Lam, Ka Han. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 77-82). / Abstracts in English and Chinese. / Acknowledgement --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Content --- p.vii / List of Abbreviations and Symbols --- p.xi / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Severity of influenza --- p.1 / Chapter 1.2 --- Introduction of influenza viruses --- p.3 / Chapter 1.2.1 --- Virion and genome structure --- p.4 / Chapter 1.2.2 --- The replication cycle of influenza viruses --- p.5 / Chapter 1.3 --- Influenza virus NP --- p.8 / Chapter 1.3.1 --- The importance of NP in RNP structure maintenance --- p.9 / Chapter 1.3.2 --- NP self oligomerization --- p.10 / Chapter 1.3.3 --- NP-RNA interaction --- p.12 / Chapter 1.3.4 --- NP and other interacting partners --- p.13 / Chapter 1.4 --- Aim of the project --- p.16 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Biological materials --- p.18 / Chapter 2.2 --- Construction of NP mutants --- p.19 / Chapter 2.3 --- Luciferase assay --- p.22 / Chapter 2.4 --- Western blot --- p.23 / Chapter 2.5 --- Protein expression and purification --- p.23 / Chapter 2.6 --- Circular dichroism spectroscopy --- p.24 / Chapter 2.7 --- Static Light scattering --- p.24 / Chapter 2.8 --- Surface plasmon resonance --- p.25 / Chapter 2.9 --- Co-immunoprecipitation (co-IP) --- p.26 / Chapter Chapter 3 --- Identification of residues crucial for NPB oligomerization and ribonucleoprotein activity / Chapter 3.1 --- Introduction --- p.27 / Chapter 3.2 --- Result --- p.31 / Chapter 3.2.1 --- NPB mutants showed deficiency in overall transcription and replication activity --- p.31 / Chapter 3.2.2 --- Expression and purification of NP mutants with low RNP activity --- p.37 / Chapter 3.2.2.1 --- Expression of MBP-tagged NP variants --- p.37 / Chapter 3.2.2.2 --- Purification of MBP-tagged NP variants --- p.38 / Chapter 3.2.3 --- Secondary structures of NP variants were comparable t o wild type NP --- p.41 / Chapter 3.2.4 --- NP variants with low RNP activity were abnormal in oligomerization in vitro --- p.42 / Chapter 3.2.5 --- NP variants with low RNP activity were impaired in homo-oligomer formation in vivo --- p.45 / Chapter 3.2.6 --- Discussion --- p.47 / Chapter Chapter 4 --- Identification of residues crucial for NP 一 RNA interaction and ribonucleoprotein activity / Chapter 4.1 --- Introduction --- p.56 / Chapter 4.2 --- Result --- p.58 / Chapter 4.2.1 --- NPB mutants showed deficiency in overall transcription and replication activity --- p.58 / Chapter 4.2.2 --- Expression and purification of NP variants with low RNP activity --- p.62 / Chapter 4.2.3 --- Secondary structures of NP variants were comparable t o wild type NP --- p.63 / Chapter 4.2.4 --- NP variants with low RNP activity were abnormal in RNA binding --- p.64 / Chapter 4.3 --- Discussion --- p.68 / Chapter Chapter 5 --- Conclusion and future prospect --- p.73 / Copyright --- p.76 / References --- p.77

Thesis (Ph. D.)--Virginia Commonwealth University, 2007. / Prepared for: Dept. of Microbiology and Immunology . Bibliography: leaves 130 - 137 . Also available online via the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "August 2007" Includes bibliographical references.

Thesis (Ph. D.)--University of Virginia, 2007. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2008. / Title from PDF title page (viewed on July 13, 2010). Includes bibliographical references.

