Lau, Shuk Wa. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 84-102). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Abstract (in English) --- p.ii / Abstract (in Chinese) --- p.iv / Acknowledgement --- p.v / Table of content --- p.vi / List of figures --- p.ix / Symbols and abbreviations --- p.x / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Structure of ovarian follicles --- p.1 / Chapter 1.2 --- Folliculogenesis and its control --- p.2 / Chapter 1.2.1 --- Ovarian follicle growth and development --- p.2 / Chapter 1.2.2 --- Follicle recruitment and regulation --- p.4 / Chapter 1.2.3 --- Oocyte maturation and ovulation --- p.9 / Chapter 1.2.4 --- Intercellular communication between oocytes and somatic cells --- p.10 / Chapter 1.3 --- Overview of proteomics --- p.12 / Chapter 1.3.1 --- Two-dimensional gel electrophoresis --- p.13 / Chapter 1.3.2 --- Mass spectrometry --- p.14 / Chapter 1.4 --- Objectives of the study --- p.15 / Chapter Chapter 2 --- Proteomic Analysis of Folliculogenesis in Zebrafish Ovary --- p.19 / Chapter 2.1 --- Introduction --- p.19 / Chapter 2.2 --- Materials and Methods --- p.21 / Chapter 2.2.1 --- Animals --- p.21 / Chapter 2.2.2 --- Isolation of ovarian follicles --- p.21 / Chapter 2.2.3 --- Protein extraction and quantification --- p.22 / Chapter 2.2.4 --- Two-dimensional electrophoresis --- p.23 / Chapter 2.2.5 --- Staining --- p.24 / Chapter 2.2.6 --- In-gel digestion --- p.24 / Chapter 2.2.7 --- Mass spectrometry --- p.25 / Chapter 2.3 --- Results --- p.25 / Chapter 2.3.1 --- Establishment of the protein profiles of different follicle stages --- p.25 / Chapter 2.3.2 --- Mass spectrometry analysis on the differentially expressed proteins --- p.26 / Chapter 2.4 --- Discussion --- p.27 / Chapter Chapter 3 --- Characterization of Y-box Binding Protein 1 (YB-1) in Zebrafish --- p.46 / Chapter 3.1 --- Introduction --- p.46 / Chapter 3.2 --- Materials and Methods --- p.49 / Chapter 3.2.1 --- Animals --- p.49 / Chapter 3.2.2 --- Isolation of ovarian follicles --- p.49 / Chapter 3.2.3 --- Protein extraction and quantification --- p.49 / Chapter 3.2.4 --- SDS polyacrylaminde gel electrophoresis (SDS-PAGE) --- p.50 / Chapter 3.2.5 --- Western blot analysis --- p.50 / Chapter 3.2.6 --- RNA isolation and reverse transcription --- p.51 / Chapter 3.2.7 --- Semi-quantitative RT-PCR quantification of expression --- p.51 / Chapter 3.2.8 --- Data analysis --- p.52 / Chapter 3.2.9 --- Immunohistochemistry --- p.52 / Chapter 3.2.10 --- Cloning of full-length ybl cDNA from zebrafish ovary and construction of recombinant plasmid for expressing ybl --- p.53 / Chapter 3.2.11 --- Expression and purification of recombinant zebrafish YB-1 protein --- p.54 / Chapter 3.2.12 --- Immunoprecipitation --- p.55 / Chapter 3.3 --- Results --- p.58 / Chapter 3.3.1 --- Confirmation of the presence of YB-1 --- p.58 / Chapter 3.3.2 --- Tissue distribution of YB-1 protein and ybl gene expression in zebrafish --- p.58 / Chapter 3.3.3 --- Stage distribution of YB-1 protein and ybl gene expression in ovarian follicles --- p.59 / Chapter 3.3.4 --- Localization of YB-1 protein within the ovarian follicle --- p.59 / Chapter 3.3.5 --- Degradation of YB-1 in the ovary --- p.60 / Chapter 3.3.6 --- Production of recombinant YB-1 (zfYB-1) --- p.60 / Chapter 3.3.7 --- Identification of YB-1 -bound partners --- p.60 / Chapter 3.4 --- Discussion --- p.61 / Chapter Chapter 4 --- General Discussion --- p.77 / References --- p.84

