Interleukin (IL)-10-secreting regulatory Bcells(B10) are acknowledged to play important roles in balancing cellular immunity and fighting against autoimmune diseases. Since the early discovery of the potential of B cells in suppressing autoimmunity by secreting IL-10 in a murine model of experimental autoimmune encephalomyelitis(EAE),accumulating evidences have revealed the existence and regulatory function of B10 cells in the progression of several autoimmune diseases including multiple sclerosis (MS), lupus and autoimmune arthritis, suggesting potential values of therapeutic intervention. Autoimmune diabetes is an autoimmune disease in animal models characterized by progressive insulitis and mass destruction of βcells in pancreatic islets. However, the role of Bregsin the development of this disease remains largely unclear. To explore whether Bregs possess a regulatory function in suppressing diabetes, B10 cells were generated from B-cell activation factor (BAFF)-stimulated B cells of Non-obese diabetic (NOD)mice. Notably, NOD mice receiving B10 transfer exhibited delayed diabetes onset and substantially reduced incidence, suggesting some therapeutic effect against autoimmune diabetes. As an important contributor to inflammation and autoimmune disorders, the pathogenic function of IL-17 producing CD4+cells (Th17) in autoimmune diabetes has been increasingly identified, which attracts me to investigate whether B10 cells can contribute to amelioration of autoimmune diabetes via suppressing Th17 cells. During the development of autoimmune diabetes in NOD mice, both B10 and Th17 significantly increased at prediabetic stage and rapidly declined after disease onset. Upon adoptive transfer of B10 cells into prediabetic NOD mice, Th17 cells in pancreatic lymph nodes and pancreas were profoundly reduced. To verify whether B10 cells can directly inhibit Th17 generation in vitro, CFSE-dilution assay combined to Th17 polarization assay was performed. Results indicated that B10 cells suppress Th17 polarization in an IL-10 independent manner, but inhibit Th17 proliferation in an partially IL-10 dependent way. Finally I transferred B10 together with naive CD4+T cells reactive to islets into lymphopenic NOD-SCID mice and detected substantially reduced Th17 frequencies in pancreatic lymph nodes and pancreas, suggesting a potential way of developing new therapeutic strategies in treating Type 1 diabetes in humans. / published_or_final_version / Pathology / Master / Master of Philosophy

Hepatocellular carcinoma (HCC) accounts for 70-85% of liver cancer, which is the sixth most common cancer in the world. Prognosis of HCC is dismal with little chance of complete recovery after diagnosis. It is of essence to discover the key molecules involved in the tumor progression. This could help earlier detection of HCC and establish targeted molecular therapies. Cyclin E1 (CCNE1) is a cyclin molecule responsible for the transition from G1 to S phase of the cell cycle and is often dysregulated in human cancers. CCNE1 is reported with overexpression in about 30-70% of HCC cases. It expresses in tumor cells as a ladder of proteins and as low molecular weight CCNE1. The study is aimed to investigate the role of CCNE1 in HCC. From the local cohort of HCC patients, 6 out of 13 patients (46.2%) of HCC tumor tissues were found with CCNE1 overexpression compared with the non-tumor tissues by western blotting. The presence of three CCNE1 isoforms in HCC was detected. The expression of total CCNE1 and each isoform varied independently among the studied HCC cell lines, with HepG2 having the highest expression and 97L the lowest. To extend our study on the regulation of CCNE1 expression, the expression of selected four genes associating with the CCNE1 expression and functions was studied by quantitative PCR (qPCR). F-box and WD repeat domain containing 7, E3 ubiquitin protein ligase (FBXW7) and cullin 3 (CUL3), the two genes responsible for CCNE1 degradation, had increased expression in the HCC cell lines with higher CCNE1 expression. Cyclin A (CCNA2), the downstream cyclin molecule of CCNE1, also had higher expression in these cell lines. In contrast, the expression of cyclin dependent kinase 2 (CDK2), the catalytic partner of CCNE1, had the least difference among the six HCC cell lines compared to other three genes. To characterize the role of CCNE1 isoforms in HCC, CCNE1 isoform 1, 2, and 3 were overexpressed in PLC cells and such overexpression remained even after 8 passages in culture. In flow cytometric analysis, GFP signal in cell culture population was viewed to observe the transduction efficiency. The vector control showed the strongest GFP signal, followed by CCNE1 isoform 3 showing dim signal. CCNE1 isoform 1 and 2 almost showed no signal. In the functional studies, the overexpression of CCNE1 isoform 3 could increase proliferation and migration of HCC cells. In summary, CCNE1 could promote proliferation and migration of HCC cells through elevated expression of CCNE1 isoform 3. / published_or_final_version / Surgery / Master / Master of Philosophy

published_or_final_version / abstract / toc / Medicine / Master / Doctor of Medicine

published_or_final_version / Ecology and Biodiversity / Doctoral / Doctor of Philosophy

