Function and Regulation of Cytochrome P450 4V2 and the Implications in Bietti’s Crystalline Dystrophy

Thesis (Ph.D.)--University of Washington, 2016-06

AS RELAÇÕES ENTRE CONFLITO TRABALHO-FAMÍLIA, BEM-ESTAR SUBJETIVO E BEM-ESTAR NO TRABALHO

O conflito trabalho-família é um fenômeno freqüente nas sociedades atuais em que homens e mulheres dividem responsabilidades familiares e profissionais. A sua direção pode ser unidirecional ou bidirecional, ou seja, da família para o trabalho, do trabalho para a família ou simultaneamente de uma esfera para outra, afetando sensivelmente as relações familiares e as relações nas organizações, sendo possível afetar diretamente o bem-estar dos indivíduos. Este é um estudo de natureza quantitativa e com desenho transversal. O critério para a escolha de participantes foi o de conveniência. O objetivo geral desta pesquisa foi analisar as relações entre conflito trabalho-família (CTF), bem-estar subjetivo (BES) e bem-estar no trabalho (BET). O estudo contou com a participação de 174 trabalhadores em uma amostra diversificada de profissionais do segmento privado, público e de ONG, sendo 52,9% do sexo feminino, com idade média de 38,29 anos (DP=11,22). Para a obtenção dos dados optou-se por um questionário abrangendo as três variáveis do estudo contendo duas escalas para aferir as dimensões de BES, uma escala para aferir BET e uma escala para aferir CTF/CFT. Para a análise dos dados foi usado o SPSS, versão 20. Foram calculadas frequências, percentuais, médias, desvios-padrão, e índices de correlação, bem como índices de precisão das medidas usadas (Alpha de Cronbach). Os resultados apontaram para uma maior interferência do trabalho na família do que da família no trabalho. Essa interferência foi significativa tanto para o bem-estar no trabalho, quanto para o bem-estar subjetivo, alterando o envolvimento com o trabalho e gerando sentimentos de infelicidade, desânimo e angústia em relação as suas realizações pessoais, e ao futuro. Quanto maior o conflito trabalho-família, menor é o bem-estar no trabalho e menor é o bem-estar subjetivo desses participantes.

A INFLUÊNCIA DA QUALIDADE DE VIDA E SUPORTE SOCIAL NO ABSENTEÍSMO POR LESÕES OCUPACIONAIS

Esta pesquisa é uma proposta de aproximação das diversas áreas de conhecimento e flexibilização de suas fronteiras, a fim de estabelecer interfaces entre os diversos campos conceituais engendrando novas práticas que ensejam formas mais abrangentes e holísticas de aproximação com a realidade. Através da avaliação da qualidade de vida, suporte social e sua relação com o absenteísmo por lesão ocupacional. Materiais e Métodos: Participaram desta pesquisa 47 trabalhadores, do sexo masculino, com idade variável entre 30 e 60 anos, que trabalham no setor de centrifugação dentro de uma fundição em uma indústria metalúrgica do ramo de autopeças em São Bernardo do Campo. Instrumentos: Foi analisado o prontuário médico de cada um desses trabalhadores, sendo analisadas como variáveis: se esses trabalhadores apresentaram lesões ocupacionais avaliadas e diagnosticadas pelos médicos. Avaliação de um questionário social, composto por quatro questões abertas: idade, nível de escolaridade, estado civil e número de dependentes. Instrumento de Avaliação da Qualidade de Vida WHOQOL Bref. e Instrumento de Avaliação de Suporte Social: Escala de percepção de suporte social (EPSS). Análise dos Resultados: Os dados coletados dos prontuários foram agrupados em quatro grupos chamados de: grupo de absenteísmo 1, grupo de absenteísmo 2, grupo de absenteísmo 3 e grupo de absenteísmo 4. Sendo o primeiro composto por 15 trabalhadores que não apresentaram lesões ocupacionais. O segundo grupo composto por 10 trabalhadores apresentou de uma hora até seiscentas horas de absenteísmo por lesão ocupacional. O terceiro grupo composto por 11 trabalhadores apresentou absenteísmo de seiscentas e uma horas até mil horas, e o quarto grupo constituído de 11 trabalhadores tiveram absenteísmo superior a mil horas. Resultados: Nas comparações entre os grupos de absenteísmo e as variáveis do questionário social não foram encontradas diferenças significantemente estatística, o mesmo tendo ocorrido na comparação entre grupos de absenteísmo e os escores do EPSS Analisando os grupos de absenteísmo e os domínios do WHOQOL Bref. Foi detectada diferença com significância estatística de P = 0,01 maior dentro do domínio físico no grupo de absenteísmo 1 que não apresentou absenteísmo em relação ao grupo de absenteísmo 4 com maior número de absenteísmo. Conclusão: Visto que as lesões ocupacionais interferem na qualidade de vida dos indivíduos, o que se pode constatar é que o modelo biomédico ainda é muito forte, principalmente dentro da saúde ocupacional que na busca de soluções ergonômicas defronta com os interesses econômicos imediatistas que não contemplam os investimentos indispensáveis à garantia da saúde do trabalhador. Sintetizando as análises conceituais e os resultados deste trabalho, mostrou-se que os problemas de saúde ocupacional são conhecidos há muito tempo. Apesar da crescente produção de trabalhos acerca da construção , conhecimento das características da saúde do trabalhador e a existência de uma tônica predominante que sugere a mudança das situações encontradas, há um escasso empenho em efetuar as transformações necessárias, num descompasso entre avanço do conhecimento e perpetuação de práticas insalubres. Para tanto é necessária uma compreensão das determinantes da saúde. Além do investimento material se faz necessário emergir um verdadeiro diálogo entre as ciências. Ressalta-se a necessidade de estudos longitudinais para elucidar a complexidade das relações entre qualidade de vida, suporte social e lesões ocupacionais. Os achados apontam para a relevância de estudos futuros acerca dessas associações.(AU)

