Cheung Ming-yan. / Thesis submitted in 2000. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 169-185). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgement --- p.vi / General abbreviations --- p.viii / Abbreviations of chemicals --- p.x / List of figures --- p.xii / List of tables --- p.xv / Table of Contents --- p.xvii / Chapter Chapter 1 --- General Introduction - Using transgenic plants as bioreactor --- p.1 / Chapter 1.1 --- Plant as Bioreactor --- p.1 / Chapter 1.1.1 --- Plant transformation historical milestones --- p.1 / Chapter 1.1.2 --- Applications of transgenic plants --- p.5 / Chapter 1.1.2.1 --- Examples of in situ Application --- p.5 / Chapter 1.1.2.2 --- Examples of ex situ application of transgenic plant --- p.9 / Chapter 1.2 --- Plant Hosts for Transformation: Arabidopsis thaliana and Glycine max --- p.18 / Chapter 1.2.1 --- Essential components for plant transformation --- p.18 / Chapter 1.2.1.1 --- Marker genes --- p.18 / Chapter 1.2.1.2 --- Promoters --- p.18 / Chapter 1.2.2 --- Arabidopsis thaliana --- p.20 / Chapter 1.2.2.1 --- Agrobacterium-mediated transformation --- p.20 / Chapter 1.2.2.2 --- Transformation methods for A. thaliana --- p.21 / Chapter 1.2.3 --- Glycine max (soybean) --- p.22 / Chapter 1.2.3.1 --- Soybean cultivars for transformation --- p.23 / Chapter 1.2.3.2 --- Soybean regeneration systems --- p.24 / Chapter 1.2.3.3 --- Soybean transformation systems --- p.26 / Chapter 1.3 --- Target Pharmaceutical and Agricultural Proteins: Lymphocytic choriomeningitis virus and Goldfish Growth hormones I and II --- p.29 / Chapter 1.3.1 --- Production of pharmaceutical proteins --- p.29 / Chapter 1.3.1.1 --- Lymphocytic choriomeningitis virus --- p.30 / Chapter 1.3.1.2 --- Nucleoprotein of LCMV --- p.33 / Chapter 1.3.2 --- Agricultural protein category --- p.34 / Chapter 1.3.2.1 --- Carassius auratus --- p.34 / Chapter 1.3.2.2 --- Growth hormones I and II --- p.35 / Chapter 1.4 --- Hypothesis and Objectives --- p.37 / Chapter Chapter 2 --- Materials and Methods --- p.38 / Chapter 2.1 --- Materials --- p.38 / Chapter 2.1.1 --- "Plants, bacterial strains and vectors" --- p.38 / Chapter 2.1.2 --- Chemicals and Regents --- p.43 / Chapter 2.1.3 --- Commercial kits --- p.44 / Chapter 2.1.4 --- Primers and Adaptors --- p.45 / Chapter 2.1.5 --- Equipments and Facilities used --- p.47 / Chapter 2.1.6 --- "Buffer, solution and medium" --- p.47 / Chapter 2.2 --- Methods --- p.48 / Chapter 2.2.1 --- Molecular Techniques --- p.48 / Chapter 2.2.1.1 --- Bacterial cultures for recombinant DNA and plant transformation --- p.48 / Chapter 2.2.1.2 --- Recombinant DNA techniques --- p.48 / Chapter 2.2.1.3 --- "Preparation and transformation of DH5a, DE3 and Agrobacterium competent cells" --- p.49 / Chapter 2.2.1.4 --- Gel electrophoresis --- p.52 / Chapter 2.2.1.5 --- "DNA, RNA and protein extractions" --- p.53 / Chapter 2.2.1.6 --- Generation of cRNA probes for Southern and Northern blot analyses --- p.56 / Chapter 2.2.1.7 --- Southern blot analysis --- p.56 / Chapter 2.2.1.8 --- Northern blot analysis --- p.57 / Chapter 2.2.1.9 --- Expression of Lymphocytic choriomeningitis virus nucleoprotein (LCMV NP) in bacterial system --- p.58 / Chapter 2.2.1.10 --- Western blot analysis for LCMV NP --- p.59 / Chapter 2.2.1.11 --- Protein dot blot for detecting the presence of recombinant LCMV-NP generated from transgenic plants --- p.62 / Chapter 2.2.1.12 --- PCR techniques --- p.62 / Chapter 2.2.1.13 --- Sequencing --- p.63 / Chapter 2.2.2 --- Plant tissue culture and transformation --- p.64 / Chapter 2.2.2.1 --- Arabidopsis thaliana --- p.64 / Chapter 2.2.2.2 --- Soybean --- p.65 / Chapter 2.2.3 --- In vitro transcription and translation of target genes in rabbit reticulocyte and wheat germ systems --- p.68 / Chapter 2.2.3.1 --- In vitro transcription of target genes with with Ribomix large scale RNA production systems-T7 and SP6 (Promega) --- p.68 / Chapter 2.2.3.2 --- In vitro translation with rabbit reticulocyte lysate and wheat germ extract --- p.69 / Chapter Chapter 3 --- Results --- p.71 / Chapter 3.1 --- Expression of Lymphocytic choriomeningitis virus nucleoprotein (LCMV NP) and goldfish growth hormones I and II (GHI and GHII) in transgenic Arabidopsis thaliana --- p.71 / Chapter 3.1.1 --- Expression of LCMV-NP in transgenic Arabidopsis thaliana --- p.71 / Chapter 3.1.1.1 --- Cloning of the gene encoding LCMV NP into the binary vector system W104 --- p.71 / Chapter 3.1.1.2 --- Transformation of W104-LCMV-NP into the Agrobacterium GV3101/pMP90 --- p.78 / Chapter 3.1.1.3 --- Transformation of LCMV-NP cDNA into Arabidopsis thaliana --- p.80 / Chapter 3.1.1.4 --- Southern blot and Northern blot analyses of transgenic plant containing the LCMV-NP cDNA --- p.83 / Chapter 3.1.1.5 --- Production of recombinant LCMV-NP protein in DE3 cells --- p.90 / Chapter 3.1.1.6 --- Detection of recombinant LCMV-NP protein in transgenic A.thaliana --- p.98 / Chapter 3.1.2 --- Expression of goldfish growth hormones I and II (GHI and GHII) in transgenic Arabidopsis thaliana --- p.105 / Chapter 3.1.2.1 --- "Screening of homozygous lines of goldfish, Carassius auratus, growth hormones transgenic Arabidopsis thaliana" --- p.105 / Chapter 3.1.2.2 --- Southern blot and Northern blot analyses of transgenic plant containing the LCMV-NP cDNA --- p.109 / Chapter 3.1.2.3 --- Detection of recombinant GHI and GHII from transgenic plant --- p.112 / Chapter 3.2 --- In vitro transcription and translation of target genes in rabbit reticulocyte and wheat germ systems --- p.117 / Chapter 3.2.1 --- Subcloning of target genes in pGEM-3Zf(+) vector --- p.117 / Chapter 3.2.1.1 --- Subcloning of LCMV-NP fragment into pGEM-3Zf(+) vector --- p.117 / Chapter 3.2.1.2 --- Subcloning of goldfish GHI and GHII fragments into pGEM-3Zf(+) vector --- p.120 / Chapter 3.2.2 --- In vitro transcription of target genes with Ribomix large scale RNA production systems-T7 and SP6 --- p.125 / Chapter 3.2.3 --- In vitro translation with rabbit reticulocyte lysate and wheat germ extract systems --- p.128 / Chapter 3.3 --- Establishment of Glycine max regeneration and transformation systems --- p.130 / Chapter 3.3.1 --- The Establishment of soybean regeneration system --- p.130 / Chapter 3.3.2 --- Establishment of soybean transformation system --- p.133 / Chapter 3.3.2.1 --- Definition of transformation efficiency --- p.133 / Chapter 3.3.2.2 --- Effects of plant hosts --- p.136 / Chapter 3.3.2.3 --- Effects of Agrobacterium strains --- p.138 / Chapter 3.3.2.4 --- The application of vacuum infiltration --- p.139 / Chapter 3.3.2.5 --- Effect of kanamycin --- p.140 / Chapter 3.3.2.6 --- Effect of cocultivation duration and light/ dark treatment during germination --- p.141 / Chapter 3.3.2.7 --- Application of the detergent Silwet-77 --- p.142 / Chapter 3.3.3 --- Verification of transformation results by PCR screening --- p.143 / Chapter Chapter 4 --- Discussion --- p.147 / Chapter 4.1 --- "Expression of LCMV-NP, GHI and GHII in A. thaliana" --- p.148 / Chapter 4.2 --- Establishing a soybean transformation system --- p.157 / Chapter 4.2.1 --- Plant hosts and explants --- p.158 / Chapter 4.2.2 --- Regeneration of explants --- p.159 / Chapter 4.2.3 --- Agrobacterium strains --- p.161 / Chapter 4.2.4 --- Bacteria-plant interaction --- p.161 / Chapter 4.2.5 --- Transient versus stable transformation --- p.165 / Chapter 4.3 --- Conclusion and perspective --- p.167 / References --- p.169 / Appendix --- p.186

