När frågan om möjligheten att patentera nya användningsområden för redan kända läkemedel aktualiserades under det Europeiska patentsystemet, kunde två hinder identifieras. Dels är den aktiva substansen i sig inte ny och dessutom förbjuder patentsystemet uttryckligen patent på medicinska förfaranden för diagnostisering eller terapeutisk och kirurgisk behandling. Genom både rättspraxis och lagstiftning har man dock försökt kringgå dessa hinder och till följd av den uttryckliga bestämmelsen om patent på den så kallade andra medicinska indikationen som EPC 2000 tillhandahåller, kan dess patenterbarhet inte längre ifrågasättas. Med anledning av den speciella karaktär som kravformuleringen för dessa patent åtnjuter och till följd av den snäva tolkning av kravformuleringen som den sparsamma praxis på detta område återspeglar, kan det däremot ifrågasättas om inte kraven för dess upprätthållande är alltför långtgående. Det utgör särskilt ett problem när läkemedelstillverkare använder möjligheten att genom "skinny labelling" utesluta patentskyddade indikationer från produktinformationen, för att kunna introducera generiska läkemedel för icke-patentskyddade indikationer på marknaden. Två motstridiga avgöranden som nyligen meddelats i Storbritannien respektive Nederländerna, vilka särskilt behandlar frågan om intrång i förhållande till "skinny labelling", återspeglar det oklara rättsläge som detta rättsområde präglas av. I Sverige kompliceras intrångsfrågan ytterligare av frågan om i vilken utsträckning dessa patent kan upprätthållas till följd av bestämmelser i folkhälsolagstiftningen om generisk substitution. Enkelt förklarat medför dessa bestämmelser att billiga generikaläkemedel, som enbart innehar försäljningsgodkännande för icke-patentskyddade indikationer, byts ut på apoteken mot originalläkemedel, oavsett för vilken indikation läkemedlet förskrivits. I praktiken innebär det att dessa läkemedelskopior även expedieras för patentskyddade indikationer. För att lösa den problematik som regelverket orsakar och till följd av de höga krav som i praxis tycks ställas upp för upprätthållandet av patent på den andra medicinska indikationen, anser författaren att lagstiftaren bör se över hur den svenska folkhälsolagstiftningen och det Europeiska patentsystemet bättre kan samspela. / Two obstacles could be identified when the question of granting patent protection for second medical use of known pharmaceutical products arose in the European patent system. On one hand, the active substance in itself is not new and on the other hand, the system explicitly prohibit method claims for treatment by surgery or therapy and diagnostic methods. However, these barriers have been circumvent in case law and legislation and due to the express provision of second medical use patents in the EPC 2000, its patentability is no longer to be questioned. Due to the special character of second medical use claims and as a result of the narrow interpretation that the sparse case law in this area reflects, it may however be questioned whether the requirement of its enforcement is far to excessive. This is particularly a problem when generic manufacturers uses the possibility of "skinny labelling", in order to exclude patented indications from the product information. As recently announced in the UK and the Netherlands, two conflicting decisions that specifically addresses the issue of patent infringement on second medical use patents in relation to "skinny labelling", reflects the legal uncertainty that characterizes this area of ​​law. In the Swedish context, the issue of enforcement of second medical use patents is further complicated as a result of mandatory provisions on generic substitution. In simple terms, these provisions causes inexpensive generic versions, which solely obtains marketing approval for off-patent indications, to exchange original pharmaceuticals at pharmacies regardless of the indication the drug has been prescribed. In practice, this means that these generic versions will also be dispensed for patented indications. In order to solve the problems that this regulatory framework causes and as a consequence of the high demands set out for the enforcement of second medical use patents, the author believes that it is advisable that the legislator performs a review on how the Swedish public health legislation and the European patent system better can interact on this issue.

