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A unique mechanism of protein oligomerization is domain swapping. It is a feature found in some proteins wherein a dimer or a higher oligomer is formed by the exchange of identical structural segments between protomers. Domain swapping is thought to have played a key role in the evolution of stable oligomeric proteins and in oligomerization of amyloid proteins. This thesis deals with studies to understand the significance of hinges involved in domain swapping for protein oligomerization and function. The stationary phase survival protein SurE from Salmonella typhimurium (StSurE) and Sesbania mosaic virus (SeMV) coat protein have been used as models for studies on domain swapping. This thesis has been divided into eight chapters. Chapter 1 provides a brief introduction to domain swapping, while Chapters 2 to 6 describes the studies carried out on StSurE protein, Chapter 7 deals with studies on SeMV coat protein. The final Chapter 8 provides brief descriptions of various experimental techniques employed during these investigations. Chapter 1 deals with a brief introduction to domain swapping in proteins. Examples where different domains are exchanged are cited. Then it describes physiological relevance of domain swapping in proteins and probable factors which promote swapping. Finally it also discusses the uncertainties that are inevitable in protein structure prediction and design. Chapter 2 describes the structure of Salmonella typhimurium SurE (StSurE; Pappachan et al., 2008) determined at a higher resolution. The chapter also deals with the sequence and structure based comparison of StSurE with other known SurE homolog structures. A comparative analysis of the relative conservation of N- and C-terminal halves of SurE protomer and variations observed in the quaternary structures of SurE homologs are presented. Then a brief introduction is provided on function of StSurE. The conserved active site of StSurE that might be important for its phosphatase activity is described. A plausible mechanism for the phosphatase activity as proposed by Pappachan et al. (2008) is presented. Crystal structures of StSurE bound with AMP, pNPP and pNP that was determined with the view of better understanding the mechanism of enzyme function is presented. These structures provide structural evidence for the mechanism proposed by Pappachan et al. (2008). Finally a substrate entry channel inferred from these structures is discussed. SurE from Salmonella typhimurium (StSurE) was selected for studies on domain swapping as there is at least one homologous structure (Pyrobaculum aerophilum - PaSurE) in which swapping of the C-terminal helices appears to have been avoided without leading to the loss of oligomeric structure or function. It was of interest to examine if an unswapped dimer of StSurE resembling PaSurE dimer could be constructed by mutagenesis. To achieve this objective, a crucial hydrogen bond in the hinge involved in C-terminal helix swapping was abolished by mutagenesis. These mutants were constructed with the intention of increasing the flexibility of the hinge which might bring the C-terminal helices closer to the respective protomer as in PaSurE. Chapter 3 presents a comparative analysis of the hinges involved in C-terminal helix swapping in PaSurE and StSurE. Based on the comparison of structure and sequence, crucial residues important for C-terminal helix swapping in StSurE were identified as D230 and H234. The chapter describes the construction of mutants obtained by substituting D230 and H234 by alanine and their biophysical characterization. Finally it describes structural studies carried out on these mutants. The mutation H234A and D230A/H234A resulted in highly distorted dimers, although helix swapping was not avoided. Comparative analysis of the X-ray crystal structures of native StSurE and mutants H234A and D230A/H234A reveal large structural changes in the mutants relative to the native structure. However the crystal structures do not provide information on the changes in dynamics of the protein resulting from these mutations. To gain better insights into the dynamics involved in the native and mutants H234A and D230A/H234A, MD simulations were carried on using GROMACS 4.0.7. Chapter 4 deals with a brief description of the theory of molecular dynamics, followed by results of simulation studies carried out on monomeric and dimeric forms of StSurE and dimeric forms of its mutants H234A and D230A/H234A. The conformational changes and dynamics of different swapped segments are discussed. Crystal structures of H234A and D230A/H234A mutants reveal that they form highly distorted dimers with altered dimeric interfaces. Chapter 5 focuses on comparison of dimeric interfaces of the native StSurE and hinge mutants H234A and D230A/H234A. Based on the analysis, three sets of interactions were selected to investigate the importance of the interface formed by swapped segments in StSurE mutants H234A and D230A/H234A. One of the selected sites corresponds to a novel interaction involving tetramerization loop in the hinge mutants H234A and D230A/H234A resulting in a salt bridge between E112 – R179’ and E112’ – H180 (prime denotes residue from the other chain of the dimeric protein). This salt bridge seems to stabilize the distorted dimer. It is shown by structural studies that the loss of this salt bridge due to targeted mutation restores symmetry and dimeric organization of the mutants. Loss of a crucial hydrogen bond in the hinge region involved in C-terminal helix swapping in SurE not only leads to large structural changes but also alters the conformation of a loop near the active site. It is of interest to understand functional consequences of these structural changes. StSurE is a phosphatase, and its activity could be conveniently monitored using the synthetic substrate para nitrophenyl phosphate (pNPP) at pH 7 and 25 ºC. Chapter 6 deals with the functional studies carried out with various StSurE mutants. The studies suggest that there is a drastic loss in phosphatase activity in hinge mutants D230A, H234A and D230A/H234A, while in the salt bridge mutants the function seems to have been restored. Few of these mutants also exhibit positive cooperativity, which could probably be due to altered dynamics of domains. Sesbania mosaic virus (SeMV) is a plant virus, belonging to genus sobemovirus. SeMV is a T=3 icosahedral virus (532 symmetry) made up of 180 coat protein (CP) subunits enclosing a positive-sense RNA genome. The asymmetric unit of the icosahedral capsid is composed of chemically identical A, B and C subunits occupying quasi-equivalent environments. Residues 48 – 59 of the N-terminal arms of the C subunits interact at the nearby icosahedral three-fold axes through a network of hydrogen bonds to form a structure called the “β-annulus”. Residues 60 – 73 form the “βA-arm” that connects the N-terminal β-annulus to the rest of the protomer. Various studies on SeMV-CP suggest that different lengths of the N-terminal segments affect the assembly of virus. It might be possible to exploit this flexibility of the N-terminus in SeMV-CP to introduce swapping of this segment between two 2-fold related C subunits as is found in Rice yellow mottle virus (RYMV), another sobemovirus, with which SeMV shares significant sequence similarity. Chapter 7 focuses on attempts made to examine the mutational effects planned to introduce domain swapping. The strategy used for introducing swapping in SeMV-CP was based on the sequence of the βA-arm or the hinge involved in swapping of β-annulus in RYMV. TEM images of the mutant virus like particles obtained suggest that they are heterogeneous. These mutants could not be crystallized, probably due to the heterogeneity. However, the assembly of the expressed proteins to virus like particles was profoundly influenced by the mutations. Chapter 8 discusses various crystallographic, biophysical and biochemical techniques used during these investigations. Finally the thesis concludes with Conclusions and Future perspectives of the various studies reported in the thesis. In summary, I have addressed the importance of amino acid residues and interactions of hinges involved in domain swapping for the quaternary structure and function of proteins.

