1α promoter after toll-like receptor signaling. For the first time, I show that IL-1α is regulated at the transcriptional level. As a second independent component of this thesis, we explore the role of the innate immune response after infection by the malaria-causing parasite, Plasmodium berghei ANKA (PbA), and how innate immune components are both beneficial and detrimental to the host depending on when and where inflammation is triggered during infection. We attempt to identify the “malarial toxin” responsible for aberrations in the immune response that is detrimental for disease outcomes and the innate signaling pathways that are involved. Many pathogens induce pathological inflammatory conditions that lead to irreparable homeostatic imbalances and become fatal to the host. Here, type I Interferon signaling is required to dampen parasite load during liver-stage infections, but leads to host mobidity if these pathways are activated in the erythrocytic phase of infection. Together, this thesis provides new insights on how components of the innate immune system are regulated, and how dysregulation of immunity can potentially lead to adverse effects during active infections.

Interferon-inducible PYHIN protein family includes the DNA-binding proteins, AIM2 and IFI16, which form ASC-caspase 1 dependent inflammasomes, important in immunity against cytosolic bacteria, DNA viruses and HIV. The role of other members of this family in the recognition of DNA and/or regulation of immune responses is unclear. We identified an immune regulatory function of p205, another member of the PYHIN family, in the transcriptional control of immune genes. Knockdown of p205 in macrophages revealed that inflammasome activation due to dsDNA and ligands that engage the NLRP3 inflammasome were severely compromised. Detailed mechanistic analysis showed that loss of p205 was associated with a decrease in Asc mRNA and protein levels. p205 knockdown resulted in reduced RNA Polymerase II-mediated endogenous Asc gene transcription and mRNA processing, suggesting a co-transcriptional control of Asc gene expression. Ectopically expressed p205 induced expression of an Asc gene-luciferase reporter and collaborated with other transcription factors, such as c/EBPβ, p65/RelA, to further enhance expression. p205 knockdown also affected the expression of the immune genes Cd86, Cox2, Cxcl2, Il1α, Il10, Il12α, Il6 and Ifnα in LPS-stimulated macrophages. Together these findings suggest that p205 regulates inflammation through control of Asc gene expression, and other immune genes. Fungal infections activate both caspase 1-dependent and -independent inflammasomes. In an independent study, we show that Paracoccidioides brasiliensis fungal infection also induces caspase 8-dependent non-canonical inflammasome. Caspase 8-dependent IL-1β processing required dectin-1, Syk and Asc. Rip3-/- Casp8-/- mice infected with P. brasiliensis displayed increased fungal load and showed worse disease progression compared to wild type and Rip3-/- mice. These results revealed the importance of caspase 8 in activating and regulating inflammasome responses during fungal infection in vivo.

Chlamydia trachomatis is the most common sexually transmitted bacterial disease agent worldwide, and, though frequently asymptomatic, can cause extreme pathology including infertility. Chlamydial species exhibit a unique biphasic developmental cycle. Once attached to a cell surface, infectious elementary bodies (EB) are internalized within an inclusion, the membrane-bound structure in which EB transform to noninfectious, replicable reticulate bodies (RB). After multiple rounds of division, RB condense to form EB, which are released and can infect new host cells. In culture, exposure to stressors, such as beta-lactam antibiotics, induce chlamydiae to reversibly detour from normal development into a noninfectious, viable state termed persistence. Cell culture data suggest that persistent forms are resistant to azithromycin (AZM), a front-line antibiotic, and are able to alter the host transcriptome. Though persistence has been described in culture for over 50 years, whether or not it: i) occurs in vivo; and ii) influences chlamydial pathogenesis, transmission and therapy has remained unresolved. To address these questions, we developed an animal model of persistent chlamydial infection using amoxicillin (AMX) treatment. AMX exposure decreased shedding of infectious chlamydiae in C. muridarum-infected mice without affecting chlamydial viability, demonstrating the presence of persistent chlamydiae. Shedding of infectious EB resumed following AMX cessation. Shedding data and microarray analyses suggested that host immunity might limit chlamydia’s exit from persistence in our model. Thus, we hypothesized that cyclophosphamide (CTX) treatment would increase the magnitude of chlamydial shedding observed after AMX-treatment cessation. CTX treatment increased post-AMX shedding by more than 10-fold compared to AMX-only controls. To determine whether persistent chlamydiae are resistant to antibiotic eradication in vivo, we induced persistence by administering AMX and treated mice with various AZM dosing regimes. Persistently infected mice demonstrated increased treatment failure following AZM therapy compared to productively infected controls. These data suggest that persistent chlamydiae are refractory to treatment in vivo and provide an explanation for the observation that treatment fails in some patients. In addition to creating the first fully characterized, experimentally tractable, in vivo model of chlamydial persistence, these experiments provide evidence that persistent/stressed chlamydial forms may serve as a long-term reservoir of infectious organisms in vivo.

