Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 121-135).

Thesis (Ph. D.)--University of Missouri-Columbia, 2005. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Not embargoed. Vita. Bibliography: 129-136.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 108-121).

La signalisation cellulaire induite par les récepteurs à sept domaines transmembranaires(R7TM) contrôle les principales fonctions physiologiques humaines. Ces R7TMs sont cibles de médicaments et initient de larges réseaux d'interactions. Nous avons modélisé dynamiquement les réseaux de signalisation du récepteur à l'hormone folliculo-stimulante(FSH) régulant la fonction de reproduction et du récepteur angiotensine, un R7TM modèle régulant la tension pour comprendre le fonctionnement de ces réseaux et prédire des données inaccessibles expérimentalement. Notre modélisation a utilisé des équations différentielles ordinaires, en assimilant une variable par espèce et un paramètre par constante cinétique. Les paramètres manquants ont été déterminés par optimisation paramétrique.Puis, nous avons développé un environnement afin de comparer plusieurs algorithmes d'optimisation et créer une nouvelle méthode hybride plus performante et adaptée à la paramétrisation des réseaux de signalisation. / Seven transmembrane receptor (7TMR) signaling controls the main human physiological functions. R7TMs are targeted by drugs and initiate large and complex networks responsible for physiological effects. In this thesis, we dynamically modelled the FSHR induced network that have critical role in reproduction and the angiotensin receptor induced network which regulates blood presure and is considered as a model 7TMR with the objective to understand their mechanisms of functionning and to predict experimentally unreachable data. Our modeling, based on ODE formalism, assumes each species as a variable and each kinetic rate as a parameter. Some parameters were unknown and requiered an adjustment. This led us to develop an environement allowing the comparaisonof existing adjustment methods and to create a novel and efficient hybrid method well-adapted to parametrization of signaling networks.

SERINC5 is a restriction factor for retroviruses, antagonized by Nef of primate lentiviruses, by glycoGag of Moloney Murine Leukaemia Virus (MoMLV) and by S2 of Equine Infectious Anaemia virus (EIAV). In addition, SERINC5 sensitizes HIV-1 to neutralizing antibodies (nAbs) targeting the MPER in gp41. However, since the identification of SERINC5 as an inhibitor of retrovirus infectivity, many features of the host factor await clarification, notably the molecular mechanisms of restriction and viral counteraction. Furthermore, SERINC5 cellular role beyond restriction is still obscure. This thesis explores multiple aspects of the mutual antagonism governing the SERINC5 interplay with retroviruses. We first describe a contribution towards the determination of the structure of SERINC5 and the identification of the determinants crucial for antiviral activity, virus sensitization to neutralization and counteraction by retroviruses. By performing a structure-based mutagenesis screening, we identified SERINC5 ECL3, ECL5 and the interface between subdomains as regions essential for inhibition of HIV-1 infectivity and virus sensitization to 4E10 and 2F5 nAbs. The simultaneous impairment of both SERINC5 antiviral effects indicates that they are mechanistically related and support the hypothesis of a SERINC5-mediated impairment of the envelope glycoproteins. We included a comparative analysis of the antiviral activity of human SERINC paralogs and their sensitivity to retroviral counteraction. It has been previously established that SERINC3 inhibits HIV-1 infectivity less potently than SERINC5, while SERINC2 has no antiviral effects. We report here that similarly to SERINC3, SERINC1 is endowed with a modest antiviral activity; in contrast, SERINC4 severely inhibits HIV-1 infectivity, despite being poorly expressed. Irrespectively of their antiretroviral potency, all SERINC proteins are incorporated into virus particles. Interestingly, we observed that virion-associated SERINC2 is specifically cleaved by the viral protease, but proteolysis does not explain the lack of antiretroviral effects. Furthermore, SERINC5 and SERINC2 have different glycomic profiles, but diverse post-translational modification is irrelevant for their opposite activity against HIV-1. In addition, we reported that human SERINCs are differently targeted by retroviral counteracting factors, with SERINC5 being the paralog most efficiently downregulated, while SERINC1 being completely resistant. A cysteines cluster within ICL4 emerged as the major determinant of SERINC5 responsiveness to different nef alleles, while it proved irrelevant for internalization by MoMLV glycoGag and EIAV S2, indicating that diverse retroviral counteractors likely target the host factor differently. Though SERINC5 ICL4 harbours multiple motifs governing SERINC5 sensitivity to antagonization, insertion of this loop within SERINC2 was not enough to transfer susceptibility to Nef activity, suggesting that the overall conformation of the protein is essential for downregulation by Nef. Importantly, the cysteine stretch within ICL4 is palmitoylated, suggesting that this modification may be important for counteraction by the lentiviral factor. SERINC5 and CD4 downregulation by Nef are functionally related, as they both require AP-2 mediated endocytosis. However, regions in Nef selectively governing SERINC5 internalization are unknown. We reported here that Phe90 within Nef αA-helix genetically uncouples the activities on SERINC5 and CD4, being selectively involved in SERINC5 downregulation. In parallel, we explored SERINC5 antagonization by different glycoGag alleles and observed that the ability to target the host factor is not conserved across divergent γ-retroviruses. Finally, we observed that HIV-1 may evade SERINC5 restriction by direct cell-to-cell infection, suggesting that the host factor may have a broader role in retroviral spreading, requiring the evolution and the conservation of active viral counteraction. To this end, we preliminary investigated a positive contribution of SERINC5 to intracellular signalling.