Borrelia burgdorferi, the causative agent of Lyme disease, exists in a defined enzootic cycle involving Ixodes scapularis ticks and various vertebrates. Humans can serve as an accidental host, if a tick colonized with B. burgdorferi happens to feed on a human. B. burgdorferi are also accidental pathogens: they do not make toxins, or destroy host tissue by other mechanisms. They merely transmit between vector and host to survive. In order to do this, they must effectively sense their current environment, and appropriately alter cellular processes. Understanding the regulatory mechanisms of how B. burgdorferi manages to do this has been a focus of the Stevenson lab for many years. Previous work identified SpoVG as a DNA-binding protein. Although a homologue of this protein had been implicated to serve a regulatory role in other bacteria, the Stevenson lab was the first to demonstrate a function for the protein, both for B. burgdorferi and two other bacteria. Studies contained in this body of work aim to provide insight into regulation of SpoVG by B. burgdorferi as well the impact that it has on gene regulation. By using genetic mutants, we determined that SpoVG is regulated at the levels of transcription and translation in culture by growth rate, temperature, and other regulatory factors. Additionally, we provide evidence that SpoVG regulates its own expression. Numerous genes are under control of SpoVG. Biochemical analyses revealed that SpoVG specifically interacts with DNAs and RNAs associated with genes found to be under its regulatory control. Finally, we provide evidence for SpoVG acting in concert with other known regulatory factors such as other DNA-binding proteins and the cyclic di-nucleotide second messengers cyclic-di-GMP and cyclic-di-AMP. All together, these studies provide insight into how B. burgdorferi broadly regulates cellular processes during different stages of the enzootic cycle. We hypothesize that SpoVG does this through globally manipulating the three-dimensional structure of the bacterial chromosome, and that exactly how SpoVG acts at any given point will be dependent on the other regulatory factors that are also present in the cell.

TNF defers apoptosis in macrophages undergoing spontaneous or pharmacologically (thapsigargin, ceramide, CCCP, etoposide or cisplatin)-induced apoptosis, as determined by measurements of caspase-3 activity and annexin-V staining (Chapter 2). The action requires TNF interaction with TNF-R1, not TNF-R2. Survival is uniquely reliant on the activity of the NF-B signaling pathway, and does not require activities arising from the PI3K/Akt, JNK, ERK, p38 MAP kinase or iNOS pathways (Chapter 3). Further, the general anti-apoptotic property of TNF and its specific antagonism of CCCP-induced apoptosis led to the finding that TNF action prevents cytochrome c release. This protection is likely mediated through effects on components of the MPTP itself, as TNF exhibited functional redundancy with the pore inhibitor cyclosporin A, and did not modify upstream events that promote MPTP opening during apoptosis, namely ROS production, cytosolic Ca2+ increase, or a reduction of total ATP (Chapter 4). Subsequent experiments with the mRNA synthesis inhibitor, actinomycin D, and the translation inhibitor, cycloheximide revealed that the protein(s) responsible for TNF-induced survival was transcribed and translated within 1 hr. However, western analyses provided no convincing evidence of the involvement of Mn-SOD, cIAP-1, XIAP, Bcl-2 or A1 in TNF cytoprotection (Chapter 5). Rather, microarray experiments identified the consistent induction of an early response gene, pim-1, within 30 min of TNF exposure (Chapter 6). This result was verified at the protein level with a specific Pim-1 antibody. Evidence was also found for induction of the anti-apoptotic protein A20, but only at mRNA level. Parthenolide, wortmannin, SP600125, PD98059, SB203580 or L-NAME1 acted against TNF-induced Pim-1 expression in a pattern that exactly matched the effects of these inhibitors on TNF-induced survival. That is, only parthenolide-mediated inactivation of NF-B abolished TNF-induced induction of Pim-1. TNF also stimulated the rapid phosphorylation (inactivation) of the pro-apoptotic BH3-only protein, Bad at Ser112 in a manner sensitive to NF-B inhibition, but not PI3K/Akt, JNK, ERK or p38 MAP kinase inhibition (Chapter 7). As Bad is a known substrate of Pim-1 and Bad 1 Parthenolide, wortmannin, SP600125, PD98059 and SB203580 are inhibitors of the NF-B, PI3K/Akt, JNK, ERK and p38 MAP kinase pathways, respectively. L-NAME inhibits iNOS. NF-B- and mitochondria-linked signaling events that contribute to TNF action in deferring physiological and chemotherapeutic drug-induced apoptosis in macrophages ii phosphorylation occurred coincident with Pim-1 upregulation, it is likely that Pim-1 kinase activity mediates the inactivation of Bad. The overall data therefore supports a model in which TNF ligation of TNF-R1 at the cell surface results in intracellular NF- B activation, leading to the induction of Pim-1 mRNA and protein, and the ensuing phosphorylation of Bad. Inactivation of pro-apoptotic Bad increases the resistance threshold of mitochondria to apoptotic insults, thereby reducing the occurrence of mitochondrial permeability transition, cytochrome c release and subsequent caspase-3 activation.

