Adipose tissue plays a crucial role in nutrient and energy homeostasis. At the time of worldwide pandemy of obesity and consequent metabolic syndrome, a great effort is made to find new treatments with potential to preserve insulin sensitivity, or even counteract development of obesity and type 2 diabetes. There are three principal possibilities how the adipose tissue biology can contribute to this goal: 1) induction of UCP1-dependent energy dissipation in brown adipose tissue; 2) conversion of white adipose depots to brown-like tissue (i.e. "browning"); and 3) stimulation of UCP1-independent thermogenesis in white adipose tissue. This thesis is based on two published works and one article under preparation. Generaly, it is focused on three different approaches targeting the above mentioned processes in adipose tissue of laboratory mouse: 1) diet supplementation with bile acids; 2) combination treatment of ω-3 polyunsaturated fatty acids and calorie restriction; and 3) cold exposure. In the experiments with administration of bile (specifically chenodeoxycholic) acid to mice, we confirm specific induction of UCP1 in both brown and subcutaneous white adipose tissue, as well as reversion of obesity in the response to the treatment. Nevertheless, most of the acute beneficial effects are mediated by...

O grande grupo heterogêneo de neuropatias periféricas hereditárias estão entre os casos mais comuns de perda sensitiva e fraqueza muscular em crianças e adolescentes. Pelo menos 84 genes estão envolvidos com neuropatias sensitivo-motoras hereditárias (NSMH), sendo suas formas de herança mais comuns as autossômico-dominantes desmielinizante e axonal e as neuropatias ligadas ao cromossomo X, e as mais raras as autossômicorecessivas desmielinizante e axonal e as formas ainda não classificadas. O gene HINT1, possuinte de 3 exons e localizado no cromossomo 5, codifica a proteína Histidine triad nucleotide binding protein 1, uma variante transcricional (mRNA) regulatória que hidroliza substratos. Recentemente mutações em HINT1 foram também relacionadas à neuropatias axonais com neuromiotonia (ARCMT2-NM), e portanto à CMT. O objetivo deste trabalho foi realizar o screening mutacional do gene HINT1 em uma amostra da população brasileira com quadro clínico de CMT recessivo (CMT2-AR), e foram encontradas 1 mutação silenciosa já previamente descrita, 1 polimorfismo exônico e 1 polimorfismo intrônico, também já conhecidos. Concluiu-se que mutações no gene HINT1 não são portanto responsáveis pela CMT-AR nesta amostra da população brasileira. / The large heterogeneous group of inherited peripheral neuropathies are among the most common causes of sensory loss and muscle weakness in children and adolescents. At least 84 genes are involved in inherited sensorymotor neuropathies (NSMH), being the demyelinating and axonal autosomaldominant and the X-linked neuropathies their most common forms of inheritance, and the demyelinating and axonal autosomal-recessive and not yet classified forms the most rare ones. The HINT1 gene, with 3 exons and located on chromosome 5, encodes the protein Histidine triad nucleotide binding protein 1, a regulatory transcriptional variant (mRNA) that hydrolyzes substrates. Recently, mutations in HINT1 were also related to axonal neuropathy with neuromyotonia (ARCMT2-NM), and therefore to CMT. The objective of this study was the mutational screening of the HINT1 gene in a sample of the Brazilian population with clinical recessive CMT (CMT2-AR), and 1 silent mutation previously described, 1 intronic polymorphism and 1 exonic polymorphism, both also known, were founded. It was then concluded that mutations in the HINT1 gene are not responsible for CMT2-AR in this particular sample of the Brazilian population.

