Hepatitis C virus (HCV) infection has become a global public health concern with over 130 million people chronically infected and over 350,000 deaths every year from HCV-related liver diseases. GB virus-B (GBV-B) infection in tamarins is a surrogate model for acute HCV infection. Whilst HCV infection commonly leads to chronicity, GBV-B is naturally cleared. To better understand this natural clearance, this project aimed to study the associated humoral immune response to GBV-B. Additionally, GBV-B-specific antibodies were produced with the aim of characterising the pathology of the virus. Previously, there was no available GBV-B neutralisation assay to identify antibodies in this animal model. Therefore, a GBV-B neutralisation assay, based on a method that is known to be successful for the closely-related HCV, was developed. This method involved producing pseudotyped retroviral particles (PV) expressing the GBV-B envelope that could infect a human hepatocarcinoma cell line. GBV-B PV production was confirmed by western blotting. Future studies can now test archived tamarin sera in this assay for the presence of neutralising antibodies. Neutralising antibodies found through this model could be epitope mapped, and incorporated into HCV vaccine design strategies. To study the pathology of GBV-B infection, GBV-B-specific antibodies were also produced using two techniques in parallel- classical hybridoma technology and ribosome display. Antibodies targeting the nucleocapsid core protein of GBV-B have been previously detected in tamarins and served as the target for production of GBV-B antibodies using both aforementioned technologies. GBV-B core-specific antibodies were successfully isolated using both technologies and can now be used in downstream techniques, such as immunohistochemistry, to characterise the pathology of GBV-B infection thereby further validating the use of the animal model.

Induced and activated as part of the innate immune response, the first line of defence against bacterial or viral infections, Inhibitor of Kappa-B Kinase ε (IKKε) triggers NF-κB and IFNβ signalling. Whilst not expressed at basal levels in healthy cells and tissue, the kinase is overexpressed in roughly 30% of human breast cancer cases, driving oncogenesis through aberrant activation of NF-κB. The impracticality of therapeutic targeting of NF-κB for cancer treatment has led to a requirement for greater understanding of IKKε's oncogenic potential to treat tumours driven by the kinase. Considering that IKKε alters cellular metabolism in dendritic cells, promoting aerobic glycolysis akin to the metabolic phenotype observed in cancer, it was hypothesised that the kinase would play a similar role in breast cancer. Using a Flp-In 293 model of IKKε induction and suppressing IKKε expression in a panel of breast cancer cell lines using siRNA, IKKε-dependent changes in cellular metabolism were characterised using labelled metabolite analysis. IKKε was found to induce serine biosynthesis, an important pathway in breast cancer development that supports glutamine-fuelling of the TCA cycle and contributes to one carbon metabolism to maintain redox balance. Promotion of serine biosynthesis occurred via a dual mechanism. Firstly, PSAT1, the second enzyme of the pathway, was found to be phosphorylated in an IKKε-dependent manner, promoting protein stabilisation. Secondly, an IKKε-dependent transcriptional upregulation of all three serine biosynthesis enzymes, PHGDH, PSAT1 and PSPH, was observed, induced by the inhibition of mitochondrial activity and the subsequent induction of ATF4-mediated mitochondria-to-nucleus retrograde signalling. These data demonstrate a previously uncharacterised mechanism of metabolic regulation by IKKε and highlight new potential therapeutic targets for the treatment of IKKε-driven breast cancer in the form of the enzymes of the serine biosynthesis pathway.

Orientador: Angelo Berchieri Junior / Coorientador: Oliveiro Caetano de Freitas Neto / Banca: Marcelo Brocchi / Banca: Jacqueline Boldrin de Paiva / Banca: Rosemeri de Oliveira Vasconcelos / Banca: Karin Werther / Resumo: Salmonella Enteritidis (SE) é responsável pela maioria dos casos de infecções alimentares em seres humanos e, geralmente, está associada ao consumo de produtos de origem avícola. Nas aves, SE coloniza o intestino, podendo sobreviver utilizando como substrato o 1,2 propanodiol (1,2-Pd) disponível neste ambiente, cujo metabolismo é viabilizado pela ação da enzima propanodiol desidratase, codificada por genes do operon pdu. A enzima propanodiol desidratase necessita de cianocobalamina como cofator enzimático, a qual é codificada pelos genes cobS e cbiA. Neste estudo, utilizou-se uma estirpe de SE e duas mutantes, sendo uma com os genes pduC, pduD, pduE defectivos (SE ΔpduCDE) e outra com os genes pduC, pduD, pduE, cobS e cbiA (SE ΔcobSΔcbiAΔpduCDE) inativados, na tentativa de avaliar a importância do 1,2-Pd para a colonização intestinal e invasão de órgãos durante a infecção de aves por SE. No primeiro experimento, estimou-se as quantidades