Abstract not available

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 104-113).

Thesis (Ph. D.)--University of Chicago, Committee on Immunology, June 2003. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 147-163).

The knowledge of the three-dimensional structures of proteins and polypeptides is essential to understand their functions. The work shown in this thesis has two objectives. The first one is to develop a new analytical method based on maximum entropy (ME) theory to analyze NMR experimental data such as NOEs and J-couplings in order to reconstitute φ,ψ Ramachandran plots of flexible biomolecules. Two model systems have been used, the flexible polypeptide motilin and the disaccharide α-D-Mannosep-(1-2)-α-D-Mannosep-O-Me (M2M). The experimental data was defined as constraints that were combined with prior information (priors) which were the φ,ψ distributions obtained from either a coil library, the Protein DataBank or Molecular Dynamics Simulations. ME theory was utilized to formulate φ,ψ distributions (posteriors) that are least committed to the priors and in full agreement with the experimental data. Reparamerization of the Karplus relation was necessary to obtain realistic distributions for the M2M. Clear structural propensities were found in motilin with a nascent α-helix in the central part (residues Y7-E17), a left handed 31 helix in the C-terminus (R18-G21) and an extended conformation in the N-terminus. The contribution of each residue to the thermodynamic entropy (segmental entropy) was calculated from the posteriors and compared favorably to the segmental entropies estimated from 15N-relaxation data. For M2M the dominating conformation of the glycosidic linkage was found to be at φH=-40° ψH=33°, which is governed by the exo-anomeric effect. Another minor conformation with a negative ψH angle was discovered in M2M. The ratio between both populations is about 3:1. The second part of the thesis is a structural study of a DNA-binding protein, the C repressor of the P2 bacteriophage (P2 C). P2 C represses the lytic genes of the P2 bacteriophage, thereby directing the P2 lifecycle toward the lysogenic lifemode. The crystal and solution structures of P2 C have been solved by X-ray crystallography and NMR, respectively. Both structures revealed a homodimeric protein with five rigid α-helices made up by residues 5-66 and a β-strand conformation in residues 69-76 in each monomer. 15N-relaxation data showed that the C-terminus (residues 85-99) is highly flexible and fully unstructured. A model representing the P2 C-DNA complex was built based on the structure and available biochemical data. In the model, P2 C binds DNA cooperatively and two homodimeric P2 C molecules are close enough to interact and bind one direct DNA repeat each. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: In press. Paper 5: Manuscript.

