Dissertations / Theses on the topic 'Immune-related gene'

L'immunothérapie basée sur le blocage des points de contrôle immunitaire (ICBs) est un traitement prometteur pour les patients atteints de mélanome ; cependant, seule une petite sous-population en tire un bénéfice à long terme. Un des défis pour améliorer l'efficacité et étendre le bénéfice des ICBs aux patients non répondeurs est de concevoir des approches innovantes permettant de transformer les tumeurs dites "froides ou désertes pour les cellules immunitaire" en tumeurs dites "chaudes ou infiltrées par les cellules immunitaires" qui sont éligibles aux ICBs. Nous avons étudié l'impact du ciblage du gène de l'autophagie Beclin1 sur le paysage immunitaire des tumeurs de mélanome B16-F10. Nos résultats ont démonté que ce ciblage inhibait significativement la croissance tumorale B16-F10 et augmentait l'infiltration des leucocytes CD45+. Le phénotypage immunitaire a révélé une augmentation de l'infiltration de cellules NK (Natural Killer) actives, de macrophages inflammatoires et résidents de type 1, de cellules dendritiques et de lymphocytes T CD8+ actifs. L’inhibition de la croissance tumorale Becn1- n'était plus observée par la déplétion des CD8+ de l'hôte, soulignant ainsi leur rôle dans le contrôle du développement de ces tumeurs. Nos résultats ont démontré que La régulation du paysage immunitaire des tumeurs Becn1- était associée à une modulation du réseau de cytokines/chémokines dans le microenvironnement tumoral (TME). Ainsi, les tumeurs Becn1- présentaient une signature de cytokines inflammatoires (comprenant CCL5, CXCL10 et IFNg) qui pourrait être responsable de l'établissement de microenvironnement inflammatoire permissif aux cellules CD8. Nous avons révélé que la surexpression de l'IFNg dans le TME des tumeurs Becn1- était responsable de l'induction de PD-L1 sur les cellules tumorales par la voie d'activation JAK/STATs. En conclusion, cette étude met en évidence Beclin1 comme une cible majeure, capable d'induire l'infiltration des cellules effectrices immunitaires dans les mélanomes en induisant une signature inflammatoire. Elle fournit également la preuve de concept pour combiner des inhibiteurs d'autophagie avec les ICBs comme une approche de pointe pour améliorer leur efficacité
Immune Checkpoint Blockades (ICBs)-based immunotherapy has emerged as a promising treatment for melanoma patients; however only a small subset of patients reaps a long term benefit. One of the major challenges to enhance the efficacy and extend the benefit of ICBs to non-responder patients is to design innovative approaches allowing the switch of “immune desert cold tumors” to “immune infiltrated hot tumors" which are eligible for ICB-based therapies. Here, we investigated the impact of targeting the early autophagy gene Beclin1 on the immune landscape of B16-F10 melanoma tumors. We found that targeting Beclin1 (Becn1-) significantly inhibited B16-F10 tumor growth and increased the infiltration of CD45+ leukocytes into the tumor bed. Immune phenotyping revealed an increased infiltration of active Natural Killer (NK) cells, inflammatory and resident type 1 macrophages, dendritic cells, and active CD8+ T lymphocytes. The inhibition of Becn1- tumor growth was no longer observed by depleting host CD8+ T cells, thus highlighting their major role in the control of Becn1- B16-F10 tumor development. We showed that Beclin1-dependent regulation of the immune landscape was associated with profound modulation of the cytokine/chemokine network in the tumor microenvironment (TME). Importantly, we revealed that Becn1- tumors displayed an inflammatory cytokine signature (comprised, but not restricted to, CCL5, CXCL10 and IFNg) that could be responsible for the switch from cold non T-inflamed to hot T-inflamed tumors. Mechanistically, we reported that the overexpression of IFNg in Becn1- TME was responsible for the induction of Programed Death ligand-1 (PD-L1) on tumor cells through the activation of JAK/STATs pathway. Overall, this study highlights Beclin1 as a valuable target, able to drive immune effectors cells into the melanoma tumors by inducing an inflammatory signature. This study provides the proof of concept for combining drugs inhibiting early autophagy process along with ICBs as a cutting-edge approach to improve their efficacy

The immune response to viral infection involves a complexity of both innate and adaptive pathways at the cellular and the molecular level. There are many approaches to begin to define the pathways at work to control viral pathogenesis. The approach favored in this thesis was to conduct a broad screen of the innate immune response at the gene expression level of infected cells. The innate immune response is critical to the control of viral infections. Type I interferons (IFN), IFNα and IFNβ, are antiviral proteins that are an integral part of the innate immune response. Furthermore, by virtue of their effects on maturation and activation of antigen-presenting cells, IFNs are a pivotal link between the innate and adaptive immune systems. Most cell types produce type-I IFN when exposed to viruses. However, viruses have evolved multiple strategies to suppress IFN production or signaling. It is imperative to understand the virus-host interaction at the molecular level in order to identify as yet unknown mechanisms of the host antiviral response; these additional pathways may be useful in counteracting the viral suppression of IFN. Type-I IFNs regulate expression of at least five hundred genes, suggesting a complex network of signaling pathways. Depending on the cell type different proteins regulate the induction of IFN or the expression of IFN-inducible genes. Identification of proteins that induce selected IFN-inducible genes may provide synergistic activity with or may have an advantage over type-I IFN for anti-viral therapy in the future. Many diseases are untreatable if identified late in their progression. In resource-limited countries, many diseases are diagnosed clinically, which can lead to incorrect or delayed diagnosis and treatment. The identification of biomarkers of disease has the potential to guide the correct therapy in a timely fashion. The objective of this thesis was to identify novel anti-viral therapies and disease biomarkers for dengue virus (DENV) infection. DENV is a mosquito-borne positive-sense single-stranded RNA virus, which causes an estimated 50 million infections annually. Most DENV infections result in a febrile illness called Dengue fever (DF). Less frequently, infections cause Dengue hemorrhagic fever (DHF), a potentially fatal vascular leakage syndrome associated with the production of pro-inflammatory cytokines. At present patients infected with DENV can only be treated by intravenous fluid support to prevent hypovolemia and hypotensive shock. This treatment is less effective in severe cases if the diagnosis is delayed. Identification of therapeutics with both antiviral and immune-modulatory activity may lower patient mortality and reduce the burden of DENV on society. DENV infection is cleared in most individuals after a short period of viremia {Libraty, 2002 #2225}. Based on in vitro and mouse models, type-I and type-II IFN signaling pathways are thought to be critical in the regulation of DENV infection. Higher serum levels of type I and type II IFNs during acute DENV infection in patients lend support to the above hypothesis {Kurane, 1993 #2152; Libraty, 2002 #2225}. To understand the DENV-human host cell interaction at the molecular level, we performed global gene expression analysis on DENV-infected primary human cells using Affymetrix GeneChips (HG-U133A). We studied dendritic cells (DC), monocytes, B cells and human umbilical vein endothelial cells (HUVECs), all of which are known to be permissive to DENV infection. We first identified genes commonly regulated in multiple cell types in response to DENV infection; we hypothesized that understanding this common gene expression profile would identify signaling pathways involved in regulation of viral spread, activation of immune cells or induction of inflammation. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), one of the 23 common response genes, was identified as a key link between type I and type II interferon response genes. Pretreatment of cells with recombinant TRAIL (rTRAIL) inhibited DENV replication in monocytes, B cells, HUVECs and DCs. Using the DC infection model, we showed that this inhibition of viral replication was apoptosis-independent. Type-I IFN receptor (IFNR) blocking experiments showed that signaling through the type-I IFN receptor played an important role in the antiviral activity of exogenous rTRAIL. Furthermore, TRAIL also significantly reduced the expression of mRNA and protein of pro-inflammatory cytokines (TNFα, MIP-1β and IFNα) and chemokines (MCP-2, IP-10 and IL-6) in response to DENV infection. The data that TRAIL inhibits both viral replication and pro-inflammatory cytokine production suggest that TRAIL has therapeutic value in dengue. The endothelial cell is the site of pathology in DENV infection in vivo (vascular permeability and plasma leakage). To understand the direct effect of DENV infection on endothelial cells and its role in the induction of genes regulating vascular permeability, we compared gene expression in DENV-infected HUVECs to that of uninfected cells and cells infected with other RNA and DNA viruses, including flaviviruses (West Nile, yellow fever, and Japanese encephalitis viruses), bunyaviruses (Sin Nombre and Hantaan viruses), Epstein-Barr virus and vaccinia virus. Among the genes confirmed for their differential expression, ST2 (Interkeukin-1 receptor-like-1 protein-IL1RL1) and indoleamine 2,3-dioxygenase (IDO) were identified to be upregulated specifically in response to DENV infection. Higher serum soluble ST2 (sST2) levels were detected in DENV-infected patients than in patients with other febrile illnesses (OFI) at the end of the febrile stage and at defervescence (p=0.0088 and p=0.0004, respectively). In addition, patients with secondary DENV infections had higher serum sST2 levels compared with patients with primary DENV infections (p=0.047 at the last day of fever and p=0.030 at defervescence). Higher levels of IDO activity (pIn conclusion, global gene expression analysis identified novel proteins with promising characteristics for the treatment and/or diagnosis of DENV infection. Although further studies will be needed to validate the clinical utility of TRAIL, sST2, and IDO, these studies demonstrate the utility of this unbiased genomics approach to identify therapies to currently incurable diseases.