Motivation Processed complementary food labels should protect and promote optimal breastfeeding and complementary feeding practices, important determinants of child survival, growth and development, and provide information regarding safe and appropriate use. However, there is a lack of formal guidelines from international normative bodies on the appropriate marketing of complementary foods. In recognition of the need for interim guidance, the Maternal, Infant and Young Child Working Group developed the Draft Guide for Marketing Complementary Foods, which provides practical guidance on how the marketing (including labelling) of processed complementary foods and supplements can be informed by the principles of the International Code of Marketing of Breast-milk Substitutes (the Code) and subsequent relevant World Health Assembly (WHA) resolutions in a way that supports optimal infant and young child feeding. Aim The aim of this study was to describe the extent to which the labelling practices (as a sub-set of marketing practices) of processed complementary food sold in South Africa comply with international guidance on the marketing of complementary foods that is fully aligned with the principles of the Code and subsequent relevant WHA resolutions (the Draft Guide for Marketing Complementary Foods). Methods Employing a cross-sectional study design, products were purchased from a sample of 17 retail grocery stores, three wholesale grocery stores, three retail pharmacies and three baby chain stores in the Gauteng, Western Cape and KwaZulu-Natal provinces from June to August 2011. Purchased products were then compared with a master list of complementary food products compiled through desk research, and missing products were identified and purchased. Label information was captured, then blinded and the order of products randomised. The Draft Guide for Marketing Complementary Foods was used to create a checklist with pre-set answers and accompanying criteria against which the captured labelling practices were then analysed. Results One hundred and sixty product labels of 35 manufacturers were analysed, none of which complied with all checklist criteria. Fifty-six (35%) labels did not provide an appropriate age of introduction, while 32 (20%) labels used phrases implying that the product was suitable for use before six months of age. Thirty-seven (23%) labels used images of infants appearing to be younger than six months. Only 20 (13%) labels carried a message regarding the importance of exclusive breastfeeding for the first six months of life, and none provided a message on the importance of the addition of complementary foods from six months together with continued breastfeeding to two years or beyond. Eight (5%) labels recommended feeding the product in a bottle and two labels (1%) used an image of a feeding bottle. Nineteen (12%) labels suggested a daily ration too large for a breastfed child, and 32 (20%) potentially promote the manufacturer’s infant formula. All labels provided label information in an appropriate language, but 102 (64%) labels relegated required label information to small text and were thus not easy to read. Only six (4%) labels failed to provide instructions for safe and appropriate use, while 44 (28%) did not include safety messages in their preparation and use instructions. Ten (6%) labels did not provide storage instructions, and 27 (17%) labels did not provide necessary warnings. Nutrient content claims, nutrient comparative claims, nutrient function/other function claims and reduction of disease risk claims were found on 126 (79%), eight (5%), 117 (73%) and 10 (6%) labels, respectively. Conclusion The labelling practices of processed complementary food labels in South Africa do not fully comply with international guidance on the marketing of complementary foods (the Draft Guide for Marketing Complementary Foods) and so do not sufficiently protect and promote optimal infant and young child feeding practices, revealing much room for improvement. Such guidance must be refined and formalised by international normative bodies and adopted into national legislation to assist manufacturers in ensuring that their complementary food labels meet an accepted standard and contribute towards the safe and appropriate use of processed complementary foods. / Thesis (MSc (Nutrition))--North-West University, Potchefstroom Campus, 2013.

