San, Wan Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 75-78). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.i / Statement --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Acknowledgements --- p.vi / Table of Contents --- p.viii / List of Tables --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- The Plant Secretory Pathway --- p.1 / Chapter 1.2 --- AtEMP70 As a Potential Candidate in PVC Proteomics Analysis --- p.4 / Chapter 1.3 --- EMP70 Protein Family --- p.6 / Chapter 1.3.1 --- Arabidopsis EMP70 Protein Family --- p.6 / Chapter 1.3.2 --- EMP70 Homologs Among Different Species --- p.9 / Chapter 1.4 --- Aims of This Study --- p.10 / Chapter Chapter 2 --- Materials and Methods --- p.12 / Chapter 2.1 --- Generation of Arabidopsis cDNA --- p.12 / Chapter 2.2 --- Plasmid Construction --- p.13 / Chapter 2.3 --- Transformation of Tobacco BY-2 Cells --- p.14 / Chapter 2.4 --- Confocal Immunofluorescence Studies --- p.15 / Chapter 2.5 --- Drug Treatments --- p.16 / Chapter 2.6 --- Transient Expression in Protoplasts --- p.16 / Chapter 2.7 --- Generation of Antibodies --- p.18 / Chapter 2.8 --- SDS-PAGE and Western Blot Analysis --- p.19 / Chapter 2.9 --- Microsomal Protein Extraction --- p.21 / Chapter 2.10 --- Subcellular Fractionation --- p.21 / Chapter 2.11 --- Membrane Strip-off --- p.23 / Chapter Chapter 3 --- Results --- p.24 / Chapter 3.1 --- Subcellular Localization Study of GFP-tagged AtEMP2 Fusions via Transient Expression --- p.24 / Chapter 3.1.1 --- AtEMP2-GFP Localized to TGN in BY-2 Protoplasts --- p.24 / Chapter 3.1.2 --- AtEMP2-GFP Localized to TGN in Arabidopsis Protoplasts --- p.30 / Chapter 3.1.3 --- N-terminal GFP-tagged AtEMP2 Fusions Localized to the Golgi Apparatus in Arabidopsis Protoplasts --- p.33 / Chapter 3.2 --- Generation and Characterization of Transgenic Tobacco BY-2 Cells and Arabidopsis PSB-L Cells Expressing AtEMP2-GFP Fusion --- p.36 / Chapter 3.2.1 --- Subcellular Localization of AtEMP2-GFP Fusion in Transgenic BY-2 Cell Lines --- p.36 / Chapter 3.2.2 --- Subcellular Localization of AtEMP2-GFP Fusion in Transgenic Arabidopsis PSB-D Cell Lines --- p.39 / Chapter 3.3 --- Immunofluorescent Labeling Study --- p.41 / Chapter 3.3.1 --- ManI Antibodies Did Not Label the Punctate Organelles --- p.41 / Chapter 3.3.2 --- AtEMP2 Antibodies Labeled the Golgi Apparatus --- p.43 / Chapter 3.4 --- Generation of AtEMP70 Antibodies --- p.46 / Chapter 3.5 --- Western Blot Analysis --- p.50 / Chapter 3.5.1 --- Heat Treatment Caused Aggregation of AtEMP2-GFP Fusion Proteins --- p.51 / Chapter 3.5.2 --- Size Change of AtEMP2-GFP Fusion Proteins in Response to Heat Treatment --- p.52 / Chapter 3.5.3 --- Aggregation Formation of AtEMP2-T7 Fusion Proteins in 95°C --- p.56 / Chapter 3.5.4 --- Distribution of Endogenous AtEMP70 in Arabidopsis Wild Type Cells --- p.58 / Chapter 3.6 --- Subcellular Fractionation --- p.61 / Chapter 3.6.1 --- C-terminal GFP- or T7-tagged Fusion Affected the Subcellular Localization of AtEMP2 --- p.61 / Chapter 3.6.2 --- Endogenous AtEMP70 Localized to the Golgi Apparatus --- p.64 / Chapter Chapter 4 --- Discussion and Future Perspectives --- p.67 / Chapter 4.1 --- Discussion --- p.67 / Chapter 4.1.1 --- ER Export Signal in the Cytosolic Tail of AtEMP70 --- p.71 / Chapter 4.1.2 --- Potential Golgi Retention Signal in the Cytosolic Tail of AtEMP70 --- p.73 / Chapter 4.2 --- Future Perspectives --- p.74 / References --- p.75

