Occasionally, crystallisation of proteins works in mysterious ways! One might obtain crystals of a protein of unknown identity in place of the protein for which crystallisation experiments were performed. If the investigator is not aware of such possibilities, valuable time and resources might be lost in attempting to determine the structure of such proteins. Instances of non-target protein getting crystallised may not come to light at all or may be realised only when attempts to determine the structure completely fail by conventional procedures after collecting and processing the diffraction data. Usually, it is not possible to reproduce the crystals of the same protein as their occurrence is serendipitous. Such rare instances of crystallisation are probably caused by fluctuating environmental or crystallisation conditions and are not reproducible. It could also be due to contaminating microbes, which is more likely when the experimentalist is not well experienced. Therefore, experimental phasing of the data collected on serendipitously obtained crystals could be a challenging task. With the rapid increase in the number of structures deposited in the protein data bank (PDB), molecular replacement has become the method of choice for structure determination in macromolecular X-ray crystallography. This is due to the fact that it is possible to select a suitable phasing model for most target proteins based on their sequence information. However, if the identity of the target protein itself is uncertain, all attempts of structure determination using phasing models selected on the basis of target protein sequence-dependent search would fail. Sequence-independent ab initio phasing techniques such as ARCIMBOLDO (Meindl et al., 2012), which has recently become available, could provide leads only if the non-target protein is an all-α-protein and the associated diffraction data extends to a resolution better than 2 Å. Even then, the success rate with this technique is low. Hence, it becomes important to employ a sequence-independent method of structure determination for such mysteriously obtained crystals. This thesis reports crystal structures of proteins which are serendipitously crystallised using a large-scale application of Molecular Replacement (MR) technique (referred in this thesis as MarathonMR). This thesis also presents an evaluation of molecular replacement strategies for structure determination. The thesis begins with an overview of crystallographic methods of structure determination with an emphasis on the method of molecular replacement (Chapter 1). The most prominent of the results obtained in the course of these investigations pertains to a crystal obtained during routine crystallisation of a viral protein mutant in the year 2011. The cell parameters were different from cell constants of crystals obtained with other known viral protein mutants crystallised earlier in the same laboratory. Unfortunately, this crystal could not be reproduced in the same form in subsequent crystallisation trials. All attempts to determine the structure through conventional molecular replacement techniques using a combination of domains from a nearly identical virus coat protein protomer as the phasing model had failed. The data was shelved as “not-solvable” in late 2011. However, the crystal had diffracted to 1.9 Å and had excellent merging statistics. Therefore, the data was retrieved recently and additional attempts were made to determine the structure through phasing techniques that have become available recently. Techniques such as AMPLE (Bibby et al., 2013) and Rosetta (DiMaio, 2013), which use large-scale homology models coupled with molecular replacement, did not lead to meaningful solutions. A couple of helices identified by ARCIMBOLDO (Meindl et al., 2012) were neither correct (retrospectively) nor sufficient to determine the entire structure. Given the excellent merging statistics of the crystal data, there was significant motivation to determine the structure, though it meant developing a fresh protocol. It was at this time that we came across the work of Stokes-Rees and Sliz (2010) in which they had demonstrated that it is possible to determine structure of proteins of unknown identity by employing almost every known protein structure as a potential phasing model. The work reported in the thesis is a result of an earlier project to examine the relationship between properties of phasing models and the quality of target protein model generated through MR by employing large scale molecular replacement runs. This project was initiated because of the realisation that the recent explosion in crystallographic structural studies has resulted in near complete exploration of the “fold-space” of proteins and PDB now has a representative structure for most plausible folds of proteins. Some folds are highly represented in the PDB. Hence, it is likely that there would be at least one homologue in the PDB which could be used as a phasing model to successfully determine the structure of a protein of unknown identity if the diffraction dataset is of excellent quality. Hence, the single dataset which had diffracted to 1.9 Å resolution was used to develop a MarathonMR procedure for structure determination. MarathonMR procedure takes sequence-independent approach to structure determination and employs large-scale molecular replacement calculations to identify the closest homologue (in structural terms initially). This protocol is described in Chapter 2 (Materials and methods) of the thesis. Through MarathonMR, structure of the dataset which had remained unsolved for 5 years was finally determined. Nearly complete sequence of the polypeptide could be deduced by inspecting the electron density map due to the high resolution and quality of the map. The protein was found to be a phosphate binding protein from a soil bacterium Stenotrophomonas maltophilia (SmPBP). The way in which the structure was determined and possible explanations for the mysterious source of this protein which had crystallised instead of the target protein is discussed in Chapter 3. Though MarathonMR procedure was developed to solve a single dataset, it was soon realised that the same procedure could be applied to other similar datasets, all of which had diffracted to reasonable resolutions with good merging statistics but had remained unsolved for unknown reasons. Among such datasets, one of the datasets which was collected in 2007 and had diffracted to 2.3 Å resolution had cell parameters very close to that of SmPBP. Hence, a poly-alanine model of the