O EFEITO DO CONTROLE RESPIRATÓRIO EM VARIÁVEIS ELETROFISIOLÓGICAS DA ATENÇÃO

A prática do ioga tem se tornado cada vez mais popular, não apenas pelos benefícios físicos, mas principalmente pelo bem-estar psicológico trazido pela sua prática. Um dos componentes do ioga é o Prãnãyama, ou controle da respiração. A atenção e a respiração são dois mecanismos fisiológicos e involuntários requeridos para a execução do Prãnãyama. O principal objetivo desse estudo foi verificar se variáveis contínuas do EEG (potência de diferentes faixas que o compõem) seriam moduladas pelo controle respiratório, comparando-se separadamente as duas fases do ciclo respiratório (inspiração e expiração), na situação de respiração espontânea e controlada. Fizeram parte do estudo 19 sujeitos (7 homens/12 mulheres, idade média de 36,89 e DP = ± 14,46) que foram convidados a participar da pesquisa nas dependências da Faculdade de Saúde da Universidade Metodista de São Paulo. Para o registro do eletroencefalograma foi utilizado um sistema de posicionamento de cinco eletrodos Ag AgCl (FPz, Fz, Cz, Pz e Oz) fixados a uma touca de posicionamento rápido (Quick-Cap, Neuromedical Supplies®), em sistema 10-20. Foram obtidos valores de máxima amplitude de potência (espectro de potência no domínio da frequência) nas frequências teta, alfa e beta e delta e calculada a razão teta/beta nas diferentes fases do ciclo respiratório (inspiração e expiração), separadamente, nas condições de respiração espontânea e de controle respiratório. Para o registro do ciclo respiratório, foi utilizada uma cinta de esforço respiratório M01 (Pletismógrafo). Os resultados mostram diferenças significativas entre as condições de respiração espontânea e de controle com valores das médias da razão teta/beta menores na respiração controlada do que na respiração espontânea e valores de média da potência alfa sempre maiores no controle respiratório. Diferenças significativas foram encontradas na comparação entre inspiração e expiração da respiração controlada com diminuição dos valores das médias da razão teta/beta na inspiração e aumento nos valores das médias da potência alfa, sobretudo na expiração. Os achados deste estudo trazem evidências de que o controle respiratório modula variáveis eletrofisiológicas relativas à atenção refletindo um estado de alerta, porém mais relaxado do que na situação de respiração espontânea.