[Truncated abstract] Osteoclasts are multinucleated giant cells responsible for the resorption of the mineralized bone matrix during the process of bone remodelling. During activation towards bone resorption, polarization of the osteoclast results in the formation of a unique plasma membrane, the ruffled border, the actual resorptive organelle of the osteoclast. Through this domain protons are actively pumped into the resorption lacuna creating an acidic microenvironment that favours the dissolution of the mineralized bone matrix. The polarised secretion of protons is carried out by the action of the vacuolar-type (H+)-ATPase (V-ATPase), composed of functionally and structurally distinct subunits of the V1 and V0 domains. The general structure of the V-ATPase complex is highly conserved from yeast to mammals, however, multiple isoforms for specific V-ATPase subunits do exist exhibiting differential subcellular, cellular and tissue-specific localizations. This study focuses on the molecular identification and characterization of V-ATPase accessory subunit Ac45 and the d2 isoform of the V0 domain d subunit in osteoclasts. Using the techniques of cDNA Subtractive Hybridization and DNA Micro-Array analyses respectively, the accessory subunit Ac45 and the d2 isoform of the V0 domain d subunit were identified in RAW264.7-cells derived OcLs. ... Using web-based computational predictions, two possible transmembrane domains, an N-terminus 'signal anchor' sequence and a C-terminus dilysine- like endoplasmic reticulum (ER) retention signal were identified. By confocal microscopy, EYFP-tagged e was found to localize to the perinuclear region of transfected COS-7 cells in compartments representing the ER and Golgi apparatus with some localization in late endosomal/lysosomal-like vesicles. ER truncation of e did not alter its subcellular localization but exhibited significantly weaker association with Ac45 compared to the wild-type as depicted by BRET analyses. Association with the other V0 subunits remain unaffected. This may hint at a possibility that Ac45 may play a role in the masking of the ER signal of e following it's incorporation into the V0 domain. Although no solid evidence for a role in the assembly of the mammalian VATPase have been established, subunit e still represents a potential candidate whose role in the V-ATPase complex requires further investigation. Collectively, the data presented in this thesis has provided further insight into the composition of the osteoclast V-ATPase proton pump by: 1) identifying an accessory subunit, Ac45 which shows promise as a potential candidate for the regulation and/or targeting of the V-ATPase complex in osteoclasts and truncation of its targeting signal impairs osteoclastic bone resorption; 2) identification and preliminary characterization of the d2 isoform of the V0 domain d subunit whose exact role in the V-ATPase complex and in osteoclasts remains to be determined, although its has been implicated to be essential for osteoclastic function; and 3) Preliminary characterization of subunit-e, a potential assembly factor candidate for the mammalian V-ATPase V0 domain.

[Truncated abstract] The design and development of effective anti-viral immunotherapies requires a comprehensive understanding of the cellular and molecular processes that are involved in the generation and regulation of immune responses. The fundamental objective of the immune system is to successfully complete the task of eliminating/controlling the invading pathogen without causing overt pathology. Cytomegaloviruses (CMVs) are large DNA viruses that are able to evade immune attack and persist lifelong within the host. In a healthy host, CMV causes an asymptomatic infection, but in instances of decreased immune functions, such as in newborns, acquired immunodeficiency syndrome (AIDS) patients and transplant recipients, the infection can result in serious morbidity and mortality. Thus, human CMV (HCMV) is a clinically important pathogen and an understanding of the pathogenesis, mechanisms of immune subversion and, importantly the cascade of immune events that ensue following infection is highly relevant. The studies presented in this thesis have provided useful insight into various aspects of viral immunity and it is hoped that they will assist in the design of more effective therapies against viruses of clinical importance. Genetic variability in humans can greatly influence anti-viral immune responses and the outcome of viral infection. ... Furthermore, these studies provide novel evidence that NK cells are also crucial for the control of virus in some organs of susceptible mice during early acute infection. The data reveals that both NK cells and CD8+ T cells utilise perforin- and IFN-? dependent control of MCMV. Furthermore, these studies provide novel evidence that protection mediated by Ly49H+ NK cells in resistant mice is dependent on perforin. Chapter 3 focuses on the biological relevance of Grz during MCMV infection. These studies found that GrzA and GrzB are essential components of the machinery involved in limiting MCMV during acute infection. These analyses also provide the first evidence suggesting that GrzM plays a role, albeit minor, in controlling MCMV replication. Furthermore, the current studies suggest that Grz can mediate direct antiviral activities independent of the induction of cell death in conjunction with perforin. Interestingly, in the absence of both GrzA and GrzB (GrzAB), mice were as susceptible to MCMV infection as perforin-deficient mice. However, unlike perforin-deficient mice, GrzAB-deficient mice controlled and survived the infection. In Chapter 4 the roles of perforin, GrzA and GrzB in anti-viral immunity and immunopathology during MCMV infection were examined. These studies show that NK cell-derived perforin is required to eliminate infected targets as well as activated effector cells, suggesting that NK cells are crucial not only in defensive immunity but also in limiting the immune activation that follows MCMV infection. In summary, the studies presented in this thesis define the significant role played by specific effector molecules in limiting MCMV replication during different stages of this viral infection. Furthermore, these studies provide novel evidence that perforin, GrzA and GrzB play distinct roles in defensive immunity and limiting immunopathology during MCMV infection.