Chiao Ying Ann. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 127-140). / Abstracts in English and Chinese. / Thesis Committee --- p.I / Statement --- p.II / Abstract --- p.III / 摘要 --- p.V / Acknowledgements --- p.VII / Abbreviations --- p.IX / Abbreviation of chemicals --- p.XI / Table of Contents --- p.XII / List of figures and tables --- p.XVIII / Chapter Chapter 1. --- Literature review / Chapter 1.1 --- Significances of manipulation of nitrogen sink-source relationship --- p.1 / Chapter 1.2 --- Nitrogen sink-source relationship in plants --- p.2 / Chapter 1.3 --- Aspartate family amino acid metabolism --- p.5 / Chapter 1.3.1 --- Asparagine metabolism --- p.9 / Chapter 1.3.1.1 --- "Asparagine synthetase (AS, EC 6.3.5.4)" --- p.9 / Chapter 1.3.1.2 --- "Asparaginase (ANS, EC 3.5.1.1)" --- p.10 / Chapter 1.3.2 --- Metabolism of aspartate-derived essential amino acids --- p.10 / Chapter 1.3.2.1 --- "Aspartate kinase (AK, EC 2.7.2.4)" --- p.10 / Chapter 1.3.2.2 --- "Homoserine dehydrogenase (HSD, EC 1.1.1.3)" --- p.12 / Chapter 1.3.2.3 --- "Dihydrodipicolinate synthase (DHPS, EC 4.2.1.52)" --- p.13 / Chapter 1.3.2.4 --- "Lysine a-ketoglutarate reductase (LKR, EC 1.5.1.7)" --- p.14 / Chapter 1.3.2.5 --- "Threonine synthase (TS, EC 4.2.3.1)" --- p.15 / Chapter 1.3.2.6 --- Cystathionine γ-synthase (CGS，EC 2.5.1.48) --- p.16 / Chapter 1.3.2.7 --- Threonine deaminase (TD，EC 4.3.1.19) --- p.17 / Chapter 1.4 --- Previous attempts to manipulate seed protein quantity and quality --- p.18 / Chapter 1.4.1 --- Enhancement of amino acids transported from source to sink --- p.18 / Chapter 1.4.2 --- Redirection of metabolic pathways to increase target amino acids --- p.19 / Chapter 1.4.2.1 --- Production of aspartate by Aspartate Aminotransferase (AAT) --- p.24 / Chapter 1.4.2.2 --- Deregulation of AK to increase the common substrate for all essential aspartate family amino acids --- p.25 / Chapter 1.4.2.3 --- Inhibition of TS and enhancement of CGS to increase Met biosynthesis --- p.25 / Chapter 1.4.2.3.1 --- Inhibition of TS --- p.26 / Chapter 1.4.2.3.2 --- Enhancement of CGS --- p.26 / Chapter 1.4.2.4 --- Deregulation of DHPS and reduction of lysine catabolism to increase lysine content --- p.27 / Chapter 1.4.2.4.1 --- Deregulation of DHPS --- p.28 / Chapter 1.4.2.4.2 --- Reduction of Lys catabolism --- p.29 / Chapter 1.4.2.3.3 --- Deregulation of DHPS and reduction of LKR --- p.29 / Chapter 1.4.3 --- Expression of seed storage proteins to entrap the free amino acids --- p.30 / Chapter 1.5 --- Expression of multiple transgenes in plants --- p.34 / Chapter 1.5.1 --- Significance of multiple genes manipulation in seed quality improvement --- p.34 / Chapter 1.5.2 --- Difficulties in introduction of multiple genes into plant genomes --- p.34 / Chapter 1.5.3 --- Recent advances in introduction of multiple genes into plant genome --- p.35 / Chapter 1.6 --- Global nitrogen regulators in plants --- p.36 / Chapter 1.6.1 --- Global regulation of nitrogen metabolism --- p.36 / Chapter 1.6.2 --- General amino acid control by GCN system --- p.38 / Chapter 1.6.3 --- General amino acid control in plants --- p.39 / Chapter 1.6.4 --- GCN system in plants --- p.41 / Chapter 1.7 --- Hypothesis and specific objectives of this study --- p.42 / Chapter Chapter 2 --- Materials and methods --- p.46 / Chapter 2.1 --- Materials --- p.46 / Chapter 2.1.1 --- "Vectors, bacterial strains and plants" --- p.46 / Chapter 