Motivation Processed complementary food labels should protect and promote optimal breastfeeding and complementary feeding practices, important determinants of child survival, growth and development, and provide information regarding safe and appropriate use. However, there is a lack of formal guidelines from international normative bodies on the appropriate marketing of complementary foods. In recognition of the need for interim guidance, the Maternal, Infant and Young Child Working Group developed the Draft Guide for Marketing Complementary Foods, which provides practical guidance on how the marketing (including labelling) of processed complementary foods and supplements can be informed by the principles of the International Code of Marketing of Breast-milk Substitutes (the Code) and subsequent relevant World Health Assembly (WHA) resolutions in a way that supports optimal infant and young child feeding. Aim The aim of this study was to describe the extent to which the labelling practices (as a sub-set of marketing practices) of processed complementary food sold in South Africa comply with international guidance on the marketing of complementary foods that is fully aligned with the principles of the Code and subsequent relevant WHA resolutions (the Draft Guide for Marketing Complementary Foods). Methods Employing a cross-sectional study design, products were purchased from a sample of 17 retail grocery stores, three wholesale grocery stores, three retail pharmacies and three baby chain stores in the Gauteng, Western Cape and KwaZulu-Natal provinces from June to August 2011. Purchased products were then compared with a master list of complementary food products compiled through desk research, and missing products were identified and purchased. Label information was captured, then blinded and the order of products randomised. The Draft Guide for Marketing Complementary Foods was used to create a checklist with pre-set answers and accompanying criteria against which the captured labelling practices were then analysed. Results One hundred and sixty product labels of 35 manufacturers were analysed, none of which complied with all checklist criteria. Fifty-six (35%) labels did not provide an appropriate age of introduction, while 32 (20%) labels used phrases implying that the product was suitable for use before six months of age. Thirty-seven (23%) labels used images of infants appearing to be younger than six months. Only 20 (13%) labels carried a message regarding the importance of exclusive breastfeeding for the first six months of life, and none provided a message on the importance of the addition of complementary foods from six months together with continued breastfeeding to two years or beyond. Eight (5%) labels recommended feeding the product in a bottle and two labels (1%) used an image of a feeding bottle. Nineteen (12%) labels suggested a daily ration too large for a breastfed child, and 32 (20%) potentially promote the manufacturer’s infant formula. All labels provided label information in an appropriate language, but 102 (64%) labels relegated required label information to small text and were thus not easy to read. Only six (4%) labels failed to provide instructions for safe and appropriate use, while 44 (28%) did not include safety messages in their preparation and use instructions. Ten (6%) labels did not provide storage instructions, and 27 (17%) labels did not provide necessary warnings. Nutrient content claims, nutrient comparative claims, nutrient function/other function claims and reduction of disease risk claims were found on 126 (79%), eight (5%), 117 (73%) and 10 (6%) labels, respectively. Conclusion The labelling practices of processed complementary food labels in South Africa do not fully comply with international guidance on the marketing of complementary foods (the Draft Guide for Marketing Complementary Foods) and so do not sufficiently protect and promote optimal infant and young child feeding practices, revealing much room for improvement. Such guidance must be refined and formalised by international normative bodies and adopted into national legislation to assist manufacturers in ensuring that their complementary food labels meet an accepted standard and contribute towards the safe and appropriate use of processed complementary foods. / Thesis (MSc (Nutrition))--North-West University, Potchefstroom Campus, 2013.