"July 2001" / Includes bibliographical references (leaves 144-183) / xv, 193 leaves : ill., plates (col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Applied and Molecular Ecology, 2002

published_or_final_version / Biological Sciences / Master / Master of Philosophy

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

published_or_final_version / abstract / toc / Microbiology / Master / Master of Philosophy

The development and optimization of anti-viral screening methods are essential to develop newer more effective, treatments against HIV. The XTT method is a widely described method for antiviral screening. Both continuous HIVinfected cells and experimentally infected T-cells have been used in the XTT assay. We compared these methods to screen several plant-derived extracts for cytotoxicity. Several considerations were taken into account when performing these tests (effect of media, solvents and plant enymes). Experiments were performed to investigate these effects. In addition, p24 and viral load quantification were compared as antiviral screening methods. The study showed that several modifications were necessary when performing the XTT assay on plant extracts, due to the effect of media, solvents and plant enymes. The XTT assays and p24 assays performed using experimentally infected cells are far more specific than those using chronically infected cells. The use of viral loads as an antiviral screening method consistently demonstrated the expected efficacy of AZT. / Thesis(MMed.)-University of KwaZulu-Natal, 2009.

Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Fish infections caused by pathogenic Flavobacterium spp. are a major problem in the aquaculture industry worldwide, often leading to large economic losses. Thirty-two Flavobacterium spp. isolates, obtained from various diseased fish species and biofilm growth, were characterised genetically using 16S rRNA gene sequencing, 16S rRNA gene PCR restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD) PCR, repetitive extragenic palindromic (REP) element PCR, plasmid profiling, whole cell protein (WCP) and outer membrane protein (OMP) analyses. The biofilm-forming capability of five genetically heterogeneous Flavobacterium spp. study isolates was investigated using a modified microtiter-plate adherence assay, as well as flow cell studies. Experimental infection studies with Mozambique tilapia (Oreochromis mossambicus) were carried out in order to determine the virulence of the Flavobacterium spp. study isolates. 16S rRNA gene sequence analysis showed the Flavobacterium spp. study isolates were closely related, and 97% sequence similarity was shared with published F. johnsoniae sequences. A high degree of genetic heterogeneity was displayed by the Flavobacterium spp. study isolates following RAPD-PCR, REP-PCR and OMP analysis, however, based on the results obtained by plasmid profiling and WCP analysis, the isolates appeared genetically very homogeneous. The biofilm phenotype was displayed by all five Flavobacterium spp. isolates tested and varied from weakly to strongly adherent. No specific correlation was observed between the RAPD, REP and/or OMP profiles and degree of adherence displayed by Flavobacterium spp. isolates. However, a specific WCP profile (profile B), exhibited by 48% of the Flavobacterium spp. isolates, was linked to strong adherence. Experimental infection studies showed that Flavobacterium spp. isolates displayed variable levels of virulence, which could not be linked to biofilm formation, nor specific genotypes. This is the first reported isolation and characterisation of Flavobacterium spp. isolated from diseased fish in Southern Africa, and there appears to be significant diversity amongst the isolates which is not geographically linked nor host related. / AFRIKAANSE OPSOMMING: Visinfeksies veroorsaak deur Flavobacterium spp. is problematies in die akwakultuur industrie wêreldwyd en lei tot groot ekonomiese verliese. Twee en dertig Flavobacterium spp. isolate, geïsoleer vanaf verskye geïnfekteerde visspesies en biofilm groei, was geneties gekarakteriseer met behulp van 16S rRNS geenvolgorde, 16S rRNS geen PKR restriksie fragment lengte polimorfisme (RFLP), toevallig geamplifiseerde polimorfiese DNS (TGPD) PKR, herhaalde ekstrageniese palindromiese (HEP) element PKR, plasmied profilering, heelsel protein (HSP) en buite membraan protein (BMP) analise. Die vermoë van vyf geneties heterogene Flavobacterium spp. isolate om biofilms te vorm was ondersoek met behulp van ‘n gemodifiseerde mikrotiterplaat vashegtings toets asook vloei-sel studies. Eksperimentele infeksie studies was uitgevoer op bloukurpers (Oreochromis mossambicus) om die virulensie van die Flavobacterium spp. studie isolate te toets. 16S rRNS geenvolgorde analise het getoon dat die Flavobacterium spp. studie isolate naby verwant was, en het 97% ooreenstemming getoon met gepubliseerde F. johnsoniae volgordes. TGPD-PKR, HEP-PKR en BMP analise het ‘n hoë graad van heterogeniteit tussen die Flavobacterium spp. studie isolate aangetoon, egter, op grond van plasmied profilering en HSP analise, was die studie isolate geneties baie homogeen. Die biofilm fenotipe was getoon deur al die getoetsde Flavobacterium spp. isolate en het gevarieer van swak tot sterk vashegting. Geen spesifieke korrelasie was waargeneem tussen die TGPD, HEP en/of BMP profiele en graad van vashegting vertoon deur Flavobacterium spp. isolate nie, maar ‘n spesifieke HSP profiel (profiel B), getoon deur 48% van die Flavobacterium spp. isolate, was verbind met sterk vashegting. Eksperimentele infeksie studies het getoon dat Flavobacterium spp. isolate varierende grade van virulensie vertoon het en wat met biofilm formasie of spesifieke genotipes geassosieer kon word nie. Hierdie is die eerste gedokumenteerde isolasie en karakterisering van Flavobacterium spp. geïsoleer van geïnfekteerde vis in Suider Afrika, en daar is beduidende diversiteit tussen die isolate wat nie geografies of gasheer geassosieerd is nie.