In most eukaryotes, chromosomes ends are protected by telomeres which are formed by repetitive DNA, specialized binding proteins, and higher order structures. Telomeres become shorter following replication due to the positioning and degradation of terminal RNA primers, as well as resection by nucleases. Extensive telomere shortening over many cell cycles elicits a DNA damage checkpoint that culminates in senescence or, in the absence of tumor suppressor pathways, apoptosis. These effects block the expansion of cells with unstable genomes, but can also precipitate disease in tissues that rely on regeneration for function. In many unicellular eukaryotes and proliferative human cells including cancer cells, telomeres can be maintained by the telomerase reverse transcriptase (TERT) and its associated RNA (TR). The elongation of telomeric DNA by telomerase depends on the telomerase essential N-terminal (TEN) and C terminal reverse transcriptase (RT) domains. We found that human TEN interacted with single-stranded telomeric DNA and restored function, in trans, to an hTERT mutant lacking hTEN. Telomerase required hTEN residues for activity, telomere maintenance, and extension of cellular replicative lifespan. Two inactive hTERT variants bearing mutations in TEN and RT domains, respectively, cooperated to regenerate telomerase activity in vitro. hTEN interacted with several regions of hTERT suggesting that dimerization may occur via TEN-TERT interactions. The in vivo defect of certain hTEN mutants may involve an inability to interact with factors that recruit the enzyme to the telomere and/or stimulate activity. Human homologs of the S. cerevisiae recruitment factor Est1 interacted with telomerase in a species-specific manner. The TPR domain of hEST1A interacted with the N-terminus of hTERT. The TPR domain of ScEst1 was required for telomere length maintenance by telomerase, and, paradoxically, also negatively regulated telomere length. In preliminary experiments, hTERT interacted with hPOT1/hTPP1. This interaction may stimulate the elongation of telomeres by telomerase. The DNA and protein interactions described herein expand our knowledge of telomerase and present new targets for the manipulation of telomerase function in human disease.

In most eukaryotes, chromosomes ends are protected by telomeres which are formed by repetitive DNA, specialized binding proteins, and higher order structures. Telomeres become shorter following replication due to the positioning and degradation of terminal RNA primers, as well as resection by nucleases. Extensive telomere shortening over many cell cycles elicits a DNA damage checkpoint that culminates in senescence or, in the absence of tumor suppressor pathways, apoptosis. These effects block the expansion of cells with unstable genomes, but can also precipitate disease in tissues that rely on regeneration for function. In many unicellular eukaryotes and proliferative human cells including cancer cells, telomeres can be maintained by the telomerase reverse transcriptase (TERT) and its associated RNA (TR). The elongation of telomeric DNA by telomerase depends on the telomerase essential N-terminal (TEN) and C terminal reverse transcriptase (RT) domains. We found that human TEN interacted with single-stranded telomeric DNA and restored function, in trans, to an hTERT mutant lacking hTEN. Telomerase required hTEN residues for activity, telomere maintenance, and extension of cellular replicative lifespan. Two inactive hTERT variants bearing mutations in TEN and RT domains, respectively, cooperated to regenerate telomerase activity in vitro. hTEN interacted with several regions of hTERT suggesting that dimerization may occur via TEN-TERT interactions. The in vivo defect of certain hTEN mutants may involve an inability to interact with factors that recruit the enzyme to the telomere and/or stimulate activity. Human homologs of the S. cerevisiae recruitment factor Est1 interacted with telomerase in a species-specific manner. The TPR domain of hEST1A interacted with the N-terminus of hTERT. The TPR domain of ScEst1 was required for telomere length maintenance by telomerase, and, paradoxically, also negatively regulated telomere length. In preliminary experiments, hTERT interacted with hPOT1/hTPP1. This interaction may stimulate the elongation of telomeres by telomerase. The DNA and protein interactions described herein expand our knowledge of telomerase and present new targets for the manipulation of telomerase function in human disease.