Cheung Siu Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 86-91). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vii / Table of Contents --- p.viii / List of Tables --- p.xiii / List of Figures --- p.xiv / Lists of Abbreviations --- p.xvii / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- The plant secretory pathways --- p.2 / Chapter 1.1.1 --- Three different protein sorting pathways to plant vacuoles --- p.3 / Chapter 1.1.2 --- VSD and VSR --- p.6 / Chapter 1.2 --- Prevacuolar compartments --- p.7 / Chapter 1.2.1 --- Lytic PVC --- p.7 / Chapter 1.2.2 --- BP-80 reporter as a lytic PVC marker --- p.8 / Chapter 1.2.3 --- PVC of PSV --- p.9 / Chapter 1.2.4 --- α-TIP CT reporter as a PVC of PSV marker --- p.10 / Chapter 1.3 --- Project objectives --- p.11 / Chapter Chapter 2 --- Development of Transgenic Tobacco BY-2 Cell Lines Expressing Fluorescent Reporters for Golgi and Prevacuolar Compartments / Chapter 2.1 --- Introduction --- p.13 / Chapter 2.2 --- Materials and Methods --- p.15 / Chapter 2.2.1 --- Chemicals --- p.15 / Chapter 2.2.2 --- Oligonucleotides: Primers and Adapters --- p.15 / Chapter 2.2.3 --- Bacterial Strains --- p.17 / Chapter 2.2.4 --- "Preparation of single-reporter constructs (GONST1 -CFP, CFP-BP-80 and CFP-a-TIP CT reporters)" --- p.17 / Chapter 2.2.4.1 --- "Cloning of pGONSTl-CFPK, a Golgi marker" --- p.17 / Chapter 2.2.4.2 --- "Cloning of pCFP-BP-80K, a lytic PVC marker" --- p.20 / Chapter 2.2.4.3 --- "Cloning of pCFP-α-TIP CTK, a putative marker for PVC of PSV" --- p.22 / Chapter 2.2.5 --- "Preparation of double-reporter constructs (CFP-BP-80-GONST1 - YFP, CFP-α-TIP CT-GONST1-YFP, CFP-BP-80-YFP-α-TIP CT and CFP-α-TIP CT-YFP-BP-80 reporters)" --- p.24 / Chapter 2.2.5.1 --- Insertion ofAdapter-XH to pCFP-BP-80K and pCFP-α-TIP CTK --- p.24 / Chapter 2.2.5.2 --- "Cloning of pCFP-BP-80-GONST 1 -YFPK, pCFP-α-TIP CT- GONST 1-YFPK, pCFP-BP-80-YFP-α-TIP CTK and pCFP- α-TIP CT-YFP-BP-80K" --- p.26 / Chapter 2.2.6 --- Agrobacterium electroporation --- p.30 / Chapter 2.2.7 --- Agrobacterium-mediated transformation of tobacco BY-2 cells --- p.30 / Chapter 2.2.8 --- Selection and screening of transformed BY-2 cells --- p.31 / Chapter 2.2.8.1 --- Antibiotic selection --- p.31 / Chapter 2.2.8.2 --- Fluorescence microscopic screening --- p.31 / Chapter 2.2.9 --- Detection of CFP and YFP reporter genes and their expressions --- p.32 / Chapter 2.2.9.1 --- CTAB genomic DNA extraction --- p.32 / Chapter 2.2.9.2 --- PCR test for CFP (and YFP) transgene in genomic DNA --- p.33 / Chapter 2.2.9.3 --- Subcellular fractionation and protein extraction --- p.33 / Chapter 2.2.9.4 --- Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis --- p.34 / Chapter 2.2.9.5 --- Confocal microscopic study --- p.35 / Chapter 2.3 --- Results --- p.36 / Chapter 2.3.1 --- Establishment of kanamycin-resistant BY-2 cells expressing CFP (and YFP) reporters --- p.36 / Chapter 2.3.2 --- Fluorescence microscopic screening of transgenic BY-2 cell lines --- p.37 / Chapter 2.3.3 --- CFP (and YFP) reporter was successfully integrated into transgenic BY-2 cell genome --- p.41 / Chapter 2.3.4 --- CFP (and YFP) reporter was expressed in transgenic BY-2 cell lines --- p.44 / Chapter 2.3.5 --- Punctate CFP (and YFP) signals were detected in transgenic BY-2 cell lines expressing single (or double) reporter --- p.48 / Chapter 2.4 --- Discussion --- p.53 / Chapter 2.4.1 --- "Transgenic BY-2 cell lines expressing single reporter marking Golgi, lytic PVC and putative