Dissertations / Theses on the topic 'Septin 4'

Septinas são proteínas ligantes a GTP encontradas desde fungos até metazoários. A primeira função identificada para septinas foi o seu papel central na organização e dinâmica do septo de divisão de leveduras. Uma das características marcantes é que septinas se organizam em heterofilamentos de 7 a 9 nm de espessura que foram purificados de diversos organismos tais como Saccharomyces cerevisiae, Drosophila e cérebro de camundongos. Hoje se sabe que septinas não estão envolvidas apenas nos processos de divisão celular, mas em uma variedade de processos como tráfico de vesículas, exocitose, interação com proteínas do citoesqueleto e com a membrana plasmática, o que resulta em alterações da morfologia celular. Neste trabalho foram desenvolvidos estudos da septina 4 humana (SEPT4) nos quais foi realizado a expressão e purificação da SEPT4 pelo uso do sistema de expressão heteróloga em E. coli e em células de insetos (Sf-9) via baculovírus. A tentativa de expressão usando o vetor pETTEV em E.coli não obteve sucesso, pois a proteína não foi expressa na forma solúvel. A construção do baculovírus recombinante AcSept4 e expressão da SEPT4 nas células de insetos foi realizada com êxito, mas o processo de purificação não foi satisfatório. Com o intuito de obter informações sobre possíveis proteínas que interagem com a SEPT4 e conseqüentemente sobre as funções desempenhadas por ela na célula, a SEPT4 foi utilizada como isca para ensaios de interação proteína-proteína pela técnica de duplo híbrido. Para isso, o gene da SEPT4 foi clonado fusionado ao domínio de ligação ao DNA Lex-A. A realização do ensaio de duplo híbrido com a proteína completa não foi possível, pois a mesma provocou a auto ativação do sistema, por isso uma nova construção foi realizada com a região GTPase e C-terminal SEPT4GC (124-478) como isca. Dentre as interações identificadas, foram encontradas apenas septinas do grupo II (SEPT6, SEPT8, SEPT10 e SEPT11) e quatro novas interações, que ainda precisam ser confirmadas. Por outro lado, uma interação já descrita na literatura envolve a proteína α-sinucleína, que é uma proteína abundantemente expressa no cérebro e associada à doença de Parkinson. O foco do estudo dessa interação foi realizar ensaios com os diferentes domínios da SEPT4 para comprovar uma interação direta e com isso tentar mapear o sítio de interação com a α-sinucleína. Os resultados obtidos pela ressonância plasmônica de superfície (SPR) indicam que o domínio C-terminal participa da interação com baixa afinidade (K,D=390 µM) e sugerem que o domínio GTPase também pode estar envolvido. Já os dados obtidos com os experimentos de RMN e anisotropia de fluorescência mostram indícios que a interação é dependente da conformação da α-sinucleína por que a interação aconteceria com maior afinidade quando a α-sinucleína está na presença de SDS.
Septins are a family of GTP binding proteins found in a great diversity of organisms. These proteins have been identified as having a central role in septum organization during yeast division. Septins are organized into heterofilaments which are 7 to 9 nm wide and these have been purified from yeast, Drosophila and mice brain. Septins are not only required for cell division, but seem to play a role also in vesicle trafficking and in the formation of diffusion barriers within cells, since they interact with cytoskeleton proteins and the plasma membrane causing changes in cell morphology. In the present work, the aim was investigate human Septin 4 (SEPT4), a septin highly expressed in the brain. One objective of this work was to find a suitable expression system and purification method for SEPT4. The protein was expressed in both E.coli and insect cells (Sf-9). Expression in E. coli with the vector pETTEV was unsuccessful because the protein was insoluble. Expression in insect cells using the recombinant baculovirus AcSept4, was obtained successfully, but the purification was difficult. Important information concerning SEPT4 function might be acquired, if interactions partners involved in cellular process were identified. With this goal in mind, a yeast two hybrid assays were performed. The sept4 gene was fused to the Lex-A DNA binding domain and used as bait in the yeast two hybrid essays. However, full length SEPT4 showed autonomous activation of reporter genes. A second construct was prepared including only GTPase domain and the carboxy terminus domain, (residues 124 to 478) and the screen of interactions were carried out only with SEPT4GC. All of the group II septins (SEPT6, SEPT8, SEPT10 and SEPT11) were identified together with four new interactions. The latter still need be confirmed. In addition, another interaction already described in the literature is between SEPT4 and α-synuclein, which is a protein highly expressed in brain and related to Parkinson\'s disease. Different spectroscopic methods and SPR were used to identify which domain of SEPT4 interacts directly with α-synuclein and in which region. The surface plasmon resonance (SPR) results indicate that the carboxy terminus participates in the interaction with low affinity (KD = 390 µM) and suggests that the GTPase domain may also be involved. The results obtained by fluorescence anisotropy and NMR studies provide evidence that the interaction is dependent on the α-synuclein conformation, because the affinity of SEPT4 and α-synuclein seemed to be higher in the presence of SDS.