structure of SmPBP, which was determined by then, was used as the phasing model to run molecular replacement and the structure was readily solved. It was surprising to note that SmPBP had crystallised serendipitously not once but twice, once in 2011 resulting in crystals that diffracted to 1.9 Å resolution and earlier in 2007 in crystals that diffracted to 2.3 Å resolution independently by two different investigators in the same laboratory. Both the structures are nearly identical and a comparison of these structures is presented in Chapter 4. Structure of SmPBP determined at 2.3 Å resolution by MarathonMR also corresponds to the dataset that had remained unsolved for the longest period of time (9 years). This success of structure determination after the lapse of such a long period emphasises the importance of carefully preserving X-ray diffraction data irrespective of its immediate outcome. In Chapter 5 of the thesis, another instance of non-target protein crystallisation, the structure of which was determined using the MarathonMR procedure is described. The crystal was obtained while carrying out crystallisation of mutants of a survival protein (SurE) expressed in Salmonella typhimurium when the bacterium is subjected to environmental or internal stresses. The original investigator had used the structure of SurE as the phasing model to determine structure of the mutant crystals and obtained a model with R and Rfree of 35% and 40%, respectively. However, the model did not refine further to lower R-factors suggesting that the solution obtained may not be correct. MarathonMR indicated that the fold of the crystallised protein could be similar to that of glycerol dehydrogenase. As SurE shares some fold similarity with one of the domains of GlyDH, the original investigator might have been able to achieve a limited success with R/Rfree factors of 35% and 40%, respectively. As the merging statistics for this diffraction data set was poor, the diffraction images were reprocessed in XDS program on Xia2 automated spot processing pipeline. The data statistics indicated merohedral twinning (14%). However, using appropriate parameters, it was possible to refine the structure obtained by MarathonMR to acceptable R/Rfree using the Refmac program. Four protomers were present in the crystal asymmetric unit (ASU). Non-crytsallographic symmetry averaging of electron density over these four molecules further improved the electron density. As the data was limited to 2.7 Å resolution, it was not possible to deduce the identity of every residue of the protein unambiguously based solely on the resulting electron density map. With the identity of the amino acids that could be deduced with certainty, it was clear that the protein belongs to glycerol dehydrogenase from a species of Enterobacteriacea family. Though a similar structure of glycerol dehydrogenase has been reported from Serratia, there are clear differences in many unambiguously determined residues which suggest that the protein is not from Serriatia. The protein has been named EnteroGlyDH as the source of the protein is likely to be from a species of Enterobacteriacea family. The structure of the protein, its biochemical implications and possible reasons for the serendipitous crystallisation of a non-target are discussed. Chapter 6 discusses the structure determination of an inorganic pyrophosphatase and catalytic domain of Succinyl transferase, the crystals of which had diffracted to 2.3 Å and 3.1 Å, respectively, but had remained unsolved. Neither of the datasets corresponds to the intended target proteins. The dataset corresponding to the protein whose structure was determined as that of an inorganic pyrophosphatase was provided by a colleague from a different laboratory in the Indian Institute of Science. It is interesting to note that the investigator had carried this dataset to one of the CCP4 workshops and had tried to determine the structure with the help of experts in the workshop. The attempts to determine its structure had however failed for reasons that are obvious now. The original investigator was unfortunately making efforts with an erroneous assumption on the identity of the target protein. As these enzymes are well studied, their structures and functions are briefly discussed. It is already well established that molecular replacement is being used with increasing frequency as the phasing technique when compared to other experimental phasing techniques. With the ever growing number of structures in the PDB, high population of certain folds and a near-plateau attained in the identification and growth of new folds, it is reasonable to expect that molecular replacement will be used even more frequently in the years to come. Therefore, for carrying out molecular replacement for a given diffraction dataset of a target protein, it is very likely that several homologous structures would be available in the PDB that could be used as potential phasing models. Hence, it becomes important to understand the influence of phasing model on the quality and accuracy of model generated through MR to achieve the best structure solution. To understand this relationship between phasing model and model obtained by MR protocol, re-determination of already known structures deposited in the PDB starting with their respective structure factors and various phasing models was initiated. Structures belonging to TIM beta/alpha-barrel (SCOPe ID: c.1) and Lysozyme-like (SCOPe ID: d.2) folds were chosen as targets. The structure of each target was re-determined serially starting with poly-alanine models of all available unique homologues as phasing models. Due to the multi-dimensional nature of this study, the results obtained were represented in a graphical form with nodes and edges. Detailed methodology of the work carried out and the data representation model are discussed in the Chapter 2 (Materials and methods). It was found that after a certain sequence identity cut-off, sequence identity between phasing model and target seems to have little influence on the quality and accuracy of the model generated through MR. Instead, other qualities of the phasing model such as Rfree and RSCC influence the quality of MR models. These results are discussed in Chapter 7. Learning from the work reported in this thesis are discussed in concluding chapter. The possible logical and programmatic upgrades to MarathonMR protocol and future path in which the relationship between phasing models and models generated through MR can be studied are discussed in Chapter 8 (Conclusion and future prospects).