Ebola virus (EBOV) is a re-emerging zoonotic virus (it is transmitted between animals and humans) that causes acute hemorrhagic fever and a high fatality rate in humans. First reported in 1976 in the Democratic Republic of the Congo (formerly Zaire), the virus is transmitted between humans through direct contact with body fluids of an infected person, causing fever, weakness, diarrhea, abdominal pain, cramping, nausea and vomiting in those affected. There is neither a licensed vaccine nor an approved treatment for Ebola virus in human patients. The reservoir species for Ebola virus is similarly unknown, as many studies have attempted yet failed to isolate living virus from potential candidates. The widely accepted and circulated hypothesis based on preliminary findings of outbreaks past is that bat species, specifically the fruit bat species Hypsignathus monstrosus, Epomops franqueti and Myonycteris torquata are potential reservoirs. Recent reports, especially concerning findings from the 2014 Ebola outbreak, have determined that insectivorous bats could similarly be reservoir species. Successful isolation of a live virus from a bat species found through a widened sampling of a variety of bat species would confirm the hypothesis that bats, either fruit or insectivorous, are the reservoir species for Ebola virus.

The mammalian intestine harbors an estimated 100 trillion microorganisms, which normally maintain a mutually beneficial relationship with the host. The intestinal epithelium consists of a single layer of intestinal epithelial cells (IECs) that provides a physical barrier as well as innate immune defense, preventing this vast community of microbes from entering host tissues. Secretory immunoglobulin A (SIgA) acts as the first line of antigen-specific immunity at the interface between the gut microbiota and the intestinal epithelium. Polymeric IgA secreted by plasma cells in the intestinal lamina propria is transported across IECs by the polymeric immunoglobulin receptor (pIgR). Defects in epithelial barrier and immune functions can lead to infections with opportunistic and pathogenic microbes and contribute to the etiology of inflammatory bowel disease (IBD). Here we investigate the ability of IEC biomarkers to define the mechanism and severity of intestinal inflammation, as well as provide insight into the function of IEC in regulating intestinal homeostasis and inflammation. Importantly, down-regulation of pIgR expression was a common feature in human IBD and mouse models of experimental colitis. One molecule of pIgR is consumed for every molecule of SIgA transported, thus high expression of pIgR is required to maintain sufficient supply of SIgA. Accordingly, we investigate the mechanisms by which IECs regulate pIgR expression in response to colonic bacteria. Cross-talk between the microbiota and IECs is mediated by pattern recognition receptors, including Toll-like receptors (TLR), leading to expression of gene products that enhance epithelial barrier function and innate immunity. The cytoplasmic adaptor protein MyD88 transduces signals from TLRs that recognize bacterial products. We show that pIgR induction by colonic bacteria is dependent on TLR4-MyD88 activation of NF-κB signaling. We examined the role of epithelial-specific MyD88 signaling in antibacterial immunity and epithelial expression of key gene products that participate in innate immunity in the gut by generating mice with an IEC-targeted deletion of the Myd88 gene (MyD88ΔIEC). MyD88ΔIEC mice display immunological and antimicrobial defects resulting in increased susceptibility to experimental colitis. We conclude that cross-talk between bacteria and IECs via MyD88-dependent signaling is crucial for maintenance of gut homeostasis.

Equine infectious anemia virus (EIAV) has been used as a model to investigate protective mechanisms against lentiviruses. Unlike other lentiviruses, EIAV replication can be eventually controlled in most infected horses leading to an inapparent carrier state free of overt clinical signs which can last for many years. Maintenance of this carrier state is absolutely dependent on active immune responses as evidenced by the fact that immunosuppressive drugs can induce the recurrence of disease. However, the immune mechanisms that are responsible for this control of infection are not yet identified. As the resolution of the initial infection is correlated with the appearance of the virus-specific cytotoxic T lymphocytes (CTL), it appears that cellular immune responses play an important role. However, most studies into this protective mechanism have been limited to the identification of specific epitopes, usually at a single time point in the infection. Few studies have examined the cellular immune responses to the viral antigens throughout the infection period. Since the virus undergoes rapid mutation following infection, the adaptive immune response must also evolve to meet this challenge. Previously, the EIAV envelope (gp90) protein was shown to be the primary determinant of vaccine efficacy. Here, we hypothesized that the maturation of cellular immune responses is a lengthy process and involves envelope-specific T cell recognition shifting from immunodominant variable determinants to conserved immunorecessive determinants during the initial stages of the EIAV infection. The first part of this dissertation was to develop a new in vivo method to identify envelope-specific T cell responses. The second part of this dissertation was to investigate whether envelope-specific T cell recognition evolved in EIAV-infected ponies. Finally, the mechanisms for this T cell immunodominant shifting were also investigated from the point of both virus sequence mutation and T cell clone expansion and contraction. Also, a new EIAV attenuated vaccine which contained a consensus gp90 sequence was tested to see if it facilitated T cell recognition of the more conserved regions early in the infection. Our results indicated that envelope-specific T cell recognition patterns changed over time. Early after infection, dominant immune responses to the peptides in the carboxyl-terminus variable region were identified. By six months post infection, the recognized peptides spanned the entire envelope sequence, with a shift to the amino-terminus conserved region. The mechanisms responsible for this change remain unclear, but analysis of T cell receptor repertoire indicated that T cell clonal expansion and contraction might be one of the reasons. Our demonstration that envelope-specific peptide recognition shifts from the variable to the more conserved regions provides evidence that the maturation of cell mediated immune response is parallelled with long-term control of this infection.