Les plaquettes jouent un rôle majeur dans l'hémostase primaire ainsi que dans l'inflammation. Elles contiennent et sécrètent une grande variété de facteurs solubles et parmi les nombreux récepteurs qu'elles expriment à leur surface, les plaquettes expriment les " Toll-Like Receptor " (TLR), récepteurs clés de l'interaction entre l'immunité innée et adaptative. En réponse à un stimulus infectieux, comme le lipopolysaccharide (LPS) des bactéries Gram-négative, ligand naturel du TLR4, ou des peptides issus d'une partie de la protéine d'enveloppe du VIH (gp41), les plaquettes vont s'activer de manière différentielle. L'activation plaquettaire est variable en fonction de leur activation par à un stimulus hémostatique (exemple : la thrombine) vs. infectieux (exemple : le LPS) ; le panel de cytokines libérées dans le surnageant plaquettaire semble en fait finement régulé. De plus, nous avons démontré la présence intra-plaquettaire de la majorité des protéines composant les voies de signalisation du TLR4 eucaryote. Nous avons ensuite montré que ces voies pouvaient être modulées. L'engagement du TLR4 plaquettaire par deux types biochimiques de LPS entraîne un relargage différentiel des facteurs solubles immunomodulateurs dans le surnageant de culture et que ce surnageant dernier génère une activation différentielle des cellules cibles, comme les cellules mononucléées du sang circulant. Ces travaux montrent que la réponse inflammatoire plaquettaire est régulée en fonction du stimulus. Ainsi, mes travaux s'inscrivent dans la ré-exploration de la fonction inflammatoire des plaquettes sanguines et l'étude du rôle des plaquettes comme cellules de l'immunité innée et inflammatoire

Sepse é a resposta inflamatória sistêmica decorrente de infecção grave, com alto índice de mortalidade, tornando-se um grave problema de saúde pública. Apesar dos inúmeros estudos realizados em busca de alternativas terapêuticas, o entendimento acerca dos mecanismos envolvidos na doença permanece restrito. A interação entre o metabolismo lipídico e a resposta inflamatória tem sido intensamente investigada. Neste estudo, avaliou-se a influência da proteína de transferência de colesterol esterificado (CETP) - glicoproteína plasmática que promove a transferência de lípides entre lipoproteínas - na resposta inflamatória. Inicialmente, foram comparados camundongos transgênicos para CETP humana (CETP) e controles irmãos não transgênicos (WT) submetidos ao modelo de sepse polimicrobiana de ligadura e perfuração do ceco (CLP), avaliando a taxa de sobrevida e o perfil inflamatório entre os grupos. Em seguida, a resposta inflamatória em macrófagos de peritônio de camundongos estimulados com LPS na ausência ou presença da CETP exógena (CETP humana recombinante) e endógena (macrófagos de animais CETP) foi analisada. Verificou-se que camundongos CETP apresentaram maior taxa de sobrevida, maior migração de linfócitos para o foco infeccioso, menores concentrações plasmáticas de IL-6 e menor expressão proteica do receptor Toll-like 4 (TLR4) e da enzima aciloxiacilo hidrolase (AOAH) no fígado, comparados aos WT. Nos macrófagos, observou-se que a presença da CETP recombinante foi capaz de se ligar ao LPS, pela análise da microscopia confocal, e, em cultura, reduziu de forma dose dependente a captação de LPS, a expressão de TLR4, a ativação do NF-kB (p65) e a secreção de IL-6 para o sobrenadante do cultivo celular. Os dados obtidos com os macrófagos de animais CETP corroboraram, em parte, os encontrados com a utilização da CETP exógena. Houve redução da captação de LPS e da ativação do NF-kB (p65), sem alteração na expressão de TLR4 e secreção de IL-6. Entretanto, apresentaram redução das concentrações de TNF-alfa celular e no sobrenadante de cultura. Dessa maneira, foi possível concluir que a CETP atua como agente modulador da resposta inflamatória induzida pela CLP e em macrófagos estimulados pelo LPS. Esses achados devem ser considerados nas doenças inflamatórias e nos futuros estudos relacionados à inibição da CETP, além de estabelecer novas perspectivas de tratamento da sepse