PVC of PSV have been developed" --- p.53 / Chapter 2.4.2 --- "Golgi, lytic PVC and putative PVC of PSV were separate and distinct organelles" --- p.53 / Chapter 2.4.3 --- Transgenic BY-2 cell lines expressing double reporter were not yet suitable for subsequent study --- p.55 / Chapter Chapter 3 --- Characterization of Transgenic Tobacco BY-2 Cell Lines Expressing Fluorescent Reporters for Prevacuolar Compartments / Chapter 3.1 --- Introduction --- p.58 / Chapter 3.2 --- Materials and Methods --- p.60 / Chapter 3.2.1 --- Confocal immunofluorescence study --- p.60 / Chapter 3.2.2 --- Drug treatment study (for single-reporter transgenic tobacco BY-2 cell line) --- p.62 / Chapter 3.2.2.1 --- Wortmannin treatment --- p.62 / Chapter 3.2.2.1.1 --- Dosage effect --- p.62 / Chapter 3.2.2.1.2 --- Time-course study --- p.62 / Chapter 3.2.2.2 --- Brefeldin A treatment --- p.63 / Chapter 3.2.2.1.1 --- Dosage effect --- p.63 / Chapter 3.2.2.1.2 --- Time-course study --- p.63 / Chapter 3.2.3 --- Drug treatment study (for double-reporter transgenic tobacco BY-2 cell line) --- p.64 / Chapter 3.2.3.1 --- Wortmannin treatment --- p.64 / Chapter 3.2.3.2 --- Brefeldin A treatment --- p.64 / Chapter 3.3 --- Results --- p.65 / Chapter 3.3.1 --- CFP-α-TIP CT reporter-marked compartment was not Golgi apparatus --- p.65 / Chapter 3.3.2 --- Wortmannin induced CFP-α-TIP CT reporter-marked compartment to vacuolate --- p.69 / Chapter 3.3.3 --- BFA induced CFP-α-TIP CT reporter-marked compartment to form aggregates --- p.72 / Chapter 3.3.4 --- Wortmannin and BFA treatment caused lytic PVC to form small vacuole and Golgi to form aggregate respectively in transgenic BY-2 cell lines expressing double-reporter --- p.75 / Chapter 3.4 --- Discussion --- p.77 / Chapter 3.4.1 --- CFP-α-TIP CT reporter-marked compartment was not Golgi apparatus --- p.77 / Chapter 3.4.2 --- CFP-α-TIP CT reporter-marked compartment was not lytic PVC --- p.77 / Chapter 3.4.3 --- Transgenic BY-2 cell lines expressing double reporter could successfully mark two compartments simultaneously in the same cell --- p.78 / Chapter Chapter 4 --- Summary and Future Prospects / Chapter 4.1 --- Summary --- p.80 / Chapter 4.1.1 --- Hypothesis --- p.80 / Chapter 4.1.2 --- Development of transgenic tobacco BY-2 cell lines --- p.81 / Chapter 4.1.3 --- Characterization of α-TIP CT reporter-marked PVC-like compartment --- p.82 / Chapter 4.2 --- Conclusions --- p.84 / Chapter 4.3 --- Future prospects --- p.85 / References --- p.86

Fung Ka Leung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 83-93). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vii / Table of Contents --- p.viii / List of Tables --- p.xii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- An overview of the secretory pathway in eukaryotic cells --- p.2 / Chapter 1.2 --- The secretory pathway in plants --- p.4 / Chapter 1.2.1 --- Plant cells contain two functionally distinct vacuoles --- p.4 / Chapter 1.2.2 --- Three vesicular pathways to two vacuole --- p.6 / Chapter 1.2.3 --- Transport vesicles in the three vesicular pathways --- p.9 / Chapter 1.2.4 --- Vacuolar sorting determinants (VSDs) --- p.10 / Chapter 1.2.5 --- Vacuolar sorting receptors (VSRs) --- p.12 / Chapter 1.3 --- The PSVs in mature seeds --- p.15 / Chapter 1.3.1 --- Biogenesis of PSV --- p.15 / Chapter 1.3.2 --- The two chimeric integral membrane reporters --- p.16 / Chapter 1.3.3 --- Subcellular localization of the two chimeric integral membrane reporters in PSVs