Abstract Atherosclerosis is the major cause of cardiovascular diseases and the leading cause of death globally. Atherosclerosis is a complex, chronic disease characterized by lipid accumulation and inflammation within the intima layer of vessel wall. Novel biomarkers and therapeutics are still being sought to provide both better diagnosis and treatment. Ghrelin represents an attractive target for studies into atherosclerosis. Ghrelin is a gastric peptide hormone, which has multiple functions, including regulation of appetite and energy metabolism. Emerging evidence suggests that it may also have a role in the cardiovascular and immune systems. The aim of the present study was to explore the role of ghrelin in atherosclerosis. The specific aims were 1) to investigate the association between the plasma ghrelin level and early atherosclerosis as determined by carotid artery intima media thickness (IMT) in a large (n =  1024) cross-sectional population-based study of middle-aged subjects, 2) to measure the associations between plasma ghrelin levels and already established risk factors of atherosclerosis in human subjects, 3) to assess the effects of ghrelin on atherogenesis in vitro by analyzing monocyte adhesion to endothelial cells, oxidized low density lipoprotein (LDL) binding and acetylated LDL uptake using macrophages, and 4) to study the influence of ghrelin on atherosclerosis using ghrelin vaccination in a mouse model of atherosclerosis. Plasma total ghrelin levels were positively associated with carotid IMT in male subjects. Association studies demonstrated plasma ghrelin levels to be negatively associated with total and LDL cholesterol, and triglyceride concentrations as well as with body mass index (BMI), and positively assocated with high density lipoprotein (HDL) cholesterol concentration in postmenopausal women and in a population-based study. In addition, estrogen increased plasma acylated ghrelin levels in postmenopausal women. Cell culture studies demonstrated that ghrelin could increase the binding of oxidized LDL and monocytes to endothelial cells. Interestingly, when endothelial cells were stimulated with tumor necrosis factor α (TNFα), then ghrelin prevented monocyte adhesion. The study with LDL receptor knockout mice, revealed that ghrelin vaccination could increase plasma ghrelin levels but had no effects on the development of atherosclerosis. However, the plasma MCP-1 level decreased in mice immunized with ghrelin vaccine. In conclusion, these studies suggest that ghrelin has modulatory functions in the vascular system and atherogenesis though the effect may not be as dominant as that of the known traditional risk factors. Whether this effect of ghrelin is positive or negative in atherogenesis will be clarified in further studies. / Tiivistelmä Sydän- ja verisuonitaudit ovat suurin kuolinsyy niin Suomessa kuin useimmissa länsimaissakin. Näiden sairauksien taustalla on yleensä valtimonkovettumatauti eli ateroskleroosi, joka voi kliinisesti ilmentyä mm. sepelvaltimotautina, aivoveritulppana ja laskimotautina. Ateroskleroosissa tulehdussoluja ja kolesterolia kertyy verisuonen seinämään muodostaen ahtauman eli ateroomaplakin valtimoon. Valtimonkovettumataudin riskitekijäitä tunnetaan jo hyvin, mutta uusia tautia ennustavia merkkiaineita sekä hoitomuotoja tarvitaan yhä. Greliini on mahalaukusta eritettävä peptidihormoni, joka osallistuu elimistössä mm. ruokahalun, energiametabolian, tulehdustekijöiden sekä sydän- ja verenkiertoelimistön toiminnan säätelyyn. Tämän työn tavoitteena oli tutkia greliinin yhteyttä ihmisen valtimonkovettumatautiin. Tutkimus toteutettiin käyttämällä kahta eri potilasaineistoa, soluviljelykokeita sekä valtimonkovettumataudin hiirimallia. Laajassa väestöpohjaisessa potilasaineistossa tutkittiin veren greliinipitoisuuden yhteyttä kaulavaltimon seinämän paksuuteen, jota pidetään valtimonkovettumista kuvaavana tekijänä. Veren greliinipitoisuuden yhteyttä valtimonkovettumataudin tunnettuihin riskitekijöihin tutkittiin myös laajassa potilasaineistossa sekä vaihdevuosi-ikäisillä naisilla, joille annettiin estrogeenikorvaushoitoa. Solukokeilla selvitettiin greliinin vaikutusta tärkeisiin valtimonkovettumataudin syntyvaiheisiin käyttäen monosyytti-, endoteelisolu- sekä makrofaagi-soluviljelmiä. Greliinin vaikutusta ateroskleroosiin in vivo selvitettiin rokottamalla LDL-reseptoripuutteiset hiiret greliini-rokotteella. Tutkimuksessa havaittiin yhteys veren korkean greliinipitoisuuden ja kaulavaltimon seinämän paksuuden välillä miehillä laajassa potilasaineistossa (n =  1024). Tulosta tukivat soluilla tehdyt kokeet, joissa greliini lisäsi hapettuneen LDL:n sitoutumista makrofaageihin sekä monosyyttien tarttumista endoteelisolujen pinnalle. Greliinin vaikutukset monosyyttien tarttumiseen endoteelisolujen pinnalle olivat päinvastaiset silloin, kun endoteelisolut käsiteltiin tulehdusta stimuloivalla tekijällä. Matalat veren greliinipitoisuudet olivat myös yhteydessä korkeisiin LDL-kolesteroli- ja triglyseriditasoihin sekä painoindeksiin ja matalaan HDL-kolesterolitasoon potilasaineistoissa. Estrogeeni nosti veren greliinipitoisuutta vaihdevuosi-ikäisillä naisilla. Greliinirokote ei vaikuttanut ateroskleroosin kehittymiseen hiirimallissa. Tutkimustulosten perusteella greliinillä näyttäisi osallistuvan valtimonkovettumataudin kehitykseen, vaikkakin sen vaikutus on pienempi kuin aiemmin tunnetuilla taudin riskitekijöillä.

Chez le jeune adulte, le substitut valvulaire aortique idéal demeure inconnu. La prothèse mécanique est durable. Cependant, elle requiert une anticoagulation à vie. De récentes études ont démontré un excès de mortalité à long terme chez les patients après un remplacement valvulaire aortique (RVA) mécanique. D’autres part, plusieurs patientes sont en âge de procréer lorsqu’une chirurgie est indiquée. La grossesse chez les patientes porteuses de RVA mécanique est à risque d’évènements thromboemboliques. Dans ce contexte, ces derniers sont mieux prévenus par la warfarine comparativement à l’héparine chez les patientes enceintes porteuses de prothèses mécaniques. Cependant, la warfarine est associée à des malformations fœtales. Les prothèses biologiques évitent l’anticoagulation. Par contre, la dégénérescence structurelle de la prothèse est plus rapide chez les patients jeunes, ce qui les expose à un haut risque de réintervention. L’objectif de ce mémoire est d’étudier les issues à long terme suivant un RVA chez le jeune adulte. Deux études ont été réalisées dans le cadre de ce travail. La première avait pour objectif de déterminer la survie et les complications à long terme dans une population de jeunes adultes ayant subi un RVA mécanique isolé. La survie de ce groupe de patient est inférieure à celle de la population générale québécoise appariée pour l’âge et le sexe. De plus, il existe un risque faible, mais constant à long terme de dysfonction de la prothèse et de réopération dans cette population. La deuxième étude a pour objectif de déterminer les complications cardiaques, maternelles et fœtales durant la grossesse chez des patientes porteuses de RVA mécanique ou biologique. Les résultats obtenus démontrent que la grossesse chez ces patientes est associée à un risque de complications maternelles et cardiaques significatives, et ce surtout chez les patientes porteuses d’une prothèse mécanique. / In young adults, the ideal aortic valve substitute remains unknown. Mechanical prostheses are durable. However, their use requires lifelong anticoagulation. Recent reports suggest an excess in long-term mortality in patients following mechanical aortic valve replacement (AVR). Furthermore, many patients are of childbearing age when the surgery is indicated. Pregnancy in patients with mechanical prostheses is associated with an increased thromboembolic event. Those are better prevented by warfarin comparatively to heparin. However, warfarin is associated with fetal malformations. Bioprostheses avoid anticoagulation. Nevertheless, young adults experience a more rapid prosthetic valve deterioration, which exposes young patients to a significantly higher risk of early reoperation. The aim of this thesis is to assess long term outcomes following AVR in young adults. For this project, two different studies were completed. The first study examines the long-term survival and complications following isolated AVR in young adults. Long-term survival was lower than what was expected in a sex- and gender-matched Quebec general population. There was a low but constant occurrence of valve dysfunction and reintervention after AVR in this population. The goal of the second study was to assess cardiac, maternal and fetal complications during and after pregnancy in women with mechanical or biological AVR. Based on our results, we concluded that pregnancies in those women carried a significant risk of cardiac and maternal adverse outcomes, particularly with mechanical prostheses.