Text in Afrikaans / Die doel van hierdie studie was om te bepaal waarom kinders uit kinderhuise wegloop en om voorstelle ter voorkoming daarvan te maak. Ten opsigte van die fenomeen "wegloop" stateer die literatuur dat kinders wegloop vanaf onaangename omstandighede en/of wegloop na aangename omstandighede. Die belewing van gesinstres, gesinskonflik, portuurgroepdruk, utopiese voorstellings van wegloop, onaangename skoolbelewing en tiener swangerskap is aangeduide rolspelers onderliggend aan bogenoemde. Die empiriese studie na wegloop uit kinderhuise bet aan die lig gebring dat kinders uit kinderhuise wegloop weens traumatiese belewing van verwydering uit die ouerhuis, etikettering en inrigtingsversadiging. Voorts is bevind dat faktore wat onderliggend aan bogenoemde is, tot 'n groot mate voorkom kan word. Sekere aanbevelings ter voorkoming van wegloop uit kinderhuise is na aanleiding van die studie gedoen en kan gebruik word in die volwassene se bemoeienis met die kinderhuiskind. / The object of the study was to identify the reasons why children run away from children's homes and to make recommendations on how to prevent them from running away. On considering "run away" as a phenomenon, the literature states that children run away from harsh circumstances to acceptable or pleasant circumstances. Experiences such as family stress, conflict in the family, peer group pressure, romanticising the idea, harsh school experiences and teenage pregnancies are roll players subjacent to the above mentioned. The empirical study of "running away from children's homes" has confirmed that children run away because of traumatic separation from parental homes, labelling and institutional intolerance. Furthermore it was also found that factors subjacent to the above mentioned can be neutralised to a large extent. Certain guidelines have been recommended for adults when confronted with children in children's homes. / Psychology of Education / M. Ed. (Voorligting)

Submitted by Maria Creuza Silva (mariakreuza@yahoo.com.br) on 2014-10-02T17:23:28Z No. of bitstreams: 1 DISS MP IZIDORIO GONÇALVES 2012.pdf: 324946 bytes, checksum: 027f5ce9ce4581dba679c4513c2310ea (MD5) / Approved for entry into archive by Maria Creuza Silva (mariakreuza@yahoo.com.br) on 2014-10-02T17:38:30Z (GMT) No. of bitstreams: 1 DISS MP IZIDORIO GONÇALVES 2012.pdf: 324946 bytes, checksum: 027f5ce9ce4581dba679c4513c2310ea (MD5) / Made available in DSpace on 2014-10-02T17:38:30Z (GMT). No. of bitstreams: 1 DISS MP IZIDORIO GONÇALVES 2012.pdf: 324946 bytes, checksum: 027f5ce9ce4581dba679c4513c2310ea (MD5) / O presente trabalho teve por objetivo analisar, quanto ao cumprimento da legislação pertinente, os rótulos dos alimentos pães e biscoitos produzidos em pequenas e médias indústrias das regiões do Recôncavo e Sudoeste da Bahia e comercializados em supermercados da cidade de Cruz das Almas - Bahia, considerando a importância da rotulagem no processo de informação em saúde para os cidadãos, tendo por base alguns dos caracteres definidos nas normas vigentes. Foram analisados 100 rótulos de biscoitos e 45 de pães totalizando 145. Os rótulos selecionados corresponderam a marcas diferentes, expostas ao consumidor em 6 supermercados com característica varejista. Os caracteres de interesse do estudo nos rótulos foram registrados em formulário específico e avaliados em relação às amostras com a base na legislação sanitária pertinente: RDC/ANVISA nº 259/02, RDC/ANVISA nº 360/03 e Lei 10.674/03. A análise dos dados foi feita através de frequência simples. Os resultados encontrados apontaram que, do total das amostras analisadas, a maioria absoluta dos rótulos, tanto de pães como de biscoitos apresentaram irregularidades quanto aos caracteres de declaração obrigatória. Entre os rótulos de pães, o maior grau de conformidade com as normas ocorreu com a variável “Conteúdo Líquido”, em 100% dos rótulos analisados. Contudo, apenas 8,8% declararam o item “Informações Nutricionais em maior destaque que outras informações” nos seus rótulos. Das 100 amostras de biscoitos, os caracteres mais satisfatórios foram “Conteúdo Líquido”, “Prazo de Validade” e “Data de Fabricação”, todos sendo informados em 98% dos rótulos. Porém a característica com maior inconformidade foi o “Lote”, com o percentual de apenas 11% de declaração nos rótulos. Conclui-se que há uma alta frequência de rótulos que não cumprem as normas vigentes, o que pode causar danos à saúde dos consumidores, apontando então para a necessidade de medidas estratégicas por parte das instituições competentes, visando a proteção da saúde através do controle sanitário dos alimentos. Considerando o alto percentual de rótulos que não declararam caracteres altamente relevantes como a “Presença de Glúten” e “Aditivos”, dentre outros, destaca-se a importância de promover ações educativas para a população, divulgando a importância das informações contidas nos rótulos, como crucial para proteção de sua saúde, ao tempo em que medidas devem ser adotadas na esfera governamental para assegurar que as indústrias cumpram o seu dever de informar conforme o que está disposto nas normas.

