Dissertations / Theses on the topic 'TNF Receptor-Associated Factor 4'

Glia are the understudied brain cells that perform many functions essential to maintain nervous system homeostasis and protect the brain from injury. If brain damage occurs, glia rapidly adopt the reactive state and elicit a series of cellular and molecular events known as reactive gliosis, the hallmark of many neurodegenerative diseases. However, the molecular pathways that trigger and regulate this process remain poorly defined. The fruit fly Drosophila melanogaster has glial cells that are strikingly similar to mammalian glia, and which also exhibit reactive responses after neuronal injury. By exploiting its powerful genetic toolbox, we are uniquely positioned to identify the genes that activate and execute glial responses to neuronal injury in vivo. In this dissertation, I use Wallerian degeneration in Drosophila as a model to characterize molecular pathways responsible for glia to recognize neural injury, become activated, and ultimately engulf and degrade axonal debris. I demonstrate a novel role for the GEF (guanine nucleotide exchange factors) complex DRK/DOS/SOS upstream of small GTPase Rac1 in glial engulfment activity and show that it acts redundantly with previously discovered Crk/Mbc/dCed-12 to execute glial activation after axotomy. In addition, I discovered an exciting new role for the TNF receptor associated factor 4 (TRAF4) in glial response to axon injury. I find that interfering with TRAF4 and the downstream kinase misshapen (msn) function results in impaired glial activation and engulfment of axonal debris. Unexpectedly, I find that TRAF4 physically associates with engulfment receptor Draper – making TRAF4 only second factor to bind directly to Draper – and show it is essential for Draper-dependent activation of downstream engulfment signaling, including transcriptional activation of engulfment genes via the JNK and STAT transcriptional cascades. All of these pathways are highly conserved from Drosophila to mammals and most are known to be expressed in mouse brain glia, suggesting functional conservation. My work should therefore serve as an excellent starting point for future investigations regarding their roles in glial activation/reactive gliosis in various pathological conditions of the mammalian central nervous system.

Glia are the understudied brain cells that perform many functions essential to maintain nervous system homeostasis and protect the brain from injury. If brain damage occurs, glia rapidly adopt the reactive state and elicit a series of cellular and molecular events known as reactive gliosis, the hallmark of many neurodegenerative diseases. However, the molecular pathways that trigger and regulate this process remain poorly defined. The fruit fly Drosophila melanogaster has glial cells that are strikingly similar to mammalian glia, and which also exhibit reactive responses after neuronal injury. By exploiting its powerful genetic toolbox, we are uniquely positioned to identify the genes that activate and execute glial responses to neuronal injury in vivo. In this dissertation, I use Wallerian degeneration in Drosophila as a model to characterize molecular pathways responsible for glia to recognize neural injury, become activated, and ultimately engulf and degrade axonal debris. I demonstrate a novel role for the GEF (guanine nucleotide exchange factors) complex DRK/DOS/SOS upstream of small GTPase Rac1 in glial engulfment activity and show that it acts redundantly with previously discovered Crk/Mbc/dCed-12 to execute glial activation after axotomy. In addition, I discovered an exciting new role for the TNF receptor associated factor 4 (TRAF4) in glial response to axon injury. I find that interfering with TRAF4 and the downstream kinase misshapen (msn) function results in impaired glial activation and engulfment of axonal debris. Unexpectedly, I find that TRAF4 physically associates with engulfment receptor Draper – making TRAF4 only second factor to bind directly to Draper – and show it is essential for Draper-dependent activation of downstream engulfment signaling, including transcriptional activation of engulfment genes via the JNK and STAT transcriptional cascades. All of these pathways are highly conserved from Drosophila to mammals and most are known to be expressed in mouse brain glia, suggesting functional conservation. My work should therefore serve as an excellent starting point for future investigations regarding their roles in glial activation/reactive gliosis in various pathological conditions of the mammalian central nervous system.