Abstract My main objectives in this thesis were to explore general patterns and mechanisms driving salmonid invasions globally and, more specifically, to examine the invasion dynamics and impacts of the North American brook trout in North European stream systems. Non-native salmonids have often spread extensively and caused many harmful impacts on their native counterparts. Among the three globally introduced salmonids, the European brown trout appeared as the 'worst' alien species (strongest impact on native fish), followed by the North American rainbow trout and brook trout. Brook trout, which is widely established in European streams, was found to be a non-aggressive species. Moreover, the growth of brown trout was unaffected by brook trout, indicating negligible interspecific interactions between the two species. Habitat niche segregation between brook trout and brown trout was evident, with brook trout occupying mainly low-velocity pool habitats, whereas brown trout resided in fast-flowing riffles. At the river-wide scale, brook trout occurred mainly in small, slightly acid headwater streams, whereas brown trout was found primarily in larger downstream river sections. Evidently, North European streams with a very low number of native fish species offer underutilized niche space for tolerant headwater specialists such as brook trout. However, the habitat niche filled by brook trout was not completely vacant, as brown trout co-occurred with brook trout in numerous small and mid-sized (3–16 m wide) streams. In these streams, brown trout reproduction was negligible presumably related to the presence of brook trout. Brook trout had also increased in density relative to brown trout during the 10-yr study period (1994 vs. 2004). Moreover, the growth rate and population densities of brook trout were high and the species had spread extensively towards the upmost headwater streams during the 10-yr study period. Thus, harmful effects on the native brown trout by brook trout are likely to occur in the core habitat of the invader, i.e. headwater streams, leaving populations of the native species unaffected elsewhere. Due to the high conservation value of the potentially impacted populations of brown trout, I strongly caution against further stocking of brook trout in European watersheds.

Magister Scientiae (Biodiversity and Conservation Biology) - MSc (Biodiv and Cons Biol) / W.H.O infant feeding options are presented as a package in the prevention of HIV transmission from mother to child. These infant feeding options are namely exclusive breastfeeding, replacement feeding and other options such as wet nursing by a tested HIV negative woman and heat treated breast milk. However, in Zambia, like many other poor countries, the cultural attitude towards breastfeeding is that the breastfeeding period generally goes up to two years. This traditional way of feeding is so much rooted in local culture that any cessation of breastfeeding or any introduction of alternative feeding would be a source of concern at community and family levels. In addition, it is a well known fact that stigma and discrimination are still high in the country. It is with this background that we decided to carry out a study in Mongu district which aimed at assessing HIV positive mothers’ knowledge of WHO infant feeding options and looking at the challenges they face vis-à-vis these recommended feeding options. DATA COLLECTION METHODS A total of 10 experienced nurses, who have been working in the HIV/AIDS programme for more than 15 years, were trained in data collection. During home visit, semistructured questionnaires were used during face- to- face interviews of each HIV positive mother who voluntarily took part in the study. SAMPLING AND SAMPLE SIZE Systematic sampling technique was used to constitute our study sample. With this technique, a complete list of 5317 HIV positive mothers was constituted by listing all HIV positive mothers whose names were in the registers of PMTCT at the selected health institutions, and who had infants whose ages ranged from 6 months to 2 years. 1636 HIV positive mothers had babies whose ages were ranging between 6 months and 2 years. Out of the 1636 we selected randomly the first participant from the complete list, and then we went on selecting every 8th HIV positive mother up to the time we constituted a sample of 200 participants. Thereafter, the selected HIV positive mothers were visited individually in their respective households for interview by trained interviewers. During home visit, 5 selected participants declined to take part in our study while 195 HIV mothers voluntarily accepted to be interviewed. RESULTS Analysis of data collected from 195 HIV positive mothers revealed that 144 study participants or 73.8 %( 95% C I 67.6-80%) of all participants knew their status through the PMTCT programme where the “opt out” approach was used to routinely screen pregnant women for HIV during ante natal visit or when admitted to labour wards. It was also established that the assessment of knowledge among study participants of exclusive breastfeeding period was good. 96.9 %( 95% CI 95.66-98.14%) of participants stated that 6 months was the recommended duration for exclusive breastfeeding when the mother is HIV positive while only 3.07 %( 95% CI 0.65-5.49%) said that exclusive breastfeeding should go beyond 6 months. It was discovered that the majority of HIV positive mothers or 166 participants representing 85.1%(95% CI 80.1-90.1%) who participated in our study considered mixed- feeding as not appropriate for infant born from HIV positive mothers while 29 participants or 14.8%(95% CI 9.8-19.8%) said that mixed feeding was recommendable. It was also found that 95 participants representing 48.7 %( 95% CI 41.6- 55.7%) opted for exclusive breastfeeding, 61 participants or 31.2% (95% CI 24.7-37.7%) participants opted for formula milk while 39 or 20 %( 95% CI 14.4- 25.6%) of participants were mixed-feeding. It was discovered that 118 participants had breastfed. Among them, 53.4 %( 95% CI 46.4-60.4%) participants said that they had breastfeed for up to 6 months while 46.6 %( 95% CI 43-50.2%) said they had breastfeed for more than 6 months. Among those who had breastfed for more than 6 months, 58.1 %( 95% CI 54.6-61.6%) said that they had done so because of financial constraints; 21.8 %( 95% CI 16-27.6%) for fear of discrimination and stigmatization; and 20 %( 95% CI 14.4-25.6%) for fear of discrimination and stigmatization and financial constraints. We also discovered during our research that for the majority of study participants or 81.5%, the decision to opt for one of the infant feeding options was a product of discussion between the HIV positive mothers and other persons such as the husband, friends, relatives and health care provider. CONCLUSION In our study we discovered that though the knowledge of PMTCT and WHO infant feeding options among study participants was good, fear of stigmatization, discrimination and abandonment was high among interviewees. This fear explains why the implementation of WHO infant feeding options is still a serious challenge amongst HIV positive mothers in Mongu, as many HIV positive mothers do not want to be seen in the community as people carrying the virus. It is also for the same reason that our study participants had to choose people to whom to talk to about their HIV positive status and with who to discuss their chosen infant feeding options. Further, due to the high level of poverty among Mongu residents, financial constraint was another major challenge in the implementation of WHO recommended infants feeding options.