Diagnosis of Alzheimer’s disease is encouraged as a first step towards people planning for their future with the condition. Despite the proposed benefits of diagnosis, it is also widely recognised that Alzheimer’s disease can expose people to stigma. Therefore, this thesis explores the relationship between stigma and future outlook, from the perspective of people affected by early and late-onset Alzheimer’s disease. In order to recognise the physicality of the condition and how psychological and social factors influence experiences, a biopsychosocial perspective is employed throughout. People with Alzheimer’s disease (n=15 people with late-onset, 7 people with early-onset) and their supporters (n=22) completed questionnaires about perceived stigma. This was followed by 14 interviews with a subsample of participants, which explored stigma and future outlook in more depth. Perceived stigma reporting across participants was low in the questionnaires; whereas interviews revealed higher levels of stigma with people discussing mixed, unpredictable reactions from a range of sources. Participants expressed awareness of the unpredictable nature of their futures with the condition. The subsequent lack of control was managed through focusing on ‘one day at a time’ and avoiding looking too far ahead. Across reflections on stigma and future outlook there was a deliberate focus on positive experiences for people affected by early and late-onset Alzheimer’s disease. The similar management of experiences across participants minimised possible age-based differences. These findings are supported by socioemotional selectivity theory, which suggests people are motivated to maintain positive emotional states when facing ‘time-limiting’ conditions irrespective of age. The research suggests people’s experiences of stigma and future outlook interact, with stigma-driven assumptions about the future affecting how people manage their daily lives. The avoidance of looking ahead suggests that policy which encourages future planning should consider its utility and explore ways of helping people to manage both exposure to stigma, and planning for the future, whilst focusing on daily living.

La majorité des composants internes de la cuve des réacteurs à eau pressurisée (REP) est fabriquée en acier inoxydable austénitique (304L ou 316L). Ces matériaux sont exposés à un milieu oxydant sous irradiation et subissent des contraintes mécaniques. Dans ces conditions, ils sont susceptibles de subir un endommagement par corrosion sous contrainte assistée par irradiation (IASCC ). La première étape des phénomènes de fissuration par IASCC est l'amorçage qui implique une rupture du film passif. La nature et la structure de l'oxyde formé sur ces aciers sont donc des paramètres clés vis-à-vis de l'amorçage de la fissuration par IASCC. Dans ce contexte, l'objectif de ce travail est d'une part de mieux appréhender les mécanismes d'oxydation des aciers inoxydables en milieu primaire et d'autre part d'étudier les effets des défauts créés par irradiation sur le film d'oxyde formé sur ces aciers. Des ions xénon ou des protons ont été implantés dans des échantillons d'acier inoxydable austénitique de type 316L, respectivement à une énergie de 240 et 230 keV, afin de simuler les défauts d'irradiation. Ces échantillons ainsi que des échantillons non implantés ont été exposés dans une boucle de corrosion, à 325°C en milieu aqueux, contenant 1000 ppm de Bore, 2 ppm de Lithium, et 1,19.10-3 mol.L-1 d'hydrogène dissous. Les échantillons ont été analysés par MET avant et après exposition en milieu primaire afin de caractériser, d'une part les défauts engendrés par l'implantation et d'autre part la nature, la structure et la morphologie de l'oxyde formé. La comparaison des échantillons implantés et non implantés a permis de montrer que la nature et la densité de défauts en sub-surface de l'alliage jouent un rôle sur la composition (principalement sur la teneur en Cr et en Mo) et l'épaisseur de la couche d'oxyde interne. L'étude de la cinétique d'oxydation par couplage de deux techniques d'analyse par faisceau d'ions (NRA et RBS) a permis de révéler des comportement différents entre les deux catégories d'échantillons : non implantés et implantés. Des essais de traçage isotopique (D et 18O) ont été menés afin d'étudier le mécanisme de formation de la couche interne ainsi que les mécanismes de transports associés. L'étude du transport de l'oxygène et de l'hydrogène à travers la couche interne et dans l'alliage sous-jacent (par SIMS et SDL) a permis d'aboutir à l'écriture d'un mécanisme de corrosion des aciers inoxydables austénitiques en milieu primaire. De plus, l'impact des défauts d'implantation sur ces phénomènes de transport a été étudié, mettant en évidence le rôle des défauts sur les propriétés de l'oxyde formé engendrant des modifications de transport de l'oxygène au sein de cette couche d'oxyde. Ces études de traçage isotopiques ont également permis de mettre en évidence une accumulation d'hydrogène dans l'alliage sous-jacent à l'oxyde. L'ensemble