TRAF4 est un gène fréquemment surexprimé dans les carcinomes suggérant qu’il y joue un rôle. Tandis que la protéine TRAF4 est majoritairement localisée dans les jonctions serrées (JS) des cellules épithéliales mammaires (CEM) normales, elle s’accumule dans le cytoplasme des CEM malignes. Dans cette étude, nous montrons que TRAF4 possède un nouveau domaine liant les phosphoinositides (PIP) et que ce dernier est requis pour son recrutement aux JS. Des analyses moléculaires et structurales ont montré que le domaine TRAF de TRAF4 forme un trimère pouvant lier jusqu’à trois molécules de lipides grâce à des résidus basiques présents à la surface. Des études cellulaires indiquent que TRAF4 régule négativement les JS et augmente la migration cellulaire. Ces deux fonctions sont dépendantes de sa capacité à lier les PIPs. Notre travail suggère que la surexpression de TRAF4 pourrait contribuer à la progression des cancers du sein en déstabilisant les JS et en favorisant la migration cellulaire
TRAF4 (tumor necrosis factor (TNF) receptor-associated factor 4) is frequently overexpressed in carcinomas suggesting a specific role in cancer. While TRAF4 protein is predominantly found at tight junctions (TJ) in normal mammary epithelial cells (MEC), it accumulates in the cytoplasm of malignant MEC. How TRAF4 is recruited and functions at TJ is unclear. Here we show that TRAF4 possesses a novel phosphoinositide (PIP)- binding domain crucial for its recruitment to TJ. Molecular and structural analyses revealed that the TRAF domain of TRAF4 exists as a trimer which binds up to 3 lipids using basic residues exposed at its surface. Cellular studies indicated that TRAF4 acts a negative regulator of TJ and increases cell migration. These functions are dependent from its ability to interact with PIPs. Our results suggest that TRAF4 overexpression might contribute to breast cancer progression by destabilizing TJ and favoring cell migration

Dissertação de Mestrado Integrado em Medicina Veterinária
The association between CTLA-4 expression and cancer prognosis has been extensively investigated in recent years, pointing to the link with inflammation, and highlighting the role of the tumor microenvironment (TME), of which inflammatory mediators like cytokines are an important non-cellular component. To the best of our knowledge, no studies on immune checkpoint regulators had been conducted on cats with mammary carcinoma before, nor had cytokine profiles been previously assessed. Thus, we investigated the serum profiles of CTLA-4 and pro-inflammatory cytokines IL-6 and TNF-α in 57 female cats with mammary carcinoma and checked for associations between CTLA-4 and cytokine serum levels. Our results clearly demonstrate that serum CTLA-4 levels are increased in cats with mammary carcinoma when compared to healthy animals (P=0.022). Furthermore, we show a strong positive correlation with TNF-α (R=0.88, P<0.001) and IL-6 levels (R=0.72, P<0.001), advancing the concept of an immunomodulatory role for this regulator in breast cancer pathogenesis. We also show a statistically significant association between higher levels of serum CTLA-4 and less aggressive clinicopathological features: smaller tumors (P<0.001), lower stage (P=0.002), absence of necrosis (P<0.001), no lymph node involvement (P=0.007), no lymphatic vessel invasion (P=0.006), positive hormone receptor status (P=0.007), non-TN status (P=0.041), non-basal status (P<0.001) and low Ki67 index (P=0.001). Our findings further expand this concept by indicating an association with specific breast cancer subtypes, namely, HER-2 positive with CTLA-4 (P<0.001) and TNF-α (P=0.004) and luminal A-like with IL-6 (P=0.020). We could not confirm an association between serum CTLA-4 and cytokines levels and survival due to the small sample size. Nevertheless, our findings suggest a potentially concentration-dependent protective role for serum CTLA-4 and IL-6, as evidenced by higher median survival times in the CTLA-4high (28 vs 22 months for the CTLA-4low group) and IL-6high (28 vs 19 months for the IL-6low group) groups. Conversely, TNF-α seems to be a negative prognostic factor, as shown by the lower median survival in the TNF-αhigh group (16.5 vs 23.5 months for the TNF-αlow group). An intriguing question that remains is how serum CTLA-4 influences or is influenced by the pro-inflammatory cytokines. Assessment of CTLA-4 tumor expression, T-lymphocyte subtypes, and tumor associated macrophages and myeloid derived suppressor cell profiles in the microenvironment, are important features to evaluate in future studies.