Prostate cancer (PCa) is the second most common cancer in men with more than 1 million new cases worldwide each year. While some of the genomic, genetic and molecular events characterizing PCa have been functionally associated with tumor onset, development and resistance to therapy, the meaning of many other molecular alterations remains poorly understood. Recent development of organoids technology and prostate organoid cultures has established an innovative and valuable model for the study of adult tissue homeostasis, physiology and disease. In this project we combined prostate organoids technology with genetic engineering and CLICK-chemistry coupled Mass Spectrometry approaches in order to better characterize molecular features of wild type and genetically engineered mouse prostate organoids modeling early steps of human prostate tumorigenesis. In details, by manipulating mPrOs to proxy ETS-related gene (ERG) precursor PIN/HGPIN lesions of human prostate, we identified possible novel pro-tumorigenic roles of ERG which unleashes cells proliferation from the tight control of growth stimuli, and, even more interesting, corrupts immune system components to escape immune surveillance. In conclusion, this project shows that coupling innovative biological systems and technological approaches can lead to significant improvements in the analysis and understanding of disease mechanisms.

Prostate cancer (PCa) is the second most common cancer in men with more than 1 million new cases worldwide each year. While some of the genomic, genetic and molecular events characterizing PCa have been functionally associated with tumor onset, development and resistance to therapy, the meaning of many other molecular alterations remains poorly understood. Recent development of organoids technology and prostate organoid cultures has established an innovative and valuable model for the study of adult tissue homeostasis, physiology and disease. In this project we combined prostate organoids technology with genetic engineering and CLICK-chemistry coupled Mass Spectrometry approaches in order to better characterize molecular features of wild type and genetically engineered mouse prostate organoids modeling early steps of human prostate tumorigenesis. In details, by manipulating mPrOs to proxy ETS-related gene (ERG) precursor PIN/HGPIN lesions of human prostate, we identified possible novel pro-tumorigenic roles of ERG which unleashes cells proliferation from the tight control of growth stimuli, and, even more interesting, corrupts immune system components to escape immune surveillance. In conclusion, this project shows that coupling innovative biological systems and technological approaches can lead to significant improvements in the analysis and understanding of disease mechanisms.

Tuberculosis (TB) disease, caused by the pathogen Mycobacterium tuberculosis (Mtb), remains a major global health problem claiming 1.5-2 million lives annually. One of the major factors contributing towards Mtb's success as a pathogen is its unique cell wall and its ability to counteract various arms of the host's immune response. Understanding these survival mechanisms will help us develop new therapeutic interventions that can enhance the capacity of the immune system to kill the pathogen. A recent genome scale study profiled a list of candidate genes that are predicted to be essential for Mtb survival of host mediated responses. One candidate was ftsEX, a protein complex comprised of an ATP binding domain, FtsE, and a transmembrane domain, FtsX. FtsEX functions through interaction with a periplasmic hydrolase, RipC. FtsEX homologs in other bacteria have been linked to a key role in regulation of PG hydrolysis during elongation and division. Using M. smegmatis as a model, we hypothesised that FtsEX and RipC are required in the regulation of PG hydrolysis during normal cell wall elongation and division under stressful conditions in vitro. Antibiotic sensitivity was confirmed using Alamar blue MIC determination assays, which showed that ftsEX and ripC had increased sensitivity to chloramphenicol and not to rifampicin, isoniazid and ethambutol. Our growth curve analysis showed that ftsEX and ripC are not essential for survival in normal growth conditions. However, ftsEX and ripC are conditionally essential for M. smegmatis in low salt media. Growth defects in this condition were characterized by short and bulgy cells, as well as elongated filamentous cells with visible chaining. Major morphological changes were seen under nitrosative stress. A higher proportion of cells struggled to divide normally and formed chains. Lateral branching was also observed in ΔftsE, ΔftsX and ΔftsEX but not in ΔripC. The protein complex was also required for survival in media containing rifampicin. Treatment with the drug exacerbated growth defects of all the mutants, which were much shorter than WT cells, indicating impairment in the elongation process. Collectively, mutants are much shorter in length with an exception of a few extremely lengthy cells, suggesting that ftsEX and ripC are required for both normal cell elongation and division and ultimately for survival in stressful conditions.