das estirpes em baço, fígado e conteúdo cecal de aves de postura de variedades vermelha e branca. Um segundo ensaio foi realizado para verificação da excreção fecal, por meio de suabes cloacais. Aliado a isso, caracterizou-se a resposta imunológica em aves SPF desafiadas com estirpes de SE acima descritas por meio da avaliação das populações de linfócitos T CD4+ e TCD8+ em fígado e tonsilas cecais e também pela quantificação de genes responsáveis pela expressão das citocinas IL-6, CCL4, CXCLi2 e IL-22 em tonsilas cecais e IL-6 e IL-18 em baço. Observou-se que as estirpes mutantes foram capazes de colonizar o intestino e invadir o baço e fígado da mesma forma que a estirpe selvagem, sendo que as aves de postura de variedade vermelha foram mais susceptíveis a infecção sistêmica. Tanto a estirpe selvagem como as mutantes estimularam o mesmo perfil de resposta imune nas aves SPF. Portanto, os genes pduC, pduD, pduE, cobS e cbiA não são necessários para... / Abstract: Salmonella Enteritidis (SE) is responsible for majority of the cases of foodborne diseases in humans and usually is associated with the consumption of poultry origin products. In birds, SE colonizes the intestine and can survive using 1,2 propanodiol (1,2-Pd) as substrate. The metabolisation of the 1,2-Pd is possible by the action of propanodiol dehydratase enzyme, encoded by genes pdu operon. Cyanocobalamin, which is encoded by genes cobS and cbiA, is the cofactor required by propanodiol dehydratase. In this study, we used one SE wild type strain and two mutants, one with mutations in pduC, pduD and pduE genes (SE ΔpduCDE) and another with mutations of pduC, pduD, pduE, cobS and cbiA genes (ΔcobSΔcbiAΔpduCDE) in attempt to evaluate the importance of 1,2-Pd for intestinal colonization and invasion of organs during infection of birds. In the first experiment, bacterial counts for all strains were obtained from spleen, liver and cecal content of white and brown varieties of commercial egg-layers. In the second assay, faecal shedding of the strains was assessed by cloacal swabs. Additionaly, immunological responses were characterised by the assessment of T CD4+ and T CD8+ cell populations in liver and cecal tonsils from SPF (Specific Pathogen Free) chicks challenged with the three strains. Complementary transcripts of genes encoding the cytokines IL-6, CCL4, CXCLi2 and IL-22 in cecal tonsils, and IL-6 and IL-18 in liver were obtained for relative quantification by qRT-PCR. It was observed that SE mutant strains were able to colonise the intestine and invade spleen and liver in the same way as the wild type strain. Brown variety of birds was more susceptible to systemic infection than white birds. Furthermore, the immune response profiles triggered by the SE wild type and the two mutants in SPF chicks were very similar. Therefore, data for the present study suggest that pduC, pduD, pduE, cobS e cbiA genes would not be essencial to ... / Doutor

Dans tous les organismes animaux la réponse immunitaire est divisée en deux composantes : la réponse humorale, qui consiste en la production d'un grand nombre de molécules toxiques pour le pathogène, et la réponse cellulaire, qui met en jeu des cellules immunitaires produites lors de l'hématopoïèse. Chez les mammifères adultes, l'hématopoïèse se déroule dans la moelle osseuse, où un microenvironnement particulier appelé " niche hématopoïétique " contrôle l'auto-renouvèlement, la prolifération et la différenciation des Cellules Souches Hématopoïétiques (CSH) à l'origine de l'ensemble des cellules sanguines/immunitaires. Suite à une infection par un pathogène, l'homéostasie du système hématopoïétique est modifiée, afin de permettre la mise en place d'une réponse immunitaire cellulaire adaptée. Le rôle de la niche hématopoïétique dans le contrôle de l'hématopoïèse suite à une infection reste à ce jour mal connu. La drosophile est utilisée comme système modèle pour étudier in vivo l'hématopoïèse et la réponse immunitaire. L'hématopoïèse a lieu chez la drosophile au stade larvaire dans un organe spécialisé appelé Glande Lymphatique (GL). Au sein de cet organe, un petit groupe de cellules, le Centre de Signalisation Postérieur (PSC), contrôle l'équilibre entre progéniteurs hématopoïétiques et cellules immunitaires différenciées, et a donc un rôle équivalent à celui de la niche hématopoïétique des mammifères. Suite à un stress immun, tel que le parasitisme par des guêpes, une différenciation massive de cellules immunitaires spécifiques, les lamellocytes, a lieu dans la GL; puis la dispersion de la GL permet la libération des lamellocytes dans la circulation lymphatique. Lors du parasitisme, la guêpe pond un œuf dans le corps de la larve de drosophile. En absence de réponse immunitaire cellulaire, l'œuf de guêpe se développe au dépend de son hôte, entraînant sa mort. En formant une capsule autour de l'œuf de guêpe, les lamellocytes neutralisent son développement et permettent la survie de l'hôte. Au cours de ma thèse, je me suis intéressée à la réponse immunitaire cellulaire de la larve de drosophile au parasitisme