Dissertation (PhD)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Since the advent of recombinant DNA technology in the 1970s, various microbial hosts have been employed for the efficient high-level heterologous production of a variety of proteins, ranging from enzymes and reagents to therapeutics and vaccines. More recent microbial hosts to be employed for these purposes are filamentous fungi, and particularly the genus Aspergillus. Aspergilli have been associated with industrial processes for many years and are used in the production of antibiotics, enzymes, citric acid and Oriental foods and beverages, and thus strains such as Aspergillus niger and Aspergillus oryzae have been afforded GRAS (Generally Regarded 8s ~afe) status. They also secrete copious amounts of homologous and heterologous proteins and are able to perform post-translational modifications effectively. Various proteins of pharmaceutical interest have been successfully expressed in Aspergillus, but the potential of these fungi to produce heterologous viral proteins has not been explored extensively. In this study, we evaluated the potential of the filamentous fungi A. niger and Aspergillus awamori as alternative hosts for the heterologous production of hepatitis B viral proteins. Hepatitis B is a serious, potentially lethal liver disease that affects 2000 million people world-wide and has a high endemicity in Southern Africa. Currently there is no effective treatment for viral hepatitis and thus a mass vaccination strategy is the only solution to curb the spread of the disease. This kind of vaccination strategy requires a cheap, safe and effective vaccine and these objectives have been achieved in the development of recombinant subunit vaccines from yeasts such as Saccharomyces cerevisiae, Hansenula polymorpha and Pichia pastoris that are commercially available. The hepatitis B virus envelope consists of a membrane fraction and three proteins, namely the major (S) protein (encoded by the S gene), the middle (M) protein (encoded by the preS2S gene) and the large (L) protein (encoded by the preS1preS2S gene). When produced in the above-mentioned yeasts, the S protein was shown to spontaneously assemble into pseudoviral particles devoid of viral DNA, which were then purified and used as vaccine. In the present study the Sand preS1preS2S genes from a local isolate of hepatitis B subtype adw2 were placed under transcriptional control of the constitutive Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpdA) promoter and the inducible A. niger glucoamylase (glaA) promoter. The respective viral genes were also fused to the region encoding the catalytic domain of the highly expressed and secreted A. niger glucoamylase, which served as a carrier moiety to possibly facilitate viral protein secretion. The various gene constructs were subsequently transformed to laboratory strains of A. niger and A. awamori and numerous transformants were obtained. One A. niger transformant carrying the S gene under control of the gpdA promoter contained approximately 7 integrated copies of the expression cassette and produced hepatitis B pseudoviral particles intracellularly at levels of 0.4 mg/I culture. These levels are approximately ten-fold higher than those initially obtained from the yeast S.cerevisiae, which showed yields of 0.01 to 0.025 mg/I. None of the other transformants could be shown to produce recombinant S or L protein and no secretion of viral protein could be demonstrated. This could be attributed to numerous factors, including vector copy number, site of integration or proteolytic activity. The most important insight emerging from this work regarding secretion of heterologous viral protein was that the addition of a carrier protein hampered, rather than enhanced secretion of the viral envelope protein, due to the inherent properties of viral protein assembly. This work also serves as a "proof of principle", showing that Aspergillus is indeed a viable alternative host for the production of hepatitis B pseudoviral particles, and could be investigated further for its potential as host for the heterologous expression of other viral proteins. / AFRIKAANSE OPSOMMING: Sedert die ontwikkeling van rekombinante DNA tegnologie in die sewentigerjare is verskeie mikroorganismes