Os insetos desenvolveram um sistema imune eficiente contra parasitas e patógenos, que compreende a resposta celular e a humoral. Os mecanismos celulares envolvem a fagocitose e a encapsulação pelos hemócitos, enquanto que as respostas humorais incluem a ativação da Profenoloxidase, e a síntese pelo corpo gorduroso dos peptídeos antimicrobianos, que são liberados na hemolinfa. Duas vias de sinalização intracelular, Toll e Imd, controlam a expressão dos genes codificadores dos peptídeos antimicrobianos. A análise do Genoma da abelha Apis mellifera permitiu a identificação dos genes dessas vias. No entanto, pouco se conhece do mecanismo de resposta imune nessas abelhas. Desta maneira, nos propusemos analisar a transcrição de genes efetores da resposta imune (abaecina, hymenoptaecina, defensina, transferrina, profenoloxidase), assim como os genes integrantes das vias de sinalização, tais como os genes de reconhecimento de microorganismos (PGRP, GNBP) e ainda, os de sinalização (cactus, relish, dorsal 1-B). Avaliamos também possíveis proteínas implicadas na resposta imune, como as proteínas de estocagem Vitelogenina, Hexamerina 70a, Lipoforina I/II e Lipoforina III. Finalmente, analisamos o efeito da nutrição e do envelhecimento sobre a imunidade em abelhas. Para análise da expressão dos genes das vias de sinalização, as abelhas foram infectadas com bactérias Serratia marcescens ou Micrococcus luteus por injeção ou via alimentação. A infecção com esses microorganismos provocou a transcrição de peptídeos antimicrobianos e de transferrina em altas quantidades após 3 e 12 horas de tratamento, além da alteração na quantidade de transcritos de outros genes. O papel dos genes profenoloxidase e dorsal na imunidade, descritos como codificadores de importantes proteínas em outros insetos, foi avaliado através da metodologia de silenciamento gênico por RNA de interferência. Observamos a diminuição da transcrição do gene alvo, mostrando a eficiência da metodologia. No entanto, a simples injeção de um RNA de fita dupla foi capaz de ativar o sistema imune de abelhas. Este efeito contribuiu para a dificuldade de atribuição do papel da Profenoloxidase na imunidade de abelhas. Contudo, os resultados de silenciamento de dorsal e suas isoformas, nos levaram a considerar que dorsal 1-A ou dorsal 2 participam da via de sinalização intracelular para produção de peptídeos antimicrobianos, principalmente de defensina. Em relação às proteínas de estocagem, tanto a quantidade de transcritos quanto de proteínas diminui após infecção com bactérias, indicando que estas proteínas estão envolvidas de alguma forma no processo de imunidade em abelhas. Além disso, consumo de alimentos ricos em proteína aumentou os níveis de transcritos das proteínas de estocagem, o que muito provavelmente favorece a manutenção da capacidade de resposta imune de abelhas. O efeito do envelhecimento no declínio da imunidade foi analisado em abelhas nutridoras (novas) e forrageiras (velhas) de uma colônia típica. Além disso, foram utilizadas abelhas de uma colônia single-cohort, que eram de uma mesma idade, mas algumas eram nutridoras, enquanto outras eram forrageiras. Todas as abelhas, independentemente da idade ou comportamento, foram capazes de ativar o sistema imune após infecção pela bactéria S. marcescens. No entanto, as abelhas com o comportamento de forrageira, independentemente da idade, sempre foram mais susceptíveis a infecções que as nutridoras. Este fato se deve, muito provavelmente, às diferenças fisiológicas entre essas abelhas, que proporciona às nutridoras maior competência à sobrevivência.
Insects have developed an efficient immune system against parasites and pathogens, which is comprised of both cellular and humoral responses. The cellular mechanisms involve phagocytosis and encapsulation by hemocytes, whereas the humoral responses include activation of prophenoloxidase and synthesis of antimicrobial peptides by the fat body, which are released into the hemolymph. Two signaling pathways, Toll and Imd, control the expression of genes encoding antimicrobial peptides. Genome-wide analyses of the honey bee, Apis mellifera, have identified predicted genes for these signaling pathways. However, immune response mechanisms in honey bees were not yet in depth studied. We analyzed the transcription of effector genes (abaecin, hymenoptaecin, defensin, transferin, prophenoloxidase), as well as other immune genes, such as pathogen recognition genes (PGRP, GNBP) and signaling genes (cactus, relish, dorsal 1- B). We also investigated the role of the storage proteins Vitellogenin, Hexamerin 70a, Lipophorin I/II and Lipophorin III in the honey bee immunity. Finally, we analyzed the effect of nutrition and aging on honey bee immunity. Gene expression of signaling pathway components was assessed in honey bees that had been infected with the bacteria Serratia marcescens or Micrococcus luteus through injection or oral challenge. Honey bees infected with these microorganisms had strong up-regulation of antimicrobial peptide genes and of transferin, and also other changes in transcript abundance after 3 and 12 hours of challenge. The roles of prophenoloxidase and dorsal in the immune response, described as genes encoding important proteins in other insects, were also investigated. In this case we used RNA interference (RNAi) to silence the expression of these genes. RNAi efficiently silenced the target genes. However, injection of doublestranded RNA in honey bees induced a reaction by the immune system. This made it difficult to determine the role of prophenoloxidase in honey bee immunity. Yet, silencing of dorsal and its isoforms led us to consider dorsal 1-A or dorsal 2 as members of the signaling pathways that produce antimicrobial peptides, especially defensin. The abundance of storage proteins transcripts and proteins was lower in infected bees than in controls, giving evidence that these proteins participate in the immune process in honey bees. Moreover, protein consumption caused up-regulation of genes encoding storage proteins, which may favor the maintenace of the immune response capacity. The effect of aging on decline in immunity was analyzed in (young) nurse bees and (old) foragers from normal free-flying colony. We also examined bees from a single-cohort colony, in which all individuals were at the same age; but some were nursing, while others were foraging. All the bees, independent of age or behavior, were able to activate the immune system after infection with S. marcescens. However, foragers, independent of age, were always more susceptible to infections than were nurse bees. This is probably due to physiological differences between bees, which confers to the nurses more competence to survivorship.

A major challenge of modern Biology is elucidating the genetic basis of adaptation. While there are many SNP-based studies trying to elucidate the genetic basis of genotype-phenotype relationships, the role of transposable element (TE)-induced mutations is understudied. Recent evidences demonstrate that TEs are a powerful tool to identify the genetic basis of adaptive phenotypic traits. Drosophila melanogaster is a good model to study adaptation because it is original from subtropical Africa and only recently colonized out-of-Africa environments. To identify and characterize the role of several candidate TEs in D. melanogaster adaptation, we have followed two different strategies: locus-specific and trait-specific. In the first chapter, we have characterized both at the molecular and phenotypic level FBti0019386, a previously identified candidate adaptive TE. We first elucidated the evolutionary history of this natural insertion and provided evidences of genomic signatures of positive selection. We then explored several phenotypes related to known phenotypic effects of nearby genes, and having plausible connections to fitness variation in nature. We found that flies with FBti0019386 insertion had a shorter developmental time and were more sensitive to stress, which are likely to be the adaptive effect and the cost of selection of this mutation, respectively. Interestingly, these phenotypic effects are not consistent with a role of FBti0019386 in temperate adaptation as has been previously suggested. Indeed, a global analysis of the population frequency of FBti0019386 showed that climatic variables explain well the FBti0019386 frequency patterns only in Australia. These results suggest that further functional validation should be gathered before concluding that a candidate loci is under spatially varying selection. Finally, although FBti0019386 insertion could be inducing the formation of heterochromatin by recruiting HP1a (Heterochromatin Protein 1a) protein, the insertion is associated with up-regulation of sra in adult females. In the second chapter of this thesis, we have studied the impact of several TE insertions in a highly conserved and ecologically relevant trait: the immune response. To do that, we first performed a new genome-wide screening in order to identify a dataset of candidate TEs involved in adaptation. By increasing the number of populations and the number of TEs analyzed compared to similar studies, we were able to increase the number of identified candidate TEs: a total of 121 TEs. Interestingly, we found that genes associated with those TEs are enriched for stress-related functions, specifically we detected a significant enrichment for immune response functions. We combined allele-specific expression (ASE), enhancer assays, and TSS detection experiments to characterize the impact of these TEs in oral immune response to the gram-negative bacteria Pseudomonas entomophila. We were also able to associate the 12 candidate TEs with gene expression changes, and determine some of the molecular mechanisms behind these expression changes. We showed that the allele with the TE was differently expressed in 13 out of the 16 analyzed genes under non-infected and/or infected conditions in at least one of the two genetic backgrounds analyzed. We also show that different TEs alter gene expression by adding promoters and enhancer regulatory sequences to their nearby genes. Although we found evidences pointing to a possible role of TEs in immune response regulation, more experiments should be performed in order to link the identified TEs with a fitness effect in this trait. Overall, our two integrative approaches allowed us to shed light on the role of TEs in generating genomic natural variation potentially underlying adaptation. The results obtained in this work illustrate that TEs are a good tool to bridge the gap between genotypic and phenotypic evolution.
Un dels reptes actuals en biologia és explicar la base genètica de l’adaptació. Mentre que hi ha molts estudis basats en SNPs intentant trobar les bases genètiques de les relacions genotip-fenotip, no s’ha estudiat tant bé el paper de les mutacions generades pels elements mòbils (TEs, de l’anglès Transposable Elements). Evidències recents demostren que els TEs són una eina potent per identificar les bases genètiques dels fenotips adaptatius. Drosophila melanogaster és un bon model per estudiar l’adaptació, ja que és originària de l’Àfrica subtropical i recentment ha colonitzat altres ambients. Per identificar i caracteritzar el paper de diversos TEs candidats per l’adaptació a D. melanogaster, hem seguit dues estratègies diferents: locus-específica i tret-específica. En el primer capítol hem caracteritzat a nivell molecular i fenotípic el TE FBti0019386, identificat prèviament com a candidat per l’adaptació. Primer hem estudiat la història evolutiva d’aquesta inserció i hem demostrat que està associada a senyals genòmiques de selecció positiva. Després hem explorat diferents fenotips relacionats amb els efectes fenotípics coneguts dels gens del costat, que poguessin tenir connexions plausibles amb la variació de la fitness a la natura. En el segon capítol, hem estudiat l’impacte de diferents TEs en la resposta immune. Per això, hem mostrejat el genoma buscant TEs candidats per l’adaptació en quatre poblacions naturals. Després, hem combinat anàlisis d’expressió específica d’al·lel, assajos d’enhancer i detecció de TSS per caracteritzar l’impacte d’aquests TEs durant la resposta a infecció amb el bacteri gram-negatiu Pseudomonas entomophila. Hem trobat que l’al·lel amb el TE s’expressa de manera diferent en 13 dels 16 gens analitzats en condicions control i/o d’infecció en almenys un dels dos fons genètics analitzats. Hem demostrat que alguns d’aquests TEs alteren l’expressió afegint promotors i enhancers als gens del costat. Tot i que les evidències assenyalen cap a un possible paper dels TEs en la regulació de la resposta immune, es requereixen més experiments per associar els TEs identificats amb un efecte en la fitness. En resum, les dues estratègies integratives seguides ens han permès mostrar el paper dels TEs en la generació de variació natural genòmica potencialment implicada en l’adaptació.