2.1.2 --- Chemicals and reagents used --- p.49 / Chapter 2.1.3 --- "Buffer, solution, gel and medium" --- p.49 / Chapter 2.1.4 --- Commercial kits used --- p.49 / Chapter 2.1.5 --- Equipments and facilities used --- p.49 / Chapter 2.2 --- Methods --- p.50 / Chapter 2.2.1 --- Molecular techniques --- p.50 / Chapter 2.2.1.1 --- DNA gel electrophoresis --- p.59 / Chapter 2.2.1.2 --- PCR technique --- p.50 / Chapter 2.2.1.3 --- Restriction digestion --- p.50 / Chapter 2.2.1.4 --- Ligation (for sticky-end ligation) --- p.51 / Chapter 2.2.1.5 --- DNA purification --- p.51 / Chapter 2.2.1.6 --- DNA sequencing --- p.51 / Chapter 2.2.1.7 --- Transformation of competent E. coli cells --- p.52 / Chapter 2.2.1.8 --- Preparation of plasmid from bacterial cells --- p.53 / Chapter 2.2.1.9 --- Transformation of competent Agrobacterium tumefaciens cells --- p.53 / Chapter 2.2.1.10 --- DNA extraction from plant tissue (Small-scale) --- p.54 / Chapter 2.2.1.11 --- RNA extraction from plant tissue --- p.55 / Chapter 2.2.2 --- Growth conditions of A. thaliana --- p.55 / Chapter 2.2.2.1 --- Surface sterilization of A. thaliana seeds --- p.55 / Chapter 2.2.2.2 --- Growing A. thaliana --- p.55 / Chapter 2.2.3 --- Characterization of transgenic A. thaliana with altered sink-source relationship --- p.57 / Chapter 2.2.3.1. --- Determination of amino acid contents in seeds --- p.57 / Chapter 2.2.3.2. --- Expression study of developing siliques of transgenic lines --- p.58 / Chapter 2.2.3.2.1 --- Tagging siliques of different developmental stages --- p.58 / Chapter 2.2.3.2.2 --- Extraction of silique RNA --- p.58 / Chapter 2.2.3.2.3 --- cDNA synthesis --- p.58 / Chapter 2.2.3.2.4 --- Real-time PCR --- p.59 / Chapter 2.2.4 --- Characterization of transgenic A. thaliana overexpressing GCN2 --- p.60 / Chapter 2.2.4.1 --- Gene expression study of vegetative tissues by real-time PCR --- p.60 / Chapter 2.2.4.2 --- Gene expression study of developing siliques by real-time PCR --- p.61 / Chapter 2.2.5 --- Making transgenic A. thaliana --- p.61 / Chapter 2.2.5.1 --- Cloning of multigene construct --- p.61 / Chapter 2.2.5.1.1 --- Subcloning of target genes into donor vectors --- p.61 / Chapter 2.2.5.1.1.1 --- Cloning of LRP into donor vector VS --- p.61 / Chapter 2.2.5.1.1.2 --- Cloning of dapA into donor vector SV --- p.64 / Chapter 2.2.5.1.1.3 --- Cloning of ansB into donor vector VS --- p.67 / Chapter 2.2.5.1.1.4 --- Cloning of antisense LKR fragment into donor vector SV --- p.70 / Chapter 2.2.5.1.2 --- Preparation of phosphorylated linkers --- p.73 / Chapter 2.2.5.1.3 --- Introduction of target genes to acceptor vector --- p.73 / Chapter 2.2.5.2 --- Agrobacterium-mediated transformation of A. thaliana via Vacuum infiltration --- p.78 / Chapter 2.2.5.3 --- Screening of transformants --- p.79 / Chapter Chapter 3. --- Results --- p.80 / Chapter 3.1 --- Characterization of transgenic lines with altered sink-source relationship --- p.80 / Chapter 3.1.1 --- Amino acid analysis of mature seeds of transgenic lines --- p.80 / Chapter 3.1.1.1 --- Aspartate family amino acids levels remain steady in seeds of transgenic plants --- p.83 / Chapter 3.1.1.2 --- Increase in seed Met content in Met-rich protein expressing transgenic plants --- p.85 / Chapter 3.1.1.3 --- Increase in seed Lys content in phas-dapA/phas-LRP transgenic plants --- p.87 / Chapter 3.1.2 --- Gene expression study of transgenic line --- p.89 / Chapter 3.1.2.1 --- Down-regulation of akthr1 and akthr2 in transgenic plants with altered N sink-source relationship --- p.89 / Chapter 3.1.2.2 --- Down regulation of GCN2 in transgenic plants with altered N