of mature tobacco seeds --- p.17 / Chapter 1.4 --- Project objectives --- p.19 / Chapter Chapter 2 --- Materials and Methods --- p.20 / Chapter 2.1 --- Construction of the YFP-BP-80 and the YFP- a -TIP reporters --- p.21 / Chapter 2.1.1 --- The pYFP-BP-80-K construct --- p.21 / Chapter 2.1.2 --- The pYFP- a -TIP-K construct --- p.22 / Chapter 2.2 --- Construction of GFP-RMR reporter --- p.23 / Chapter 2.2.1 --- Cloning of pGFP-RMR --- p.23 / Chapter 2.2.2 --- Cloning of pGFP-RMR-K --- p.23 / Chapter 2.3 --- Construction of pGONST1-YFP construct --- p.26 / Chapter 2.3.1 --- The pGONSTl-YFP construct --- p.26 / Chapter 2.4 --- Transformation of Agrobacterium by electroporation --- p.27 / Chapter 2.5 --- Tobacco transformation and selection --- p.28 / Chapter 2.5.1 --- Plant materials --- p.28 / Chapter 2.5.2 --- Tobacco transformation --- p.28 / Chapter 2.6 --- Screening of transgenic tobacco plants expressing YFP fusion proteins --- p.30 / Chapter 2.6.1 --- Kanamycin screening --- p.30 / Chapter 2.6.2 --- Extraction of genomic DNA from leaves --- p.30 / Chapter 2.6.3 --- PCR of genomic DNA --- p.31 / Chapter 2.7 --- Southern blot analysis of genomic DNA --- p.32 / Chapter 2.8 --- Western blot analysis of transgenic tobacco plants --- p.33 / Chapter 2.8.1 --- Extraction of total protein from tobacco leaves or seeds --- p.33 / Chapter 2.8.2 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis --- p.34 / Chapter 2.9 --- Confocal immunofluorescence studies --- p.35 / Chapter 2.9.1 --- Preparation of sections --- p.35 / Chapter 2.9.2 --- Single labeling --- p.35 / Chapter 2.9.3 --- Double labeling with one polyclonal and one monoclonal antibodies --- p.36 / Chapter 2.9.4 --- Double labeling with two polyclonal antibodies --- p.36 / Chapter 2.9.5 --- Collection of images --- p.37 / Chapter 2.10 --- Chemicals --- p.38 / Chapter 2.11 --- Primers --- p.38 / Chapter 2.12 --- Bacterial strain --- p.38 / Chapter 2.13 --- Antibodies --- p.39 / Chapter 2.14 --- Growing condition of transgenic plants and determining the developmental stage of tobacco flowers --- p.39 / Chapter Chapter 3 --- Results --- p.41 / Chapter 3.1 --- Generation of transgenic tobacco plants --- p.42 / Chapter 3.2 --- PCR screening of transgenic tobacco plants --- p.46 / Chapter 3.3 --- Southern blot analysis --- p.48 / Chapter 3.4 --- Detection of the YFP fusion proteins in transgenic tobacco plants by western blot analysis --- p.50 / Chapter 3.4.1 --- Detection of the YFP fusion proteins in leaves --- p.50 / Chapter 3.4.2 --- Western blot analysis of vegetative tissues --- p.57 / Chapter 3.4.3 --- Western blot analysis of mature seeds --- p.59 / Chapter 3.5 --- Confocal immunofluorescence studies --- p.61 / Chapter 3.5.1 --- Detection of YFP signals in root tip cells --- p.61 / Chapter 3.5.2 --- Detection of YFP signals in developing seeds --- p.65 / Chapter 3.5.3 --- Subcellular localization of the YFP fusion proteins in mature seeds --- p.67 / Chapter Chapter 4 --- Discussion --- p.72 / Chapter Chapter 5 --- Summary and Future Perspectives --- p.77 / Chapter 5.1 --- Summary --- p.78 / Chapter 5.1.1 --- Generation of transgenic tobacco plants expressing the YFP fusion proteins --- p.78 / Chapter 5.1.2 --- Full-length fusion proteins and cleaved soluble YFP were detected in vegetative tissues --- p.79 / Chapter 5.1.3 --- Only cleaved soluble YFP was detected in mature seeds --- p.79 / Chapter 5.1.4 --- The two fusion proteins might localized in different compartments in developing seeds --- p.79 / Chapter 5.1.5 --- Both fusion proteins were localized within the PSVs of mature seeds --- p.80 / Chapter 5.2 --- Future perspectives --- p.81 / References --- p.83