O grande grupo heterogêneo de neuropatias periféricas hereditárias estão entre os casos mais comuns de perda sensitiva e fraqueza muscular em crianças e adolescentes. Pelo menos 84 genes estão envolvidos com neuropatias sensitivo-motoras hereditárias (NSMH), sendo suas formas de herança mais comuns as autossômico-dominantes desmielinizante e axonal e as neuropatias ligadas ao cromossomo X, e as mais raras as autossômicorecessivas desmielinizante e axonal e as formas ainda não classificadas. O gene HINT1, possuinte de 3 exons e localizado no cromossomo 5, codifica a proteína Histidine triad nucleotide binding protein 1, uma variante transcricional (mRNA) regulatória que hidroliza substratos. Recentemente mutações em HINT1 foram também relacionadas à neuropatias axonais com neuromiotonia (ARCMT2-NM), e portanto à CMT. O objetivo deste trabalho foi realizar o screening mutacional do gene HINT1 em uma amostra da população brasileira com quadro clínico de CMT recessivo (CMT2-AR), e foram encontradas 1 mutação silenciosa já previamente descrita, 1 polimorfismo exônico e 1 polimorfismo intrônico, também já conhecidos. Concluiu-se que mutações no gene HINT1 não são portanto responsáveis pela CMT-AR nesta amostra da população brasileira. / The large heterogeneous group of inherited peripheral neuropathies are among the most common causes of sensory loss and muscle weakness in children and adolescents. At least 84 genes are involved in inherited sensorymotor neuropathies (NSMH), being the demyelinating and axonal autosomaldominant and the X-linked neuropathies their most common forms of inheritance, and the demyelinating and axonal autosomal-recessive and not yet classified forms the most rare ones. The HINT1 gene, with 3 exons and located on chromosome 5, encodes the protein Histidine triad nucleotide binding protein 1, a regulatory transcriptional variant (mRNA) that hydrolyzes substrates. Recently, mutations in HINT1 were also related to axonal neuropathy with neuromyotonia (ARCMT2-NM), and therefore to CMT. The objective of this study was the mutational screening of the HINT1 gene in a sample of the Brazilian population with clinical recessive CMT (CMT2-AR), and 1 silent mutation previously described, 1 intronic polymorphism and 1 exonic polymorphism, both also known, were founded. It was then concluded that mutations in the HINT1 gene are not responsible for CMT2-AR in this particular sample of the Brazilian population.

Chez les eucaryotes pluricellulaires, la différenciation des cellules repose en partie sur l’activation oula répression des gènes. Les profils d’expression génique mis en place vont perdurer d’une générationcellulaire à l’autre. Ce phénomène met en jeu des mécanismes épigénétiques qui remodèlentlocalement la structure de la chromatine. Chez Drosophila melanogaster, les protéines des groupesPolycomb (PcG) et Trithorax (TrxG) participent au maintien du profil d’expression des gènes au coursdu développement. Les protéines PcG maintiennent les gènes réprimés tandis que les protéines TrxGmaintiennent les gènes activés. Une troisième classe de protéines nommée Enhancers of Trithoraxand Polycomb (ETP) module l’activité des PcG et TrxG. Dorsal Switch Protein 1 (DSP1) est uneprotéine HMGB (High Mobility Group B) classée comme une ETP. Par tamisage moléculaire, nousavions montré que la protéine DSP1 était présente au sein de complexes de poids moléculaire de 100kDa à 1 MDa. Le travail de thèse présenté ici a pour but d’identifier les partenaires de la protéineDSP1 dans l’embryon et de mieux connaître les propriétés biochimiques de DSP1. Premièrement, j’aimis en place puis effectué l’immunopurification des complexes contenant DSP1 dans des extraitsprotéiques embryonnaires. Cette approche nous a permis d’identifier 23 partenaires putatifs de laprotéine DSP1. Parmi ces protéines, nous avons identifié la protéine Rm62 qui est une ARN hélicaseà boîte DEAD. Les relations biologiques entre DSP1 et Rm62 ont été précisées. Deuxièmement, j’aidéterminé, par une approche biochimique, de nouvelles caractéristiques physico-chimiques de laprotéine DSP1. / In multicellular organism, the identity of cell is determined by several factors playing on genesexpression. Once established, the gene expression pattern is transmitted to daughter cells through aprocess involving epigenetic mechanisms that locally reshape the structure of chromatin. In Drosophilamelanogaster, the Polycomb (PcG) and trithorax (trxG) group genes are involved in the maintenanceof gene expression profile during development. Inside multimeric complexes, PcG proteins maintaingenes in repressed state whereas TrxG maintain genes active. A third class of proteins, calledEnhancers of Trithorax and Polycomb, regulate PcG and TrxG activities. Dorsal Switch Protein 1(DSP1) is a High Mobility Group B protein acting as an ETP. But DSP1 has not yet been identified inPcG or TrxG complexes. On the basis of gel filtration analysis of protein complexes in embryo nuclearextracts, it appears that the majority of DSP1 is present in complex(es) from 100 kDa to 1MDa. Aimsof present work are the identification of DSP1 protein partners in drosophila embryo and thecharacterization of biochemical properties of DSP1. Firstly, I used immunopurification from drosophilaembryonic nuclear extracts. The proteins purified with DSP1 were characterized through sequencingof peptides from individual protein bands by mass spectrometry. Among identified proteins, wefocused on the DEAD Box RNA helicase, Rm62. The role of interaction between DSP1 and Rm62 hasbeen characterized. Secondly, I have identified a new physicochemical aspect of DSP1 protein.