sink-source relationship --- p.90 / Chapter 3.1.2.4 --- Expression study of other genes in aspartate family pathway --- p.90 / Chapter 3.2 --- Characterization of GCN2 overexpressing line --- p.93 / Chapter 3.2.1 --- Gene expression study of seedlings of GCN2 overexpressing plants --- p.93 / Chapter 3.2.1.1 --- Increased GCN2 expression by azaserine treatment --- p.93 / Chapter 3.2.1.2 --- Increased akthrl and akthr2 expression in GCN2 overexpressing plants --- p.96 / Chapter 3.2.1.3 --- Expression study of other genes in aspartate family pathway --- p.96 / Chapter 3.2.2 --- Gene expression study of GCN2 overexpressing plants during seed development --- p.98 / Chapter 3.3 --- Construction of transgenic plants by multigene assembly system --- p.100 / Chapter 3.3.1 --- Successful construction of recombinant plasmid carrying four target genes --- p.100 / Chapter 3.3.2 --- Transformation of A. thaliana with multigene vector --- p.103 / Chapter Chapter 4 --- Discussion --- p.104 / Chapter 4.1 --- Characterization of transgenic plants with altered sink-source relationship of aspartate family amino acid metabolism --- p.104 / Chapter 4.1.1 --- Total content of aspartate family amino acids remains steady in transgenic lines --- p.105 / Chapter 4.1.2 --- Methionine content increases in phas-PN2S and phas-MetL transgenic plants --- p.106 / Chapter 4.1.3 --- Relative lysine content increases in phas-dapA/phas-LRP transgenic plants --- p.107 / Chapter 4.1.4 --- Coordinated regulation of gene expressions of akthrl and akthr2 with GCN2 expression in transgenic plants with altered sink-source relationship --- p.109 / Chapter 4.2 --- GCN system in plants --- p.110 / Chapter 4.2.1 --- Transcriptional regulation of GCN2 in A. thaliana --- p.110 / Chapter 4.2.2 --- Regulation of amino acid biosynthesis by GCN system --- p.111 / Chapter 4.2.2.1 --- Regulation of akthrl and akthr2 by GCN2 --- p.111 / Chapter 4.2.2.2 --- GCN4 homolog in plants? --- p.112 / Chapter 4.2.2.3 --- Regulation of amino acid metabolism by GCN system --- p.113 / Chapter 4.3 --- Generation of transgenic plants with a combination of altered sink- source relationship --- p.114 / Chapter Chapter 5. --- Conclusion and Future Prospective --- p.116 / Appendix I: The major chemicals and reagents used in this research --- p.118 / "Appendix II: Major buffers, solutions and mediums used in this research" --- p.120 / Appendix III: Commercial kits used in this research --- p.125 / Appendix IV: Major equipment and facilities used in this research --- p.126 / References --- p.127

Cette thèse se place dans le cadre de l'étude in silico, c'est-à-dire assistée par ordinateur, des liens qui unissent la séquence d'une protéine à la (ou aux) structure(s) tri-dimensionnelle(s) qu'elle adopte. Le décryptage de ces liens présente de nombreuses applications dans divers domaines et constitue sans doute l'une des problématiques les plus fascinantes de la recherche en biologie moléculaire.<p><p>Le premier aspect de notre travail concerne le développement de potentiels statistiques dérivés de bases de données de protéines dont les structures sont connues. Ces potentiels présentent plusieurs avantages: ils peuvent être aisément adaptés à des représentations structurales simplifiées, et permettent de définir un nombre limité de fonctions énergétiques qui incarnent l'ensemble complexe d'interactions gouvernant la structure et la stabilité des protéines, et qui incluent également certaines contributions entropiques. Cependant, leur signification physique reste assez nébuleuse, car l'impact des diverses hypothèses nécessaires à leur dérivation est loin d'être