MicroRNA-155 promotes resolution of hypoxia-inducible factor 1α activity during prolonged hypoxia

The hypoxia-inducible factor (HIF) is a key regulator of the transcriptional response to hypoxia. While the mechanism underpinning HIF activation is well understood, little is known about its resolution. Both the protein and the mRNA levels of HIF-1a (but not HIF-2a) were decreased in intestinal epithelial cells exposed to prolonged hypoxia. Coincident with this, microRNA (miRNA) array analysis revealed multiple hypoxiainducible miRNAs. Among these was miRNA-155 (miR-155), which is predicted to target HIF-1a mRNA. We confirmed the hypoxic upregulation of miR-155 in cultured cells and intestinal tissue from mice exposed to hypoxia. Furthermore, a role for HIF-1a in the induction of miR-155 in hypoxia was suggested by the identification of hypoxia response elements in the miR-155 promoter and confirmed experimentally. Application of miR-155 decreased the HIF-1a mRNA, protein, and transcriptional activity in hypoxia, and neutralization of endogenous miR-155 reversed the resolution of HIF-1a stabilization and activity. Based on these data and a mathematical model of HIF-1a suppression by miR-155, we propose that miR-155 induction contributes to an isoform-specific negative-feedback loop for the resolution of HIF-1a activity in cells exposed to prolonged hypoxia, leading to oscillatory behavior of HIF-1a-dependent transcription. © 2011, American Society for Microbiology.

Some Mean-Value Theorems of the Cauchy Type

2000 Mathematics Subject Classification: Primary 26A24, 26D15; Secondary 41A05

Cocaine Enhances HIV-1 Infectivity in Monocyte Derived Dendritic Cells by Suppressing microRNA-155

Cocaine and other drugs of abuse increase HIV-induced immunopathogenesis; and neurobiological mechanisms of cocaine addiction implicate a key role for microRNAs (miRNAs), single-stranded non-coding RNAs that regulate gene expression and defend against viruses. In fact, HIV defends against miRNAs by actively suppressing the expression of polycistronic miRNA cluster miRNA-17/92, which encodes miRNAs including miR-20a. IFN-g production by natural killer cells is regulated by miR-155 and this miRNA is also critical to dendritic cell (DC) maturation. However, the impact of cocaine on miR-155 expression and subsequent HIV replication is unknown. We examined the impact of cocaine on two miRNAs, miR-20a and miR-155, which are integral to HIV replication, and immune activation. Using miRNA isolation and analysis, RNA interference, quantitative real time PCR, and reporter assays we explored the effects of cocaine on miR-155 and miR-20 in the context of HIV infection. Here we demonstrate using monocyte-derived dendritic cells (MDCCs) that cocaine significantly inhibited miR-155 and miR-20a expression in a dose dependent manner. Cocaine and HIV synergized to lower miR-155 and miR-20a in MDDCs by 90%. Cocaine treatment elevated LTR-mediated transcription and PU.1 levels in MDCCs. But in context of HIV infection, PU.1 was reduced in MDDCs regardless of cocaine presence. Cocaine increased DC-SIGN and and decreased CD83 expression in MDDC, respectively. Overall, we show that cocaine inhibited miR-155 and prevented maturation of MDDCs; potentially, resulting in increased susceptibility to HIV-1. Our findings could lead to the development of novel miRNA-based therapeutic strategies targeting HIV infected cocaine abusers.

miRNAS circulantes como biomarcadores para fibrilação atrial aguda

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Some non-coding RNAs (miRNAs) have been involved in regulatory activity in arrhythmogenesis, targeting genes that contribute to the development of AF. The present study aimed to evaluate the expression of candidate miRNAs in plasma from patients with AF and new-onset AF and its application as future markers for diagnosis and monitoring of disease. miR-21, miR-133a, miR-133b, miR-150, miR-328 and miR-499 were selected as targets in this study through a prior literature review. They were isolated from plas-ma of individuals aged from 20 to 85 years old with AF (n = 17), new-onset AF (n = 5) and without AF (n = 15), where the latter was the control group. The results were ana-lyzed by Real-Time PCR (RT-PCR) with miScript SYBR Green PCR. We observed that miR-21, miR-133b, miR-328 and miR-499 had different levels of expression be-tween the three groups (p <0.05). Increased expression of miR-21 (0.6-fold), miR-133b (1.4-fold), miR-328 (2.0-fold) and miR-499 (2.3-fold) in patients with new-onset AF when compared to AF and control subjects. The miR-133a and miR-150 expression did not differ among the groups. miR-21, miR-133b, miR-328 and miR-499 may be potential biomarkers for AF as well as for new-onset AF, for monitoring and for the di-agnosis. These findings may contribute to the understanding of the process that trig-gers AF and suggest application these molecules as future biomarkers for AF.