In eukaryotes, the defined loci on each chromosome, the centromeres, accomplish the critical task of correct cell division. In some organisms, centromeres are composed of a euchromatic central core region embedded in a stretch of heterochromatin and the inheritance and maintenance of centromeres are controlled by dynamic epigenetic phenomena. Although the size of centromeres differs between organisms, its organization, and the placement of euchromatic and heterochromatic regions is conserved from the fission yeast, Schizosaccharomyces pombe, to humans, Homo sapiens. However, relatively little is known about centromeres in the filamentous fungi from the Ascomycota, representing the largest group of fungi and fungal pathogens. Further, studies from humans, flies, yeast and plants have shown that the inheritance of centromeres is not strictly guided by centromeric DNA content, which is highly AT-rich, repetitive and constantly evolving. Therefore, it is difficult to align ans assemble the sequenced contigs of centromeric regions of higher eukaryotes, including most filamentous fungi. A genetic technique, tetrad (or octad) analysis has helped to map the centromeres of the filamentous fungus Neurospora crassa early on. The research presented in this dissertation used N. crassa as a model to focus on characterizing different features of centromeres with an emphasis on the centromere-specific histone H3 (CenH3) protein. Data included here represent the first study on centromere-specific proteins in Neurospora, and demonstrate that the central core of the centromeres are heterochromatic, showing enrichment of silent histone marks, which is in contrast to the centromere arrangement in fission yeast. The CenH3 protein, whose deposition on the genome licenses formation or maintenance of centromeres, shows highly divergent N-terminal regions and a conserved histone fold domain (HFD) in all eukaryotes. This bipartite nature of CenH3 is also observed in the Ascomycota, which provides an opportunity for functional complementation assays by replacing Neurospora CenH3 (NcCenH3) with CenH3 genes from other species within the Ascomycota. The results from this experimental approach provide good measures for (1) determining the specific regions of CenH3 required for the assembly of centromeres during meiotic and mitotic cell divisions and (2) analyzing the resistance to changes in the organization of centromeres in N. crassa. The genetic analysis showed that the divergent N-terminal region is essential for the proper assembly of centromeres, and that the conserved carboxy-terminus of CenH3 is important for the process of meiosis but not mitotic cell division. ChIP-seq analyses suggest that the observed loss of Podospora anserina CenH3 (PaCenH3- GFP) from certain N. crassa centromeres does not result in obvious phenotypic defects, e.g. diminished growth or evidence for aneuploidy. Further, the low enrichment of PaCenH3-GFP at certain centromeres is possibly predetermined during meiosis, which results in irreversible and progressive decreases in enrichment. It remains to be determined if this process is random as far as selection of centromeres is concerned. Together the results presented here suggest that during meiosis more stringent structural requirements for centromere assembly apply and that these are dependent on CenH3, and that depletion of CenH3 from centromeres does not critically affect mitosis in the asynchronously dividing nuclei of Neurospora hyphae. / Graduation date: 2013