Text in Afrikaans / This research describes the coats of arms and emblems of hospitals and nursing colleges in the Republic of South Africa, an aspect of nursing's heritage and history have not yet been described. Data were collected by means of questionnaires. An extensive literature review on heraldics and an analysis of the meanings contained in the symbols appearing on these devices were done. The analysis resulted in the following; the uniqueness of the individual devices, the development of nursing and nursing education, the influence of values, norms, ideals, culture, religion and history on the selection of symbols. The heraldic meanings of symbols and the meanings indicated in the questionnaires were compared with the identified core concepts for nursing. The overall finding was that any congruence was purely circumstantial. / Hierdie navorsing beskryf die wapens en embleme van hospitale en verpleegkolleges in die Republiek van Suid-Afrika, 'n aspek van die erfenis en geskiedenis van verpleging wat nog nie vantevore beskryf is nie. Inligting was deur middel van vraelyste ingesamel. 'n Uitgebreide literatuuroorsig oor die heraldiek en 'n ontleding van die betekenisse van die simbole soos aangetref op die wapens/embleme, is gedoen.Uit die analisering is die volgende bevind; die uniekheid van die individuele wapens/embleme, die ontwikkeling van verpleging en verpleegonderrig, die invloed van waardes, norme, ideale, kultuur, godsdiens en geskiedenis op die keuse van simbole. Die heraldiese betekenis van simbole en die betekenis wat in die vraelyste daaroor aangedui is, is met die geidentifiseerde kembegrippe vir verpleging, vergelyk. Die algemene bevinding is dat enige ooreenkoms bloot toevallig is. / Health Studies / M.A. (Verpleegkunde)