Sepsis is a systemic inflammatory response due to serious infection with high mortality rate, which has become a serious problem for public health. Despite numerous studies seeking for therapeutic alternatives, the understanding of the mechanisms involved in this disease remains limited. The interaction between lipid metabolism and inflammatory response has been intensively investigated. In the present study it was evaluated the influence of CETP (cholesteryl ester transfer protein) - plasma glycoprotein that promotes the transfer of lipids between lipoprotein - in the inflammatory response. Initially transgenic mice for human CETP (CETP) were compared to non transgenic control mice (WT) after polymicrobial sepsis induced by cecal ligation and puncture (CLP), to determine survival rate and the inflammatory profile between groups. Then, macrophages isolated from peritoneal cavity stimulated with LPS in the presence or absence of exogenous CETP (recombinant human CETP) and endogenous CETP (macrophages from CETP mice) were analyzed. It was found that CETP mice showed a higher survival rate, a greater lymphocyte migration to infectious focus, a lower IL-6 plasma concentration and a decrease in Toll-like receptor 4 (TLR4) and acyloxyacyl hydrolase enzyme (AOAH) protein expression in the liver in comparison to WT mice. In macrophages, recombinant CETP was able to bind to LPS, by confocal microscopy analysis and in cell culture, it was observed that in the presence of the recombinant CETP macrophages presented decreased in LPS uptake, TLR4 expression, NF-kB activation (p65) and IL-6 secretion into the cell culture medium. Furthermore, the results with macrophages from animals CETP corroborate partly with what was found in the exogenous experiments. LPS uptake and NF-kB activation (p65) were reduced, but no difference regarding the expression of TLR4, nor the IL-6 secretion to the cell culture medium. However, the CETP group also showed reduced levels of TNF-alfa both in macrophages and in the culture supernatant. Thus, we conclude that CETP acts as modulator of the inflammatory response induced by CLP and in the macrophages stimulated by LPS. In addition, new therapeutic perspectives could be established

INTRODUÇÃO: Apesar do contínuo progresso no tratamento e suporte clínico a sepse continua sendo uma das principais causas de morbidade e mortalidade nas unidades de terapia intensiva, com desfechos semelhantes ao longo dos últimos 50 anos. A suscetibilidade à infecção grave no recém-nascido é parcialmente devida à imaturidade do sistema imune inato associado à mínima em exposição antigênica in utero e à ação ineficaz das células T efetoras e das célula B. Embora a ativação do sistema imune inato por padrões de reconhecimento (PRR) como os dos receptores Toll-like (TLR) tenham sua importância amplamente reconhecida nos últimos anos, seu comportamento frente a uma infecção in vivo ainda não foi completamente compreendido. Neste trabalho nós analisamos a expressão dos TLR-2 e TLR-4 em células apresentadoras de antígeno em recém-nascidos com e sem sepse. CAUSUÍSTICA E MÉTODO: Trata-se de um estudo prospectivo realizado no período entre fevereiro de 2011 e janeiro de 2013 onde foram incluídos quarenta e cinco recém-nascidos a termo, sem malformação congênita, admitidos na Unidade de Cuidados Intensivos Neonatal do Instituto da Criança-HCFMUSP e divididos em grupos 1 e 2. O grupo 1 consistiu em 27 recém-nascidos com diagnóstico clínico e laboratorial de sepse tardia enquanto que o grupo 2 foi composto por 18 recém-nascidos sem quadro séptico vigente. As citocinas foram determinadas por teste de CBA em sangue periférico. A expressão e MFI dos TLR-2 e TLR-4 foi determinado por imunofenotipagem em APCs e linfócitos no sangue periférico total através de análise pelo citômetro de fluxo