Graduation date: 1994

Brefeldin A (BFA) has been a useful tool for studying organelle dynamics and protein trafficking in plant cells. Using several Golgi (MAN1 and GONST1) and TGN (SCAMP1 and SYP61) fluorescent protein markers as tools, I have showed that BFA-induced aggregates from Golgi apparatus and TGN are morphologically distinct in the same plant cells. In addition, the internalized endosomal marker FM4-64 colocalized with the TGN-derived aggregates but separated from the Golgi aggregates. In the presence of the endocytosis inhibitor tyrphostin A23, SCAMP1 and FM4-64 are largely excluded from the TGN SYP61-positive BFA-induced aggregates, indicating homotypic fusion of TGN rather than de novo endocytic trafficking is important for the formation of TGN/EE-derived BFA-induced aggregates. Since the TGN also serves as an EE receiving materials from plasma membrane continuously, these data therefore support the notion that the secretory Golgi organelle is distinct from the endocytic TGN/EE in response to BFA treatment in plant cells. / Little is known about the trafficking mechanism of plasma membrane (PM) proteins in the endomembrane system of plant cells that contain several membrane-bound organelles including the endoplasmic reticulum (ER), Golgi, trans-Golgi network (TGN) of early endosome (EE), prevacuolar compartment (PVC) or late endosome (LE). Here, I study the transport pathway and sorting signals of secretory carrier membrane protein 1 (SCAMP1) by following its transient expression in tobacco BY-2 protoplasts and show that SCAMP1 reaches the PM via an ER-Golgi-TGN-PM pathway. Loss-of-function and gain-of-function analysis of various GFP fusions with SCAMP1 mutations further demonstrates that: (1) the cytosolic N terminus of SCAMP1 contains an ER export signal; (2) the transmembrane domain 2 (TMD2) and TMD3 of SCAMP1 are essential for Golgi export; and (3) SCAMP1 TMD1 is essential for TGN-to-PM targeting. Therefore, both the cytosolic N-terminus and TMD sequences of SCAMP1 play integral roles in mediating its transport to the PM via an ER-Golgi-TGN pathway. / Cai, Yi. / Adviser: Liwen Jiang. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 93-102). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

by Tse Yu Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 133-138). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vi / Table of Contents --- p.vii / List of Tables --- p.xi / List of Figures --- p.xii / List of Abbreviations --- p.xv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1. --- The Plant secretory pathway --- p.2 / An overview on the secretory pathway --- p.2 / Vesicular pathways and transport vesicles --- p.4 / Chapter 2. --- Vacuolar sorting receptors --- p.6 / BP-80 and its homologues --- p.6 / RMR proteins --- p.7 / Chapter 3. --- Prevacuolar compartments --- p.8 / PVCs in mammalian and yeast cells --- p.8 / PVCs for seed protein storage vacuoles --- p.9 / PVCs for lytic vacuoles --- p.11 / Chapter Chapter 2 --- Development of Transgenic Tobacco BY-2 Cell Lines Expressing Fluorescent Markers for Golgi and Prevacuolar Compartments --- p.15 / Chapter 1. --- Introduction --- p.16 / Chapter 1.1 --- Fluorescent proteins are useful tools in studying protein trafficking and subcellular localization in living cells --- p.16 / Chapter 1.2 --- Tobacco BY-2 cells --- p.18 / Chapter 1.3 --- Plant prevacuolar compartments --- p.19 / Chapter 2. --- Materials and Methods --- p.21 / Chapter 2.1 --- Construction of RFP-BP-80 and RFP-α-TIP reporters --- p.21 / Chapter 2.2 --- Construction of YFP-BP-80 and YFP-α-TIP reporters --- p.27 / Chapter 2.3 --- Construction of YFP markers for Golgi organelles --- p.32 / Chapter 2.4 --- Agrobacterium electroporation --- p.33 / Chapter 2.5 --- Transformation of tobacco BY-2 cells --- p.34 / Chapter 2.6 --- Screening of transgenic BY-2 cells expressing RFP markers --- p.35 / Chapter 2.8 --- Production of anti-BP-80 CT antibody --- p.43 / Chapter 2.9 --- Chemicals --- p.45 / Chapter 2.10 --- Primers --- p.45 / Chapter 2.11 --- Bacterial strain --- p.46 / Chapter 3. --- Results --- p.47 / Chapter 3.1 --- Generation and characterization of transgenic BY-2 cell lines expressing RFP reporters --- p.47 / Chapter 3.2 --- Generation and preliminary characterization of transgenic BY-2 cell lines expressing YFP reporters --- p.55 / Chapter 3.3 --- Confocal detection ofYFP reporters in transgenic cell lines --- p.64 / Chapter 3.4 --- Characterization of anti-BP-80 CT antibody --- p.66 / Chapter 4. --- Discussion --- p.68 / Chapter Chapter 3 --- Dynamic of Plant Prevacuolar Compartments in Transgenic Tobacco BY-2 Cells --- p.72 / Chapter 1. --- Introduction --- p.73 / Chapter 1.1 --- The plant secretory pathway --- p.73 / Chapter 1.2 --- Organelle markers in plant secretory pathway --- p.74 / Chapter 1.3 --- Markers for Lytic PVCs --- p.75 / Chapter 2. --- Materials and Methods --- p.77 / Chapter 2.1 --- Confocal immunofluorescence studies --- p.77 / Chapter 2.2 --- FM4-64 uptake study --- p.79 / Chapter 2.3 --- Brefeldin A treatment --- p.79 / Chapter 2.4 --- Wortmannin treatment --- p.80 / Chapter 2.5 --- Movement study of YFP-marked PVC --- p.82 / Chapter 3. --- Results --- p.83 / Chapter 3.1 --- Different internal organelles were labeled by two different YFP reporters --- p.83 / Chapter 3.2 --- The YFP-BP-80 reporter localized with endogenous VSR proteins --- p.86 / Chapter 3.3 --- Brefeldin A enlarged PVC organelles --- p.89 / Chapter 3.4 --- Identity of PVC-derived BFA-induced compartments --- p.99 / Chapter 3.5 --- Wortmannin induced PVCs to form small vacuoles --- p.102 / Chapter 3.6 --- PVCs are mobile organelles in living cells --- p.112 / Chapter 4. --- Discussion --- p.114 / Chapter Chapter 4 --- Summary and Future Perspectives --- p.123 / Chapter 1. --- Summary --- p.124 / The hypothesis --- p.124 / Development of three transgenic cell lines --- p.125 / Distinct organelles were marked by two different YFP reporters --- p.126 / The YFP-BP-80 reporter defined the lytic PVCs --- p.126 / Response of YFP-marked PVCs to Brefeldin A treatment --- p.127 / Response of YFP-marked PVCs to Wortmannin treatment --- p.127 / PVCs are mobile organelles in living cells --- p.129 / Chapter 2. --- Future perspectives --- p.130 / References --- p.133