RESUMO - RELEVÂNCIA CLÍNICA DA PROTEÍNA 4 ASSOCIADA AO LINFÓCITO T CITOTÓXICO 4 (CTLA-4) E SUA CORRELAÇÃO COM AS CITOQUINAS PRÓ-INFLAMATÓRIAS INTERLEUCINA 6 (IL-6) E FACTOR DE NECROSE TUMORAL ALFA (TNF-α) NO CARCINOMA MAMÁRIO FELINO - A associação entre a expressão da CTLA-4 e o prognóstico no cancro tem sido amplamente investigada, valorizando o papel da inflamação e do microambiente tumoral (TME), do qual mediadores inflamatórios como as citoquinas são uma importante componente não celular. Até à data, não existem estudos sobre reguladores de checkpoint imunológico em gatos com carcinoma mamário, nem foram avaliados perfis de citoquinas. Assim, foram investigados pela primeira vez, os perfis séricos da CTLA-4 e das citoquinas pró-inflamatórias IL-6 e TNF-α em 57 gatas com carcinoma mamário e verificada a existência de associações entre os níveis séricos da CTLA-4 e das referidas citoquinas. Os resultados obtidos demonstram que os níveis de CTLA-4 estão aumentados no soro das gatas com carcinoma mamário, quando comparadas com animais saudáveis (P=0.022). Foi também demonstrada uma correlação forte com os níveis séricos do TNF-α (R=0.88, P<0.001) e da IL-6 (R=0.72, P<0.001), reforçando o papel imunomodulatório deste regulador. Adicionalmente foi encontrada uma associação significativa entre os níveis séricos elevados da CTLA-4, e várias características clinicopatológicas menos agressivas: tumores mais pequenos (P<0.001), estadiamento precoce, (P=0.002), ausência de necrose tumoral (P<0.001), sem envolvimento dos linfonodos (P=0.007), sem invasão linfática (P=0.006), com positividade para os receptores hormonais (P=0.007), subtipo não-TN (P=0.041), subtipo não-basal (P<0.001), e baixo índice Ki67 (P=0.001). Os resultados obtidos ainda revelaram uma associação com subtipos específicos de cancro da mama, nomeadamente o HER-2 positivo com sobre-expressão da CTLA-4 (P<0.001) e do TNF-α (P=0.004) e o luminal A com sobre-expressão da IL-6 (P=0.020). Não foi possível confirmar a associação entre os níveis séricos da CTLA-4 e das citoquinas e o tempo de sobrevivência, devido ao tamanho reduzido da amostra. No entanto, os resultados obtidos sugerem um efeito protetor dependente da concentração da CTLA-4 e IL-6 séricos, como evidenciado pelos tempos medianos de sobrevivência mais altos nos grupos CTLA-4high (28 vs 22 meses para o grupo CTLA-4low) e IL-6high (28 vs 19 meses para o grupo IL-6low). Em contraste, o TNF-α parece ser um fator de prognóstico negativo, como sugere o tempo mediano de sobrevivência mais baixo no grupo TNF-αhigh (16.5 vs 23.5 meses para o grupo TNF-αlow). Permanece a questão de como o CTLA-4 influencia ou é influenciado pelas citoquinas pró-inflamatórias. A avaliação da expressão tumoral da CTLA-4, dos subtipos de linfócitos T, e dos perfis de macrófagos associados ao tumor e células supressoras da linha mieloide no microambiente tumoral, são aspetos importantes a avaliar em estudos futuros.
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Thesis (Ph.D. in Immunology) -- University of Colorado at Denver and Health Sciences Center, 2005.
Typescript. Includes bibliographical references (leaves 144-178). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

The tumor necrosis factor (TNF) receptor-associated factors (TRAFs) were initially discovered as proteins that inducibly interact with the intracellular region of TNF receptors (TNFRs). Because the TNFRs lack intrinsic catalytic activity, the TRAFs are hypothesized to orchestrate signaling activation downstream of the TNFR superfamily, however their mechanism of activation remains unclear (Inoue et al., 2000; Bishop, 2004). Originally, the TRAFs were compared to the signal transducers and activators of transcription (STAT) protein family, due to their sequence homology, and the presence of multiple RING- and zinc-finger domains, suggesting that their function may be to regulate transcriptional activity (Rothe et al., 1994; Hu et al., 1994; Sato et al. 1995; Cheng et al., 1995). However, subsequent research focused predominantly on their cytoplasmic functions, and more recently on their function as E3 ubiquitin ligases (Pineda et al., 2007). In my research, I analyzed the subcellular localizations of the TRAFs following CD40 ligand (CD40L)-stimulation, and found that TRAF2 and 3 rapidly translocate into the nucleus of primary neurons and Neuro2a cells. Interestingly, similar analysis conducted in pre-B lymphocytes (Daudi cells) revealed a different response to CD40L-stimulation, with TRAF2 and 3 being rapidly degraded within 5-minutes of stimulation. These findings are significant because they demonstrate for the first time that the TRAFs translocate into the nucleus and suggest that they may function within the nucleus in a cell-specific manner. I next analyzed the ability of TRAF2 and 3 to bind to DNA, and found that they both bind to chromatin and the NF-kappaB consensus element in Neuro2a cells, following CD40L-stimulation. Similar analyses of the chromatin binding of TRAF2 and 3 in Daudi cells revealed that they were rapidly degraded, similar to the results from my analysis of their subcellular localization. These findings show for the first time that the TRAFs interact with DNA, and therefore support the hypothesis that the TRAFs may function within the nucleus as transcriptional regulators. Finally, I analyzed the ability of the TRAFs to regulate transcriptional activity by luciferase assay. Previous studies showed that overexpression of TRAF2 and 6 could induce NF-kappaB transcriptional activity; however researchers have not been able to determine the mechanism by which they do so. In my studies, I found that every TRAF can directly regulate transcriptional activity either as co-activators or co-repressors of transcription, in a cell- and target protein-specific manner. Additionally, I found that TRAF2 can act as a transcriptional activator, and that its ability to regulate transcription is largely dependent upon the presence of its RING-finger domain. In conclusion, these studies have revealed an entirely novel function for the TRAFs as immediate-early transcriptional regulators. Future research into the genes that are regulated by the specific TRAF complexes will further elucidate how the TRAFs regulate TNFR signaling, as well as whether dysfunctions in TRAF signaling may be associated with known disorders. If specific TRAF complexes are found to regulate specific genes, then pharmacological targeting of the individual TRAF complexes may allow for the highly specific inhibition of signaling events downstream of the TNFRs, without compromising overall receptor signaling, transcription factor pathways, or cellular systems.