Sur la cinquantaine de maladies rares lysosomales, seules 8 peuvent être traitées par enzymothérapie substitutive avec plus ou moins d’efficacité. Il y a donc un réel besoin de développer de nouveaux traitements mais aussi de mieux caractériser les causes de ces maladies. Durant cette thèse nous nous sommes intéressés à la maladie de Pompe qui résulte de l’absence ou de la carence en enzyme lysosomale alpha-glucosidase acide (GAA) responsable de la dégradation du glycogène en glucose dans de nombreux tissus. Actuellement seule la forme infantile de cette maladie peut être traitée alors que la forme juvénile/adulte est faiblement améliorée par le traitement Myozyme®. Cette thèse a eu pour but de développer une nouvelle enzymothérapie qui, à terme, permettrait d’empêcher la progression de la maladie et de soigner, de manière satisfaisante, les formes juvéniles et adultes de la maladie de Pompe. Dans ce but, nous avons utilisé des dérivés monosaccharidiques « Analogues du Mannose-6-phosphate (M6P) Fonctionnalisés sur l’Aglycone (AMFA) », qui sont ensuite greffés sur la GAA recombinante humaine (rhGAA) afin d’améliorer son adressage aux lysosomes obtenant la rhGAA-AMFA.Une première étude in vitro, sur des fibroblastes de patients atteints de la forme adulte de la maladie, a démontré que le greffage des AMFA sur la rhGAA produite en cellules d’insectes Sf9 améliorait significativement l’affinité pour le récepteur du M6P (RM6P), l’internalisation et l’activité de l’enzyme et lui conférait une efficacité sur les souris GAA-/-, modèles de la maladie de Pompe, par rapport au traitement actuel (Article 1). Puis nous avons pu démontrer, pour la première fois, l’efficacité de la rhGAA-AMFA produite en cellules CHO sur des souris GAA-/- âgées. Ces résultats suggèrent, ainsi, la possibilité d’utiliser cette néo-enzyme dans le traitement de la forme adulte de la maladie (Article2). Enfin, le greffage des AMFA permet d’obtenir une maturation intracellulaire complète de la rhGAA sous forme active dans des myoblastes et myotubes de patients adultes et dans les quadriceps de souris âgées modèles Pompe, ce qui n’a pas été observé pour Myozyme® (Article 3). Lors de cette thèse, nous avons également démontré que de nouveaux analogues disaccharidiques, ayant une meilleure affinité que les monosaccharides pour le RM6P, peuvent efficacement cibler la rhGAA pour le traitement de la maladie de Pompe. Un brevet a été déposé sur ces résultats (Brevet PCT/FR2016/052339).En conclusion, ce travail a permis de développer une nouvelle technologie de ciblage plus efficace des enzymes lysosomales par des analogues synthétiques. La désignation de médicament orphelin pour l’alpha glucosidase acide conjuguée aux analogues du mannose-6-phosphate a été obtenue suite à ces travaux auprès de l’Agence Européenne du Médicament pour le traitement de la maladie de Pompe (EMA/OD/098/16).Mots clés: maladies lysosomales, maladie de Pompe, enzymothérapie, récepteur du mannose 6-phosphate / On 53 known rare lysosomal diseases, only 8 can be treated by enzyme replacement therapy with more or less efficiency. There is therefore a need to develop new treatments but also to better characterize these diseases. During this thesis, we focused on Pompe disease which results from the absence or deficiency of the lysosomal enzyme alpha-glucosidase acid (GAA), responsible for the degradation of glycogen in glucose in many tissues. Currently only the infantile form of this disease can be treated while the juvenile/adult form is slightly improved by Myozyme® treatment. This thesis aimed to devel a new enzyme replacement therapy which could prevent the progression of the disease and satisfactorily treat the late onset form of the disease. To do that, we used monosaccharide derivatives “Mannose-6-phosphate analogues (M6P) Functionalized on Aglycone (AMFA)”, which were grafted onto human recombinant GAA (rhGAA) in order to improve its lysosome addressing obtaining the rhGAA-AMFA.A first in vitro study on adult patient fibroblasts showed that the addition of AMFA to rhGAA, produced in Sf9 insect cells, significantly improved its affinity for the M6P receptor (RM6P), its internalization and activity. It was also more efficient on the GAA-/- Pompe mouse model compared to current treatment (Article 1). Then, we demonstrated for the first time the efficiency of rhGAA-AMFA produced in CHO cells in aged mice model. These results suggest the possibility to use this neo-enzyme in the treatment of the adult form that still resists to treatment (Article 2). Finally, the addition of AMFA allows a complete maturation of rhGAA into its active form in myoblasts and myotubes of adult patients and in the quadriceps of aged mice Pompe model. This was not observed for Myozyme® (Article 3). In this thesis we have also demonstrated that novel disaccharide analogues with a better affinity than monosaccharides for RM6P can efficiently target GAA for the treatment of Pompe disease. A patent has been filed on these results (Patent PCT / FR2016 / 052339).In conclusion, this work has led to the development of a new technology more efficient in targeting lysosomal enzymes by mean of new synthetic analogues. An orphan drug designation for the recombinant human acid alpha-glucosidase conjugated with mannose-6-phosphate analogues was obtained on the basis of this work at the European Medicines Agency for the treatment of Pompe disease (EMA/OD/098/16).Key words: lysosomal diseases, Pompe disease, enzyme replacement therapy, mannose 6-phosphate receptor

