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This thesis is concerned with the development of new synthetic routes for the asymmetric syntheses of a range of β- and γ-amino acids. Chapter 1 introduces the various biological activities displayed by cyclic β-amino acid containing compounds together with their occurrence in pharmaceutical molecules and β-peptides. Some of the most commonly used synthetic strategies for the preparation of carbocyclic β-amino acids are briefly described, with the focus on the formation and functionalisation of a five-membered carbocyclic ring. Chapter 2 describes a full investigation into a highly diastereoselective Ireland-Claisen rearrangement of stereodefined allyl β-amino esters to access enantiopure α-substituted β-amino acid products. The synthetic utility of this methodology is highlighted by its application in the asymmetric syntheses of five previously inaccessible C(5)-substituted 1,2-anti-1,5-syn-transpentacins. Chapter 3 delineates investigations into a highly diastereoselective conjugate additionelimination protocol for the preparation of a cyclic β'-amino-α,β-unsaturated ester. Subsequent chemo- and diastereoselective conjugate addition reactions of Grignard reagents and lithium amides to this substrate enabled the asymmetric syntheses of four C(5)-substituted 1,2-anti-1,5-syn-transpentacins and two five-membered β,β'-diamines. Chapter 4 details the extension of the protocol developed in Chapter 3 for the conjugate addition of Grignard reagents to a range of acyclic γ-(N,N-dibenzylamino)-substituted α,β-unsaturated esters. Elaboration of the β,γ-disubstituted γ-amino ester products culminated in the asymmetric syntheses of six β,γ-disubstituted γ-amino acids. Chapter 5 chronicles the preparation of an azabicyclic α,β-unsaturated ester, following which attempts towards the asymmetric synthesis of various substituted pyrrolizidines using a conjugate addition protocol are subsequently described. Chapter 6 contains full experimental procedures and characterisation data for all compounds synthesised in Chapters 2, 3, 4 and 5.

by Daron Adam Fincham. / Bibliography: leaves [84-99] / Thesis (M.Phil.) -- Chinese University of Hong Kong, 1983

Digitized by Kansas Correctional Industries

BURP域家族是植物特有的一個蛋白家族，結構多樣，共同點是在羧基端都有一個保守的BURP域。迄今為止，關於BURP域家族成員的功能及細胞定位的研究非常有限。部分RD22-like的亞家族成員由於顯示出受非生物脅迫而誘導表達的特性，因此被認為功能可能與非生物脅迫回應相關。本研究對克隆到的一個受非生物脅迫誘導表達的大豆基因(GmRD22)進行了生物進化分析，并詳細分析了其在不同的大豆品種以及不同非生物脅迫下的表達模式，揭示了其表達豐度與大豆的抗非生物脅迫能力有關，並且使用不同的轉基因系統(細胞水準跟植物水準)揭示了其過量表達有助於減輕非生物脅迫對植物造成的影響。研究利用GFP融合蛋白追蹤技術和免疫電鏡技術揭示GmRD22蛋白定位於細胞壁，其中BURP域對於GmRD22定位于細胞壁起到關鍵作用。研究也揭示了GmRD22能夠與細胞外的一種過氧化物酶GmPer1相互作用，GmRD22在轉基因擬南芥和轉基因水稻中的過量表達能夠顯著提高脅迫條件下轉基因植株中木質素的含量。我們認為GmRD22通過與細胞壁過氧化物酶的相互作用來提高植物在脅迫條件下細胞壁的完整性從而增強植株的抗性。 / The BURP-domain protein family comprises a diverse group of plant-specific proteins that share a conserved BURP domain at the C terminus. However, there have been only limited studies on the functions and subcellular localization of these proteins. Members of the RD22-like subfamily are postulated to associate with stress responses due to the stress-inducible nature of some RD22-like genes. In this report, different expression patterns of a stress-inducible RD22-like protein from soybean (GmRD22) either in different soybean species or under different osmotic stress conditions were analyzed, different transgenic systems (cells and in planta) were used to show that the ectopic expression of GmRD22 can alleviate salinity and osmotic stress. The detailed microscopic studies were also performed using both fusion proteins and immuno-electron microscopic techniques to demonstrate the apoplast localization of GmRD22, for which the BURP domain is a critical determinant of the subcellular localization. The apoplastic GmRD22 interacts with a cell wall peroxidase and the ectopic expression of GmRD22 in both transgenic A. thaliana and transgenic rice resulted in increased lignin production when subjected to salinity stress. It is possible that GmRD22 regulates cell wall peroxidase and hence strengthens cell wall integrity under osmotic stress conditions. / Detailed summary in vernacular field only. / Wang, Hongmei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 124-136). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / Acknowledgements --- p.iii / Table of contents --- p.v / List of tables --- p.x / List of figures --- p.xi / General abbreviations --- p.xiii / Chemical abbreviations --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Abiotic stress in the world --- p.2 / Chapter 1.2 --- The advances of plant abiotic stress resistance mechanisms --- p.5 / Chapter 1.2.1 --- Sensor of salt stress --- p.7 / Chapter 1.2.2 --- Reestablishment of ionic homeostasis --- p.9 / Chapter 1.2.3 --- Osmoregulation by compatible osmolytes --- p.11 / Chapter 1.2.4 --- Oxidative stress management --- p.12 / Chapter 1.2.5 --- Transcription regulation of gene expression in osmotic stress --- p.14 / Chapter 1.3 --- The BURP-domain protein family --- p.19 / Chapter 1.3.1 --- Introduction of BURP-domain protein family --- p.19 / Chapter 1.3.2 --- Advances of BURP-domain protein studies --- p.20 / Chapter 1.3.3 --- The BURP-domain protein and osmotic stress --- p.21 / Chapter 1.4 --- Background information of this project --- p.22 / Chapter 1.5 --- Hypothesis and objectives --- p.25 / Chapter Chapter 2 --- Materials and Methods --- p.26 / Chapter 2.1 --- Bacterial strains, vectors and plasmids, cell lines and plant materials --- p.27 / Chapter 2.2 --- Chemicals and reagents --- p.32 / Chapter 2.3 --- Primers used in this study --- p.35 / Chapter 2.4 --- Molecular cloning of GmRD22 --- p.38 / Chapter 2.5 --- DNA and RNA extraction and Northern blot --- p.40 / Chapter 2.5.1 --- DNA and plasmid extraction --- p.40 / Chapter 2.5.2 --- RNA extraction from plant --- p.41 / Chapter 2.5.3 --- Generation of DIG-labeled PCR probe --- p.41 / Chapter 2.5.4 --- Northern blot --- p.43 / Chapter 2.6 --- Reverse transcription and Real-time analysis --- p.44 / Chapter 2.7 --- Phylogenetic analysis --- p.45 / Chapter 2.8 --- Basic molecular techniques --- p.46 / Chapter 2.8.1 --- Recombinant DNA --- p.46 / Chapter 2.8.2 --- Transformation of E. coli competent cells --- p.46 / Chapter 2.8.3 --- Transformation of A. tumefacien competent cells --- p.47 / Chapter 2.8.4 --- Gel electrophoresis --- p.48 / Chapter 2.8.5 --- Sequencing --- p.48 / Chapter 2.9 --- Establishment of transgenic models --- p.49 / Chapter 2.9.1 --- Establishment of transgenic BY-2 cell --- p.49 / Chapter 2.9.2 --- Establishment of transgenic A. thaliana --- p.50 / Chapter 2.9.3 --- Establishment of transgenic rice --- p.51 / Chapter 2.10 --- Cell viability assay under osmotic stress treatment --- p.51 / Chapter 2.11 --- Root elongation assay of transgenic A. thaliana --- p.52 / Chapter 2.12 --- Osmotic stresses treatment of transgenic rice lines --- p.52 / Chapter 2.13 --- Protein expression, production of antibodies and Western blot --- p.53 / Chapter 2.14 --- Subcellular localization of fusion protein by confocal microscopic study --- p.55 / Chapter 2.15 --- Electron microscopic study --- p.56 / Chapter 2.16 --- Immunoprecipitation and mass spectrometry --- p.57 / Chapter 2.17 --- Cell wall components analysis --- p.60 / Chapter 2.18 --- Statistical analysis --- p.61 / Chapter Chapter 3 --- Results --- p.62 / Chapter 3.1 --- GmRD22 gene --- p.63 / Chapter 3.1.1 --- GmRD22 encodes a BURP-domain protein in soybean --- p.63 / Chapter 3.1.2 --- Phylogenetic analysis of GmRD22 --- p.65 / Chapter 3.2 --- GmRD22 gene expression --- p.73 / Chapter 3.2.1 --- GmRD22 shows a biphasic induction by salinity stress and ABA treatment --- p.73 / Chapter 3.2.2 --- GmRD22 is also inducible by osmotic stress --- p.76 / Chapter 3.2.3 --- GmRD22 shows stronger and faster induction in WF 7 than Union --- p.76 / Chapter 3.3 --- Functional study --- p.78 / Chapter 3.3.1 --- Construction of GmRD22 transformants --- p.78 / Chapter 3.3.2 --- Ectopic expression of GmRD22 improve osmotic stresses tolerance in transgenic BY-2 cells --- p.80 / Chapter 3.3.3 --- Ectopic expression of GmRD22 alleviates osmotic stresses in transgenic A. thaliana --- p.83 / Chapter 3.3.4 --- Ectopic expression of GmRD22 alleviates osmotic stresses in transgenic rice --- p.86 / Chapter 3.4 --- GmRD22 is an apoplastic protein --- p.90 / Chapter 3.4.1 --- Western blot analysis in different soybean extracts --- p.90 / Chapter 3.4.2 --- Subcellular localization of GmRD22-GFP fusion protein in onion epidermal and A. thaliana root system --- p.93 / Chapter 3.4.3 --- GmRD22 localization in native soybean --- p.96 / Chapter 3.5 --- BURP domain is essential for the subcellular localization --- p.99 / Chapter 3.6 --- GmRD22 interacts with a putative apoplastic peroxidase --- p.103 / Chapter 3.6.1 --- Identification of GmRD22 interacting protein --- p.103 / Chapter 3.6.2 --- GmPer1 