PTSD is a moderately heritable disorder that causes intense and chronic suffering in many afflicted individuals. The pathogenesis of PTSD is not well understood, and genetic mechanisms are particularly elusive. Neurotransmitter systems are thought to contribute to PTSD etiology and are the targets of most pharmacotherapies used to treat PTSD, including the only two FDA approved options and a wide array of off-label options. However, the degree to which variation in genes which encode for and regulate neurotransmitter receptors increase risk of developing PTSD is unclear. Recently, large collaborative groups of PTSD genetics researchers have completed genome-wide association studies (GWAS) using massive sample sizes and have made summary statistics available for public use. In 2018, a new technique for high-powered analysis of GWAS summary statistics called GSA-SNP2 was introduced. In order to explore the relationship between PTSD and genetic variants in widely theorized molecular targets, this study applied GSA-SNP2 to manually curated neurotransmitter receptor gene-sets. Curated gene-sets included nine total "neurotransmitter receptor group" gene-sets and 45 total "receptor subtype" gene-sets. Each "neurotransmitter receptor group" gene-sets was designed to capture concentration of genetic risk factors for PTSD within genes which encode for all receptor subtypes that are activated by a given neurotransmitter. In contrast, "receptor subtype" gene-sets focused on specific subtypes and also accounted for intracellular signaling; each was designed to capture concentration of genetic risk factors for PTSD within genes which encode for specific receptor subtypes and the intracellular signaling proteins through which they exert their effects. Due to practical considerations, this work used summary statistics derived from a GWAS with far fewer participants (2,424 cases; 7,113 controls) than initially planned (23,212 cases; 151,447 controls). Prior to controlling for multiple comparisons, 7 of the investigated gene-sets reached statistical significance at the p ≤ .05 level. However, after controlling for multiple comparisons, none of the investigated gene-sets reached statistical significance. Due to limited statistical power of the current work, these results should be interpreted very cautiously. The current study is best interpreted as a preliminary study and is most informative in relation to refining study design. Implications for next steps are emphasized in discussion and nominally significant results are synthesized with the literature to demonstrate the types of research questions that might be addressed by applying a refined version of this study design to a larger sample. / Doctor of Philosophy / Though nearly all individuals will be exposed to a potentially traumatic event in their lifetime, only a small percentage will experience PTSD, which is a severe psychological disorder. Though genetics are known contribute to an individual's level of risk for developing PTSD, relatively little is known about which particular genetic differences are key. Neurotransmitter receptors are thought to contribute to the risk for PTSD and are a key aspect of medications for PTSD. However, little is known about whether genetic differences in neurotransmitter receptors contribute to risk for developing PTSD. Recently, large collaborative groups of PTSD genetics researchers have completed studies which investigate genetic risk factors from across the genome using massive sample sizes and have made the statistical output of these studies available to the public. In 2018, a new technique called GSA-SNP2 was created to help assist with efforts to analyze aspects of that statistical output that have not been previously analyzed. This study used GSA-SNP2 to analyze the degree to which groups of neurotransmitter receptor genes contribute to the risk of developing PTSD. Due to the coronavirus pandemic, the researcher did not have access to the computing power needed to analyze the initially planned data which included 23,212 individuals with PTSD and 151,447 individuals without PTSD. As a substitute, the current work is an analysis using statistical output data from a study which included 2,424 individuals with PTSD and 7,113 individuals without PTSD. Based on a level of statistical significance that is typically used in most psychological studies, seven of the investigated gene-sets contribute highly to the risk for PTSD. However, it was necessary to use a different threshold for statistical significance due to the testing of many different groups of genes. After making that adjustment, none of the investigated gene-sets reached statistical significance. Due to limited statistical power of the current work, these results should be interpreted very cautiously. The current study is best interpreted as a preliminary study and is most informative in relation to refining study design. Implications for next steps are emphasized in discussion and nominally significant results are synthesized with the literature to demonstrate the types of research questions that might be addressed by applying a refined version of this study design to a larger sample.