clairement établi. Nous nous sommes attachés à l'étude de certaines limitations des ces potentiels: leur dépendance en la taille des protéines incluses dans la base de données, la non-additivité des termes de potentiels, et l'importance souvent négligée de l'environnement protéique spécifique ressenti par chaque résidu. Nous avons ainsi mis en évidence que l'influence de la taille des protéines de la base de données sur les potentiels de distance entre résidus est spécifique à chaque paire d'acides aminés, peut être relativement importante, et résulte essentiellement de la répartition inhomogène des résidus hydrophobes et hydrophiles entre le coeur et la surface des protéines. Ces résultats ont guidé la mise au point de fonctions correctives qui permettent de tenir compte de cette influence lors de la dérivation des potentiels. Par ailleurs, la définition d'une procédure générale de dérivation de potentiels et de termes de couplage a rendu possible la création d'une fonction énergétique qui tient compte simultanément de plusieurs descripteurs de séquence et de structure (la nature des résidus, leurs conformations, leurs accessibilités au solvant, ainsi que les distances qui les séparent dans l'espace et le long de la séquence). Cette fonction énergétique présente des performances nettement améliorées par rapport aux potentiels originaux, et par rapport à d'autres potentiels décrits dans la littérature.<p><p>Le deuxième aspect de notre travail concerne l'application de programmes basés sur des potentiels statistiques à l'étude de protéines qui adoptent des structures alternatives. La permutation de domaines est un phénomène qui affecte diverses protéines et qui implique la génération d'un oligomère suite à l'échange de fragments structuraux entre monomères identiques. Nos résultats suggèrent que la présence de "faiblesses structurales", c'est-à-dire de régions qui ne sont pas optimales vis-à-vis de la stabilité de la structure native ou qui présentent une préférence marquée pour une conformation non-native en absence d'interactions tertiaires, est intimement liée aux mécanismes de permutation. Nous avons également mis en évidence l'importance des interactions de type cation-{pi}, qui sont fréquemment observées dans certaines zones clés de la permutation. Finalement, nous avons sélectionné un ensemble de mutations susceptibles de modifier sensiblement la propension de diverses protéines à permuter. L'étude expérimentale de ces mutations devrait permettre de valider, ou de raffiner, les hypothèses que nous avons proposées quant au rôle joué par les faiblesses structurales et les interactions de type cation-{pi}. Nous avons également analysé une autre protéine soumise à d'importants réarrangements conformationnels: l'{alpha}1-antitrypsine. Dans le cas de cette protéine, les modifications structurales sont indispensables à l'exécution de l'activité biologique normale, mais peuvent sous certaines conditions mener à la formation de polymères insolubles et au développement de maladies. Afin de contribuer à une meilleure compréhension des mécanismes responsables de la polymérisation, nous avons cherché à concevoir rationnellement des protéines mutantes qui présentent une propension à polymériser contrôlée. Des tests expérimentaux ont été réalisés par le groupe australien du Professeur S.P. Bottomley, et ont permis de valider nos prédictions de manière assez remarquable.<p><p><p><p>The work presented in this thesis concerns the computational study of the relationships between the sequence of a protein and its three-dimensional structure(s). The unravelling of these relationships has many applications in different domains and is probably one of the most fascinating issues in molecular biology.