par des guêpes. Je me suis plus particulièrement intéressée au rôle de la " niche hématopoïétique " dans cette réponse. Pour cela, j'ai initié une approche transcriptomique ayant pour but d'identifier les gènes spécifiquement exprimés dans le PSC en réponse au parasitisme. En parallèle, j'ai caractérisé le rôle de la voie de signalisation Toll/NF?B dans la GL lors de la réponse au parasitisme. La voie Toll/NF?B joue un rôle essentiel dans la réponse immunitaire humorale et son rôle dans la réponse immunitaire cellulaire reste à définir. Mes travaux indiquent que la voie Toll/NF?B est activée dans le PSC suite au parasitisme. Son activation est médiée par le facteur de transcription NF?B "Dorsal-related Immunity Factor" (Dif), qui est requis dans le PSC pour permettre la différenciation rapide et massive de lamellocytes et la dispersion des cellules de la GL. De plus, j'ai établi un réseau génique, impliquant les deux voies de signalisation Toll/NF?B et EGFR ainsi que les espèces réactives de l'oxygène (ROS) dans le contrôle de la réponse au parasitisme. Une augmentation du niveau de ROS dans le PSC et l'activation de la voie EGFR dans les cellules immunitaires ont été décrits comme nécessaires à l'encapsulation des œufs de guêpe après parasitisme. Mes données établissent qu'ils sont en plus requis respectivement dans les cellules du PSC et dans les progéniteurs hématopoïétiques pour permettre la dispersion de la GL après parasitisme. Basé sur la forte conservation des voies de signalisation et processus moléculaires contrôlant l'hématopoïèse entre les mammifères et la drosophile, mes résultats posent la question de la conservation du réseau génique établi chez la drosophile et du rôle de la voie NF?B dans la niche hématopoïétique des mammifères lors d'une réponse à une infection. / In all organisms, the immune response is divided into two parts: the humoral response, which consists of producing a large number of molecules to combat the pathogen, and the cellular response, which relies on immune cells produced during hematopoiesis. In adult mammals, hematopoiesis occurs in the bone marrow, where a particular microenvironment called the "hematopoietic niche" controls self-renewal, proliferation and differentiation of Hematopoietic Stem Cells (HSCs), which give rise to all blood cell types. Following a pathogenic infection, the hematopoietic system's homeostasis is modified in order to obtain an adapted cellular immune response. The role that the hematopoietic niche plays during an immune response remains unclear. Drosophila is used as a model system to study in vivo hematopoiesis and the immune response. In drosophila, hematopoiesis occurs at the larval stage in a specialized organ called the Lymph Gland (LG). Within this organ, a small group of cells termed the Posterior Signalling Center (PSC), controls the balance between hematopoietic progenitors and differentiated immune/blood cells, a role similar to the mammalian hematopoietic niche. Following an immune challenge, especially in response to wasp parasitism, a massive differentiation of specific immune cells called lamellocytes occurs in the LG. The LG subsequently disperses to release lamellocytes into the hemolymph. During parasitism, the wasp lays an egg in the drosophila larva. In the absence of a cellular immune response, the wasp egg will develop and kill its host. By forming a capsule around the wasp egg, lamellocytes impede the pathogen's development and permit the host's survival. During my PhD, I studied the drosophila larva cellular immune response to wasp parasitism. I focused my research on the role of the "hematopoietic niche". I therefore initiated a transcriptomic study, in order to identify genes expressed by the PSC in response to parasitism. In parallel, I characterized the role of the Toll/NF?B signalling pathway in the LG during parasitism. The Toll/NF?B pathway plays a key role in the humoral response both in drosophila and mammals; however its role in the cellular immune response remains unknown. My results indicate that the Toll/NF?B pathway is activated in the PSC following parasitism. Its activation is mediated by the NF?B transcription factor " Dorsal-related Immunity Factor " (Dif), which is required in the PSC for rapid lamellocyte production and LG dispersion. Furthermore, I established the existence of a genetic network comprising the Toll/NFkB and EGFR signalling pathways and Reactive Oxygen Species (ROS), in order to control the immune response to parasitism. An increase in ROS levels in the PSC and EGFR pathway activation in the immune cells, have been described as required for wasp egg encapsulation. My data suggest that the ROS and the EGFR pathway are also required for LG dispersion following wasp parasitism, in PSC cells and in hematopoietic progenitors, respectively. Based on the high conservation of signalling pathways and molecular processes controlling hematopoiesis, my results raise the question of whether such a network is conserved in the mammalian hematopoietic niche in response to pathogenic infections.