miRNAS circulantes como biomarcadores para fibrilação atrial aguda

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Some non-coding RNAs (miRNAs) have been involved in regulatory activity in arrhythmogenesis, targeting genes that contribute to the development of AF. The present study aimed to evaluate the expression of candidate miRNAs in plasma from patients with AF and new-onset AF and its application as future markers for diagnosis and monitoring of disease. miR-21, miR-133a, miR-133b, miR-150, miR-328 and miR-499 were selected as targets in this study through a prior literature review. They were isolated from plas-ma of individuals aged from 20 to 85 years old with AF (n = 17), new-onset AF (n = 5) and without AF (n = 15), where the latter was the control group. The results were ana-lyzed by Real-Time PCR (RT-PCR) with miScript SYBR Green PCR. We observed that miR-21, miR-133b, miR-328 and miR-499 had different levels of expression be-tween the three groups (p <0.05). Increased expression of miR-21 (0.6-fold), miR-133b (1.4-fold), miR-328 (2.0-fold) and miR-499 (2.3-fold) in patients with new-onset AF when compared to AF and control subjects. The miR-133a and miR-150 expression did not differ among the groups. miR-21, miR-133b, miR-328 and miR-499 may be potential biomarkers for AF as well as for new-onset AF, for monitoring and for the di-agnosis. These findings may contribute to the understanding of the process that trig-gers AF and suggest application these molecules as future biomarkers for AF.

Acurácia dos indicadores clínicos do diagnóstico de enfermagem falta de adesão em pessoas vivendo com AIDS

In the early 1990s, a major milestone in the treatment of Acquired Immune Deficiency Syndrome was the development of highly active combination antiretroviral therapy. The great benefit generated by the use of this therapy was prolonging the survival of the people who got this disease, since it is no longer considered fatal, becoming a chronic condition. Despite improvements generated by this therapy, there are still many difficulties to be overcome. One is the patient adherence to their treatment, bringing challenges to services and health professionals. Hence the need for early identification of nursing diagnosis Lack of Accession so that solutions are sought by the nurse with the patient and his family. With this problem, adds to the difficulty of hospital nurses in inferring that diagnosis, especially in identifying their defining characteristics. In this context, the objective was to evaluate the accuracy of clinical indicators of nursing diagnosis Lack of Adherence to antiretroviral treatment for people living with the Acquired Immunodeficiency Syndrome. The research took place in two stages. The first consists of the evaluation of the diagnostic indicators in the study; and second, the diagnostic inference performed by specialist nurses. The first step took place in a referral hospital in the treatment of infectious diseases in the Northeast of Brazil, and data were collected through an instrument for carrying out history and physical examination and analyzed for the presence or absence of the diagnostic indicators. In the second stage, the data were sent to experts, who judged the presence or absence of the diagnosis in the studied clientele. The project was submitted to the Ethics Committee of the Federal University of Rio Grande do Norte, obtaining approval with the General Certificate for Ethics Assessment (CAAE) No 46206215.3.0000.5537. Data were analyzed using descriptive and inferential statistics. Test were used Fisher's exact, chi-square test of Pearson and logistic regression. Since the accuracy of clinical indicators was measured by sensitivity, specificity, predictive values, likelihood ratios. As a result, we identified the presence of diagnosis Lack of Accession on 69% (n = 78) of the study patients. The defining characteristics that showed statistically significant association with the diagnosis studied were: lack of adherence behavior, complications related to development, missing scheduled appointments, failure to achieve results, and exacerbation of symptoms. The characteristic with greater sensitivity was missing scheduled appointments and the highest specificity behavior of noncompliance. The logistic regression showed as predictors for the diagnosis Lack of Accession: lack of adherence behavior, missing scheduled appointments, failure to achieve results, and exacerbation of symptoms. It was concluded that the identification of clinical indicators accurately enabled a good prediction of the nursing diagnosis Lack of Accession on people living with the Acquired Immune Deficiency Syndrome, helping nurses develop early on strategies for promoting adherence to the use of antiretrovirals.