Memoria para optar al título de Químico Farmacéutico
Autorizada por el autor, pero con restricción para ser publicada a texto completo hasta diciembre de 2015, en el Portal de Tesis Electrónicas.
La diferenciación de fibroblastos cardiacos (FC) a miofibroblastos cardiacos (MFC) es gatillada por TGF-β1, la cual señaliza principalmente a través de las proteínas Smad. Los MFC muestran como principal característica la presencia de microfilamentos citoplásmicos de α-SMA, estructuradas como fibras de estrés, lo que les permite la contracción. Por otro lado, LPS es un ligando del receptor TLR4, que señaliza de manera dependiente e independiente de MyD88 teniendo como principal efector el NF-κB, induciendo así la expresión de genes de citoquinas proinflamatorias. Sin embargo, aunque se ha descrito un efecto antagónico entre la señalización inducida por LPS y la señalización canónica de TGF- β, a la fecha no se ha estudiado si en FC y en MFC la expresión de α-SMA inducida por TGF-β1 es antagonizada por LPS. Para responder estas interrogantes se utilizaron FC y MFC de ratas adultas, y se determinó in vitro la capacidad de LPS de inhibir la expresión de α-SMA. Además, utilizando TAK- 242, un inhibidor de las vías intracelulares dependientes de TLR4, se determinó que los efectos gatillados por LPS ocurrían a través de este receptor. La utilización de los inhibidores PD98059, LY29002 y BAY 11-7082 permitió determinar que en FC LPS a través de la vía de señalización PI3K/Akt, disminuye la expresión de α-SMA. Por lo que nuestros resultados demuestran que LPS inhibe la expresión de α-SMA en FC, a través del receptor TLR4 mediante la activación de vía de señalización PI3K/Akt y que LPS es capaz de disminuir la expresión de α-SMA en MFC a través de TLR4 inhibiendo la mantención del fenotipo miofibroblasto
The difference between cardiac fibroblasts (CF) and cardiac myofibroblast (CMF) is triggered by TGF-β1, which mainly signals via Smad proteins. The main characteristic of CMF is cytoplasmic microfilaments of α-SMA; this are structured as stress fibers, which allow the contraction of CMF. Also, LPS is a ligand of the TLR4 receptor that signals via a dependant and independent pathway of MyD88 whose main factor is NF-κB; this induces the expression of inflammatory cytokine genes. However, despite an opposite effect has been described between the signaling induced by LPS and the canonical signaling of TGF-β, the opposite effect that LPS may have on the expression of α-SMA on CF and CMF induced by TGF-β1 has not been studied so far. In order to answer these questions, CF and CMF of adult rats were used; this showed in vitro evidence that LPS is capable of inhibiting the expression of α-SMA. Also, with the use of TAK-242, which is an inhibitor of the dependant intracellular domain of TLR4, we determined that the effects triggered by LPS occurred through said receptor. Using PD98059, LY29002 and BAY11-7082 inhibitors allowed us to determine that, via the PI3K/Akt signaling pathway, LPS decreases the expression of α-SMA in CF. Therefore, our results show that LPS inhibit the expression of α-SMA in CF through the TLR4 receptor via the activation of the PI3K/Akt signaling pathway, and that LPS is capable of decreasing the expression of α-SMA in CMF through TLR4 receptor, which inhibits the maintenance of the myofibroblast phenotype

Ankylosing spondylitis (AS) is a common inflammatory arthritis of the spine and other affected joints, which is highly heritable, being strongly influenced by the HLA-B27 status, as well as hundreds of mostly unknown genetic variants of smaller effect. The aim of my research was to confirm some of the previously observed genetic associations and to identify new associations, many of which are in biological pathways relevant to AS pathogenesis, most notably the IL-23/T_H17 axis (IL23R) and antigen presentation (ERAP1 and ERAP2). Studies presented in this thesis include replication and refinement of several potential associations initially identified by earlier GWAS (WTCCC-TASC, 2007 and TASC, 2010). I conducted an extended study of IL23R association with AS and undertook a meta-analysis, confirming