The object of my diploma is focused in the pension system of The Czech Republic and the perspective of it in the future. At present there is still no problem in the financial balance of the pay-as-you-go pillar but due to the ageing of population the Czech dominant pillar will certain face to serious problems in case of no modifications. This conclusion arises from the first part of the analysis which also consists of the possible parameter modifications that could keep the system in balanced in long term. The next part is the analysis of the savings in the pension funds as the main alternative to the PAYGs. Each approach has its advantages and disadvantages which are main objects of both analyses in this diploma. Beside of the ability to keep the system stabled in financial terms I examine also the equivalence of contributions to the level of pensions that is important for the motivation of people to be responsible for their financial security in their old-age. The last part deals with the experiences of the pension systems in Chile, Slovak Republic and Sweden from where we could gain the real results of the reformed systems in different way in each country. From the results of these analyses I also give some possible recommendations for the elementary modifications in Czech system in the final part.

Internet est un véritable écosystème. Il se développe, évolue et s’adapte aux besoins des utilisateurs en termes de communication, de connectivité et d’ubiquité. Dans la dernière décennie, les modèles de communication ont changé passant des interactions machine-à-machine à un modèle machine-à-contenu. Cependant, différentes technologies sans-fil et de réseaux (tels que les smartphones et les réseaux 3/4G, streaming en ligne des médias, les réseaux sociaux, réseaux-orientés contenus) sont apparues pour améliorer la distribution de l’information. Ce développement a mis en lumière les problèmes liés au passage à l’échelle et à l’efficacité énergétique; d’où la question: Comment concevoir ou optimiser de tels systèmes distribués qui garantissent un accès haut débit aux contenus tout en (i) réduisant la congestion et la consommation d’énergie dans le réseau et (ii) s’adaptant à la demande des utilisateurs dans un contexte connectivité quasi-permanente? Dans cette thèse, nous nous intéressons à deux solutions proposées pour répondre à cette question: le déploiement des réseaux de caches et l’implantation des protocoles économes en énergie. Précisément, nous proposons des modèles analytiques pour la conception de ces réseaux de stockage et la modélisation de la consommation d’énergie dans les réseaux d’accès sans fil. Nos études montrent que la prédiction de la performance des réseaux de caches réels peut être faite avec des erreurs relatives absolues de l’ordre de 1% à 5% et qu’une proportion importante soit 70% à 90% du coût de l’énergie dans les cellules peut être économisée au niveau des stations de base et des mobiles sous des conditions réelles de trafic. / Internet is a real ecosystem. It grows, evolves and adapts to the needs of users in terms of communication, connectivity and ubiquity of users. In the last decade, the communication paradigm has shifted from traditional host-to-host interactions to the recent host-to-content model; while various wireless and networking technologies (such as 3/4G smartphones and networks, online media streaming, social networks, clouds, Big-Data, information-centric networks) emerged to enhance content distribution. This development shed light on scalability and energy efficiency issues which can be formulated as follows. How can we design or optimize such large scale distributed systems in order to achieve and maintain high-speed access to contents while (i) reducing congestion and energy consumption in the network and (ii) adapting to the temporal locality of users demand in a continuous connectivity paradigm? In this thesis we focus on two solutions proposed to answer this question: In-network caching and Power save protocols for scalability and energy efficiency issues respectively. Precisely, we propose analytic models for designing core cache networks and modeling energy consumption in wireless access networks. Our studies show that the prediction of the performance of general core cache networks in real application cases can be done with absolute relative errors of order of 1%–5%; meanwhile, dramatic energy save can be achieved by mobile devices and base stations, e.g., as much as 70%–90% of the energy cost in cells with realistic traffic load and the considered parameter settings.