O Vírus da Imunodeficiência Humana (HIV) é o agente etiológico da Síndrome da Imunodeficiência Adquirida (AIDS). A AIDS é uma doença de distribuição mundial, e estima-se que existam atualmente pelo menos 36,9 milhões de pessoas infectadas com o vírus. Durante o seu ciclo replicativo, o HIV promove diversas alterações na fisiologia da célula hospedeira a fim de promover sua sobrevivência e potencializar a replicação. A rápida progressão da infecção pelo HIV-1 em humanos e em modelos animais está intimamente ligada à função da proteína acessória Nef. Dentre as diversas ações de Nef está a regulação negativa de proteínas importantes na resposta imunológica, como o receptor CD4. Sabe-se que esta ação resulta da indução da degradação de CD4 em lisossomos, mas os mecanismos moleculares envolvidos ainda são totalmente elucidados. Nef forma um complexo tripartite com a cauda citosólica de CD4 e a proteína adaptadora 2 (AP-2), em vesículas revestidas por clatrina nascentes, induzindo a internalização e degradação lisossomal de CD4. Pesquisas anteriores demonstraram que o direcionamento de CD4 aos lisossomos por Nef envolve a entrada do receptor na via dos corpos multivesiculares (MVBs), por um mecanismo atípico, pois, embora não necessite da ubiquitinação de carga, depende da ação de proteínas que compõem os ESCRTs (Endosomal Sorting Complexes Required for Transport) e da ação de Alix, uma proteína acessória da maquinaria ESCRT. Já foi reportado que Nef interage com subunidades dos complexos AP-1, AP-2, AP-3 e Nef não parece interagir com subunidades de AP-4 e AP-5. Entretanto, o papel da interação de Nef com AP-1 e AP-3 na regulação negativa de CD4 ainda não está totalmente elucidado. Ademais, AP-1, AP-2 e AP-3 são potencialmente heterogêneos devido à existência de isoformas múltiplas das subunidades codificadas por diferentes genes. Todavia, existem poucos estudos para demonstrar se as diferentes combinações de isoformas dos APs são formadas e se possuem propriedades funcionais distintas. O presente trabalho procurou identificar e caracterizar fatores celulares envolvidos na regulação do tráfego intracelular de proteínas no processo de regulação negativa de CD4 induzido por Nef. Mais especificamente, este estudo buscou caracterizar a participação do complexo AP-1 na modulação negativa de CD4 por Nef de HIV-1, através do estudo funcional das duas isoformas de ?-adaptina, subunidades de AP-1. Utilizando a técnica de Pull-down demonstramos que Nef é capaz de interagir com ?2. Além disso, nossos dados de Imunoblot indicaram que a proteína ?2-adaptina, e não ?1-adaptina, é necessária no processo de degradação lisossomal de CD4 por Nef e que esta participação é conservada para degradação de CD4 por Nef de diferentes cepas virais. Ademais, por citometria de fluxo, o silenciamento de ?2, e não de ?1, compromete a diminuição dos níveis de CD4 por Nef da membrana plasmática. A análise por imunofluorêsncia indireta também revelou que a diminuição dos níveis de ?2 impede a redistribuição de CD4 por Nef para regiões perinucleares, acarretando no acúmulo de CD4, retirados por Nef da membrana plasmática, em endossomos primários. A depleção de ?1A, outra subunidade de AP-1, acarretou na diminuição dos níveis celulares de ?2 e ?1, bem como, no comprometimento da eficiente degradação de CD4 por Nef. Além disso, foi possível observar que, ao perturbar a maquinaria ESCRT via super-expressão de HRS (uma subunidade do complexo ESCRT-0), ocorreu um acumulo de ?2 em endossomos dilatados contendo HRS-GFP, nos quais também detectou-se CD4 que foi internalizado por Nef. Em conjunto, os resultados indicam que ?2-adaptina é uma importante molécula para o direcionamento de CD4 por Nef para a via ESCRT/MVB, mostrando ser uma proteína relevante no sistema endo-lisossomal. Ademais, os resultados indicaram que as isoformas ?-adaptinas não só possuem funções distintas, mas também parecem compor complexos AP-1 com diferentes funções celulares, já que apenas a variante AP-1 contendo ?2, mas não ?1, participa da regulação negativa de CD4 por Nef. Estes estudos contribuem para o melhor entendimento dos mecanismos moleculares envolvidos na atividade de Nef, que poderão também ajudar na melhor compreensão da patogênese do HIV e da síndrome relacionada. Em adição, este trabalho contribui para o entendimento de processos fundamentais da regulação do tráfego de proteínas transmembrana no sistema endo-lisossomal.
The Human Immunodeficiency Virus (HIV) is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS). AIDS is a disease which has a global distribution, and it is estimated that there are currently at least 36.9 million people infected with the virus. During the replication cycle, HIV promotes several changes in the physiology of the host cell to promote their survival and enhance replication. The fast progression of HIV-1 in humans and animal models is closely linked to the function of an accessory protein Nef. Among several actions of Nef, one is the most important is the down-regulation of proteins from the immune response, such as the CD4 receptor. It is known that this action causes CD4 degradation in lysosome, but the molecular mechanisms are still incompletely understood. Nef forms a tripartite complex with the cytosolic tail of the CD4 and adapter protein 2 (AP-2) in clathrin-coated vesicles, inducing CD4 internalization and lysosome degradation. Previous research has demonstrated that CD4 target to lysosomes by Nef involves targeting of this receptor to multivesicular bodies (MVBs) pathway by an atypical mechanism because, although not need charging ubiquitination, depends on the proteins from ESCRTs (Endosomal Sorting Complexes Required for Transport) machinery and the action of Alix, an accessory protein ESCRT machinery. It has been reported that Nef interacts with subunits of AP- 1, AP-2, AP-3 complexes and Nef does not appear to interact with AP-4 and AP-5 subunits. However, the role of Nef interaction with AP-1 or AP-3 in CD4 down-regulation is poorly understood. Furthermore, AP-1, AP-2 and AP-3 are potentially heterogeneous due to the existence of multiple subunits isoforms encoded by different genes. However, there are few studies to demonstrate if the different combinations of APs isoforms are form and if they have distinct functional properties. This study aim to identify and characterize cellular factors involved on CD4 down-modulation induced by Nef from HIV-1. More specifically, this study aimed to characterize the involvement of AP-1 complex in the down-regulation of CD4 by Nef HIV-1 through the functional study of the two isoforms of ?-adaptins, AP-1 subunits. By pull-down technique, we showed that Nef is able to interact with ?2. In addition, our data from immunoblots indicated that ?2- adaptin, not ?1-adaptin, is required in Nef-mediated targeting of CD4 to lysosomes and the ?2 participation in this process is conserved by Nef from different viral strains. Furthermore, by flow cytometry assay, ?2 depletion, but not ?1 depletion, compromises the reduction of surface CD4 levels induced by Nef. Immunofluorescence microscopy analysis also revealed that ?2 depletion impairs the redistribution of CD4 by Nef to juxtanuclear region, resulting in CD4 accumulation in primary endosomes. Knockdown of ?1A, another subunit of AP-1, resulted in decreased cellular levels of ?1 and ?2 and, compromising the efficient CD4 degradation by Nef. Moreover, upon artificially stabilizing ESCRT-I in early endosomes, via overexpression of HRS, internalized CD4 accumulates in enlarged HRS-GFP positive endosomes, where co-localize with ?2. Together, the results indicate that ?2-adaptin is a molecule that is essential for CD4 targeting by Nef to ESCRT/MVB pathway, being an important protein in the endo-lysosomal system. Furthermore, the results indicate that ?-adaptins isoforms not only have different functions, but also seem to compose AP-1 complex with distinct cell functions, and only the AP-1 variant comprising ?2, but not ?1, acts in the CD4 down-regulation induced by Nef. These studies contribute to a better understanding on the molecular mechanisms involved in Nef activities, which may also help to improve the understanding of the HIV pathogenesis and the related syndrome. In addition, this work contributes with the understanding of primordial process regulation on intracellular trafficking of transmembrane proteins.