<p><p>The first part of our work is devoted to the development of statistical potentials derived from databases of known protein structures. These potentials allow to define a limited number of energetic functions embodying the complex ensemble of interactions that rule protein folding and stability (including some entropic contributions), and can be easily adapted to simplified representations of protein structures. However, their physical meaning remains unclear since several hypotheses and approximations are necessary, whose impact is far from clearly understood. We studied some of the limitations of these potentials: their dependence on the size of the proteins included in the database, the non-additivity of the different potential terms, and the importance of the specific environment of each residue. Our results show that residue-based distance potentials are affected by the size of the database proteins, and that this effect can be quite strong, is residue-specific, and seems to result mostly from the inhomogeneous partition of hydrophobic and hydrophilic residues between the surface and the core of proteins. On the basis of these observations, we defined a set of corrective functions in order to take protein size into account while deriving the potentials. On the other hand, we developed a general procedure of derivation of potentials and coupling terms and consequently created an energetic function describing the correlations between several sequence and structure descriptors (the nature of each residue, the conformation of its main chain, its solvent accessibility, and the distances that separate it from other residues, in space and along the sequence). This energetic function presents a strongly improved predictive power, in comparison with the original potentials and with other potentials described in the literature.<p><p>The second part describes the application of different programs, based on statistical potentials, to the study of proteins that adopt alternative structures. Domain swapping involves the exchange of a structural element between identical proteins, and leads to the generation of an oligomeric unit. We showed that the presence of “structural weaknesses”, regions that are not optimal with respect to the folding mechanisms or to the stability of the native structure, seems to be intimately linked with the swapping mechanisms. In addition, cation-{pi} interactions were frequently detected in some key locations and might also play an important role. Finally, we designed a set of mutations that are likely to affect the swapping propensities of different proteins. The experimental study of these mutations should allow to validate, or refine, our hypotheses concerning the importance of structural weaknesses and cation-{pi} interactions. We also analysed another protein that undergoes large conformational changes: {alpha}1-antitrypsin. In this case, the structural modifications are necessary to the proper execution of the biological activity. However, under certain circumstances, they lead to the formation of insoluble polymers and the development of diseases. With the aim of reaching a better understanding of the mechanisms that are responsible for this polymerisation, we tried to design mutant proteins that display a controlled polymerisation propensity. An experimental study of these mutants was conducted by the group of Prof. S.P. Bottomley, and remarkably confirmed our predictions.<p> / Doctorat en sciences appliquées / info:eu-repo/semantics/nonPublished