O sistema imune neonatal tem sido considerado funcionalmente imaturo e recentes estudos sugerem que a suscetibilidade do neonato às infecções pode ser devido a alterações funcionais de células apresentadoras de antígenos que podem levar a deficiências secundárias nas respostas adaptativas. A ativação das células apresentadoras de antígenos é desencadeada pela estimulação de receptores, como os Toll-like Receptors (TLRs) e alterações na ativação desses receptores podem levar a uma subsequente redução da ativação de proteínas da via de sinalização intracelular e consequente alterações dos níveis das citocinas pró- e anti-inflamatórias, contribuindo assim, para uma resposta imune ineficiente do neonato. O Toll-like receptor 2 (TLR-2) é um receptor essencial para o reconhecimento seletivo de vários antígenos bacterianos e virais, em especial, o peptideoglicano, que compreende cerca de 50% da parede celular de bactérias Grampositivas, como os estafilococos, que são agentes infecciosos que prevalecem nas Unidades de Terapia Intensiva Neonatal. O objetivo deste estudo foi avaliar a ativação e resposta de monócitos de sangue do cordão umbilical de recém-nascidos pré-termo saudáveis < 34 semanas de gestação (Grupo 1), recém-nascidos pré-termo : 34 e < 37 semanas de gestação (Grupo 2) e recém-nascidos a termo (Grupo 3) e de adultos saudáveis, como controles, após a estimulação de TLR-2 ex-vivo com Pam3CSK4. Após a estimulação dos monócitos, foram determinados os níveis de expressão dos marcadores de ativação celular, os níveis das citocinas pró- e anti-inflamatórias e a expressão de moléculas envolvidas na sinalização intracelular. A caracterização das populações leucocitárias, bem como a capacidade fagocítica de Staphylococcus aureus e geração de burst oxidativo por monócitos e neutrófilos foram analisados por Citometria de Fluxo. Os resultados demonstraram que as células dendríticas e monócitos de neonatos expressam TLR-2 em níveis semelhantes aos de adultos. A expressão adequada de TLR-2 sugere um reconhecimento antigênico eficiente que é refletido em uma ativação apropriada das moléculas da cascata de sinalização e uma potente produção de citocinas pró-inflamatórias, apesar da reduzida produção de IL-10. Fagócitos neonatais apresentaram capacidade fagocítica de S. aureus reduzida em relação aos adultos e geração do burst oxidativo semelhante entre os grupos, no entanto neonatos prétermo apresentaram produção de peróxido de hidrogênio deficiente, o que poderia contribuir com uma reduzida morte intracelular deste microrganismo. Em conclusão, o recém-nascido não apresenta uma imaturidade funcional, mas sim, um desequilíbrio em sua resposta imune inata, com uma aparente menor produção de fatores antiinflamatórios, o que pode levar a predisposição à sepse / The neonatal immune system has been considered functionally immature and recent studies suggest that susceptibility of the neonate to infections may be due to functional alterations in antigen-presenting cells that can prompt to secondary deficiencies in adaptive responses. The activation of antigen-presenting cells is triggered by stimulation of receptors such as Toll-like receptors (TLRs) and changes in the activation of these receptors may lead to a subsequent reduction in the activation of intracellular signaling pathway proteins and consequent changes in pro- and anti-inflammatory cytokine levels, thus contributing to an inefficient immune response of the neonate. Toll-like Receptor 2 (TLR-2) is an essential receptor for the selective recognition of several bacterial and viral antigens, in particular, peptidoglycan, which comprises about 50% of the Gram-positive bacteria cell wall, such as staphylococci, which are infectious agents that prevail in Neonatal Intensive Care Units. The aim of this study was to evaluate the activation and response of monocytes derived from umbilical cord blood of healthy preterm newborns <34 weeks of gestation (Group 1), preterm newborns :34 and <37 weeks of gestation (Group 2) and term newborns (Group 3) and from healthy adults, as controls, after ex-vivo TLR-2 stimulation with Pam3CSK4. After monocyte stimulation, it was determined the expression levels of cellular activation markers, pro- and anti-inflammatory cytokine levels and the expression of molecules involved in downstream intracellular signaling. The characterization of leukocyte