Acurácia dos indicadores clínicos do diagnóstico de enfermagem falta de adesão em pessoas vivendo com AIDS

In the early 1990s, a major milestone in the treatment of Acquired Immune Deficiency Syndrome was the development of highly active combination antiretroviral therapy. The great benefit generated by the use of this therapy was prolonging the survival of the people who got this disease, since it is no longer considered fatal, becoming a chronic condition. Despite improvements generated by this therapy, there are still many difficulties to be overcome. One is the patient adherence to their treatment, bringing challenges to services and health professionals. Hence the need for early identification of nursing diagnosis Lack of Accession so that solutions are sought by the nurse with the patient and his family. With this problem, adds to the difficulty of hospital nurses in inferring that diagnosis, especially in identifying their defining characteristics. In this context, the objective was to evaluate the accuracy of clinical indicators of nursing diagnosis Lack of Adherence to antiretroviral treatment for people living with the Acquired Immunodeficiency Syndrome. The research took place in two stages. The first consists of the evaluation of the diagnostic indicators in the study; and second, the diagnostic inference performed by specialist nurses. The first step took place in a referral hospital in the treatment of infectious diseases in the Northeast of Brazil, and data were collected through an instrument for carrying out history and physical examination and analyzed for the presence or absence of the diagnostic indicators. In the second stage, the data were sent to experts, who judged the presence or absence of the diagnosis in the studied clientele. The project was submitted to the Ethics Committee of the Federal University of Rio Grande do Norte, obtaining approval with the General Certificate for Ethics Assessment (CAAE) No 46206215.3.0000.5537. Data were analyzed using descriptive and inferential statistics. Test were used Fisher's exact, chi-square test of Pearson and logistic regression. Since the accuracy of clinical indicators was measured by sensitivity, specificity, predictive values, likelihood ratios. As a result, we identified the presence of diagnosis Lack of Accession on 69% (n = 78) of the study patients. The defining characteristics that showed statistically significant association with the diagnosis studied were: lack of adherence behavior, complications related to development, missing scheduled appointments, failure to achieve results, and exacerbation of symptoms. The characteristic with greater sensitivity was missing scheduled appointments and the highest specificity behavior of noncompliance. The logistic regression showed as predictors for the diagnosis Lack of Accession: lack of adherence behavior, missing scheduled appointments, failure to achieve results, and exacerbation of symptoms. It was concluded that the identification of clinical indicators accurately enabled a good prediction of the nursing diagnosis Lack of Accession on people living with the Acquired Immune Deficiency Syndrome, helping nurses develop early on strategies for promoting adherence to the use of antiretrovirals.

Winter lovers: engajamento e participação no universo de game of thrones da internet

The formation of groups acquired new features with the arrival of the internet. The links before straitened between family, work groups and close friends today reach long distances through online communities. These communities represent groups that have affinities and common interests, and use the community space to discuss these. Examples of these communities are those related to franchise Game of Thrones, a literary phenomenon that has expanded by various media, including the social, television, and communities. This report aims to present the work steps and the theoretical reflection, necessary for the achievement of a final product in file format, which aimed to measure the engagement and participation of GOT fans on the Internet, especially in two Facebook GOT communities during the fifth season of the series aired on HBO.

Molecular Insights of CD4+ T Cell Differentiation, Effector Formation and Helper Function

CD4+ T cells play a crucial in the adaptive immune system. They function as the central hub to orchestrate the rest of immunity: CD4+ T cells are essential governing machinery in antibacterial and antiviral responses by facilitating B cell affinity maturation and coordinating the innate and adaptive immune systems to boost the overall immune outcome; on the contrary, hyperactivation of the inflammatory lineages of CD4+ T cells, as well as the impairments of suppressive CD4+ regulatory T cells, are the etiology of various autoimmunity and inflammatory diseases. The broad role of CD4+ T cells in both physiological and pathological contexts prompted me to explore the modulation of CD4+ T cells on the molecular level.

microRNAs (miRNAs) are small RNA molecules capable of regulating gene expression post-transcriptionally. miRNAs have been shown to exert substantial regulatory effects on CD4+ T cell activation, differentiation and helper function. Specifically, my lab has previously established the function of the miR-17-92 cluster in Th1 differentiation and anti-tumor responses. Here, I further analyzed the role of this miRNA cluster in Th17 differentiation, specifically, in the context of autoimmune diseases. Using both gain- and loss-of-function approaches, I demonstrated that miRNAs in miR-17-92, specifically, miR-17 and miR-19b in this cluster, is a crucial promoter of Th17 differentiation. Consequently, loss of miR-17-92 expression in T cells mitigated the progression of experimental autoimmune encephalomyelitis and T cell-induced colitis. In combination with my previous data, the molecular dissection of this cluster establishes that miR-19b and miR-17 play a comprehensive role in promoting multiple aspects of inflammatory T cell responses, which underscore them as potential targets for oligonucleotide-based therapy in treating autoimmune diseases.