Introdução: As Mucopolissacaridoses (MPS) são doenças lisossômicas causadas pela deficiência de enzimas envolvidas na degradação dos glicoaminoglicanos. O acúmulo anormal dessa molécula compromete a função celular e orgânica, levando a um espectro de manifestações clínicas, de caráter multissistêmico e progressivo. Manifestações gastrointestinais, tais como episódios frequentes de fezes amolecidas e diarréia, têm sido frequentemente evidenciadas e relatadas por pacientes com MPS. Ainda que os sintomas gastrointestinais sejam frequentemente ofuscados pelos fenótipos neurológicos graves, eles podem afetar a qualidade de vida dos pacientes e de seus familiares. Objetivo: Avaliar as manifestações gastrointestinais em pacientes com MPS que estavam ou não recebendo Terapia de Reposição Enzimática (TRE). Adicionalmente, para melhor compreender o assunto, avaliamos a histologia da mucosa intestinal em camundongos com MPS I. Métodos: Estudo transversal com amostragem de conveniência. Foram incluídos no estudo pacientes com diagnóstico de qualquer tipo de MPS que estavam ou não em TRE. Os sujeitos foram avaliados através de inquérito alimentar, questionamentos quanto ao aparecimento de sintomas gastrointestinais e realização de uma série de exames bioquímicos. Adicionalmente, foram realizados testes de bioimpedância elétrica (para avaliação da composição corporal) e teste molecular para hipolactasia primária a partir de amostras de DNA dos pacientes, assim como análise da histologia da mucosa intestinal em um modelo de camundongo com MPS I. Resultados: Foram incluídos 27 pacientes com diferentes tipos de MPS, sendo 15 (55,6%) do sexo feminino e 12 (44,4%) do sexo masculino, com mediana de idade de 12 (1-28) anos. Os sintomas gastrointestinais mais prevalentes foram flatulência, distensão abdominal, dor abdominal e fezes amolecidas. Houve diferença significativa na prevalência de flatulência entre os tipos de MPS (p=0,004). A prevalência de flatulência e de distensão abdominal foi significativamente maior no grupo dos que não recebiam TRE, em comparação ao grupo dos que recebia TRE (p = 0,04 e 0,03, respectivamente). A maioria dos exames bioquímicos realizados para investigação e/ou exclusão de alguma patologia específica apresentaram resultados normais, sugerindo que o aparecimento de sintomas gastrointestinais nesses pacientes possa ser decorrente da própria MPS através de mecanismo fisiopatológico independente. A análise da histologia do intestino delgado de camundongos MPS I identificou células aumentadas de volume, sugerindo algum tipo de acúmulo intracelular. Em relação ao Teste Molecular para Hipolactasia, 58,8% dos pacientes apresentaram genótipo CC, o qual é compatível com intolerância à lactose. Os exames de Bioimpedância Elétrica sugerem que, os pacientes em TRE apresentam menor proporção de massa gorda (MG) em relação aos pacientes que não estavam em TRE. Conclusão: Este foi o primeiro estudo sobre manifestações gastrointestinais de pacientes brasileiros com MPS realizado em nosso meio. Inúmeros sinais e sintomas foram observados, com maior prevalência relativa nas MPS I, II, III e IV do que na MPS VI. O sintoma mais frequentemente relatado foi flatulência. Embora não tenha sido estatisticamente significativo a diferença, a proporção de pacientes com sintomas gastrointestinais foi menor nos pacientes que estavam em TRE. A Intolerância à lactose observada em maior proporção nos pacientes com MPS com manifestações gastrointestinais, a avaliação da composição corporal e as alterações observadas na mucosa intestinal do camundongo com MPS I devem ser levadas em consideração na interpretação dos resultados. Acreditamos que mais estudos, dirigidos para as manifestações gastrointestinais, devam ser realizados para comprovar esses achados e melhor compreender os mecanismos fisiopatológicos dos sintomas relacionados nos pacientes aferidos. / Introduction: The mucopolysaccharidoses (MPS) are lysosomal storage disorders characterized by a deficiency in enzymes involved in glycosaminoglycan degradation. Abnormal accumulation of this molecule compromises cellular and organic function, leading to a spectrum of progressive, multisystem clinical manifestations. Gastrointestinal manifestations, such as frequent episodes of loose stools and diarrhea, are often reported by patients with MPS. Although these gastrointestinal symptoms are often overshadowed by severe neurological phenotypes, they can have a negative impact on the quality of life of patients and their family members. Objective: To assess gastrointestinal manifestations in patients with MPS who were or not receiving enzyme replacement therapy (ERT). Furthermore, we sought to assess bowel mucosa histology in a mouse model of MPS I. Methods: Cross-sectional study with a convenience sampling strategy. The sample comprised patients with a diagnosis of MPS of any type and regardless of ERT status. Patients were assessed by means of a dietary recordatory and an interview focused on gastrointestinal symptoms, as well as a battery of biochemical tests. Bioelectrical Impedance was performed for body composition assessment.Patient DNA samples were also tested by molecular tests for primary lactase hypolactasia, and bowel mucosa specimens from MPS I mice underwent histological examination. Results: A total of 27 MPS patients were included, 15 (55.6%) female and 12 (44.4%) male, with a median age of 12 (1-28) years. The most prevalent gastrointestinal symptoms were flatulence, abdominal distension, abdominal pain, and loose stools. A significant difference in the prevalence of flatulence was observed among different MPS types (p=0.004). The prevalence of flatulence and abdominal distension was significantly higher in the non-ERT group than in the ERT group (p = 0.04 and 0.03 respectively). Most biochemical tests performed to work up and/or rule out specific conditions were within normal limits, which suggests that the development of gastrointestinal systems in these patients may be due to MPS itself through an independent pathophysiological mechanism. Histological analysis of smallbowel tissue from MPS I mice found increased cell volume indicative of some form of intracellular accumulation. On molecular testing for lactase deficiency, 58.8% of patients had the CC genotype, which is consistent with lactose intolerance. Bioelectrical Impedance analysis suggest that patients with TRE have lower proportion of fat mass (FM) compared to patients who were not on ERT. Conclusion: This was the first study to assess gastrointestinal manifestations in Brazilian patients with different MPS subtypes.Several signs and symptoms were observed with higher relative prevalence in MPS I, II, III and IV to that in MPS VI. The most frequently reported symptom was flatulence. Although was no statistical difference the proportion of patients with gastrointestinal symptoms was lower in patients on ERT.The lactose intolerance observed in a higher proportion of MPS patients with gastrointestinal manifestations, body composition assessment and observed changes in the bowel mucosa of MPS I mice should be taken into account when interpreting the results. Further studies focusing on the gastrointestinal manifestations of MPS are warranted to corroborate our findings and provide a better understanding of the pathophysiological mechanisms associated with these symptoms in affected patients.