Thylakoid organization and the distribution of photosystem (PS) I and II proteins in the green alga Coleochaete scutata were analyzed by electron microscopy. Thylakoids were observed to associate in varied conformations. Extended bands of thylakoids were present, as in other algae, but numerous grana, characteristic of higher plants, were also detected. Immunolabelling experiments were conducted with two heterologous antisera raised against PS proteins in the cyanobacterium Synechococcus elongatus: one antiserurn against the 60 and 62 kDa PSI reaction centre proteins, the other against the 47 kDa PSII core antenna protein. PSI was 2.6 times more concentrated in the nonappressed membranes (NAM) than in the appressed (AM), with 62% of labelling on NAM. The concentration of PSII in AM was 1.6 times that of the NAM, accounting for 75% of PSII. Thus, in C scutata, PSI and PSII are located in both appressed and nonappressed thylakoid membranes, but with a trend towards the lateral heterogeneity of PS proteins observed in higher plants. These results support the body of ultrastructural and molecular data linking charophytes with the ancestry of higher plants.

Leishmania is a dimorphic parasitic protozoan which exists as a flagellated promastigote in the sandfly vector and as an intracellular amastigote in the phagolysosomal compartment of mammalian host macrophages. It is the amastigote form that is responsible for the pathology in susceptible vertebrate hosts. Leishmania donovani is responsible for visceral leishmaniasis, the most severe form of the leishmanial diseases. We have investigated the antibody response against an amastigote-specific protein, A2, which is developmentally expressed in L. donovani during promastigote-to-amastigote cytodifferentiation. A2 is conserved in L. donovani and L. mexicana species but not in other Leishmania species tested. We have shown that this characteristic contributes to its potential as a useful specific diagnostic antigen for visceral leishmaniasis. Developmental expression of A2 involves A2 mRNA untranslated regions (UTRs) and we have demonstrated that A2 UTRs can regulate expression of exogenous suicide genes throughout the Leishmania life cycle. We have shown that the A2 gene regulatory system has potential for the generation of developmentally attenuated L. donovani strains. Finally, we have performed a preliminary characterization of a gene, A2rel, that is tandemly associated with A2 genes in the genome. Contrary to A2 genes, the A2rel gene is well conserved in the Leishmania species. Although A2rel does not share sequence similarity with any known leishmanial genes characterized to date, it does appear to share characteristics with membrane-bound glycoproteins.

Bloom's syndrome is a rare autosomal recessive disorder characterised by an early onset of cancer of many types, erythematous lesions on sun-exposed skin, retarded growth, immunodeficiency and sub- or infertility. Cells from Bloom's syndrome patients have replication defects and an abnormally unstable genome manifested in chromosomal breaks and deletions and in an increased mutation rate. Most characteristically, these cells show elevated levels of sister-chromatid exchanges which probably result from homologous recombination events. Since the cells are not hypersensitive to DNA damaging agents, the defect is unlikely to be in one of the common DNA repair pathways. The gene mutated in Bloom's syndrome, BLM, was cloned in 1995 and found to encode a helicase from the RecQ family. This family is named after its E. coli member, RecQ, and includes at least five human genes. Three of these are mutated in inherited disorders; Bloom's syndrome, Werner's syndrome and Rothmund-Thomson syndrome. In my DPhil project, I have investigated the enzymatic properties of the BLM protein. I have purified the protein in recombinant form and shown that it is a DNA-dependent ATPase and an ATP-dependent helicase with 3'-5' polarity. It binds and unwinds a variety of DNA structures, with a preference for tetraplex (G4)-DNA, Holliday junctions (recombination intermediates) and internal DNA bubbles. Furthermore, it is capable of branch migration, an activity distinct from its helicase activity. BLM forms oligomeric rings with fourfold and sixfold symmetry, both in a cell extract and as purified protein. These results, in combination with the cellular phenotype of Bloom's syndrome and with evidence from the analysis of other RecQ homologues in model organisms such as yeast and E. coli, point to a role for BLM in somatic recombination (recombinational repair). Models for this function are discussed in this thesis.

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"May 2002" Includes bibliographical references (leaves 229-251) Describes the investigation of the alteration of ABH antigen expression on the surface of red blood cells in patients with haematological malignancies.