To systematically study miRNA regulation in effector CD4+ T cells, I devised a large-scale miRNAome profiling to track in vivo miRNA changes in antigen-specific CD4+ T cells activated by Listeria challenge. From this screening, I identified that miR-23a expression tightly correlates with CD4+ effector expansion. Ectopic expression and genetic deletion strategies validated that miR-23a was required for antigen-stimulated effector CD4+ T cell survival in vitro and in vivo. I further determined that miR-23a targets Ppif, a gatekeeper of mitochondrial reactive oxygen species (ROS) release that protects CD4+ T cells from necrosis. Necrosis is a type of cell death that provokes inflammation, and it is prominently triggered by ROS release and its consequent oxidative stress. My finding that miR-23a curbs ROS-mediated necrosis highlights the essential role of this miRNA in maintaining immune homeostasis.

A key feature of miRNAs is their ability to modulate different biological aspects in different cell populations. Previously, my lab found that miR-23a potently suppresses CD8+ T cell cytotoxicity by restricting BLIMP1 expression. Since BLIMP1 has been found to inhibit T follicular helper (Tfh) differentiation by antagonizing the master transcription factor BCL6, I investigated whether miR-23a is also involved in Tfh differentiation. However, I found that miR-23a does not target BLIMP1 in CD4+ T cells and loss of miR-23a even fostered Tfh differentiation. This data indicate that miR-23a may target other pathways in CD4+ T cells regarding the Tfh differentiation pathway.

Although the lineage identity and regulatory networks for Tfh cells have been defined, the differentiation path of Tfh cells remains elusive. Two models have been proposed to explain the differentiation process of Tfh cells: in the parallel differentiation model, the Tfh lineage is segregated from other effector lineages at the early stage of antigen activation; alternatively, the sequential differentiation model suggests that naïve CD4+ T cells first differentiate into various effector lineages, then further program into Tfh cells. To address this question, I developed a novel in vitro co-culture system that employed antigen-specific CD4+ T cells, naïve B cells presenting cognate T cell antigen and BAFF-producing feeder cells to mimic germinal center. Using this system, I were able to robustly generate GC-like B cells. Notably, well-differentiated Th1 or Th2 effector cells also quickly acquired Tfh phenotype and function during in vitro co-culture, which suggested a sequential differentiation path for Tfh cells. To examine this path in vivo, under conditions of classical Th1- or Th2-type immunizations, I employed a TCRβ repertoire sequencing technique to track the clonotype origin of Tfh cells. Under both Th1- and Th2- immunization conditions, I observed profound repertoire overlaps between the Teff and Tfh populations, which strongly supports the proposed sequential differentiation model. Therefore, my studies establish a new platform to conveniently study Tfh-GC B cell interactions and provide insights into Tfh differentiation processes.

microRNA Regulation of Cellular Immunity

Immunity is broadly defined as a mechanism of protection against non-self entities, a process which must be sufficiently robust to both eliminate the initial foreign body and then be maintained over the life of the host. Life-long immunity is impossible without the development of immunological memory, of which a central component is the cellular immune system, or T cells. Cellular immunity hinges upon a naïve T cell pool of sufficient size and breadth to enable Darwinian selection of clones responsive to foreign antigens during an initial encounter. Further, the generation and maintenance of memory T cells is required for rapid clearance responses against repeated insult, and so this small memory pool must be actively maintained by pro-survival cytokine signals over the life of the host.

T cell development, function, and maintenance are regulated on a number of molecular levels through complex regulatory networks. Recently, small non-coding RNAs, miRNAs, have been observed to have profound impacts on diverse aspects of T cell biology by impeding the translation of RNA transcripts to protein. While many miRNAs have been described that alter T cell development or functional differentiation, little is known regarding the role that miRNAs have in T cell maintenance in the periphery at homeostasis.