Many bacteria, viruses and other parasites cause severe morbidity or mortality in their host populations, creating strong selection for physiological or behavioural mechanisms to avoid disease. Likewise, changes in host susceptibility and contact patterns can dramatically influence the spread of infectious diseases, and hence selection for traits such as virulence and infectivity range in parasites. Understanding how ecological and evolutionary changes in one population affect selection in another represents a key challenge for theoreticians and empiricists alike, and is essential for gaining further insights into host-parasite relationships. This thesis contains theoretical models that explore how genetic and environmental factors shape the evolutionary and coevolutionary dynamics of hosts and parasites. In particular, the roles of genetic specificity (i.e. genotype-by-genotype interactions) and population mixing patterns are investigated, using both mathematical models and computer simulations. A broad range of scenarios are covered, including the coevolution of broad resistance and infectivity ranges (generalism), the persistence of coevolutionary cycling and the maintenance of sex, the effects of mating behaviour on disease prevalence and evolution, and the evolution of sexual and social behaviour. The models presented herein develop our understanding of host-parasite relationships and highlight the importance of genetic interactions and ecological feedbacks.

IL-6 is an inflammatory cytokine that contributes to the pathogenesis of many immunological diseases including rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, allergic asthma, as well as the protection against infections caused by various pathogens. These are linked to its role in regulating CD4 T cell differentiation and effector function. Most of these functions are dependent on the IL-6-mediated signaling through the transcription factor Stat3. In this thesis, we identify a novel molecular mechanism by which IL-6 regulates CD4 T cell effector function. We show that IL-6-dependent signal raises the levels of mitochondrial Ca2+ late during activation of CD4 T cells. This is further used to prolong the expression of effector cytokines IL-4 and IL-21. The modulation of mitochondrial Ca2+ is mediated by the regulation of mitochondrial Stat3 and the formation of respiratory supercomplexes. Thus, in addition to the canonical signaling of IL-6 through Stat3 as a transcription factor, IL-6 also modulates mitochondrial Stat3 to regulate mitochondrial function in CD4 T cells. This could be an alternative pathway by which IL-6 regulates effector function of CD4 T cells and it could contribute to the pathogenesis of inflammatory disease. Little is known about the effects of IL-6 on CD8 T cells. In this thesis, we reveal a paradigm-shifting mechanism by which IL-6 regulates antibody production by converting CD8 T cells into B cell helpers through IL-21. Briefly, IL-6 promotes the differentiation of a subset of naïve CD8 T cells into a unique population of effector CD8 T cells characterized by the production of high levels of IL-21. IL-21-producing CD8 T cells provide help to B cells to induce isotype switching and protective antibody production during infection. In summary, this thesis provides new insights into both mechanistic and functional aspects of IL-6 in regulating T cell function. These findings may shed light on the development of new therapeutic approaches in treating autoimmune disorders and preventing infectious diseases.

The advent of high-throughput sequencing has brought about the creation of an unprecedented amount of research data. Analytical methodology has not been able to keep pace with the plethora of data being produced. Two assays, ImmunoSEQ and the cytokinesisblock micronucleus (CBMN), that both produce count data and have few methods available to analyze them are considered. ImmunoSEQ is a sequencing assay that measures the beta T-cell receptor (TCR) repertoire. The ImmunoSEQ assay was used to describe the TCR repertoires of patients that have undergone hematopoietic stem cell transplantation (HSCT). Several different methods for spectratype analysis were extended to the TCR sequencing setting then applied to these data to demonstrate different ways the data set can be analyzed. The different methods include CDR3 distribution perturbation, Oligoscores, Simpson's diversity, Shannon diversity, Kullback-Liebler divergence, a non-parametric method and a proportion logit transformation method. Herein we also demonstrate adapting compositional data analysis methods to the TCR sequencing setting. The various methods were compared when analyzing a set of 13 subjects who underwent hematopoietic stem cell transplantation. The eight subjects who developed graft versus host disease were compared to the five who did not. There was no little overlap in the results of the different methods showing that researchers must choose the appropriate method for their research question of interest. The CBMN assay measures the rate of micronuclei (MN) formation in a sample of cells and can be paired with gene expression or methylation assays to determine association between MN formation and other genetic markers. Herein we extended the generalized monotone incremental forward stagewise (GMIFS) method to the situation where the response is count data and there are more independent variables than there are samples. Our Poisson GMIFS method was compared to a popular alternative, glmpath, by using simulations and applying both to real data. Simulations showed that both methods perform similarly in accurately choosing truly significant variables. However, glmpath appears to overfit compared to our GMIFS method. Finally, when both methods were applied to two data sets GMIFS appeared to be more stable than glmpath.