Host proteins involved in defence against parasites are expected to evolve rapidly and adaptively due to selective pressure from pathogens. Social insect taxa, which face an increased threat from pathogens due to demographic attributes such as high genetic similarities amongst nestmates and high population densities, differ in ways that allow us to examine the impact that variable pathogen loads have on the evolution of immune system genes. Social insects which nest in subterranean habitats come into contact with a wide range and large number of parasites. In contrast, many social insects nest in arboreal or lithocolous localities and so have less contact with the soil and the microbes within it. There is mounting evidence that social insect species which nest in subterranean habitats are associated with greater levels of parasitism. It was predicted that immunity genes will have evolved at a greater rate, as evidenced by positive selection, in social insect taxa that inhabit subterranean nests, as compared to their arboreal and lithocolous counterparts. Transferrins are single polypeptide chains that play an important role in iron metabolism and resistance to infection in a variety of organisms. In order to limit the amount of iron microbial fauna receive, transferrin is up-regulated following infection in all insects studied to date. As the ability of a microbe to grow and develop within a host’s body depends upon the availability of iron, conflict has arisen between host and parasite for procuring this essential element. For example, evidence of the battle to keep and acquire iron has been detected in salmon transferrins, which are evolving rapidly and adaptively. It has been proposed that transferrins in these fish are evolving in this manner because they occupy many different habitats during their lifecycle, supporting the notion that the type and number of parasites encountered by an organism can affect the strength of selection on their immune system genes. Polyrhachis is one of the most species rich ant genera in the world and is very well represented in Australia. Species within this genus exhibit extensive variation in nesting habit, making them a suitable and useful model for examining the evolution of immune genes under different selective regimes. Additionally, Polyrhachis ants lack metapleural glands, which secrete substances with antibiotic properties able to kill a wide range of pathogenic organisms. This lack enhances their use in an analysis of immune gene evolution, as without these secretions to help eliminate pathogenic microorganisms, other aspects of their immune system are likely to have experienced stronger selective pressure from pathogens than in the case of many other ants, due to a reduced armoury. I used different models of evolution, based on the nonsynonymous to synonymous substitution ratio (dN/dS ratio), to compare the evolution of the immune related gene transferrin in 14 Polyrhachis species with different nesting habits. The type of selection in this protein across lineages ranged from strong purifying selection to positive selection, demonstrating that certain species of Polyrhachis have experienced different selective pressure to change the amino acid composition of their transferrin. Three lineages (with consensus between models) have a dN/dS ratio greater than one, indicative of positive selection. Of these three species, two nest in subterranean and one in arboreal localities. However, while predominantly arboreal, the third species is known to also nest in the ground. I suggest that the increased level of evolution in certain lineages was brought about by variable loads and types of pathogens encountered by Polyrhachis ants as they radiated. Transferrins of five species with subterranean nesting habits have a dN/dS ratio < 1, indicative of purifying selection. It may be that in these species other factors are more important than nesting habit in influencing parasite loads. Overall, predictions as to the parasite load a given species is likely to encounter are sound, however it seems that these predictions need to be inclusive of other factors, as many features are likely to influence parasite exposure. As well as models designed to detect variable selective intensity across lineages, models that assign a dN/dS ratio to each site were used. Three individual sites were assigned a dN/dS ratio greater than one using consensus among models. These sites are located in regions that are likely to be bound by bacterial iron binding proteins, as they align with regions in vertebrate transferrins known to be so and that have also been subject to adaptive evolution. The occurrence of this type of evolution in equivalent sites and regions in such phylogenetically distant organisms suggests that these sites are important targets for microorganisms that seek to acquire iron from host transferrin. Using transferrins of distantly related insect taxa, including three hymenopteran genera, I tested for positive selection across lineages and at individual sites. Positive selection was not detected at any site or in any lineage with consensus across models. Rather, the molecule appears to be under strong and significant purifying selection, suggesting that the molecule is under selective pressure not to change its amino acid composition. In this analysis of distantly related taxa I included the transferrin of a species of Polyrhachis that had a dN/dS ratio greater than one in the analysis Polyrhachis transferrins. I propose an explanation as to why positive selection was detected in transferrin of this species in one analysis and not another. Over time synonymous changes accumulate and can mask high rates of nonsynonymous change when it has occurred, as any detrimental nonsynonymous change is expected to be rapidly eliminated from the population. The insects used in this analysis are distantly related, therefore even if certain lineages have experienced selective pressure to change (and we know that at least one has), looking for evidence of this at this scale is likely to miss it, unless selection has been very strong and/or constant. As such, I suggest that where possible it is advisable to search for adaptive evolution in genes within shorter evolutionary time scales, such as in the analysis of Polyrhachis transferrins. Most transferrins consist of two homologous lobes (the N and C lobes) and iron binding in each lobes involves six amino acid residues. With the exceptions of a termite and cockroach, insect transferrins studied to date are not generally conserved for binding motifs in their C termini, thus the capacity to bind iron in this region appears to have been lost. The degeneration of this region is thought to be due to antagonistic interactions between host transferrin and iron scavenging proteins of pathogenic bacteria. It is noteworthy that the positively selected sites in transferrin of Polyrhachis ants are located in the N -terminal, which is expected, if the C- terminal of transferrin in these insects is unable to bind iron. Based on alignments of transferrins from distantly related animals (mammals to insects) doubt has been expressed as to as to whether most insect transferrins can bind iron at all {Lambert, 2005 #757}. My alignment differs to that presented by Lambert et al. {Lambert, 2005 #757}, and I conclude that there is no basis for suggesting that insect transferrins are unable to bind iron in the N-terminal.