BD FACSDiva. RESULTADOS: Os dados clínicos foram semelhantes entre os grupos 1 e 2, exceto para o estado infeccioso. Microrganismos foram identificados em 37 % no grupo 1 e estes tiveram níveis mais elevados de citocinas pró-inflamatórias (IL-8, IL-6, IL-1beta) e de citocina anti-inflamatória (IL-10). Nas células dendríticas, a expressão de TLR-2 e 4 foi semelhante entre os grupos enquanto que houve menor expressão nos pacientes infectados da molécula co-estimuladora CD86 (p < 0,05) e expressão semelhante de CD1a e CD80 em relação aos RN não infectados. No monócito, o MFI para TLR-2 e a freqüência de expressão do TLR-4 foi maior no grupo 1 (p = 0,01). Apesar da frequência de linfócitos totais ter sido mais baixa no grupo 1 (p = 0,002), não foi observada diferença quanto as suas subpopulações exceto em relação a maior frequência de LT efetor no grupo infectado com menor expressão da molécula CD28. Houve maior frequência de LB ativados no grupo 1 enquanto que a população total e as demais subpopulações foram semelhantes em número, moléculas de ativação e na expressão dos TLR-2 e 4 em ambos os grupos. CONCLUSÃO: Este estudo analisou a resposta imune inata no recém-nascido com e sem sepse. As IL-6, IL-8 e IL-10 foram bons indicadores desta doença. Recém-nascidos sépticos, que dependem quase exclusivamente do sistema imune inato, apresentaram pouca resposta in vivo na ativação de células dendríticas e monócitos propiciando uma resposta imune deficiente e maior susceptibilidade à infecção
INTRODUCTION: Despite continuous progress in the clinical treatment and other supportive care therapies, sepsis remains a leading cause of morbidity and mortality in the intensive care unit with similar outcome throughout the past 50 years. The susceptibility to severe infection is partially due to newborn immature innate immune system associated to minimal in utero antigen exposure and effector T and B cell impaired function. Although the importance of pattern recognition domains such as Toll-like receptors (TLR) in the innate immune system activation has been fully acknowledged within the last few years its behavior in front of an in vivo infection scenario is still not completely understood. Here we analyzed the TLR-2 and TLR-4 expression in antigen-presenting cell in healthy and septic newborns. PATIENTS AND METHODS: This prospective study was conducted during the period from February 2011 until January 2013 at Sao Paulo University, Sao Paulo, Brazil. Forty-five term newborns without congenital malformation were included from the Newborn Intensive Care Unit at Children\'s Hospital. As group 1, 27 newborns who had clinical and laboratory diagnostic of late onset sepsis were included while 18 newborns were evaluated in a non-septic status and were included at group 2. Cytokines were measured by cytometric bead array in peripheral blood. TLR-2 and TLR-4 expression and MFI were determined by immunophenotyping at peripheral whole blood in APC cells and lymphocytes and analyzed on a BD FACSDiva flow cytometer. RESULTS: Clinical data was similar between septic and non-septic groups except for the infectious status. Group 1 had microorganisms identified in 37 % septic newborns associated with higher levels of pro-inflammatory (IL-8, IL-6, IL-1beta) and anti-inflammatory interleukins (IL-10). When it comes to dendritic cells, the expression of TLR-2 and 4 was similar between groups whereas there was lower expression of co-molecule CD86 (p < 0,05) and similar expression of CD1a and CD80 between infected and non-infected patients. At monocytes, the MFI for TLR-2 and the frequency of TLR-4 expression was higher in infected newborn (p=0,01). There were lower levels of total lymphocytes in infected patients (p=0,002) but no difference was observed in T cells subtypes frequency except for higher levels of effector T cell in infected group with lower expression of CD28 molecule. Group 1 had higher levels of activated B cell whereas total population and the other subsets were similar in number, activation molecules and TLR-2 and 4 expressions in both groups. CONCLUSION: This study investigated the innate immune response in septic and non-septic newborn. Interleukin levels 6, 8 and 10 were good indicators of sepsis. Septic newborns, which count most exclusively with innate immune system, had little in vivo response at dendritic cell and monocyte activation leading to an impaired immune response and increased susceptibility to infection