Fu Lai Hong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 106-110). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vi / Lists of Figures --- p.x / Lists of Tables --- p.xiii / List of Abbreviations --- p.xiv / Amino acid abbreviation --- p.xvi / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Human α-L-iduronidase (hIDUA) --- p.2 / Chapter 1.1.1 --- Lysosomal storage disease --- p.2 / Chapter 1.1.2 --- Treatments of MPS 1 --- p.4 / Chapter 1.2 --- Plant cells as bioreactors --- p.5 / Chapter 1.3 --- The Plant secretary pathway --- p.7 / Chapter 1.3.1 --- Transport of soluble proteins --- p.9 / Chapter 1.3.2 --- Transport of integral membrane proteins --- p.10 / Chapter 1.4 --- Differences between plant and human proteins --- p.11 / Chapter 1.5 --- Reducing the differences between plant and human proteins --- p.12 / Chapter 1.6 --- Previous study: Expression of IDUA in transgenic tobacco plant --- p.13 / Chapter 1.7 --- Project objectives --- p.14 / Chapter 1.8 --- Long term significance --- p.14 / Chapter Chapter 2 --- Materials and Methods --- p.15 / Chapter 2.1 --- Introduction --- p.16 / Chapter 2.2 --- Materials --- p.18 / Chapter 2.2.1 --- Chemical --- p.18 / Chapter 2.2.2 --- Plant materials --- p.18 / Chapter 2.2.3 --- Plasmid vectors and bacterial strains --- p.18 / Chapter 2.2.4 --- Human a-iduronidase (hIDUA) cDNA --- p.19 / Chapter 2.2.5 --- Primers --- p.20 / Chapter 2.3 --- Methods --- p.22 / Chapter 2.3.1 --- Generation of IDUA antibodies --- p.22 / Chapter 2.3.1.1 --- Synthetic peptide raised IDUA antibodies --- p.23 / Chapter 2.3.1.1.1 --- Design of synthetic peptides --- p.23 / Chapter 2.3.1.1.2 --- Immunization of rabbits --- p.25 / Chapter 2.3.1.2 --- E. coli-derived rhIDUA protein --- p.25 / Chapter 2.3.1.2.1 --- Cloning and expression of rhIDUA --- p.25 / Chapter 2.3.1.2.2 --- Western analysis of E. coli-derived rhIDUA --- p.29 / Chapter 2.3.1.2.3 --- MS/MS analysis of rhIDUA protein --- p.29 / Chapter 2.3.1.2.4 --- Immunization of rabbits --- p.31 / Chapter 2.3.2 --- Affinity-purified antibodies --- p.33 / Chapter 2.3.3 --- Characterization of affinity-purified IDUA antibodies --- p.33 / Chapter 2.3.4 --- Construction of chimeric gene constructs --- p.34 / Chapter 2.3.5 --- Expression of IDUA in tobacco BY-2 cells --- p.39 / Chapter 2.3.5.1 --- Electropoartion of Agrobacteria --- p.39 / Chapter 2.3.5.2 --- Agrobacterium-mediated transformation --- p.39 / Chapter 2.3.5.3 --- Screening of positive trans formants --- p.40 / Chapter 2.3.6 --- Characterization of transgenic BY-2 cell expressing IDUA fusion --- p.40 / Chapter 2.3.6.1 --- Genomic DNA polymerase chain reaction (Genomic DNA PCR) --- p.40 / Chapter 2.3.6.1.1 --- Genomic DNA extraction from BY-2 callus --- p.40 / Chapter 2.3.6.1.2 --- Genomic DNA PCR of tobacco BY-2 callus --- p.41 / Chapter 2.3.6.2 --- Reverse transcription-PCR (RT-PCR) --- p.42 / Chapter 2.3.6.2.1 --- Total RNA extraction from BY-2 cell --- p.42 / Chapter 2.3.6.2.2 --- RT-PCR of BY-2 cell --- p.42 / Chapter 2.3.6.3 --- Western blot analysis of BY-2 cell and medium --- p.43 / Chapter 2.3.6.3.1 --- Protein extraction from tobacco BY-2 cells and culture medium --- p.43 / Chapter 2.3.6.3.2 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.44 / Chapter 2.3.6.3.3 --- Immunodetection and Coomassie blue stain --- p.44 / Chapter 2.3.7 --- Purification of IDUA from culture media --- p.46 / Chapter Chapter 3 --- Results --- p.47 / Chapter 3.1 --- Generation of IDUA antibodies --- p.48 / Chapter 3.1.1 --- Cloning and expression of rhIDUA in E. coli --- p.48 / Chapter 3.1.2 --- Characterization of IDUA antibodies --- p.51 / Chapter 3.1.2.1 --- Specificity of IDUA antibodies towards hIDUA protein. --- p.51 / Chapter 3.1.2.2 --- Cross-reactivity of IDUA antibodies with wild type tobacco BY-2 cell --- p.55 / Chapter 3.2 --- Chimeric gene constructs construction and confirmation --- p.58 / Chapter 3.3 --- Screening of transformed tobacco BY-2 callus with kanamycin-resistance --- p.66 / Chapter 