de ces résultats a permis d'apporter de nouveaux éléments de compréhension relatifs à la formation de la couche d'oxyde et à l'absorption d'hydrogène dans les aciers inoxydables austénitiques exposés en milieu primaire, et de mettre en avant l'effet des défauts d'implantation sur les propriétés de l'oxyde formé. / Internal parts of pressurized water reactor (PWR) vessels are often made of austenitic stainless steels (304L and 316L). These structural materials are exposed to an oxidizing medium under irradiation and mechanical stresses. Under these conditions, they can suffer damages by IASCC (Irradiation-Assisted Stress Corrosion Cracking). The first step in this cracking phenomenon is the initiation, which implies the breakdown of the passive layer. The nature and the structure of the oxide film formed on these steels are key factors in initiation of IASCC cracks. In this context, the objective of this work is first to better understand the oxidation mechanisms of stainless steels in primary medium and second to study the effects of irradiation induced defects on the oxide film formed on stainless steels in primary medium. Xenon ions and protons, were implanted in 316L-type austenitic stainless steel samples, respectively at an energy of 240 and 230 keV in order to simulate the irradiation defects. Implanted and non-implanted samples were exposed in a corrosion loop at 325°C to an aqueous medium containing 1000 ppm of boron, 2 ppm of lithium and 1,19.10-3 mol.L-1 of dissolved hydrogen. The samples were analyzed by TEM before and after exposure to primary medium in order to characterize both the defects generated by the implantation and the nature, structure, and morphology of the formed oxide. Comparing implanted and non-implanted samples has shown that the nature and the density of defects in the alloy subsurface played an important role on the composition (mainly on the content of Cr and Mo) and on the thickness of the inner layer. The study of the oxidation kinetics by coupling two ion beam analysis techniques (NRA and RBS) has revealed different behavior between the two types of samples: non-implanted and implanted. Tracer experiments (using D and 18O) were conducted to study the growth mechanism of the inner oxide layer and the associated transport mechanisms. The study of the oxygen and hydrogen transport through the inner layer and the underlying alloy, by SIMS and GD-OES, has resulted in writing a corrosion mechanism for austenitic stainless steels exposed to primary medium and linking this mechanism to hydrogen absorption in the alloy. Furthermore, the impact of implantation defects on these transport phenomena has been studied, highlighting the role of defects on oxide layer properties generating modification of the oxygen transport in the oxide scale. These results have helped to shed some light on the mechanism and kinetics involved in the formation of the oxide layer and on the hydrogen absorption in austenitic stainless steels exposed to primary medium and to point out the effect of implantation defects on the oxidation processes.

Svanen är en av världens ledande miljömärkningar och firade nyligen 20 års jubileum. Det har under ett antal år varit möjligt att förutom produkter även svanenmärka tjänster och verksamheter, så som exempelvis restauranger och hotell. För att miljöarbetet inom en verksamhet ska fungera så bra som möjligt är kommunikationen i denna av yttersta vikt. Det har inte utförts några kända studier kring hur kommunikationen på Svanenmärkta hotell fungerar vilket är motivet bakom denna studie. Det finns en misstänksamhet i samhället mot miljömärkta varor och greenwashing har blivit ett allt vanligare begrepp, vilket innebär att företag utnyttjar miljömärkningar för att få ett bättre anseende på marknaden och inte för att egentligen gynna miljön. Syftet med denna studie har varit att titta på hur miljökommunikationen fungerar inom Svanenmärkta hotell. Ett delsyfte har även varit att ta reda på huruvida medarbetarna på hotellen uppfattar att verksamheten genomsyras av en miljömedvetenhet, något som Svanen strävar efter med sin miljömärkning. En fallstudie av två Svanencertifierade hotell har utförts genom intervjuer av personal från olika avdelningar på dessa hotell. Intervjumaterialet har sedan transkriberats och analyserats för att kunna dra slutsatser utifrån den fakta som framkommit. Även en litteraturstudie har genomförts i syfte att studera tidigare forskning i ämnet. Dessutom har material gällande hotellens miljörutiner granskats och analyserats. Resultaten från fallstudien visar att medarbetarna på hotellen är nöjda med kommunikationen över lag, detta trots att det i princip inte finns någon kommunikation mellan avdelningarna på hotellen. Visst utrymme för förbättring finns dock och både koncernen samt inom hotellen skulle det förmodligen tjänas på att decentralisera miljöfrågorna. På båda hotellen anses även att en miljömedvetenhet genomsyrar verksamheterna. Detta skulle kunna vara en följd av Svanencertifieringen, det kan även bero på andra orsaker.