The multi-million-dollar medical uniform industry has not utilized advancements in garment and textile technology that could positively impact the protection of healthcare professionals and patients. In most cases the uniforms meet basic requirements – they clothe the professional in a recognizable way. Little innovation in design, function and performance, has been applied to these garments. This is particularly evident in the case of the stereotypical white lab coat worn by many physicians, despite evidence indicating that these lab coats may carry contamination and play a role in the spread of deadly bacteria. Healthcare Associated Infections (HAIs) are among the most serious problems facing modern medical care, costing millions of lives and dollars annually worldwide. This research investigates the design and use of the physician’s lab coat – an immediately recognizable symbol of Western medicine. The research identifies the medical, functional, cultural and symbolic roles of the lab coat within the hospital environment and beyond, to the larger the global society. This thesis examines the extent to which the design of medical wear can impact the effect of hospital-acquired infections, support doctor/patient relationships and enhance the performance and behavior of the healthcare professional by envisioning a future lab coat which offers increased protection for physician and patient, aids in communication and enhances the performance of the doctor by utilizing digital technologies incorporated into the lab coat whereby the lab coat becomes the only tool necessary for the physician.

Ce travail porte sur la vie psychique des populations sans domicile fixe, des exclus ou des sujets gravement précaires. Malgré les différents profils parsemant ce public, l’errance n’est pas un épiphénomène survenant autour de pathologies psychiatriques mais une maladie psychique profonde, singulière, et non réductible à d’autres entités nosographiques. C’est une configuration psychodynamique qui prend son origine dans une incapacité à se loger dans l’objet primaire, c’est-à-dire à se sentir contenu dans sa préoccupation et dans sa fonction de rêverie, créant un « trou noir » dans lequel l’errant menace sans cesse de tomber. Les mécanismes défensifs associés à cette psychodynamique ont des effets sur l’aménagement de l’extériorité : l’extériorité est un prisme où s’articulent l’espace, le corps, la sensorialité et l’objet. Si d’ordinaire, l’extériorité fonctionne de manière silencieuse, dans l’errance elle se révèle bruyante et symptomatique, si bien que les différents espaces qui constituent l’extériorité se collusionnent. Pour analyser ces emmêlements de l’extériorité, l’investigation clinique s’est effectuée sur les champs de la grande exclusion par la participation à des équipes mobiles psychiatrie-précarité et au Samu Social, mais également par la création de groupes de parole et à médiation au sein d’accueils de jour. La présentation des pratiques cliniques et du travail d’accompagnement social tentant de faire émerger un lien durable et sécurisé, fait partie des préoccupations de cette recherche. Au terme de ce travail, deux concepts sont proposés : celui de Manteau Cloacal, pouvant se définir comme une corruption autocalmante et autostimulante des enveloppes psychiques dans une recherche de contact avec l’objet primaire ; et celui d’Effondrement scénique, tentative pour forcer un « domicile psychique fixe » en l’objet par l’usage des sensations, de la mise en scène et d’une certaine forme de séduction. / This work concerns the psychic life of the populations without fixed residence, excluded or the seriously precarious subjects. In spite of the various profiles strewing this public, the wandering is not an epiphenomenon occurring around psychiatric pathologies but a major, singular psychic disease non-reducible with other nosographic entities. It is a psychodynamic configuration which takes its origin in incapacity to place itself in the primary object, i.e. to feel contained in its concern and its function of daydream, creating a « black hole » into which unceasingly the subject is likely to fall. The defensive mechanisms associated with this psychodynamic have effects on the externality: the externality is a prism where space, the body, the sensoriality and the object are articulated. So usually, the externality functions in a quiet way. But in the psychic wandering it appears noisy and symptomatic so that various spaces which constitute the externality mix. To analyze these mixings up of the externality, the clinical investigation was carried out on the fields of the great exclusion by the participation in psychiatry-precariousness mobile teams and Samu Social, but also by the creation of therapeutic group into social structures. Presentation of the clinical practices and the work of social accompaniment aiming to make emerge a durable and safe bond belonged to the concerns of this research. At the end of this work, two concepts are proposed: the Cloacal Coat, which can be defined as an anesthetic and stimulative psychic envelope in a search for contact with the primary object; and the Scenic Collapse, which attempt to force by the use of the sensoriality and a certain form of seduction, a « psychic residence » in the object.