the association between AS and IL23R (non-synonymous SNP rs11209026, p=1.5 x 10-9, OR=0.61). An extensive re-sequencing and fine mapping project, including a meta-analysis, to replicate and refine the association of TNFRSF1A with AS was also undertaken; a novel variant in intron 6 was identified and a weak association with a low frequency variant, rs4149584 (p=0.01, OR=1.58), was detected. Somewhat stronger associations were seen with rs4149577 (p=0.002, OR=0.91) and rs4149578 (p=0.015, OR=1.14) in the meta-analysis. Associations at several additional loci had been identified by a more recent GWAS (WTCCC2-TASC, 2011). I used in silico techniques, including imputation using a denser panel of variants from the 1000 Genomes Project, conditional analysis and rare/low frequency variant analysis, to refine these associations. Imputation analysis (1782 cases/5167 controls) revealed novel associations with ERAP2 (rs4869313, p=7.3 x 10-8, OR=0.79) and several additional candidate loci including IL6R, UBE2L3 and 2p16.3. Ten SNPs were then directly typed in an independent sample (1804 cases/1848 controls) to replicate selected associations and to determine the imputation accuracy. I established that imputation using the 1000 Genomes Project pilot data was largely reliable, specifically for common variants (genotype concordence~97%). However, more accurate imputation of low frequency variants may require larger reference populations, like the most recent 1000 Genomes reference panels. The results of my research provide a better understanding of the complex genetics of AS, and help identify future targets for genetic and functional studies.

碩士
國立臺灣大學
流行病學研究所
93
Background – Patients with severe acute respiratory syndrome (SARS) exhibits signs and symptoms of respiratory and systemic infection that follows with clinical course of varying severity, including death due to acute respiratory distress syndrome (ARDS). In addition to advanced age, comorbidity, and high viral load, genetic predisposition has been postulated to influence the immune response and clinical course. This study aims to identify potential genetic effectors involved in the pathogenesis of SARS. Method – This is a population-based case-control study that enrolled 108 unrelated laboratory-confirmed SARS patients and 333 healthy Taiwanese as reference. Gene products that are known or predicted to be involved in host response were selected for study of polymorphism. SARS patients are divided into 3 groups based on the duration of illness as index for severity: <15 days for mild, 15~28 days for intermediate, and >28 days for severe case. Result – Clinical severity was significantly associated with certain genotype of three SNPs: a nonsynonymous substitution in the coding region of Toll-like receptor 3, TLR3(L412F) and in the promoter regions of TNF receptor-associated factor 6- TRAF6 (A-9423C) and heme oxygenase-1 - HO-1(T-495A). TLR3 is directly upstream to TRAF6 in the interferon-inducing pathway, and the joint effect of TLR3 or TRAF6 in the pathway was considered, termed TLR3-TRAF6. The severe SARS patients had 4.06-folds (95% CI=1.69~9.74, p=0.002) increased odds of possessing any susceptible genotypes of TLR3-TRAF6 (TLR3(6300T/T), TRAF6(-9423C/C) or (A/C)) and 2.77-folds (95% CI=1.69~9.74, p=0.002) of possessing susceptible genotype of HO-1 (HO-1(-495A/A) or (A/T)) than patients with shorter clinical course in a cumulative logit model. This genetic effect is more profound for the younger SARS patients (<40 yr) than the elder group. Conclusion - TLR3 recognizes double-stranded RNA, and together with TRAF6, activates interferon production; HO-1 can protect tissues from immune-mediated injury. Along with our previous report of two interferon-γ- inducible genes, FGL2 - a prothrombinase and CXCL10 - a chemokine activating TH1 response, our results strongly support the hypothesis that genetic predisposition to certain host responses participate in the pathogenesis of severe SARS-CoV infection. Identifying pathways participating in viral pathogenesis can provide insights to formulating therapeutic interventions for SARS, and probably ARDS from other causes.