The transmission of sex pheromone-mediated signals is essential for goldfish reproduction. However, the neural pathways underlying this reproductive signalling pathway in the goldfish brain is not well described. Lesioning experiments have shown previously that two brain areas, the preoptic area (POA) and the ventral telencephali pars ventralis (Vv) in particular, are important for reproduction. We used patch clamp electrophysiology to study the electrical activities of POA and Vv neurons. Based on the intrinsic properties of these neurons, we suggest there are five different functional classes of POA neurons and a single class of Vv neurons. In addition, by electrically stimulating the olfactory bulb (OB), we were able to show that this primary sensory structure makes monosynaptic glutamatergic connections with both POA and Vv neurons. While electrophysiology measures signalling events occurring at short time scales on the order of milliseconds to minutes, we were also interested in studying sex pheromone signalling in the goldfish brain over a long time scale. Thus, we describe changes in gene expression in male goldfish exposed to waterborne sex pheromones (17alpha,20beta dihydroxy-4-pregene-3-one and Prostaglandin-F2alpha) over 6 hours. We perform cDNA microarrays on Prostaglandin-F2alpha-treated fish to study the rapid modulation of transcription and define the signalling pathways affected. Our microarrays showed that 71 genes were differentially regulated (67 up and 4 down). Through gene ontology enrichment analysis, we found that these genes were involved in various biological processes such as RNA processing, neurotransmission, neuronal development, apoptosis, cellular metabolism and sexual reproduction. RT-PCRs were performed to validate our microarrays and to facilitate direct comparisons of the effects of the two sex pheromones, 17alpha,20beta dihydroxy-4-pregene-3-one and Prostaglandin-F2alpha. By combining electrophysiology and gene expression analyses, we were able to study sex-pheromone signalling on two different time scales. One short, occurring on the order of milliseconds to minutes, that involves electrical activities in the brain through the glutamatergic amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-D-aspartate receptors; and the other long occurring several hours later that involves changes in the gene expression levels of calmodulin and ependymin among other genes underlying neuroplasticity. Reproductive neuroplasticity in the goldfish may therefore require the activation of glutamatergic receptors which then activate downstream signals like calmodulin and ependymin to transform the sex pheromones-mediate signal into gene expression.

碩士
中國醫藥大學
中西醫結合研究所
93
Sepsis is induced by bacterial infection. The underlying reason of sepsis many resulted from uncontrolled inflammatory response. Host immune responses can be recruited by pathogens detected through TLRs. The study trys to reveal the relationship between TLRs and sepsis. Materials and methods: Peritoneal injection of Klebsiella Pneumoniae into mouse. Feed the mouse with herb drug. Immunohistochemistry for the specimen from stomach, intestine, ileum, liver, and lung of mouse, and evaluate the distribution of TLR4-positive portions. Results: The septic mouse recruits many inflammatory cells and reveals TLR4 positive, dominate present on subserosal area. No obvious changes can be showed on both herb groups.