reeds gebruik vir die doeltreffende produksie van 'n verskeidenheid proteïne teen hoë vlakke; onder andere ensieme, reagense, terapeutiese middels en vaksiene. Onlangs is filamentagtige swamme, veral van die genus Aspergillus, ontwikkel vir heteroloë proteïenproduksie. Aspergilli word al vir baie jare in nywerheidsprosesse gebruik, onder andere in die vervaardiging van antibiotika, ensieme, sitroensuur en sekere Oosterse voedsel- en drankprodukte. As gevolg van hierdie jarelange gebruik van rasse soos Aspergillus niger en Aspergillus oryzae, word hulle algemeen aanvaar as veilig vir menslike gebruik. Hierdie swamme besit veral die vermoë om hoë vlakke van homoloë en heteroloë proteïene uit te skei en die na-translasiemodifisering van proteïene korrek uit te voer. Verskeie proteïene van farmaseutiese belang is al suksesvol in Aspergillus uitgedruk, maar die potensiaal van hierdie swamme om virale proteïene te vervaardig is nog nie deeglik ondersoek nie. Hierdie studie ondersoek die geskiktheid van die filamentagtige swamme A. niger en Aspergillus awamori om as alternatiewe gashere vir die heteroloë produksie van hepatitis B proteïene te dien. Hepatitis B is 'n ernstige en selfs dodelike lewersiekte. Omtrent 2000 miljoen mense wêreld-wyd is met die virus geïnfekteer en dit is veral endemies in Suiderlike Afrika. Daar is tans geen doeltreffende behandeling vir virale hepatitis en dus is wêreld-wye inentingsprogramme die enigste oplossing om die verspreiding van die siekte te bekamp. Hierdie inentingsstrategie vereis die beskikbaarheid van 'n bekostigbare, veilige en doeltreffende vaksien. Die rekombinante subeenheidvaksiene wat ontwikkel is deur van gashere soos Saccharomyces cerevisiae, Hansenula polymorpha en Pichia pastoris gebruik te maak, voldoen aan hierdie vereistes en is kommersieel beskikbaar. Die omhulsel van die hepatitis B virus bestaan uit 'n membraangedeelte en drie proteïene, naamlik die hoofproteïen (S) (gekodeer deur die S-geen), die middelproteïen (M) (gekodeer deur die preS2S-geen) en die grootproteïen (L) (gekodeer deur die preS1preS2S-geen). Wanneer die S-proteïen in bo-genoemde giste uitgedruk word, vorm dit spontaan pseudovirale partikels wat nie virale DNA bevat nie. Hierdie partikels word dan gesuiwer en as vaksien gebruik. In hierdie studie is die S- en preS1preS2S-gene, vanaf 'n plaaslike isolaat van hepatitis B subtipe adw2, onder transkripsionele beheer van die konstitutiewe Aspergillus nidulans gliseraldehied-3-fosfaat-dehidrogenasepromoter (gpdA) en die induseerbare A. niger glukoamilasepromoter (glaA) geplaas. Die onderskeie virale gene is ook aan die koderende gedeelte vir die katalitiese domein van A. niger glukoamilase gelas om fusieproteïene te vorm. Glukoamilase word teen hoë vlakke deur Aspergillus vervaardig en uitgeskei en kan dus moontlik dien as draerproteïen om sekresie van die proteïne te bevorder. Transformasie van die geenkonstrukte na laboratoriumrasse van A. niger en A. awamori het verskeie transformante gelewer. Een A. niger transformant bevattende die S-geen onder transkripsionele beheer van die gpdA promoter het minstens sewe kopieë van die uitdrukkingskaset in sy genoom geïntegreer en het hepatitis B pseudovirale partikels intrasellulêr teen vlakke van 0.4 mg/I swamkultuur vervaardig. Hierdie vlakke is omtrent tienvoudig hoër as die vlakke van 0.01 - 0.025 mg/I wat S.cerevisiae oorspronklik opgelewer het. Nie een van die ander transformante het rekombinante S of L proteïene vervaardig nie en sekresie van virale proteïen kon nie getoon word nie. Hierdie verskynsel mag te wyte wees aan verskeie faktore insluitende vektor-kopiegetal, setel van integrasie en proteolitiese aktiwiteit. Die belangrikste insig uit hierdie studie aangaande sekresie van heteroloë virale proteïene is dat die koppeling van die virale omhulsel-proteïen aan 'n draerproteïen sekresie benadeel het, eerder as om dit te bevorder. Hierdie verskynsel is te wyte aan die inherente geneigdheid van virale omhulselproteïene om 'n kompleks te vorm. Die studie dien ook as "bewys van beginsel" dat Aspergillus wel 'n werkbare alternatiewe gasheer vir die produksie van hepatitis B pseudovirale partikels is, en dat dit verder ondersoek sou kon word as potensiële gasheer vir die heteroloë uitdrukking van ander virale proteïene.