Thromboxane A2 receptor (TP) has been shown to play important roles in multiple aspects of cancer development including regulation of tumor growth, survival and metastasis. Molecular mechanisms of TP mediated cancer cell invasion remain to be identified. TP agonist, I-BOP, significantly elevated several matrix metalloproteinases (MMPs) including MMP-1, MMP-3, MMP-9 and MMP-10 in A549 human lung adenocarcinoma cells overexpressing TPα (A549-TPα) or TPβ (A549-TPβ). Signaling pathways of I-BOP-induced MMP-1 expression were examined in further detail as a model system for MMPs induction. Signaling molecules involved in I-BOP-induced MMP-1 expression were identified by using specific inhibitors including small interfering (si)-RNAs of signaling molecules and promoter reporter assay. The results indicate that I-BOP-induced MMP-1 expression is mediated by protein kinase C (PKC), extracellular signal-regulated kinase (ERK)-activator protein-1(AP-1) and ERK-CCAAT/enhancer-binding protein β (C/EBPβ) pathways. I-BOP-induced cellular invasiveness of A549-TPα cells was blocked by, GM6001, a general inhibitor of MMPs. Knockdown of MMP-1 and MMP-9 by their respective siRNA partially reduced I-BOP-stimulated A549-TPα cells invasion suggesting that other MMPs induced by I-BOP were also involved. Furthermore, secreted MMP-1 in conditioned media from I-BOP-treated A549-TPα cells (CM-I-BOP) autocrinely induced monocyte chemoattractant protein-1 (MCP-1) expression. The induction of MCP-1 by MMP-1 in A549 cells was via activation of protease-activated receptor 2 (PAR2) instead of commonly assumed PAR1. This conclusion was reached from the following findings: (1) expression of MCP-1 induced by trypsin, a PAR2 agonist, was inhibited by a PAR2 antagonist. (2) expression of MCP-1 induced by MMP-1 and by CM-I-BOP was blocked by a PAR2 antagonist but not by other PAR antagonists; (3) expression of MCP-1 induced by MMP-1 and by CM-I-BOP was attenuated significantly by pretreatment of cells with PAR2-siRNA. Finally, MCP-1 also can be induced by direct activation of TP in a SP1 involved mechanism. CM-I-BOP enhanced MCP-1-dependent migration of RAW 264.7 macrophages. Co-culture of A549 cells with RAW 264.7 macrophages induced expression of MMPs, VEGF and MCP-1 genes, and increased the invasive potential in A549 cells. My studies provide molecular mechanisms by which TP-mediated cancer cell invasion and suggest that TP is a potential anti-cancer drug target.