Tese de Doutoramento em Psicologia Aplicada
Children’s emotional and behavioural functioning is increasingly recognized as critical for children's success, in school as well as in other contexts, and in later phases of life into adulthood. Emotional and behavioural disorders during early childhood has been vastly studied, with recent considerable interest in expanding the research from environmental (e.g., maternal care and contextual experience), to genetic predictors (e.g., polymorphisms in neurotransmission system, given its link to behaviour). Despite this growing interest, research is still in many ways preliminary. The goal of the present doctoral dissertation was to study preschoolers’ emotional and behavioural disorders using a Gene-X-Environment interaction (GxE) approach, combining environmental variables and child’s genetic background related to the immune system. Specifically, its two main aims were to investigate environmental determinants and their action mechanisms on child’s internalizing and externalizing problems; and, on the other hand, to examine whether an immune-related gene interact with environment in the prediction of withdraw behaviour (internalizing symptoms). Therefore, Chapter 1 introduced the state of the art behind the proposed field of study. Chapter 2 then focus the first aim of this dissertation: to examine the effect of family risks and maternal care and their action mechanisms on child’s internalizing and externalizing problems in preschool age. Specifically, Chapter 2 analyzed whether the relationship between family risk and child’s internalizing and externalizing problems is differentially mediated by maternal insensitivity and intrusiveness. Based on a sample of 205 children, their mothers and their preschool teachers, results revealed that only maternal intrusiveness mediated the relation between family risk and children’s internalizing behaviours, in way that children display higher internalizing symptoms when mothers were more intrusive particulatly in the case of higher risk families. Chapter 3 provided a systematic review on GxE research focused on internalizing or/and externalizing problems in early childhood in order to provide a critical overview of the literature. Based on a sample of 14 studies, results concluded that the prevalence of G×E effects in predicting early emotional and behaviour problems is salient, with parental variables, namely, parental psychopathology and parental interactive behaviours, as important moderators. This review also revealed a prevalence of ‘usual suspect’ candidate genes, mostly integrated in the dopaminergic and serotonergic systems (neurotransmission-related genes), disregarding other putatively relevant systems, such as the immune system. Finally, Chapter 4 took into consideration the link between the immune system and some psychiatric conditions, and analyzed the role of the child’s polymorphism (rs2430561) on the Interferon Gamma Gene (IFNG) on withdrawn behaviour, testing maternal psychological distress as a possible moderator of such association. Results proved consistent with the differential susceptibility model of person-X-context interaction. This dissertation underlines the relevance of considering the interaction between individual and environmental factors in shaping emotional and behavioural functioning in a preschool age. Building on this work, future studies should consider larger scale samples, and follow approaches which include different biological, psychological and environmental aspects for a broader comprehension of early emotional and behavioural problems.
O funcionamento emocional e comportamental infantil tem sido amplamente reconhecido como critício para o sucesso das crianças, no contexto escolar, mas também em outros contextos, e em fases posteriores do ciclo de vida, até à idade adulta. As perturbações emocionais e comportamentais durante a infância têm sido muito estudadas, recentemente com considerável interesse na expansão da investigação de preditores ambientais (e.g., cuidados maternos e experiências contextuais), para preditores genéticos (e.g., polimorfismos em genes implicados no sistema de neurotransmissão, dada a sua relação com o comportamento). Apesar deste interesse crescente, a investigação é ainda preliminar em múltiplos aspetos. O objetivo da presente dissertação de doutormaneto foi estudar as perturbações emocionais e comportamentais em idade pré-escolar, usando uma abordagem de intereção entre Genes e Ambiente (Gene-X-Ambiente) combinando variáveis ambientais e informação genética relacionada com o sistema imunitário da criança. Especificamente, os dois objetivos principais foram estudar determinantes ambientais e os seus mecanismos de interação relativamente a problemas de internalização e externalização da criança; e, por outro lado, investigar se um gene relacionado com o sistema imunitário interage com o ambiente na predição de comportamento de isolamento (sintomas de internalização). Assim, o Capítulo 1 introduz o estado da arte acerca da área de estudo proposta. O Capítulo 2 foca o primeiro objetivo desta dissertação: estudar o efeito do risco familiar e dos cuidados maternos e os seus mecanismos de interação nos problemas de internalização e externalização em idade pré-escolar. Especificamente, o Capítulo 2 analisa se a relação entre risco familiar e problemas de internalização e externalização é mediada de forma diferencial pela insensibilidade ou intrusividade maternas. Baseado numa amostra de 205 crianças, as suas mães e professores, os resultados revelaram que a relação entre o risco familiar e os problemas de internalização foi apenas mediada pela inrusividade, de forma que as crianças apresentaram mais problemas de internalização quando as suas mães eram mais intrusivas, particularmente no caso de elevado risco familiar. O Capítulo 3 forneceu uma revisão sistemática da literatura sobre estudos de interação Gene-X-Ambiente focados em problemas de internalização e/ou externalização na infância, de forma a oferecer uma visão crítica da literatura neste âmbito. Baseado numa amostra de 14 estudos, os resultados concluiram que a prevalência do efeito de interação Gene-X-Ambiente na predição de problemas emocionais e comportamentais é saliente entre os estudos, com variáveis parentais a funcionarem como moderadores importantes, nomeadamente a psicopatologia parental e os comportamentos interativos parentais. Esta revisão sistemática também realçou a prevalência dos genes candidatos ‘suspeitos do costume’, integrados maioritariamente nos sistemas dopaminérgico e serotonérgico (genes relacionados com a neurotransmissão), desconsiderando outros sistemas putativamente relevantes, como o sistema imunitário. Finalmente, o Capítulo 4 teve em consideração a relação entre o sistema imunitário e algumas condições psiquiátricas, e analisou o papel de um polimorfismo (rs2430561) no Gene do Interferon Gama (IFNG) no comportamento de isolamento da criança, testando o bem-estar psicológico materno como possível moderador. Os resultados provaram consistência com o modelo da susceptibilidade diferencial. Esta dissertação sublinha a relevância de considerar a interação entre fatores individuais e ambientais no funcionamento emocional e comportamental em idade préescolar. Com base neste trabalho, estudos futuros deverão considerar amostras de maior escala e seguir abordagens que incluam diferentes aspectos biológicos, psicológicos e ambientais, para uma compreensão mais ampla dos problemas emocionais e comportamentais manifestados em idades tão precoces.
This research was conducted at the Psychology Research Centre (UID/PSI/01662/2013), University of Minho. It was supported by the Portuguese Foundation for Science and Technology (PhD Fellowship SFRH/BD/85536/2012) and the Portuguese Ministry of Science, Technology and Higher Education through national funds, and in the scope of QREN – POPH – Typology 4.1 – Advanced Training, reimbursed by the European Social Found and MSTHE funds. It was conducted as part of the PTDC/PSI-PCL/116897/2010 project, which was financed by national funds through the FCT/MEC (PIDDAC) and cofunded by the European Regional Development Fund (FEDER) through COMPETE – Operational Programme Competitiveness Factors (POFC).

碩士
國立屏東科技大學
生物科技系所
101
Polysaccharides may contain pathogen-associated moleculer patterns (PAMPS) stimulated innate immune response in white shrimp (Litopenaeus vannamei). There are total of 5 experimental diets, each was fed to 200 shrimps per tank (initial wt: 0.84 g) for 2 months. Weight gain of shrimp fed control diet, 0.2, 0.4 and 0.6 and 0.8 g kg-1 polysaccharide containing diet increased 669.58 %, 812.58 %, 774.25%, 740.70% and 707.21% (p<0.05), respectively. Quantitative PCR analysis showed that expression mRNA level of LvToll 2, and Lvtoll 3 and interferon gamma inducible lysosomal thiol enzyme (Lv GILT) was clearly up-regulated in hemocyte, hepatopancrea, gill, muscle and stomach of both pellet feeding supplemented with polysaccharide at 20th, 40th and 60th day culturing, compared to shrimp fed control diet. In another experiment, L. vannamei, which had been fed control diet, or polysaccharide-containing diets after 60 days culturing, were challenged with V. alginolyticus at 105 colony-forming units (CFU) shrimp-1 and then placed in sea water of 25‰. The survival of shrimp fed a diet containing 0.2 and 0.4 g kg -1 after 11 days injected increased significantly, as compared to that of shrimp fed control diet. It is therefore concluded that administration of polysaccharide in the diet at 0.2 and 0.4 g kg-1 could enhance the immune ability of L. vannamei and increase its resistance to V. alginolyticus infection