Infectious disease is a major issue facing the livestock industry. Further understanding of the role of genetic factors in the observed phenotypic variability of the immune response to pathogens and vaccination could assist in designing improved preventative measures such as more efficacious vaccines and targeted breeding strategies to select for disease resistance. Major candidate genes for controlling immune responsiveness are located within the major histocompatibility complex (MHC). The highly polymorphic classical MHC genes are key determinants in the strength and type of immune response. However, it has proved difficult to establish genotyping approaches to define functionally relevant allelic variations for outbred species such as cattle, not least because the peptide binding clefts (PBC) of classical MHC molecules are highly polymorphic, and the genes within the MHC complex are closely linked. The overall aim of this project was to investigate the role of MHC genes in immune responsiveness in approximately 200 F2 and backcross Holstein-Charolais cattle. These animals were generated as part of the Roslin Bovine Genome (RoBoGen) herd, established through a quantitative trait loci (QTL) project, in which a number of phenotypic traits including immune traits were measured. The immune traits included responses to a Foot-and-mouth disease virus (FMDV) peptide, and vaccines against bovine respiratory syncytial virus (BRSV), para-influenza virus 3 (PIV-3) and bovine herpes virus-1 (BHV-1), as well as T cell response to Staphylococcus aureus. The immune phenotypes measured included IgG and interferon- (IFN- ) levels and T cell proliferation. The cattle MHC region, known as bovine leukocyte antigens (BoLA), resides on bovine chromosome 23. The BoLA region contains approximately 200 genes most of which are immune-related. Class II gene polymorphisms were considered to be the most likely to influence the immune traits measured, and the project primarily focused on the best defined gene, BoLA-DRB3. A sequence-based typing technique was successfully improved to facilitate genotyping of the PBC of BoLA-DRB3 in all generations of the RoBoGen herd (approximately 400 animals) and identified 24 distinct alleles. The sequence information obtained also enabled further analysis of the role of defined ‘pockets’ within the PBC, which directly determine peptide binding affinity. All datasets were statistically analysed using a residual maximum likelihood (REML) model and it was shown that several of the DRB3 alleles within the RoBoGen herd had highly significant (p<0.05) associations with the immune response to the FMDV peptide. In addition DRB3 alleles were identified which had significant associations with the response to the respiratory pathogen vaccinations and exposure to S. aureus. The pocket analysis also enabled the identification of several amino acid positions within the PBC which were significantly associated with the immune response traits. In order to explore whether DQ Class II gene polymorphisms also played a role in the variability of responses and whether BoLA Class I-Class II haplotypes could be discerned, microarrays which utilized allele specific oligonucleotides for BoLA Class I and Class II DQ genes were employed. In addition, to investigate whether the number of DQ gene pairs per chromosome influenced the responses, a quantitative polymerase chain reaction (qPCR) assay to determine DQA gene dosage was developed. However, due to the extremely complex nature of the BoLA region both, techniques would require improving to be used for large-scale studies. Nonetheless, information about haplotypes was determined from the microarray results and the qPCR technique lays the foundations for future development to investigate DQ gene dosage. The MHC region in cattle is very complex due the high level of polymorphisms and gene duplications. It is likely that many genes play a role in the immune response to both pathogens and vaccines. However, from the evidence presented here, polymorphisms in the PBC of BoLA-DRB3, particularly within the pockets, are significantly associated with variation in immune response to many different antigens and this information could be exploited in the design of vaccines or breeding cattle for improved disease resistance.

Parasitic helminths infect millions of people and animals worldwide. A key feature of their lifecycle is the longevity of survival within a single host, which is often attributed to the ability of the parasite to divert or modulate the immune response against it. The excretory-secretory (ES) products released by helminths are of interest as the mediators of such immunomodulation. Heligmosomoides polygyrus is an excellent model of gastrointestinal (GI) helminth infection in rodents, and has been used here to investigate several aspects of the immune response, and the manipulation of these, in mice. Firstly, the roles of B cells and antibodies in infection with H. polygyrus and towards the adult ES (HES) were investigated. Using several B cell-deficient mouse strains, a minimal effect on immunity to primary infection with H. polygyrus was demonstrated. However, primary infection serum binds to a select set of highly immunodominant components of the complex protein mixture of HES, which were identified as venom allergen-like proteins (VALs). Utilising four strains of mice that vary in their resistance phenotype to H. polygyrus, several aspects of immunity towards the worm were investigated. Increased levels of markers of alternatively activated macrophages, which are a key component of the granulomatous inflammatory response around invading H. polygyrus larvae, were found in the most resistant strains, SJL and BALB/c. Depletion of macrophages, by administration of clodronate, severely disrupted the granuloma and parasite clearance. Numbers of innate lymphoid cells and the subsequent Th2 response, specificity range and titre of antibody, and activation of regulatory T cells all correlate with a resistant phenotype. A deficiency in the cytokine macrophage migration inhibitory factor (MIF) renders a resistant BALB/c mouse completely susceptible to infections with H. polygyrus, and Nippostronygylus brasiliensis, an acute model of GI helminth infection. This is accompanied by a failure to induce both ILCs and an early myeloid-derived cell population upon infection. The influx of alternatively activated macrophages around larvae in the mucosa of the small intestine is delayed in MIF-/- mice, although all immunological parameters are comparable to wild-type by day 14 post-infection. The susceptible phenotype of MIF-/- mice can be replicated using a chemical inhibitor of MIF in BALB/c mice. Finally, the previously documented transforming growth factor-β (TGF-β) activity of HES was dissected out further using two methods of fractionation. Distinct fractions with TGF-β activity were subjected to mass spectrometry to identify protein components that could be potential candidates for this activity.