Este trabalho teve por objetivo avaliar a modulação autonômica da freqüência cardíaca (FC) durante o repouso, nas posições supina e sentada, e durante teste de esforço físico dinâmico descontínuo do tipo degrau (TEFDD-D) em mulheres jovens e pós-menopausa sem (PMSRH) e com reposição hormonal (PMCRH); determinar o limiar de anaerobiose (LA) a partir da análise das respostas de FC e pela análise dos índices de RMSSD (raiz quadrada da média dos quadrados das diferenças entre os intervalos R-R normais sucessivos), em milissegundos (ms), e comparar o grau de correlação entre estas duas metodologias de análise. Foram estudadas 11 jovens (24 ± 2,77 anos), 13 PMSRH (57 ± 5,28) e 9 PMCRH (55 ± 5,40 anos). O TEFDD-D foi realizado em cicloergômetro, sendo iniciado com a potência de 15 W e com incrementos de 5 em 5 W. A FC (bpm) e os intervalos R-R (ms) foram captados em tempo real, por um período de 360s em repouso, em cada posição, e durante 60s em repouso sentado no cicloergômetro, 240s em exercício e 60s em recuperação, em cada potência do TEFDD-D. Foram calculados as médias da FC (bpm) e os índices de RMSSD dos intervalos R-R (ms) para as condições de repouso e durante 180s do exercício nas potências estudadas; cálculo da variação da FC (bpm) no início do exercício e do tempo (s) desta variação. A determinação do LA foi feita pelo ajuste do modelo matemático e estatístico semiparamétrico (SPM) aos dados de FC e pelos índices de RMSSD dos intervalos R-R (ms). Os testes estatísticos utilizados foram: Wilcoxon, Kruskall-Wallis, Friedman, Dunn e o teste de correlação de Spearman, nível de significância de 5%. Durante o repouso, as jovens apresentaram valores dos índices de RMSSD significativamente (p<0,05) superiores em relação aos outros 2 grupos. As variações da FC das jovens no início do exercício foram maiores que as dos grupos PMSRH e PMCRH, enquanto que o tempo de variação da FC foi similar entre os 3 grupos. Na transição do repouso para o exercício, a FC aumentou progressivamente, enquanto que a variabilidade da freqüência cardíaca (VFC) diminuiu. Na comparação intergrupo dos índices de RMSSD, obtidos em cada nível de potência, foi observada diferença significativa (p<0,05) apenas em 35W. Tanto pelo modelo SPM, como pela análise dos índices de RMSSD, as jovens atingiram o LA em potências superiores comparativamente as PMSRH e PMCRH. Os grupos PMSRH e PMCRH apresentaram resultados similares. Não foram observadas diferenças significativas (p>0,05) na comparação dos 2 métodos. O teste de correlação de Spearman mostrou uma associação significativa (p<0,05) entre os mesmos. Estes dados sugerem que após a menopausa ocorre uma diminuição da modulação vagal sobre o coração tanto em repouso como durante o exercício físico, decorrente do processo do envelhecimento e da redução da capacidade física. A terapia de reposição hormonal não teve nenhuma influência sobre os resultados. As duas metodologias de análise do LA se mostraram similares, sugerindo que a mudança de inclinação da resposta da FC ocorre em níveis de esforço em que a VFC se encontra significativamente reduzida. / The objectives of the present study were to assess the autonomic modulation of the heart rate (HR) at rest, in the supine and sitting position, and during a step type discontinuous dynamic physical effort (STDDPE) in young and postmenopausal women not receiving (PMWtHR) and receiving hormonal replacement treatment (PMWHR); to determine the anaerobiosis threshold (AT) based on the analysis of HR response and the RMSSD indices (square root of the mean squared differences of successive R-R intervals), in milliseconds (ms), and to compare the degree of correlation between these two analysis methodologies. The study was conducted on 11 young women (24 ± 2.77 years), 13 PMWtHR (57 ± 5.28) and 9 PMWHR (55 ± 5.40 years). The STDDPE was performed on a bicycle ergometer at an initial power of 15 W, followed by power increments of 5 W. HR (bpm) and R-R intervals (ms) were obtained in real time over a period of 360 s under resting conditions in each position, during 60 s in the sitting rest position on the bicycle ergometer, 240 s during exercise and 60 s during recuperation at each STDDPE power. Mean HR (bpm) and RMSSD indices of the R-R intervals (ms) were calculated for the resting condition and during 180 s of exercise in the powers studied; the HR variation (bpm) and its time (s) were also calculated in the beginning of exercise. AT was determined by the semiparametric mathematical and statistical model (PMS) and by the RMSSD indices of the R-R intervals (ms). Data were analyzed statistically by the Wilcoxon, Kruskal-Wallis, Friedman, Dunn and Spearman correlation tests, with the level of significance set at 5%. During rest, young women presented significantly higher RMSSD indices (p<0.05) than the other 2 groups. The HR variations in young women in the beginning of the exercise were higher than the ones from the PMWtHR and PMWHR groups, whereas HR variations time was similar for the 3 groups. During the transition from rest to exercise HR increased progressively and HRV decreased. Intergroup comparison of RMSSD indices, obtained in each level of power, showed a significant difference (p<0.05) only at 35 W power. On both PMS model and RMSSD indices analysis, young women reached AT at a higher power compared to PMWtHR and PMWHR groups. The PMWtHR and PMWHR groups presented similar results. No significant differences (p>0.05) were observed when the methods were compared. The Spearman correlation test showed a significant association (p<0.05) between methods. These data suggest that after menopause there is a decrease in vagal modulation of the heart both at rest and during physical exercise due to the aging process and the reduction in physical capacity. Hormonal replacement therapy had no effect on the results. Both methodologies of AT analysis were similar, suggesting that the change in the HR response occurs in levels of effort in which the HRV is significantly reduced.