Malaria is a major health problem and an effective vaccine is essential for the eradication of the disease. Despite extensive efforts, a malaria vaccine remains elusive due to the parasite's complex life cycle, diverse morphology, and immune system evasion mechanisms. Antibodies against C terminal domain of merozoite surface protein 1 (MSP1-19), a highly conserved protein and the main vaccine candidate for blood-stage malaria, can inhibit erythrocyte invasion by the parasite and alleviate the disease symptoms. However, MSP1-19 is poorly immunogenic and classic protein-in-adjuvant MSP1-19-based vaccine formulations failed to induce strong immune responses due to low immunogenicity and generation of ineffective antibodies. The aim of this study was to use hepatitis B virus core (HBc) particles to increase the immunogenicity of MSP1-19. HBc forms particles with protruding spikes and induces a strong and specific immune response against foreign epitopes inserted at the tips of the spikes. In addition, positioning of MSP1-19 on the particle can influence the accessibility of certain antibody binding sites, possibly altering elicited antibody fine specificity and vaccine efficiency. MSP1-19 domain was inserted into the middle of the HBc sequence so that it is displayed at the tips of the HBc particle. Two HBc-MSP1-19 constructs, having different insert flanking linkers, displayed soluble particle formation after bacterial expression and lysis optimization. The particles were purified and the suitability of these two constructs as malaria vaccine candidates was assessed. Firstly, binding of the conformational anti-MSP1-19 antibodies indicated that MSP1-19 domain in the chimeric proteins has the correct disulphide bond pattern which is crucial for the protective properties of an MSP1-19-based vaccine. Furthermore, electron microscopy imaging and determination of initial 3D structures confirmed that both HBc MSP1-19 constructs form particles resembling the wild-type HBc particles, meaning the insertion of MSP1-19 did not heavily distort the overall HBc particle structure. In addition, it was shown that MSP1-19 domains are displayed at the tips of the particle spikes. Particle formation and foreign epitope display are important for the epitope's immunogenicity improvement. The immunogenicity of the chimeric particles was then assessed in mice. Both constructs elicited similar high antibody titres without the use of additional adjuvants, but no difference was observed between the particulate constructs and a non-particulate control (an MSP1-19-based protein). Interestingly, although both HBc-MSP1-19 and non-particulate MSP1-19-elicited antibodies recognized native malarial parasite, only the particulate construct antibodies demonstrated a moderate parasite growth inhibition while the antibodies from the control group did not show parasite inhibition above the background levels. In conclusion, it was shown that MSP1-19 can be expressed in bacteria as a soluble correctly folded protein fused to HBc. More importantly, the fusion protein is capable of forming immunogenic particles which generate antibodies that recognize native MSP1 and inhibit parasite growth more effectively than the protein without the HBc. Therefore, this work lays grounds and supports further chimeric HBc-MSP1-19 research and development.

Miu Fei Fei. / On t.p. "42" are subscripts following the word "MSP-1" in the title. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 117-125). / Abstracts in English and Chinese. / ACKNOWLEGEMENTS --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.v / LIST OF FIGURE --- p.vii / LIST OF ABBREVIATIONS --- p.ix / CHAPTERS: / Chapter 1. --- BACKGROUND OF MALARIA / Chapter 1.1 --- Epidemilogy --- p.2 / Chapter 1.2 --- Mode of Infection --- p.4 / Chapter 1.3 --- Conventional Control & Vaccination --- p.9 / Chapter 1.4 --- Vaccine Candidate PfMSV-142 --- p.16 / Chapter 1.5 --- Cloning and Expression of pfMSP-142 --- p.26 / Chapter 1.6 --- Aims of Study --- p.32 / Chapter 2. --- Materials and Methods / Chapter 2.1 --- Materials --- p.32 / Chapter 2.2 --- Methods --- p.38 / Chapter 3. --- Construction of recombinants N-PfMSP-142 and C- PfMSP-142 / Chapter 3.1 --- Construction of C-PfMSP-l42 --- p.51 / Chapter 3.2 --- Construction ofN-PfMSP-l42 --- p.56 / Chapter 4. --- Purification with IMAC / Chapter 4.1 --- Immobilized Metal Affinity Chromatography (IMAC) --- p.58 / Chapter 4.2 --- Purification ofN-PfMSP-l42 --- p.61 / Chapter 4.3 --- Purification profile of N-PfMSP-142 --- p.68 / Chapter 4.4 --- Purification of C-PfMSP-l42 --- p.70 / Chapter 4.5 --- Purification profile of C-PfMSP-142 --- p.73 / Chapter 4.6 --- Expression pattern of recombinants PfMSP-142 --- p.76 / Chapter 5. --- Purification combined with other chromatography method / Chapter 5.1 --- Affinity chromatography --- p.78 / Chapter 5.2 --- Gel filtration chromatography --- p.80 / Chapter 5.3 --- Ion exchange chromatography --- p.83 / Chapter 5.4 --- Conclusion --- p.93 / Chapter 6. --- Characteristic of IMAC products --- p.94 / Chapter 7. --- Characteristic of N-hisPfMSP-l42 & C-hisPfMSP-l --- p.42 / Chapter 7.1 --- Immunogenitcity of N-PfMSP-l42 and C-PfMSP-142 --- p.100 / Chapter 7.2 --- Competitive ELISA --- p.105 / Chapter 8. --- Discussion --- p.107 / REFERENCE --- p.117

Leung Wai-hang. / "November 2002." / On t.p. "33" and "42" are subscripts following the word "MSP-1" in the title. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 162-171). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