by Ma Suk Ling.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2003.
Includes bibliographical references (leaves 106-129).
Abstracts in English and Chinese.
Abstract --- p.i
摘要 --- p.iii
Acknowledgements --- p.v
Publications --- p.vi
Abbreviations --- p.vii
Chapter Chapter 1 --- General Introduction --- p.1
Chapter 1.1 --- Epidemiology of AD --- p.2
Chapter 1.2 --- Clinical and pathological features of AD --- p.3
Chapter 1.2.1 --- Clinical features of AD --- p.3
Chapter 1.2.2. --- Pathological features of AD --- p.3
Chapter 1.3 --- Diagnosis of AD --- p.4
Chapter 1.4 --- Classification of AD --- p.5
Chapter 1.5 --- Causes of AD --- p.5
Chapter 1.6 --- Risk factors --- p.5
Chapter 1.6.1 --- Age --- p.5
Chapter 1.6.2 --- Family history --- p.6
Chapter 1.6.3 --- Genetics --- p.6
Chapter 1.6.3.1 --- Autosomal dominant mutations --- p.6
Chapter 1.6.3.2 --- Genotypes of Apolipoprotein E --- p.6
Chapter 1.6.4 --- Environmental factors --- p.7
Chapter Chapter 2 --- Pathology in Alzheimer's disease --- p.8
Chapter 2.1 --- Overview of Alzheimer's disease pathology --- p.8
Chapter 2.2 --- Amyloid plaques --- p.8
Chapter 2.2.1 --- Amyloid precursor protein --- p.8
Chapter 2.2.2 --- Processing ofAPP --- p.9
Chapter 2.2.3 --- Amyloid β (Aβ) --- p.12
Chapter 2.2.4 --- APP mutations and AD --- p.12
Chapter 2.3 --- Neurofibrillary tangles (NFT) --- p.15
Chapter 2.3.1 --- Tau --- p.15
Chapter 2.3.2 --- Tau mutation and neurodegeneration --- p.17
Chapter 2.4 --- Hypotheses for AD pathology --- p.18
Chapter 2.4.1 --- Amyloid cascade hypothesis --- p.18
Chapter 2.4.2 --- Inflammatory hypothesis --- p.20
Chapter 2.4.2.1 --- Microglia and astrocytes --- p.21
Chapter 2.4.2.2 --- Inflammatory cytokines --- p.23
Chapter 2.4.2.3 --- Inflammation and AD --- p.25
Chapter 2.4.3 --- ApoE hypothesis --- p.27
Chapter 2.4.3.1 --- Apolipoprotein E --- p.27
Chapter 2.4.3.2 --- ApoE and AD --- p.28
Chapter 2.5 --- Theory towards the pathology of AD --- p.30
Chapter Chapter 3 --- ApoE genotyping --- p.32
Chapter 3.1 --- Introduction --- p.32
Chapter 3.2 --- Materials and methods --- p.32
Chapter 3.2.1 --- Patients and control subjects --- p.32
Chapter 3.2.2 --- Blood sampling --- p.33
Chapter 3.2.3 --- DNA genotyping --- p.34
Chapter 3.2.4 --- Statistical analysis --- p.35
Chapter 3.3 --- Results --- p.35
Chapter 3.. --- Discussion --- p.38
Chapter Chapter 4 --- IL-1β polymorphism in relation to the risk of ADin Chinese --- p.39
Chapter 4.1 --- Introduction --- p.39
Chapter 4.2 --- Materials and methods --- p.44
Chapter 4.2.1 --- Patients and control subjects --- p.44
Chapter 4.2.2 --- Blood sampling --- p.44
Chapter 4.2.3 --- DNA genotyping --- p.44
Chapter 4.2.4 --- Statistical analysis --- p.48
Chapter 4.3 --- Results --- p.48
Chapter 4.4 --- Discussion --- p.53
Chapter Chapter 5 --- TNFα polymorphism in relation to the risk of ADin Chinese --- p.63
Chapter 5.1 --- Introduction --- p.63
Chapter 5.2 --- Materials and methods --- p.66
Chapter 5.2.1 --- Patients and control subjects --- p.66
Chapter 5.2.2 --- Blood sampling --- p.66
Chapter 5.2.3 --- DNA genotyping --- p.66
Chapter 5.2.4 --- Haplotype determination --- p.70
Chapter 5.2.5 --- Statistical analysis --- p.70
Chapter 5.3 --- Results --- p.70
Chapter 5.4 --- Discussion --- p.75
Chapter Chapter 6 --- LRP8 polymorphism in relation to the risk of ADin Chinese --- p.81
Chapter 6.1 --- Introduction --- p.81
Chapter 6.2 --- Materials and methods --- p.87
Chapter 6.2.1 --- Patients and control subjects --- p.87
Chapter 6.2.2 --- Blood sampling --- p.87
Chapter 6.2.3 --- DNA genotyping --- p.87
Chapter 6.2.4 --- Statistical analysis --- p.89
Chapter 6.3 --- Results --- p.91
Chapter 6.4 --- Discussion --- p.98
Chapter Chapter 7 --- Conclusions and prospects for future work --- p.102
Chapter 7.1 --- Conclusion --- p.102
Chapter 7.2 --- Prospects for future work --- p.105
Reference --- p.106

碩士
中山醫學大學
微生物免疫研究所
103
In mammary glands, epithelial cells contact basement membrane (BM) to form glandular structures with a central lumen. In vitro cultures of mammary cells on Matrigel display this morphology. By contrast, cells cultured on tissue culture plastic form monolayers. Owing to the importance of the microenvironment, we compared gene expression in cells cultured on plastic and Matrigel. Many immune-related genes, including complement component 3 (C3), lipopolysaccharide-binding protein (LBP), CD14, growth arrest-specific 6 (GAS6) and milk fat globule-EGF factor 8 (MFG-E8) were upregulated in cells cultured on BM. We speculate that these genes play a role in lumen formation in mammary acini since cell death and the subsequent clearance of the corpses are essential to establish this tissue architecture. Here we have confirmed our microarray data and found that kinetics of gene expression parallels with that of lumen formation, suggesting that these genes might have a role in luminal clearance of the mammary gland. In addition to substratum type, substratum rigidity, cell shape or their related signaling pathways (Rho/Rac pathways) differentially influence the expression of immune-related genes. Necroptosis positively regulates the expression of these genes since inhibition of cathepsin blocks gene expression and lumen formation. Thus, extracellular matrix might control tissue architecture via modulation of immune-related genes.

碩士
國立臺灣海洋大學
水產養殖學系
104
Progranulins (PGRN) is a multifunctional growth factor that regulate cell proliferation, cell survival, tumorigenesis, inflammation, embryonic development, and tissue repair. When organisms are injured or infected, PGRN can be digested into granulin units (GRN) by elastases. GRN units have the function of innate immunity regulation. We had isolated several types of tilapia PGRN genes, PGRN1 and PGRN2, which encode 206 and 199 amino acids composed of two GRN units, respectively. In this study, we isolated one OnPGRN2 gene from Nile tilapia (Oreochromis niloticus). OnPGRN2 was abundantly expressed in spleen and head kidney of male and female adult Nile tilapia. Intraperitoneal injection with S. iniae can induce OnPGRN2 mRNA expression in Nile tilapia. We used Tol2 transposon system to establish two transgenic zebrafish lines expressing different levels of secretory OnPGRN2 (OnPGRN2-S, OnPGRN2-W) by muscle-specific promoter. Expressions of innate immune-related genes had no significant difference in OnPGRN2 transgenic zebrafish lines. Upon intraperitoneal injection with S. agalactiae, the survival rate was enhanced in OnPGRN2-S transgenic zebrafish line, and the relative survival rate was 40%. Pro-inflammatory genes of OnPGRN2 transgenic zebrafish lines were highly activated after challenge with S. agalactiae. It suggests OnPGRN2-S can activate pro-inflammatory factors including TNFa, IL-6, COX-2a at 24 hpi to enhance innate immunity against bacterial pathogen. We detected mRNA expressions of OnPGRN2 and 2.6 kb upstream sequences in two Nile tilapia strains, NT1 and NT2. OnPGRN2 mRNA expression in NT1 which is resistant strain was higher than NT2 which is sensitive strain to Streptococcus, and five immune-related transcription factor binding sites were identified to bear sequence variations between two strains. The relationship between OnPGRN2 promoter sequence and expression will be further investigated. OnPGRN2 gene will be a novel molecular marker for marker-assisted selection of disease-resistant Nile tilapia.