In Chapter 3 of this dissertation, tools to study miRNA biology and function were developed. First, to understand the effect that miRNA overexpression had on T cell responses, a novel overexpression system was developed to enhance the processing efficiency and ultimate expression of a given miRNA by placing it within an alternative miRNA backbone. Next, a conditional knockout mouse system was devised to specifically delete miR-191 in a cell population expressing recombinase. This strategy was expanded to permit the selective deletion of single miRNAs from within a cluster to discern the effects of specific miRNAs that were previously inaccessible in isolation. Last, to enable the identification of potentially therapeutically viable miRNA function and/or expression modulators, a high-throughput flow cytometry-based screening system utilizing miRNA activity reporters was tested and validated. Thus, several novel and useful tools were developed to assist in the studies described in Chapter 4 and in future miRNA studies.

In Chapter 4 of this dissertation, the role of miR-191 in T cell biology was evaluated. Using tools developed in Chapter 3, miR-191 was observed to be critical for T cell survival following activation-induced cell death, while proliferation was unaffected by alterations in miR-191 expression. Loss of miR-191 led to significant decreases in the numbers of CD4+ and CD8+ T cells in the periphery lymph nodes, but this loss had no impact on the homeostatic activation of either CD4+ or CD8+ cells. These peripheral changes were not caused by gross defects in thymic development, but rather impaired STAT5 phosphorylation downstream of pro-survival cytokine signals. miR-191 does not specifically inhibit STAT5, but rather directly targets the scaffolding protein, IRS1, which in turn alters cytokine-dependent signaling. The defect in peripheral T cell maintenance was exacerbated by the presence of a Bcl-2YFP transgene, which led to even greater peripheral T cell losses in addition to developmental defects. These studies collectively demonstrate that miR-191 controls peripheral T cell maintenance by modulating homeostatic cytokine signaling through the regulation of IRS1 expression and downstream STAT5 phosphorylation.

The studies described in this dissertation collectively demonstrate that miR-191 has a profound role in the maintenance of T cells at homeostasis in the periphery. Importantly, the manipulation of miR-191 altered immune homeostasis without leading to severe immunodeficiency or autoimmunity. As much data exists on the causative agents disrupting active immune responses and the formation of immunological memory, the basic processes underlying the continued maintenance of a functioning immune system must be fully characterized to facilitate the development of methods for promoting healthy immune function throughout the life of the individual. These findings also have powerful implications for the ability of patients with modest perturbations in T cell homeostasis to effectively fight disease and respond to vaccination and may provide valuable targets for therapeutic intervention.

MicroRNA expression and function in neonatal hypoxic ischaemic encephalopathy

Hypoxic ischaemic encephalopathy (HIE) is a devastating neonatal condition which affects 2-3 per 1000 infants annually. The current gold standard of treatment - induced hypothermia, has the ability to reduce neonatal mortality and improve neonatal morbidity. However, to be effective it needs to be initiated within the therapeutic window which exists following initial insult until approximately 6 hours after birth. Current methods of assessment which are relied upon to identify infants with HIE are subjective and unreliable. To overcome this issue, an early and reliable biomarker of HIE severity must be identified. MicroRNA (miRNA) are a class of small non-coding RNA molecules which have potential as biomarkers of disease state and potential therapeutic targets. These tiny molecules can modulate gene expression by inhibiting translation of messenger RNA (mRNA) and as a result, can regulate protein synthesis. These miRNA are understood to be released into the circulation during cellular stress, where they are highly stable and relatively easy to quantify. Therefore, these miRNAs may be ideal candidates for biomarkers of HIE severity and may aid in directing the clinical management of these infants. By using both transcriptomic and proteomic approaches to analyse the expression of miRNAs and their potential targets in the umbilical cord blood, I have confirmed that infants with perinatal asphyxia and HIE have a significantly different UCB miRNA signature compared to UCB samples from healthy controls. Finally, I have identified and investigated 2 individual miRNAs; both of which show some potential as classifiers of HIE severity and predictors of long term outcome, particularly when coupled with their downstream targets. While this work will need to be validated and expanded in a new and larger cohort of infants, it suggests the potential of miRNA as biomarkers of neonatal pathological conditions such as HIE.