In the second part of the project, the immunology of PfMSP-133 was studied. During the invasion of merozoites, PfMSP--142 is processed into two fragments with molecular weight of 33kDa and 19kDa. The 19kDa fragment (PfMSP-119) originating from the carboxyl--terminal of PfMSP--142 is relatively more immuno-dominant in different malarial species such as P. falciparum, P. vivax and P. yoelii. In the past, only limited researches about PfMSP-1 33 were performed. Apart from its difficulty in expression, PfMSP-1 33 was also believed to be incapable of inducing protection. / Nevertheless, following the breakthrough of expressing recombinant PfMSP-1 33 in our laboratory, we have demonstrated in this study that recombinant MSP-133 can elicit antibodies with a titer up to a million. Also, we observed that MSP-133 can help MSP-119 to induce protective immunity and such effect is independent from the covalent linkage between these two proteins. Most importantly, our results show that recombinant PfMSP-133 can elicit the production of antibodies that can potentiate the inhibitory effect of anti-MSP-142 serum at high serum dilution. Results of this study give new insights in malarial vaccine development in terms of optimizing the use of adjuvant and immunization regimens. / The 42kDa carboxyl terminal fragment of Plasmodium falciparum Merozoite Surface Protein-1 (PfMSP--142) is one of the most promising candidate antigens in the development of malarial vaccine. In vivo experiments in the 1990's showed that Aotus monkeys immunized with PfMSP--142 were protected from malarial challenge. Later on, other experiments also demonstrated the possibility of using recombinant PfMSP-142 as candidate antigen for malarial vaccine. Previously, recombinant PfMSP-142 (Bvp42) was expressed with the baculovirus expression system and characterized in our laboratory. / The aim of the first part of this project is to improve the production of Bvp42. Experimental results have shown that the expression level of Bvp42 was increased under a BMN compatible baculovirus expression vector---pVL1393. Besides, a codon optimized MSP-142 nucleotide is constructed for the construction of a baculovirus carrying codon optimized MSP-142 gene and aimed for higher expression level. Unfortunately, no Bvp42 expression is observed in the transfection samples and the reason of this observation is unclear. Meanwhile, the purification of Bvp42 was also improved. Pretreatment of the hemolymph with Q--sepharose before affinity chromatography could enhance the purity of the final product. / Yuen, Sai-hang Don. / "July 2007." / Adviser: Walter K. K. Ho. / Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0220. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 183-195). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

The ultimate goal of mitosis is the equal distribution of chromosomes between the two daughter cells. One of the key players that ensures faithful chromosome segregation is the chromosomal passenger complex (CPC). CPC localisation to mitotic centromeres is complex, involving interactions with Shugoshin and binding to phosphorylated histone H3T3. It was recently reported that Heterochromatin Protein 1 (HP1) has a positive impact on CPC function during mitosis. The interaction between HP1 and the CPC appears to be perturbed in cancer-­‐derived cell lines, resulting in decreased HP1 levels at mitotic centromeres and may be a potential cause for increased chromosome mis-­‐segregation rates. In this study, I tethered HP1α to centromeres via the DNA-­‐binding domain CENP-­‐B. However, instead of improving the rate of chromosome mis-­‐segregation, HP1α tethering resulted in activity of the spindle assembly checkpoint and destabilisation of kinetochore-­‐microtubule attachments, most likely caused by the robust recruitment of the CPC. Tethered HP1α even traps the CPC at centromeres during mitotic exit, resulting in a catalytically active CPC throughout interphase. However, it was not clear whether endogenous HP1 contributes to CPC localisation and function prior to mitosis. Here I also describe a substantial interaction between endogenous HP1 and the CPC during the G2 stage of the cell cycle. The two isoforms HP1α and HP1γ contribute to the clustering of the CPC into active foci in G2 cells, a process that is independent of CDK1 kinase activity. Furthermore, the H3S10ph focus formation in the G2 phase appears to be independent of H3T3ph and H2AT120ph, the two histone marks that determine the CPC localisation in early mitosis. Together, my results indicate that HP1 contributes to CPC concentration and activation at pericentromeric heterochromatin in G2. This novel mode of CPC localisation occurs before the Aurora B-­‐driven methyl/phos switch releases HP1 from chromatin, which possibly enables the H3T3ph and H2AT120ph driven localisation of the CPC during mitosis.