博士
國立臺灣大學
免疫學研究所
105
Human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced PSCs (iPSCs), represent a potentially unlimited cell sources for clinical applications. hESCs are derived from the inner cell mass (ICM) of the early stage human blastocyst. The iPSCs are reprogrammed from somatic cells to pluripotent cells by transduction of the factors Oct4, Sox2, Klf4, and c-Myc. Previous studies have suggested that hPSCs may benefit from immune privilege and limited immunogenicity reflected by the reduced expression of major histocompatibility complex class (MHC)-related molecules. However, the immunogenicity of PSCs is still controversial. Some evidence shows that PSCs do activate immune response in immunocompetent mice. Moreover, before the application of hPSCs for clinical application, the most important issue is the immunogenicity of cells derived from PSCs. Here, we investigated the global immune-related gene expression profiles of hESCs, hiPSCs and somatic cells, and identified candidate immune-related genes that may alter their immunogenicity. The expression levels of global immune-related genes were determined by comparing undifferentiated and differentiated stem cells and in three types of human somatic cells: dermal papilla cells, ovarian granulosa cells, and foreskin fibroblast cells. We identified the differentially-expressed genes CD24, GATA3, PROM1, THBS2, LY96, IFIT3, CXCR4, IL1R1, FGFR3, IDO1, and KDR which overlapped with selected immune-related gene lists. Among these genes, CD24, PROM1, FGFR3, IDO1 and KDR were consistently significant upregulated, and LY96, IFIT3 and IL1R1 were consistently significant downregulated in undifferentiated stem cells compared with somatic cells. The genes GATA3, CXCR4, PROM1, FGFR3 and KDR were consistently upregulated, and only THBS2 was uniformly downregulated in 15-day differentiated stem cells compared with somatic cells. The mammalian target of rapamycin complex (mTOR) signaling pathway has been identified as a key mediator of human immunity and has been leveraged as a therapeutic strategy in tissue transplantation by using rapamycin. In further analyses, mTOR signaling was investigated in the differentiated stem cells following treatment with rapamycin. We found that the inhibition of mTOR signal pathways significantly downregulated immunogenicity of differentiated stem cells. CD24 expression was significantly reduced by rapamycin treatment in the differentiated stem cells. The data indicated mTOR pathway regulated CD24 in stem cell derivatives. Furthermore, reduced expression of CD24 and GATA3 by lentiviral transduction with specific shRNAs in stem cell derivatives leaded to differential regulation of multiple immune-related genes. We also tested the immune responses induced by differentiated stem cells by mixed lymphocyte reactions. We found that CD24- and GATA3-deficient differentiated stem cells including neural lineage cells had limited abilities to activate human lymphocytes. By analyzing the transcriptome signature of immune-related genes, we showed a tendency of the hPSCs to differentiate toward an immune cell phenotype. Taken together these data indicated candidate immune-related genes, in our case, CD24 and GATA3 that might constitute a valuable target for clinical applications.

博士
國立中山大學
海洋生物研究所
95
The present study investigated the expression profiles of nine genes involved in immune defense of the Pacific white shrimp Litopenaeus vannamei and their responses to oral administration of beta-1,3-glucan. The nine immune related genes were beta-glucan binding protein-high density lipoprotein (BGBP-HDL), lipopolysaccharide/beta-glucan binding protein (LGBP), hemocyanin, prophenoloxidase (proPO), transglutaminase (TGase), penaeidin-3 (PEN-3), crustin, cytosolic manganese superoxide dismutase (cMnSOD), and lysozyme. A series of experiments were carried out in the study including: (1) cDNA cloning and characterization of proPO and LGBP; (2) tissue mRNA expressions of the nine genes in adult shrimp; (3) expression and localization of the nine genes during larval and postlarval ontogenic development; (4) the effects of dietary beta-1,3-glucan on the expression of the nine genes. The cDNA cloning study showed that the proPO cDNA contains an open reading frame of 2061 bp and encodes a 686 amino-acid peptide. The protein sequence of the proPO has a similarity of 85% with those of Penaeus monodon and P. semisulcatus and has an identity of between 58 and 77% with other crustaceans. Northern blot analysis revealed that proPO was constitutively expressed mainly in hemocytes. Its transcripts were observed in hemocytes and many other tissues when detected with RT-PCR. The results of in situ hybridizations showed that the hemocytes that infiltrated in tissues were responsible for the positive signals. The LGBP cDNA contains an open reading frame of 1104 bp and encodes a 367 amino-acid protein with a 17 a. a. signal peptide. The protein sequence of the LGBP has a similarity of 97% with LGBP of L. stylirostris, >90% identity with BGBP of P. monodon and LGBP of Fenneropenaeus chinensis and has an identity of between 63 and 86% with other crustaceans. Northern blot analysis revealed that LGBP was constitutively expressed mainly in hepatopancreas. The results of in situ hybridizations showed that the hepatopancreatic F cells might be the major cell type for LGBP production. Using the complete cDNAs of proPO and LGBP and partial fragments of the other seven genes, their tissue expressions were analyzed by conventional RT-PCR, quantitative real time PCR (qPCR) and in situ hybridization. The results demonstrated that BGBP-HDL, LGBP and hemocyanin were mainly expressed in the hepatopancreas and their expressions levels were about 10 to 30% those of

碩士
國立屏東科技大學
水產養殖系所
101
Trichlorfon is an organophosphorous pesticide and has been used in agriculture as a pesticide and as a human medicine to combat internal parasites. It is also use as an ectoparasiticide in the livestock and aquaculture industries, which was pollutant to prawn industry. Thus, the aim of this study is to assessed the effects of trichlorfon during short-term exposure in immunological responses, immune related genes expressions and acetylcholinesterase (AChE) enzyme activity of the giant freshwater prawn, Macrobrachium rosenbergii at 0, 0.2, 0.4 mg L-1 for 0, 3, 6, 12 and 24 hrs, respectively. Total hemocyte count (THC), differential hemocyte count (DHC), respiratory burst activity, phenoloxidase (PO) activity, superoxide dismutase (SOD) activity, glutathion peroxidase (GPx) activity, transglutaminase (TGase) activity, and clotting time of hemocytes were conducted to assessed the immunological responses. The results showed that THC increased after 12 and 24 hrs exposed of trichlorfon at both concentration. DHC showed that Granular cells value increased and Hyaline cells reduced whereas PO activity increased at 6 and 12 hrs under 0.2 mg L-1 TRF. SOD activity increased at 3, 12, and 24 hrs for 0.2 mg L-1 TRF treatment and at 6 and 12 hrs for 0.4 mg L-1 TRF whereas, GPx activity decreased in under exposed of time-dependent in both TRF concentration compared with control. Production of respiratory burst increased generally after 12-24 hrs exposed trichlorfon at both concentration compared to the control. TGase activity and clotting showed TGase activity reduced whereas clotting time of hemocytes increased were indicates that coagulation system inhibited. The potential biomarker of organophosphorous pesticide, AChE revealed significant decreases after exposing for six hours, and showed a concentration-dependent tendency. Immune related genes expressions including PO, LGBP, peroxinectin, α2-macroglubulin, transglutaminase, Cu,Zn-SOD, and GPx of the prawn were also carried in the evaluation. All the immune genes expression decreased significantly when the prawn exposed to 0.4 mg L-1 for 24 hours. The prawn exposed to trichlorfon for short-term not only decrease the circulating hemocyte, but also induced the oxidative stress, which caused the subsequent damage to DNA formation of the immune genes. These concluded that the immunological responses and immune genes expressions of the prawn exposed to trichlorfon at concentration 0.4 mg L-1 for 24 hour were perturbed and then caused the deficiency of immunity and the increase of susceptibility to pathogens infection subsequently.

碩士
國立臺灣海洋大學
水產養殖學系
104
In this study, we explore the effects of ten kinds of Chinese herbal medicine (Si-Jun-Zi Decoction, Paeonia lactiflora, Epimedium sagittatum, Lycium barbarum, Polygonatum odoratum, Rehmannia glutinosa, Fallopia multiflora, Glycyrrhiza uralensis, Ligustrum lucidum, Poria cocos) induced immune-related genes expression and GIV resistant of Epinephelus coioides. Frist, we used different concentration of Chinese herbal medicine to treat grouper kidney cell. Observation cytotoxic of cells and analyzed the immune-related genes expression. In addition, fish were fed diets containing 4% Chinese herbal medicine for 30 days. To analyzed effect of immune-related genes expression and against GIV infection. Results of this study showed that extract of Paeonia lactiflora, Epimedium sagittatum, Lycium barbarum, Rehmannia glutinosa, Fallopia multiflora and Poria cocos treatment grouper kidney cell, induced immune-related genes such as IL-6 and IL-1β was significantly up-regulated, and TNF-α was not significantly up-regulated. Results of feeding Epinephelus coioides with Chinese herbal medicines supplementation diet the IL-6, TNF-α, Mx, IL-1β and IgM gene in the head kidney was significantly expression higher in the group Paeonia lactiflora, Epimedium sagittatum, Lycium barbarum and Fallopia multiflora then in the control. Challenged with GIV, the survival rate was significantly higher in the group Si-Jun-Zi Decoction, Paeonia lactiflora, Epimedium sagittatum, Lycium barbarum, Fallopia multiflora and Rehmannia glutinosa then in the control. However, use grouper kidney cell selected Chinese herbal medicine was inaccuracy. In this study, six kind of Chinese herbal medicines could promote the immune ability in group, which suggested that these herbs are potential immune-stimulant and vaccine adjuvant for aquaculture.