published_or_final_version / abstract / Microbiology / Doctoral / Doctor of Philosophy

Poon Wing Ming Jodie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 207-233). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Abbreviations --- p.v / List of Figures --- p.x / List of Tables --- p.xiii / Contents --- p.xiv / Chapter CHAPTER ONE --- INTRODUCTION --- p.1 / Chapter 1.1. --- Severe Acute Respiratory Syndrome (SARS) --- p.1 / Chapter 1.1.1. --- Background of SARS --- p.1 / Chapter 1.1.2. --- Etiology and pathology of SARS --- p.3 / Chapter 1.1.3. --- Genome organization and expression of SARS-CoV --- p.5 / Chapter 1.1.4. --- Current molecular advances of SARS-CoV --- p.13 / Chapter 1.1.5. --- Current research advances on SARS-CoV nucleocapsid --- p.18 / Chapter 1.1.6. --- Current diagnostic assays of SARS-CoV infection --- p.23 / Chapter 1.1.7. --- Current treatment --- p.25 / Chapter 1.1.8. --- Vaccine development --- p.27 / Chapter 1.2. --- Aims of study --- p.30 / Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.33 / Chapter 2.1. --- Subcellular localization study of the SARS-CoV nucleocapsid protein --- p.33 / Chapter 2.1.1. --- "Cloning of SARS-CoV nucleocapsid cDNA into the green fluorescence protein (GFP) mammalian expression vector, pEGFP-C1" --- p.33 / Chapter 2.1.1.1. --- Amplification of SARS-CoV nucleocapsid gene by polymerase chain reaction (PCR) --- p.33 / Chapter 2.1.1.2. --- Purification of PCR products --- p.35 / Chapter 2.1.1.3. --- Restriction digestion of purified PCR products and the circular pEGFP-C 1 vector --- p.36 / Chapter 2.1.1.4. --- Ligation --- p.36 / Chapter 2.1.1.5. --- Preparation of chemically competent bacterial cell E.coli strain DH5a for transformation --- p.37 / Chapter 2.1.1.6. --- Transformation of ligation product into chemically competent bacterial cells --- p.38 / Chapter 2.1.1.7. --- Small-scale preparation of bacterial plasmid DNA --- p.39 / Chapter 2.1.1.8. --- Screening for recombinant clones --- p.40 / Chapter 2.1.1.9. --- DNA sequencing of cloned plasmid DNA --- p.41 / Chapter 2.1.1.10. --- Midi-scale preparation of recombinant plasmid DNA --- p.42 / Chapter 2.1.2. --- Cell culture --- p.44 / Chapter 2.1.2.1. --- Sub-culture of VeroE6 and HepG2 cell lines --- p.44 / Chapter 2.1.2.2. --- Transient transfection of GFP fusion construct --- p.45 / Chapter 2.1.3. --- Epi-fluorescent microscopy --- p.46 / Chapter 2.2. --- Study on differential gene expression patterns upon SARS-CoV nucleocpasid induction by cDNA microarray analysis --- p.48 / Chapter 2.2.1. --- Cloning of SARS-CoV N gene into mammalian expression vector pCMV-Tagl --- p.48 / Chapter 2.2.2. --- Cell culture --- p.50 / Chapter 2.2.2.1. --- Sub-culture of VeroE6 cell line --- p.50 / Chapter 2.2.2.2. --- Transient transfection of pCMV-Tag1 -SAR-CoV N construct --- p.50 / Chapter 2.2.3. --- Total RNA isolation --- p.51 / Chapter 2.2.3.1. --- Total RNA isolation by RNeasy Mini Kit --- p.51 / Chapter 2.2.3.2. --- Checking of RNA integrity --- p.53 / Chapter 2.2.3.3. --- Checking of RNA purity --- p.54 / Chapter 2.2.3.4. --- Determinations of total RNA concentrations and precipitation --- p.54 / Chapter 2.2.4. --- cDNA microarray (done by Affymetrix Inc. as a customer service) --- p.55 / Chapter 2.2.4.1. --- Precipitation of RNA --- p.55 / Chapter 2.2.4.2. --- Quantification of RNA --- p.56 / Chapter 2.2.4.3. --- Synthesis of double-stranded cDNA from total RNA --- p.56 / Chapter (i) --- First stand cDNA synthesis --- p.56 / Chapter (ii) --- Second cDNA synthesis --- p.57 / Chapter 2.2.4.4. --- Clean-up of double stranded cDNA --- p.58 / Chapter (i) --- Phase lock gel-phenol/ chloroform extraction --- p.58 / Chapter (ii) --- Ethanol precipitation --- p.58 / Chapter 2.2.4.5. --- Synthesis of biotin-labeled cRNA --- p.59 / Chapter 2.2.4.6. --- Clean-up and quantification of in vitro transcription (IVP) products --- p.59 / Chapter (i) --- In vitro transcription clean-up --- p.59 / Chapter (ii) --- Ethanol precipitation --- p.60 / Chapter (iii) --- Quantitation of cRNA --- p.60 / Chapter (iv) --- Sample checking --- p.60 / Chapter 2.2.4.7. --- cRNA fragmentation for target preparation --- p.60 / Chapter 2.2.4.8. --- Eukaryotic target hybridization --- p.61 / Chapter 2.2.4.9. --- "Probe array washing, staining and scanning" --- p.62 / Chapter 2.2.5. --- Confirmation of results by RT-PCR --- p.62 / Chapter 2.2.5.1. --- First-strand cDNA synthesis --- p.62 / Chapter 2.2.5.2. --- RT-PCR of candidate gene --- p.63 / Chapter 2.3. --- In vitro RNA interference of SARS-CoV nucleocapsid --- p.66 / Chapter 2.3.1. --- siRNA target site selection --- p.66 / Chapter 2.3.2. --- Cloning of target siRNA sequences into pSilencer 3.1-H1 vector --- p.71 / Chapter 2.3.3. --- Cell culture --- p.72 / Chapter 2.2.3.1. --- Sub-culture ofVeroE6 cells --- p.72 / Chapter 2.3.3.2. --- Transient co-transfection --- p.72 / Chapter 2.3.4. --- Detection of SARS-CoV nucleocapsid mRNA expression level by RT-PCR --- p.73 / Chapter 2.3.4.1. --- Total RNA isolation by TRIzol reagent --- p.73 / Chapter 2.3.4.2. --- First-strand cDNA synthesis --- p.74 / Chapter 2.3.4.3. --- RT-PCR assays --- p.74 / Chapter 2.3.5. --- Detection of SARS-CoV nucleocapsid protein expression level by Western blotting --- p.75 / Chapter 2.3.5.1. --- Total protein extraction --- p.75 / Chapter 2.3.5.2. --- Protein quantification --- p.75 / Chapter 2.3.5.3. --- Protein separation by SDS-PAGE and Western blot --- p.76 / Chapter 2.3.5.4. --- Western blot analysis --- p.78 / Chapter 2.4. --- Human fgl2 prothrombinase promoter analyses --- p.80 / Chapter 2.4.1. --- Cloning of the full-length human fgl2 prothrombinase promoter construct into a promoterless mammalian expression vector-pGL3-Basic --- p.80 / Chapter 2.4.2. --- Cloning of SARS-CoV Membrane gene into the mammalian expression vector pCMV-Tagl --- p.82 / Chapter 2.4.3. --- Cell culture --- p.84 / Chapter 2.4.3.1. --- Sub-culture of HepG2 and VeroE6 cell lines --- p.84 / Chapter 2.4.3.2. --- "Transient co-transfection of the full-length human fgl2 prothrombinase promoter construct with the pCMV-Tagl empty vector, pCMV-Tagl-SARS-CoV M expression vector, or pCMV-Tag1 -SARS-CoV N expression vector" --- p.84 / Chapter 2.4.4. --- Dual-luciferase reporter assay --- p.85 / Chapter 2.4.5. --- Detection of fgl2 mRNA expression level under the induction of SARS-CoV nucleocapsid protein by RT-PCR --- p.86 / Chapter 2.4.5.1. --- Total RNA isolation by TRIzol reagent --- p.86 / Chapter 2.4.5.2. --- First strand cDNA synthesis --- p.86 / Chapter 2.4.5.3. --- RT-PCR of fgl2 gene --- p.87 / Chapter CHAPTER THREE --- RESULTS --- p.88 / Chapter 3.1. --- Computer analysis of SARS-CoV Nucleocapsid --- p.88 / Chapter 3.2. --- Subcellular localization of SARS-CoV nucleopcasid protein in VeroE6 cells and HepG2 cells --- p.102 / Chapter 3.3. --- cDNA microarray analysis on differential gene