Choi, Man Wing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 126-139). / Abstracts in English and Chinese. / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Antigen presenting cells (APC) in Immune systems --- p.1 / Chapter 1.1.1 --- Dendritic cells --- p.2 / Chapter 1.1.1.1 --- Differentiation of dendritic cells in mice --- p.2 / Chapter 1.1.1.2 --- Maturation of dendritic cells --- p.3 / Chapter 1.1.1.3 --- Stimulation and polarization of T cells stimulated by dendritic cells --- p.6 / Chapter 1.1.2 --- Monocyte and macrophage --- p.7 / Chapter 1.1.2.1 --- Differentiation of monocyte and macrophage in humans --- p.7 / Chapter 1.1.2.2 --- Changes involved in differentiation of monocytes into macrophages --- p.9 / Chapter 1.2 --- "Isolation, structure and activity of mushroom polysaccharides" --- p.13 / Chapter 1.2.1 --- Sources of mushroom polysaccharides --- p.13 / Chapter 1.2.2 --- Extraction methods --- p.14 / Chapter 1.2.3 --- Structure-Activity Relationship (SAR) of mushroom polysaccharides --- p.15 / Chapter 1.2.4 --- Previous studies on immunomodulatory effects of mushroom sclerotial polysaccharides --- p.18 / Chapter 1.3 --- Recognition of β-glucan by specific receptors --- p.20 / Chapter 1.3.1 --- Complement Receptor 3 (CR3) --- p.22 / Chapter 1.3.1.1 --- Introduction of CR3 --- p.22 / Chapter 1.3.1.2 --- Expressions of CR3 to recognize fungi --- p.22 / Chapter 1.3.2 --- Dectin-1 --- p.24 / Chapter 1.3.2.1 --- Introduction of Dectin-1 --- p.24 / Chapter 1.3.2.2 --- Structure of Full-length Dectin-1 --- p.26 / Chapter 1.3.2.2.1 --- Isoforms of Dectin-1 in Mice --- p.28 / Chapter 1.3.2.2.2 --- Isoforms of Dectin-1 in Humans --- p.28 / Chapter 1.3.2.3 --- Immune responses triggered by of Dectin-1 --- p.29 / Chapter 1.3.3 --- Toll-like 2 receptor (TLR2) --- p.31 / Chapter 1.3.3.1 --- Introduction of TLR2 --- p.31 / Chapter 1.3.3.2 --- Structure of TLR2 --- p.33 / Chapter 1.3.3.3 --- Immune responses triggered by TLR2 --- p.34 / Chapter 1.4 --- Research Objectives --- p.35 / Chapter Chapter 2 --- Materials and Methods --- p.38 / Chapter 2.1 --- Materials --- p.38 / Chapter 2.1.1 --- Mushroom sclerotia --- p.38 / Chapter 2.1.1.1 --- Polysaccharide extraction from mushroom sclerotia --- p.38 / Chapter 2.1.2 --- Antibodies and reagents --- p.41 / Chapter 2.1.3 --- Human acute leukocyte monocytic cell line and culture medium --- p.42 / Chapter 2.1.4 --- Preparation of murine bone marrow-derived immature dendritic primary cells (immature BMDCs) --- p.43 / Chapter 2.2 --- Methods --- p.45 / Chapter 2.2.1 --- Chemical Analysis --- p.45 / Chapter 2.2.1.1 --- Measurement of monosaccharide profile --- p.45 / Chapter 2.2.1.1.1 --- Acid depolymerisation --- p.45 / Chapter 2.2.1.1.2 --- Neutral sugar derivatization --- p.45 / Chapter 2.2.1.1.3 --- Gas chromatography (GC) --- p.46 / Chapter 2.2.1.2 --- Determination of total sugar by phenol-sulfuric acid method --- p.47 / Chapter 2.2.1.3 --- Determination of protein content by Lowry-Folin Method --- p.48 / Chapter 2.2.1.4 --- Size exclusion chromatography by high pressure liquid chromatography (HPLC) --- p.49 / Chapter 2.2.1.5 --- Endotoxin detection --- p.50 / Chapter 2.2.2 --- Measurement of Bioactivities --- p.51 / Chapter 2.2.2.1 --- Trypan blue exclusion assay --- p.51 / Chapter 2.2.2.2 --- MTT cell proliferation assay --- p.51 / Chapter 2.2.2.3 --- BrdU cell proliferation assay --- p.53 / Chapter 2.2.2.4 --- Expression of cell surface markers --- p.54 / Chapter 2.2.2.5 --- Phagocytosis / Endocytosis of FITC-labeled dextrans --- p.55 / Chapter 2.2.2.6 --- Nitric oxide production assay --- p.55 / Chapter 2.2.2.7 --- Reactive oxygen species production --- p.57 / Chapter 2.2.2.8 --- Determination of cytokine profile using cytokine antibody array --- p.58 / Chapter 2.2.2.9 --- Cell cycle analysis --- p.59 / Chapter 2.2.2.10 --- Expression of surface receptors --- p.60 / Chapter 2.2.2.11 --- Statistical analysis --- p.61 / Chapter Chapter 3 --- Results and Discussion --- p.61 / Chapter 3.1 --- Chemical characteristics of sclerotial polysaccharides --- p.61 / Chapter 3.1.1. --- The yield of sclerotial polysaccharides --- p.61 / Chapter 3.1.2 --- Total carbohydrate content of sclerotial polysaccharides --- p.65 / Chapter 3.1.3 --- Protein content of sclerotial