3.4 --- Genomic DNA PCR screening of transformed tobacco BY-2 callus . --- p.67 / Chapter 3.5 --- RT-PCR screening of transformed BY-2 cells --- p.70 / Chapter 3.6 --- Western blot analysis of transformed tobacco BY-2 cells and culture media --- p.72 / Chapter 3.6.1 --- Tobacco BY-2 cells --- p.72 / Chapter 3.6.2 --- Tobacco BY-2 cell culture media --- p.76 / Chapter 3.7 --- Purification of IDUA protein in culture media --- p.81 / Chapter Chapter 4 --- Discussion --- p.82 / Chapter Chapter 5 --- Summary and Future Perspectives --- p.89 / Chapter 5.1 --- Summary --- p.90 / Chapter 5.2 --- Future perspectives --- p.92 / Appendix Identification and Characterization of an Unknown Protein by 1B Antibody --- p.93 / Chapter 6.1 --- Introduction --- p.94 / Chapter 6.2 --- Objectives --- p.94 / Chapter 6.3 --- Materials and Methods --- p.95 / Chapter 6.3.1 --- Western blot analysis of different plant species --- p.95 / Chapter 6.3.2 --- Subcellular localization of the unknown protein --- p.95 / Chapter 6.3.3 --- Affinity-purification of the unknown protein --- p.95 / Chapter 6.4 --- Results --- p.97 / Chapter 6.4.1 --- Western blot analysis of different plant species --- p.97 / Chapter 6.4.2 --- Subcellular localization of an unknown protein --- p.98 / Chapter 6.4.3 --- Affinity-purification of 1B protein --- p.104 / Chapter 6.5 --- Summary and Future Perspectives --- p.105 / Chapter 6.5.1 --- Summary --- p.105 / Chapter 6.5.2 --- Future Perspectives --- p.105 / References --- p.106

Submitted in fulfillment of the requirements for the Degree of Master of Technology: Biotechnology, Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, South Africa,2009. / Nearly half of all individuals living with HIV worldwide at present are woman and the best current strategy to prevent sexually transmitted HIV is antiretrovirals (ARVs). Microbicides are ARV’s which directly target viral entry and avert infection at mucosal surfaces. However, most promising ARV entry inhibitors are biologicals which are costly to manufacture and deliver to resource-poor areas. Microbicides formulated as simple gels, which are currently not commonly used in ARV therapy, show immense potential for use in prevention and treatment of multidrug-resistant viral infections in developing countries. Among the most potent HIV entry inhibitory molecules are lectins, which target the high mannose N-linked glycans which are displayed on the surface of HIV envelope glycoproteins. Of the microbicides, the red algal protein griffithsin (GRFT) has potent anti-HIV inhibitory activity and is active by targeting the terminal mannose residues on high mannose oligosaccharides. It has a total of 6 carbohydrate binding sites per homodimer, which likely accounts for its unparalleled potency. The antiviral potency of GRFT, coupled with its lack of cellular toxicity and exceptional environmental stability make it an ideal active ingredient of a topical HIV microbicide. v Scytovirin (SVN) is an equally potent anti-HIV protein, isolated from aqueous extracts of the cyanbacterium, Scytonema varium. Low, nanomolar concentrations of SVN have been reported to inactivate laboratory strains and primary isolates of HIV- 1. The inhibition of HIV by SVN involves interactions between the protein and HIV-1 envelope glycoproteins gp120, gp160 and gp41. Current recombinant production methods for GRFT and SVN molecules are unfortunately hampered by inadequate production capacities. This project therefore aimed to determine