碩士
中興大學
化學系所
94
IL-4 is a pleiotropic cytokine which exerts its actions on various lineages of hematopoietic and nonhematopoients cells. This cytokine is one of the central regulators of immunity in health and disease states. IL-4 is a 15-kD polypeptide with widespread effects on many cell types and secreted by helper T cell. IL-4 participates in a lot of inflammatory responses. The IL-4δ2 is a naturally occurring splice variant of human IL-4 with exons 1, 3, and 4 in an open reading frame. It is known to antagonize many biological activities of IL-4. Mycobacterial infections are chiefly controlled by cellular immunity that is through to be the activation of infected macrophages by Th1-type cytokines particularly interferon (INF)-γ. Sepsis is a complicated syndrome in which pro-inflammatory and anti-inflammatory cytokines are expressed simultaneously. Many of the components of the innate immune response are cause cell and tissue damage and hence multiple organ failure. However, it is still unclear for the expression of IL-4 and IL-4δ2 in patients with severe sepsis and TB. Does IL-4 gene polymorphism increase the organic susceptibility to inflammability and IL-4 gene expression associate with sepsis or TB？This is we want to discussion. Many factors determined capacity of cytokine production and release; the hereditary factor is a important ones of them. Genomic variations also may affect gene expression profiles, as well as the structure and production of proteins. In this study, we investigate the IL-4 gene expression polymorphism, and we also investigate the quantification of IL-4 and IL-4δ2 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR) techniques. We observed no significant differences in genotype distribution and allele frequency of the IL-4 gene. But IL-4 and IL-4 δ 2 mRNA expressions were significantly decreased in patients (p<0.05). The IL-4/IL-4δ2 ratio is no significantly lower than controls (p>0.05). These results suggest that there is no association with IL-4 encoding gene polymorphisms in TB and sepsis. Although the expression of IL-4 and IL-4δ2 were decreased in sepsis, they can not be used to predict the outcome of sepsis. Differences in cytokine expression likely determine whether disease progresses, resolves, or becomes latent. Greater IL-4δ2 expression trends to good reason in TB. The ratio of IL-4/IL-4δ2 is as a possible marker of the phase of disease in TB. The relative roles of IL-4 and IL-4δ2 in the pathogenesis of sepsis and TB deserve further investigation.

碩士
國立陽明大學
臨床醫學研究所
97
OBJECTIVES - We compared changes of serum biomarkers during right ventricular septal (RVSP) and apical pacing (RVAP) in patients with permanent pacemakers. BACKGROUD - Conventional right ventricular apical pacing might be detrimental to left ventricular (LV) function. These observated results presumbly because bypass of the His-Purkinje system produces dysynchronous LV contraction. Changes in inflammatory biomarkers between patients with novel right ventricular septal and conventional apical pacing remain unknown. METHODS - 42 patients undergoing permanent pacemaker implantation were divided prospectively into 2 groups with RV apical pacing (RVAP group, n=30) or RV septal pacing (RVSP group, n=12). Baseline serum was collected for biomarkers then the lower pacing rate was programmed up to 70BPM for each patient. After 4 weeks of pacing, the compared serum was collected again for analysis. The primary endpoints were the changes of these biomarkers from baseline to four weeks. RESULTS - In the RVAP patients group, levels of TNF-α and IL-6 were increased statistically significantly (p=0.001 and 0.03). Changes of hsCRP, ADMA, and BNP were not significant. In patients with RVSP, TNF-α level was increased with statistical significance (p=0.03) and changes of hsCRP, IL-6, ADMA, and BNP still remained insignificant. Between the two groups, the BNP and TNF-α levels of RVSP patients were significantly lower as compared to RVAP patients (p=0.03 and 0.01) at the end of the study. CONCLUSIONS - Compared the conventional RVAP of permanent pacemakers with novel RVSP, this study demonstrates lower levels of BNP and TNF-α of septal pacing at the end of the study after 4 weeks (p<0.05). Further large-scale, prospective, randomized clinical trials are needed.