polysaccharides --- p.66 / Chapter 3.1.4 --- Monosaccharide profiles of sclerotial polysaccharides from PR by gas chromatography (GC) --- p.66 / Chapter 3.1.5 --- Molecular weight of sclerotial polysaccharides from PR by size exclusion chromatography (SEC) --- p.69 / Chapter 3.1.6 --- Endotoxin test --- p.73 / Chapter 3.2 --- Immune responses for human monocytic cell line THP-1 --- p.74 / Chapter 3.2.1 --- MTT cell viability assay --- p.74 / Chapter 3.2.2 --- BrdU cell proliferation assay --- p.75 / Chapter 3.2.3 --- Change in cell morphology of THP-1 --- p.79 / Chapter 3.2.4 --- Phenotypic maturation of THP-1 --- p.81 / Chapter 3.2.5 --- Up-regulated phagocytic ability of THP-1 --- p.84 / Chapter 3.2.6 --- Increased nitrite production in THP-1 --- p.86 / Chapter 3.2.7 --- Production of reactive oxygen species --- p.88 / Chapter 3.2.8 --- Human cytokines profile array --- p.90 / Chapter 3.2.9 --- Cell cycle analysis --- p.93 / Chapter 3.2.10 --- Surface receptors expression --- p.95 / Chapter 3.2.11 --- Summary --- p.98 / Chapter 3.3 --- Immune responses for murine immature BMDCs --- p.102 / Chapter 3.3.1 --- Inhibition effects on murine immature BMDCs --- p.102 / Chapter 3.3.2 --- Change in cell morphology of murine immature BMDCs --- p.103 / Chapter 3.3.3 --- Phenotypic maturation of murine immature BMDCs --- p.105 / Chapter 3.3.4 --- Down-regulation of endocytosis in murine immature BMDCs --- p.106 / Chapter 3.3.5 --- Increased nitrite production --- p.109 / Chapter 3.3.6 --- Decreased expression of CD 11c in PRW-treated immature BMDCs --- p.109 / Chapter 3.3.7 --- Cytokine profile detection --- p.112 / Chapter 3.3.8 --- Surface receptors expression --- p.116 / Chapter 3.3.9 --- Summary --- p.119 / Chapter Chapter 4 --- Conclusion and future works --- p.123 / Appendix --- p.125 / References --- p.126

Guo, Cuixia. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 75-85). / Abstracts in English and Chinese. / Thesis committee --- p.ii / Abstract --- p.iii / 摘要 --- p.iv / Acknowledgment --- p.v / List of Tables --- p.vi / List of Figures --- p.vii / List of Abbreviations --- p.viii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Introduction to immune system --- p.1 / Chapter 1.2 --- Immune effecter cells --- p.1 / Chapter 1.2.1 --- Macrophage --- p.1 / Chapter 1.2.2 --- Dendritic Cells (DCs) --- p.5 / Chapter 1.3 --- Immunomodulatory and antitumor activities of mushrooms --- p.8 / Chapter 1.3.1 --- Introduction to mushroom --- p.11 / Chapter 1.3.2 --- Mushroom polysaccharides --- p.11 / Chapter 1.3.3 --- Mushroom β-glucan --- p.14 / Chapter 1.4 --- The receptors for polysaccharides associated with immune effecter cells --- p.16 / Chapter 1.4.1 --- CR3 --- p.16 / Chapter 1.4.2 --- Dectin-1 --- p.18 / Chapter 1.4.3 --- TLR2 --- p.19 / Chapter 1.5 --- Nuclear factor-kappa B (NF-kB) activation --- p.19 / Chapter 1.6 --- Previous studies on mushroom sclerotium --- p.20 / Chapter 1.6.1 --- Pleurotus tuber-regium (PT) --- p.20 / Chapter 1.6.2 --- Polyporus rhinocerus (PR) --- p.21 / Chapter 1.7 --- Objectives --- p.21 / Chapter 2. --- Materials and Methods --- p.23 / Chapter 2.1 --- Materials --- p.23 / Chapter 2.1.1 --- Mushroom sclerotia --- p.23 / Chapter 2.1.2 --- Animal --- p.23 / Chapter 2.1.3 --- Cell lines --- p.24 / Chapter 2.2 --- Methods --- p.24 / Chapter 2.2.1 --- Hot water extraction --- p.24 / Chapter 2.2.2 --- Measurement of monosaccharide profile --- p.25 / Chapter 2.2.2.1 --- Acid depolymerization --- p.25 / Chapter 2.2.2.2 --- Neutral sugar derivatization --- p.25 / Chapter 2.2.2.3 --- Gas chromatography (GC) --- p.26 / Chapter 2.2.3 --- Determination of molecular weight by size exclusion chromatography (SEC) --- p.27 / Chapter 2.2.4 --- Determination of total sugar by phenol-sulfuric acid method --- p.28 / Chapter 2.2.5 --- Determination of protein content by Lowry-Folin method --- p.28 / Chapter 2.2.6 --- Detection of endotoxin --- p.29 / Chapter 2.2.7 --- Immunomodulatory activities induced in RAW264.7 cell line and murine peritoneal macrophages (PMs) --- p.30 / Chapter 2.2.7.1 --- Isolation of murine peritoneal macrophages (PMs) --- p.30 / Chapter 2.2.7.2 --- Detection of cell surface antigens on RAW 264.7 cells and PMs --- p.30 / Chapter 2.2.7.3 --- Phagocytic uptake --- p.31 / Chapter 2.2.7.4 --- Reactive Oxygen Species (ROS) generation --- p.32 / Chapter 2.2.7.5 --- Nitric Oxide (NO) production --- p.32 / Chapter 2.2.7.6 --- Inducible Nitric Oxide Synthase (iNOS) expression --- p.32 / Chapter 2.2.7.6.1 --- Cell lysates preparation --- p.33 / Chapter 2.2.7.6.2 --- Determination of protein concentrations --- p.33 / Chapter 2.2.7.6.3 --- Western blot --- p.34 / Chapter 2.2.7.7 --- Tumor Necrosis Factor-alpha (TNF-α) production --- p.36 / Chapter 2.2.8 --- DC cell marker determination --- p.37 / Chapter 2.2.9 --- Nuclear factor kappa B (NF-kB) activation --- p.37 / Chapter 2.2.10 --- Determination of the expression of existing cell surface β-glucan receptors --- p.37 / Chapter 2.2.11 --- Statistical methods --- p.38 / Chapter 3. --- Results --- p.39 / Chapter 3.1 --- Yield and chemical composition of mushroom sclerotial polysaccharides --- p.39 / Chapter 3.2 --- Endotoxin examination --- p.41 / Chapter 3.3 --- Monosaccharide profiles of PTW and PRW by GC --- p.41 / Chapter 3.4 --- Molecular weight profile by size exclusion chromatography (SEC) --- p.43 / Chapter 3.5 --- Immunomodulatory activities induced in RAW264.7 cells and murine peritoneal macrophages (PMs) --- p.46 / Chapter 3.5.1 --- Detection of cell surface antigens on RAW 264.7 cells and PMs --- p.46 / Chapter 3.5.2 --- Phagocytic uptake --- p.49 / Chapter 3.5.3 --- ROS generation --- p.53 / Chapter 3.5.4 --- NO production --- p.56 / Chapter 3.5.5 --- iNOS expression --- p.59 / Chapter 3.5.6 --- TNF-α production --- p.60 / Chapter 3.5.7 --- Morphological changes of cells --- p.62 / Chapter 3.5.8 --- DC cell marker determination --- p.64 / Chapter 3.6 --- Receptors expression on RAW 264.7 cells and PMs --- p.66 / Chapter 3.7 --- NF-kB activation --- p.68 / Chapter 3.8 --- Discussion --- p.70 / Chapter 4. --- Conclusions and Future Works --- p.73 / Chapter 5. --- References --- p.75

A desnutrição protéica e a obesidade são os maiores problemas alimentares na atualidade, afetando bilhões de pessoas em todo o mundo. A transição nutricional, que vem ocorrendo nas últimas décadas está mudando o perfil nutricional, diminuindo a desnutrição e aumentando a porcentagem de indivíduos obesos. A resposta imune inata é extremamente influenciada pela dieta, apresentando alterações significativas tanto em desnutrição quanto na obesidade. Portanto, nos propusemos a avaliar os efeitos da desnutrição protéica e da obesidade nos parâmetros nutricionais, bioquímicos e imunológicos em camundongos. Os animais desnutridos tiveram perda de peso, além de diminuição nas proteínas totais, albumina sérica, glicose, insulina, colesterol, triacilgliceróis e sensibilidade à insulina. Os animais desnutridos também apresentaram valores hematológicos (hemoglobina, hematócrito e hemácias) reduzidos, além de apresentarem pancitopenia e hipocelularidade medular. As funções de adesão, espraiamento, fagocitose, atividade fungicida, produção de espécies reativas de oxigênio, nitrogênio e citocinas inflamatórias, apresentaram-se diminuídas, evidenciando alterações na resposta imune inata. Os animais submetidos a dieta hiperlipídica apresentaram consumo reduzido de ração, no entanto não houve diferença nas taxas de glicose, insulina, proteínas totais, albumina, hemoglobina, hematócrito e número de hemácias. Os animais hiperlipídicos tiveram aumento de colesterol, diminuição do triacilglicerol e aumento da sensibilidade à insulina. A função macrofágica de adesão, espraiamento, fagocitose, atividade fungicida, produção de espécies reativas de oxigênio, nitrogênio e citocinas inflamatórias foi levemente aumentada. / Protein malnutrition and obesity are major food problems nowadays, affecting billions of people around the world. The nutritional transition that has occurred in recent decades is changing the nutritional profile, reducing malnutrition and increasing the percentage of obese individuals. The innate immune response is highly influenced by diet, with significant changes in both malnutrition and in obesity. Therefore, we set out to evaluate the effects of malnutrition and obesity on nutritional parameters, biochemical and immunological in mice. The malnourished animals presented weight loss, and a decrease in total protein, albumin, glucose, insulin, cholesterol, triglycerides