is a putative extracellular class III peroxidase --- p.107 / Chapter 3.6.3 --- Overexpression of GmRD22 affected lignin metabolism in transgenic rice and A. thaliana under salinity stress --- p.109 / Chapter 3.6.4 --- GmPer1 homologues increased expression under salinity stress --- p.113 / Chapter Chapter 4 --- Discussion and Conclusion --- p.115 / Chapter 4.1 --- GmRD22 as a member of RD22-like subfamily --- p.116 / Chapter 4.2 --- Induction mechanism of GmRD22 expression is related to ABA --- p.117 / Chapter 4.3 --- Biological function of GmRD22 providing protective effect under osmotic stress --- p.118 / Chapter 4.4 --- The BURP domain of GmRD22 plays a key role in its apoplastic targeting --- p.119 / Chapter 4.5 --- The interaction between GmRD22 and apoplastic peroxidase provides the clue for the mechanism of enhanced osmotic stress tolerance in GmRD22 transgenic plants --- p.120 / Chapter 4.6 --- Conclusion --- p.123 / References --- p.124 / Chapter Appendix I --- Restriction and modifying enzymes --- p.137 / Chapter Appendix II --- Chemicals --- p.138 / Chapter Appendix III --- Buffer, solution, gel and medium formulation --- p.143 / Chapter Appendix IV --- Equipment and facilities --- p.146

Introduction The association of the HLA shared epitope with rheumatoid arthritis (RA) in black South Africans is well established. The aims of the thesis were to identify non-HLA risk loci associated with RA using the Immunochip array and to assess the association of specific amino acids at specific positions of the DRB1 locus with the risk for developing RA in black South Africans. Methods Ethnically and geographically matched RA cases (n=435) and controls (n=463) were genotyped using the Immunochip array which has ~196 000 single nucleotide polymorphisms (SNPs) previously associated with 12 autoimmune diseases. After quality control, 103 700 SNPs were tested for association in 263 cases and 374 controls. For the amino acid study, DNA sequencing of exon 2 of the DRB1 locus was performed on the seropositive cases (n=261) using the Allele SEQR HLA-DRB1 (Abbot) assay and four digit HLA typing was assigned using the Assign software (Conexio Genomics). The amino acid sequences were inferred from the called allele type. Association testing and conditional analysis were performed. Results A total of 72 HLA SNPs reached statistical significance (p< 5x10-8) of which 71 SNPs locate to the HLA-DR or DQ genes or to the intergenic region between these two genes. Specifically, 4 SNPs in the intergenic region between HLA-DRB1 and HLA-DQA1 (rs3104413, OR=3.88, p=5.49x10-21; rs3129769, OR=3.91, p=4.60x10-21; rs6931277, OR=3.97, p=1.03x10-21; rs9272353, OR=4.1, p=3.01x10-21) were highly associated. Ten non-HLA SNPs on chromosome 6 reached significance of which 7 SNPs locate to the intergenic region between BTNL2 and HLA-DRA and confer protection. Four “SNPs” on chromosome 1 locating to a copy number variant region of the intergenic region between PLD5 and LOC400723 were significantly associated with RA in this study (rs2809867, OR=0.33, p=5.01x10-08; rs12738883, OR=0.33, p=3.98x10-08; rs78352781, OR=0.33, p=3.98x10-08; rs12739315, OR=0.33, p=3.98x10-08) and showed an even stronger association with the rheumatoid factor positive subgroup. Valine at amino acid position 11 of DRB1 demonstrated the strongest associated with RA (OR=5.1(3.7, 7.0), p=1.63x10 -27) and serine at this position was protective (OR=0.4(0.3, 0.5), p=2.46x10 -16). Interestingly, the frequency of valine in black South African RA cases was much lower than in Caucasians with RA. Using principal component analysis, black South Africans were found to be genetically distinct from west and east African populations. Conclusion This study demonstrated both similarities and differences in the risk for RA between Caucasians and black South Africans. Similar to Caucasians, the HLA region confers the strongest genetic risk for RA in black South Africans. More specifically, valine at position 11 of DRB1 confers the strongest risk for RA in black South Africans and serine confers protection. Four novel non-HLA RA-associated SNPs in the intergenic region between LOC400723 and PLD5 were identified. Variants of the PTPN22 gene, although strongly associated with RA in Caucasians, are not associated with RA in this population. Since this population is genetically distinct, the findings need to be independently validated in other African populations.