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

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Despite recent advances in antiretroviral therapy, nearly 37 million people continue to live with human immunodeficiency virus (HIV). Basic and applied research on the assembly of HIV could be enhanced by using a genetically tractable organism, such as yeast, rather than mammalian cells. While previous studies showed that expression of the HIV Gag polyprotein in Saccharomyces cerevisiae spheroplasts resulted in the production of virus-like particles (VLPs), many questions regarding the utility of yeast in HIV assembly remain uninvestigated. Here, we report use of S. cerevisiae for both the production of VLPs with selectively packaged RNA and to evaluate the human Y-box-binding protein 1 (YB-1) in selective RNA packaging into VLPs. Our data reveal: (1) When co-expressed alongside HIV-1 Gag, an RNA mammalian expression cassette is selectively encapsidated and released in VLPs produced from spheroplasts; (2) Inclusion of the 5’UTR-5’Gag RNA upstream of the mammalian expression cassette greatly increased the selectivity to which non-viral RNA was packaged into VLPs; and (3) heterologous expression of the human YB-1 protein in S. cerevisiae did not facilitate the selective packaging of viral RNA into VLPs, likely due to inability to bind upstream elements in the HIV-1 viral RNA. Overall, this research provides a key first step in the use of yeast for the production of viral vectors used in gene therapy, and lays a foundation for further experiments investigating the role of YB-1 and other host proteins in selective RNA packaging.

Inflammation and an influx of macrophages are common elements in many diseases. Among pro-inflammatory cytokines, tumor necrosis factor (TNF) plays a central role by amplifying the cytokine network. Progranulin (PGRN) is a growth factor that binds to TNF receptors and interferes with TNF-mediated signaling. Extracellular PGRN is processed into granulins by proteases released from immune cells. PGRN exerts anti-inflammatory effects, whereas granulins are pro-inflammatory. The factors coordinating these ambivalent functions remain unclear. In our study, we identify Y-box binding protein-1 (YB-1) as a candidate for this immune-modulating activity. Using a yeast-2-hybrid assay with YB-1 protein as bait, clones encoding for progranulin were selected using stringent criteria for strong interaction. We demonstrate that at physiological concentrations, YB-1 interferes with the binding of TNF to its receptors in a dose-dependent manner using a flow cytometry-based binding assay. We show that YB-1 in combination with progranulin interferes with TNF-mediated signaling, supporting the functionality with an NF-B luciferase reporter assay. Together, we show that YB-1 displays immunomodulating functions by affecting the binding of TNF to its receptors and influencing TNF-mediated signaling via its interaction with progranulin.