and insulin sensitivity. The malnourished animals also showed hematological (hemoglobin, hematocrit and red blood cells) reduced, besides having pancytopenia and hypocellular bone marrow. The functions of adhesion, spreading, phagocytosis, fungicidal activity, production of reactive oxygen species, nitrogen and inflammatory cytokines, showed up diminished , showing changes in the innate immune response. The animals subjected to high-fat diet showed reduced feed intake, however there was no difference in the rates of glucose, insulin, total protein, albumin, hemoglobin, hematocrit and erythrocyte counts. Hyperlipidaemic animals had increased cholesterol and triglyceride decreased and increased insulin sensitivity. The role of macrophage adhesion, spreading, phagocytosis, fungicidal activity, production of reactive oxygen species, nitrogen and inflammatory cytokines was slightly increased.

Infections in the central nervous system pose a considerable challenge to the host. On one hand, a quick and rapid immune response is important to control the infection, while on the other hand, too robust a response can damage the CNS, which has poor regenerative properties. Therefore, nowhere else in the body is such a balance between pro- and anti-inflammatory mediators as important. Mice infected with the coronavirus, mouse hepatitis virus strain J2.2-V-1, are a useful model for understanding the two sides of the immune system. In this neurotropic viral infection, demyelination occurs secondary to the immune response's control of the viral infection. Thus, J2.2-V-1 infection also functions as an infectious animal model for multiple sclerosis (MS). Many arms of the pro-inflammatory immune system have been studied during J2.2-V-1 infection but the anti-inflammatory immune response has not been thoroughly investigated prior to this study. The data demonstrated here represent an in-depth look into the role of regulatory T cells and IL-10 during J2.2-V-1 infection. Specifically, by adoptive transfer of Tregs, I show that there is a relative paucity of Tregs during J2.2-V-1 infection in C57BL/6 mice and their addition decreases clinical scores, demyelination and the T cell response during infection without affecting viral clearance. A RAG1-/- adoptive transfer model demonstrates clinical results consistent with results obtained in B6 mice, while further demonstrating that Tregs function in the draining cervical lymph node by dampening dendritic cell activation and pro-inflammatory chemokine and cytokine release. There is also a relative decrease in T cell proliferation. Thus, Tregs are protective in J2.2-V-1-induced encephalomyelitis and their enhancement is a potential therapy for MS. Additionally, IL-10 is an important anti-inflammatory component of the immune response, as its absence causes increased immunopathology with increased demyelination in J2.2-V-1-infected B6 mice. Through the development of a recombinant J2.2-V-1 virus that produces IL-10, I also demonstrate that increasing the level of IL-10 at the site of infection is protective early in the immune response. Antigen-specific IFN-γ+ CD4 and CD8 T cells produce IL-10 at the height of the inflammation. CD8 T cells require a high level of antigen stimulation and the most recently activated CD69+CD8 T cells express high levels of IL-10. Additionally, this IL-10 expression is transient in both CD4 and CD8 T cells, presumably only by the recently stimulated cells. Through microarray analysis, protein expression and cytolytic assay, I show that IL-10+CD8 T cells are more activated than IL-10-CD8 T cells. Nonetheless, the IL-10 produced is anti-inflammatory and its production in CD8 T cells is protective in J2.2-V-1-infected mice. Thus, the most activated and cytotoxic CD8 T cells self-regulate the immune response through the production of IL-10. Overall, these studies show that the anti-inflammatory component of the immune system is vital to protecting the host from the immunopathology that occurs during J2.2-V-1 virus clearance. Specifically, the addition of Tregs and IL-10 helps ameliorate clinical disease and demyelination. These studies suggest that increasing Tregs and/or increasing the cytokine IL-10 in patients with MS may have therapeutic potential.