La compréhension de la fonction des protéines et des systèmes biologiques passe par une connaissance fine des mécanismes moléculaires sous-jacents. La cristallographie et la résonance magnétique nucléaire permettent d'appréhender ces mécanismes au niveau atomique en fournissant des informations sur la structure et sur la dynamique des macromolécules biologiques. Nous nous sommes ainsi intéressés à deux protéines, la lipase lip2 de la levure Yarrowia lipolytica et la protéine membranaire OmpA de la bactérie Klebsiella pneumoniae. Nous avons recherché des conditions d'expression de la protéine lip2 marquée uniformément ou spécifiquement sur une boucle (appelée " lid ") afin d'en étudier la dynamique. Des conditions de marquage uniforme à l'azote 15 de lip2 recombinante dans Yarrowia lipolytica ont été mises au point, mais le marquage acide aminé spécifique n'a pu être réalisé à cause de phénomènes de dilution isotopique trop importants dans cette levure. Nous avons résolu par cristallographie aux rayons X la structure du domaine C-terminal de la protéine OmpA et étudié sa dynamique en solution par RMN (techniques de relaxation 15N). Nous avons caractérisé la dynamique de son domaine N-terminal membranaire reconstitué en liposomes par RMN du solide : en utilisant la rotation à l'angle magique à 60kHz et à la détection 1H sur un spectromètre 1 GHz, nous avons pu attribuer une majorité des résonances du tonneau ? et établir un profil de paramètre d'ordre des vecteurs NH. Des expériences de protéolyse ménagée ont révélé par ailleurs un site de coupure unique à la trypsine au sein de la boucle extracellulaire L3. Enfin, une première caractérisation de la protéine complète exprimée dans la membrane externe d'Escherichia coli a été entreprise par RMN du solide sur membranes externes natives. / Understanding the function of proteins and biological systems requires an accurate knowledge of the underlying molecular mechanisms. Crystallography and nuclear magnetic resonance provide a detailed description of these mechanisms, with an atomic resolution, by providing data on both structures and motions. We investigated two proteins, the lip2 lipase from the yeast Yarrowia lipolytica and the membrane protein OmpA from the bacteria Klebsiella pneumoniae. We tried to produce lip2 with uniform and amino-acid specific stable isotope labelling on its functional loop (the lid) for NMR experiments. The homologous recombinant expression in Yarrowia lipolytica turned out to be the most efficient for uniform labelling but failed for specific labelling due to extensive isotope scrambling. We solved the structure of OmpA C-terminal domain by X-ray crystallography, and analyzed its dynamics in solution by NMR (15N relaxation techniques). We characterized its transmembrane N-terminal domain in proteoliposomes by solid state NMR: using state of the art ultra-fast MAS (60 kHz), 1H detection and a 1 GHz spectrometer, we could assign most ?-barrel resonances and establish a NH order parameter profile. In a complementary approach, we used proteolysis to reveal a unique trypsin cleavage site on the extracellular loop 3. Finally, a first characterization of the full-length protein expressed in the outer membrane of Escherichia coli was initiated by solid state NMR on intact outer membranes.

No description available.