expression pattern upon the over-expression of SARS-CoV Nucleocapsid gene --- p.114 / Chapter 3.4. --- In vitro RNA Interference of SARS nucleocapsid --- p.129 / Chapter 3.5. --- Transactivation of fgl2 prothrombinase gene promoter by SARS-CoV nucleocapsid protein in HepG2 and VE6 cells --- p.138 / Chapter CHAPTER FOUR --- DISCUSSION --- p.155 / Chapter 4.1. --- "The EGFP-tagged SARS-CoV N protein was localized in the cytoplasm only in VE6 cells, but translocated into both cytoplasm and nucleus in HepG2 cellsin the epi-fluorescence microscopy study" --- p.155 / Chapter 4.2. --- cDNA microarray demonstrated alternations of mRNA transcript level on a number of genes belonging to various functional classes upon over expression of SARS-CoV nucleocapsid gene --- p.162 / Chapter 4.3. --- RNA interference demonstrated effective gene silencing of SARS-CoV nucleocapsid gene --- p.171 / Chapter 4.4. --- SASR-CoV nucleocapsid protein induced the promoter activity of the prothrombinase fibrinogen-like protein2/ fibroleukin (fgl2) gene --- p.191 / Chapter 4.5. --- Conclusion --- p.196 / Chapter 4.6. --- Future work --- p.198 / Appendices --- p.199 / References --- p.207

The role of viral proteins in the pathogenesis of infectious hematopoietic necrosis virus (IHNV) was studied at the molecular level. The expression of the viral genes at the protein and RNA level, and their cellular localization, were characterized to further our understanding of viral pathogenesis. The pathogenic effect of individual viral proteins was also investigated and a method for detecting viral RNA in infected fish tissues was developed. The polarity of transcription was confirmed in terms of the relative amounts of each viral protein. Also, cells treated with glycosylation inhibitors did not exhibit cytopathic effect, demonstrating that a functioning host glycosylation system is necessary for viral replication. These studies also revealed a previously undescribed non-glycosylated protein, S, which appeared to be virus-encoded. The expression of the nonvirion protein (NV), was also detected in infected kidney tissues. The location of M2 and NV in the cell was found to be the nucleus and cytoplasm. The expression of the NV gene was further analyzed at the level of transcription and the regulation signals for IHNV transcription were investigated. Unique transcriptional initiation and terminational signals for the fish lyssa-like rhabdoviruses were identified. The transcriptional initiation signal, 3'-CGUG-5', was distinctly different from that of the other rhabdoviruses, 3'-UUGU-5'. The role of the M2 and NV proteins in viral pathogenesis was investigated by transient expression of these proteins individually in cultured fish cells. The M2 protein alone resulted in inhibition of host-directed gene expression at the level of transcription and induction of nuclear fragmentation. The NV protein was not involved in the regulation of the host gene expression, but was involved in another type of cytopathic effect characterized as cell rounding. This is the first biological function attributed to the NV protein. A PCR method was developed for detecting IHNV N-specific RNA in formalin-fixed, paraffin-embedded fish tissues. The method is sensitive and specific. The technique is capable of detecting viral RNA in samples that have been remained at room temperature in 10% buffered formalin for over 2 years. / Graduation date: 1997

Ph.D.