populations, as well as the phagocytic ability of Staphylococcus aureus and generation of oxidative burst by monocytes and neutrophils were analyzed by flow cytometry. The results demonstrated that neonatal dendritic cells and monocytes express TLR- 2 at similar levels to those of adults. The proper expression of TLR-2 suggests an efficient antigen recognition which is reflected in an appropriate activation of downstream signaling molecules and potent production of pro-inflammatory cytokines, in spite of the reduced production of IL-10. Neonatal phagocytes showed reduced phagocytic capacity of S. aureus compared to adults and similar generation of oxidative burst between groups, however preterm neonates showed deficient production of hydrogen peroxide, which could contribute to a reduced intracellular killing of this microorganism. In conclusion, the newborn does not present a functional immaturity, but an imbalance in its innate immune response, with an apparent lower production of antiinflammatory factors, which can lead to a predisposition to sepsis

O sistema imune neonatal tem sido considerado funcionalmente imaturo e recentes estudos sugerem que a suscetibilidade do neonato às infecções pode ser devido a alterações funcionais de células apresentadoras de antígenos que podem levar a deficiências secundárias nas respostas adaptativas. A ativação das células apresentadoras de antígenos é desencadeada pela estimulação de receptores, como os Toll-like Receptors (TLRs) e alterações na ativação desses receptores podem levar a uma subsequente redução da ativação de proteínas da via de sinalização intracelular e consequente alterações dos níveis das citocinas pró- e anti-inflamatórias, contribuindo assim, para uma resposta imune ineficiente do neonato. O Toll-like receptor 2 (TLR-2) é um receptor essencial para o reconhecimento seletivo de vários antígenos bacterianos e virais, em especial, o peptideoglicano, que compreende cerca de 50% da parede celular de bactérias Grampositivas, como os estafilococos, que são agentes infecciosos que prevalecem nas Unidades de Terapia Intensiva Neonatal. O objetivo deste estudo foi avaliar a ativação e resposta de monócitos de sangue do cordão umbilical de recém-nascidos pré-termo saudáveis < 34 semanas de gestação (Grupo 1), recém-nascidos pré-termo : 34 e < 37 semanas de gestação (Grupo 2) e recém-nascidos a termo (Grupo 3) e de adultos saudáveis, como controles, após a estimulação de TLR-2 ex-vivo com Pam3CSK4. Após a estimulação dos monócitos, foram determinados os níveis de expressão dos marcadores de ativação celular, os níveis das citocinas pró- e anti-inflamatórias e a expressão de moléculas envolvidas na sinalização intracelular. A caracterização das populações leucocitárias, bem como a capacidade fagocítica de Staphylococcus aureus e geração de burst oxidativo por monócitos e neutrófilos foram analisados por Citometria de Fluxo. Os resultados demonstraram que as células dendríticas e monócitos de neonatos expressam TLR-2 em níveis semelhantes aos de adultos. A expressão adequada de TLR-2 sugere um reconhecimento antigênico eficiente que é refletido em uma ativação apropriada das moléculas da cascata de sinalização e uma potente produção de citocinas pró-inflamatórias, apesar da reduzida produção de IL-10. Fagócitos neonatais apresentaram capacidade fagocítica de S. aureus reduzida em relação aos adultos e geração do burst oxidativo semelhante entre os grupos, no entanto neonatos prétermo apresentaram produção de peróxido de hidrogênio deficiente, o que poderia contribuir com uma reduzida morte intracelular deste microrganismo. Em conclusão, o recém-nascido não apresenta uma imaturidade funcional, mas sim, um desequilíbrio em sua resposta imune inata, com uma aparente menor produção de fatores antiinflamatórios, o que pode levar a predisposição à sepse / The neonatal immune system has been considered functionally immature and recent studies suggest that susceptibility of the neonate to infections may be due to functional alterations in antigen-presenting cells that can prompt to secondary deficiencies in adaptive responses. The activation of antigen-presenting cells is triggered by stimulation of receptors such as Toll-like receptors (TLRs) and changes in the activation of these receptors may lead to a subsequent reduction in the activation of intracellular signaling pathway proteins