if these molecules can be produced in plant cell culture systems. The transgenic tobacco cell culture system was evaluated to determine if it can be an alternative, cost effective production system for these molecules. Results of the study show that the microbicide genes can be cloned into plant transformation vectors, used to successfully transform SR1 tobacco cell lines and adequately produce 3.38ng and 10.5ng of GRFT and SVN protein respectively, per gram of SR1 tobacco callus fresh weight. The promising results attained in this study form the basis for further work in optimising plant cell based production systems for producing valuable anti-HIV microbicides, a possible means to curbing the elevated HIV infection rates worldwide. / CSIR

Submitted in fulfillment of the requirements for the Degree of Master of Technology: Biotechnology, Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, South Africa,2009. / Nearly half of all individuals living with HIV worldwide at present are woman and the best current strategy to prevent sexually transmitted HIV is antiretrovirals (ARVs). Microbicides are ARV’s which directly target viral entry and avert infection at mucosal surfaces. However, most promising ARV entry inhibitors are biologicals which are costly to manufacture and deliver to resource-poor areas. Microbicides formulated as simple gels, which are currently not commonly used in ARV therapy, show immense potential for use in prevention and treatment of multidrug-resistant viral infections in developing countries. Among the most potent HIV entry inhibitory molecules are lectins, which target the high mannose N-linked glycans which are displayed on the surface of HIV envelope glycoproteins. Of the microbicides, the red algal protein griffithsin (GRFT) has potent anti-HIV inhibitory activity and is active by targeting the terminal mannose residues on high mannose oligosaccharides. It has a total of 6 carbohydrate binding sites per homodimer, which likely accounts for its unparalleled potency. The antiviral potency of GRFT, coupled with its lack of cellular toxicity and exceptional environmental stability make it an ideal active ingredient of a topical HIV microbicide. v Scytovirin (SVN) is an equally potent anti-HIV protein, isolated from aqueous extracts of the cyanbacterium, Scytonema varium. Low, nanomolar concentrations of SVN have been reported to inactivate laboratory strains and primary isolates of HIV- 1. The inhibition of HIV by SVN involves interactions between the protein and HIV-1 envelope glycoproteins gp120, gp160 and gp41. Current recombinant production methods for GRFT and SVN molecules are unfortunately hampered by inadequate production capacities. This project therefore aimed to determine if these molecules can be produced in plant cell culture systems. The transgenic tobacco cell culture system was evaluated to determine if it can be an alternative, cost effective production system for these molecules. Results of the study show that the microbicide genes can be cloned into plant transformation vectors, used to successfully transform SR1 tobacco cell lines and adequately produce 3.38ng and 10.5ng of GRFT and SVN protein respectively, per gram of SR1 tobacco callus fresh weight. The promising results attained in this study form the basis for further work in optimising plant cell based production systems for producing valuable anti-HIV microbicides, a possible means to curbing the elevated HIV infection rates worldwide.