The purpose of this research work was to develop a nozzle capable of depositing dense CoNiCrAlY coatings via cold gas dynamic spray (CGDS) as well as compare the oxidation performance of bond coats manufactured by CGDS, high-velocity oxy-fuel (HVOF) and air plasma spray (APS) at temperatures of 1000°C and 1100°C. The work was divided in two sections, the design and manufacturing of a CGDS nozzle with an optimal profile for the deposition of CoNiCrAlY powders and the comparison of the oxidation performance of CoNiCrAlY bond coats. Throughout this work, it was shown that the quality of coatings deposited via CGDS can be increased by the use of a nozzle of optimal profile and that early formation of protective α-Al2O3 due to an oxidation temperature of 1100°C as opposed to 1000°C is beneficial to the overall oxidation performance of CoNiCrAlY coatings.

The purpose of this research work was to develop a nozzle capable of depositing dense CoNiCrAlY coatings via cold gas dynamic spray (CGDS) as well as compare the oxidation performance of bond coats manufactured by CGDS, high-velocity oxy-fuel (HVOF) and air plasma spray (APS) at temperatures of 1000°C and 1100°C. The work was divided in two sections, the design and manufacturing of a CGDS nozzle with an optimal profile for the deposition of CoNiCrAlY powders and the comparison of the oxidation performance of CoNiCrAlY bond coats. Throughout this work, it was shown that the quality of coatings deposited via CGDS can be increased by the use of a nozzle of optimal profile and that early formation of protective α-Al2O3 due to an oxidation temperature of 1100°C as opposed to 1000°C is beneficial to the overall oxidation performance of CoNiCrAlY coatings.

The purpose of this research work was to develop a nozzle capable of depositing dense CoNiCrAlY coatings via cold gas dynamic spray (CGDS) as well as compare the oxidation performance of bond coats manufactured by CGDS, high-velocity oxy-fuel (HVOF) and air plasma spray (APS) at temperatures of 1000°C and 1100°C. The work was divided in two sections, the design and manufacturing of a CGDS nozzle with an optimal profile for the deposition of CoNiCrAlY powders and the comparison of the oxidation performance of CoNiCrAlY bond coats. Throughout this work, it was shown that the quality of coatings deposited via CGDS can be increased by the use of a nozzle of optimal profile and that early formation of protective α-Al2O3 due to an oxidation temperature of 1100°C as opposed to 1000°C is beneficial to the overall oxidation performance of CoNiCrAlY coatings.

Text in Afrikaans / This research describes the coats of arms and emblems of hospitals and nursing colleges in the Republic of South Africa, an aspect of nursing's heritage and history have not yet been described. Data were collected by means of questionnaires. An extensive literature review on heraldics and an analysis of the meanings contained in the symbols appearing on these devices were done. The analysis resulted in the following; the uniqueness of the individual devices, the development of nursing and nursing education, the influence of values, norms, ideals, culture, religion and history on the selection of symbols. The heraldic meanings of symbols and the meanings indicated in the questionnaires were compared with the identified core concepts for nursing. The overall finding was that any congruence was purely circumstantial. / Hierdie navorsing beskryf die wapens en embleme van hospitale en verpleegkolleges in die Republiek van Suid-Afrika, 'n aspek van die erfenis en geskiedenis van verpleging wat nog nie vantevore beskryf is nie. Inligting was deur middel van vraelyste ingesamel. 'n Uitgebreide literatuuroorsig oor die heraldiek en 'n ontleding van die betekenisse van die simbole soos aangetref op die wapens/embleme, is gedoen.Uit die analisering is die volgende bevind; die uniekheid van die individuele wapens/embleme, die ontwikkeling van verpleging en verpleegonderrig, die invloed van waardes, norme, ideale, kultuur, godsdiens en geskiedenis op die keuse van simbole. Die heraldiese betekenis van simbole en die betekenis wat in die vraelyste daaroor aangedui is, is met die geidentifiseerde kembegrippe vir verpleging, vergelyk. Die algemene bevinding is dat enige ooreenkoms bloot toevallig is. / Health Studies / M.A. (Verpleegkunde)