and consequent changes in pro- and anti-inflammatory cytokine levels, thus contributing to an inefficient immune response of the neonate. Toll-like Receptor 2 (TLR-2) is an essential receptor for the selective recognition of several bacterial and viral antigens, in particular, peptidoglycan, which comprises about 50% of the Gram-positive bacteria cell wall, such as staphylococci, which are infectious agents that prevail in Neonatal Intensive Care Units. The aim of this study was to evaluate the activation and response of monocytes derived from umbilical cord blood of healthy preterm newborns <34 weeks of gestation (Group 1), preterm newborns :34 and <37 weeks of gestation (Group 2) and term newborns (Group 3) and from healthy adults, as controls, after ex-vivo TLR-2 stimulation with Pam3CSK4. After monocyte stimulation, it was determined the expression levels of cellular activation markers, pro- and anti-inflammatory cytokine levels and the expression of molecules involved in downstream intracellular signaling. The characterization of leukocyte populations, as well as the phagocytic ability of Staphylococcus aureus and generation of oxidative burst by monocytes and neutrophils were analyzed by flow cytometry. The results demonstrated that neonatal dendritic cells and monocytes express TLR- 2 at similar levels to those of adults. The proper expression of TLR-2 suggests an efficient antigen recognition which is reflected in an appropriate activation of downstream signaling molecules and potent production of pro-inflammatory cytokines, in spite of the reduced production of IL-10. Neonatal phagocytes showed reduced phagocytic capacity of S. aureus compared to adults and similar generation of oxidative burst between groups, however preterm neonates showed deficient production of hydrogen peroxide, which could contribute to a reduced intracellular killing of this microorganism. In conclusion, the newborn does not present a functional immaturity, but an imbalance in its innate immune response, with an apparent lower production of antiinflammatory factors, which can lead to a predisposition to sepsis

Neste estudo investigamos os efeitos dos ácidos graxos sobre a função e sinalização intracelular de linfócitos humanos. Os ácidos oléico (OA) e linoléico (LA), em baixas concentrações, estimularam a proliferação celular induzida pela IL-2 através do aumento da fosforilação da proteína PKC-<font face=\"symbol\">Z que levou a um aumento da fosforilação de ERK 1/2. Já os ácidos palmítico (PA), esteárico (SA), DHA e EPA diminuíram a proliferação destas células e inibiram a fosforilação de JAK1 e 3, STAT5, ERK e Akt. Os resultados obtidos são sugestivos de que o efeito inibitório promovido por PA, SA, DHA e EPA sobre a proliferação de linfócitos ocorreu devido à diminuição da fosforilação de proteínas fundamentais para a proliferação celular. Por outro lado, OA e LA estimularam a proliferação de linfócitos aumentando a fosforilação de ERK 1/2 através da ativação de PKC-<font face=\"symbol\">Z, efeito dependente da PI3K. O efeito inibitório promovido pelo DHA está associado a uma alteração na quantidade de lipid rafts na membrana plasmática nos quais o receptor de IL-2 está localizado. / The effect of fatty acids (FA) on interleukin -2 (IL-2) signaling pathway in human lymphocytes was investigated. Docosahexaenoic (DHA), eicosapentaenoic (EPA), palmitic (PA) and stearic (SA) acids decreased lymphocyte proliferation in concentrations above 50 <font face=\"symbol\">mM. However, oleic (OA) and linoleic (LA) acids increase lymphocyte proliferation at 25 <font face=\"symbol\">mM. PA, SA, DHA and EPA decreased JAK 1, JAK 3, STAT 5 and AKT phosphorylation induced by IL-2 but OA and LA did not cause any effect. OA and LA increased ERK1/2 phosphorylation whereas the other FA caused a marked decrease. PKC-<font face=\"symbol\">Z phosphorylation was decreased by OA and LA only. In conclusion, the inhibitory effect of PA, SA, DHA and EPA on lymphocyte proliferation observed in our previous study was due to a decrease in protein phosphorylation activated by IL-2. Probably, OA and LA stimulated lymphocyte proliferation by increasing ERK 1/2 phosphorylation throught PKC-<font face=\"symbol\">Z activation. The inhibition of JAK 1, JAK3, STAT 5, ERK1/2 and Akt phosphorylation caused by DHA is associated to a decrease in membrane lipid rafts contend.