Masters Research - Master of Education / In recent years a considerable literature has accumulated to establish that western society is confronting a monumental crisis in health care. In the thesis that follows we shall see that the nature of the growing crisis is multifaceted and includes scientific, socio-cultural and philosophical dimensions, all of which figure prominently in the way in which we educate people of all ages for health. My aim in the first part of the thesis will be to explore and reflect upon a number of these facets, with an aim to showing that the epistemological framework they presuppose represents a valuable but incomplete understanding of newly emerging health problems which are themselves, partly the outcome of the highly technologised societies in which we live. Once this preliminary objective of the thesis has been completed, I shall contend in Part II of the thesis that one area of crisis which remains insufficiently understood is the relationship between food and nutrition. I deliberately use the word relationship between food and nutrition, because the traditional emphasis of such concerns within the context of health has focused primarily on nutrition, largely in the quantitative sense of encouraging people to obtain enough vitamins, minerals, proteins, carbohydrates and fats required to keep them healthy. My contention is that without broadening the discussion to encompass the relationship between food and nutrition, the answers we give to the quantitative questions are inevitably myopic and limited. My goal in the third and final part of this thesis will be to make clear that in light of the importance of the connection we have with our food and recent developments made in the philosophy of quantum mechanics, an exciting new discipline is emerging which Professor Ronald S. Laura has called the ‘Metaphysics of Food’. As part of my elaboration of this area, I shall weave together strands of insight from the research of Professor Laura and Dr. Masaru Emoto, two major pioneers in advancing this field of knowledge. Professor Laura’s theory of ‘participatory consciousness����, drawn from his elaboration of quantum entanglement and his theory of ‘empathetic epistemology’ provides a fruitful conceptual framework, I shall argue, for the philosophical elucidation of Emoto’s research on the metaphysics of vii water. Using the confluent theoretical interpretative heuristics of Laura and Emoto, my objective will be to argue that in principle, the foods we eat can themselves be impacted favourably or adversely in health terms by entanglements of consciousness which in turn affect our own health in subtle but important ways. Although the articulation of the pedagogic implications of this insight would be beyond the remit of this Master Thesis, a conceptual foundation will have been laid here upon which a new edifice for further research at the doctoral level can be built.

Masters Research - Master of Education / In recent years a considerable literature has accumulated to establish that western society is confronting a monumental crisis in health care. In the thesis that follows we shall see that the nature of the growing crisis is multifaceted and includes scientific, socio-cultural and philosophical dimensions, all of which figure prominently in the way in which we educate people of all ages for health. My aim in the first part of the thesis will be to explore and reflect upon a number of these facets, with an aim to showing that the epistemological framework they presuppose represents a valuable but incomplete understanding of newly emerging health problems which are themselves, partly the outcome of the highly technologised societies in which we live. Once this preliminary objective of the thesis has been completed, I shall contend in Part II of the thesis that one area of crisis which remains insufficiently understood is the relationship between food and nutrition. I deliberately use the word relationship between food and nutrition, because the traditional emphasis of such concerns within the context of health has focused primarily on nutrition, largely in the quantitative sense of encouraging people to obtain enough vitamins, minerals, proteins, carbohydrates and fats required to keep them healthy. My contention is that without broadening the discussion to encompass the relationship between food and nutrition, the answers we give to the quantitative questions are inevitably myopic and limited. My goal in the third and final part of this thesis will be to make clear that in light of the importance of the connection we have with our food and recent developments made in the philosophy of quantum mechanics, an exciting new discipline is emerging which Professor Ronald S. Laura has called the ‘Metaphysics of Food’. As part of my elaboration of this area, I shall weave together strands of insight from the research of Professor Laura and Dr. Masaru Emoto, two major pioneers in advancing this field of knowledge. Professor Laura’s theory of ‘participatory consciousness����, drawn from his elaboration of quantum entanglement and his theory of ‘empathetic epistemology’ provides a fruitful conceptual framework, I shall argue, for the philosophical elucidation of Emoto’s research on the metaphysics of vii water. Using the confluent theoretical interpretative heuristics of Laura and Emoto, my objective will be to argue that in principle, the foods we eat can themselves be impacted favourably or adversely in health terms by entanglements of consciousness which in turn affect our own health in subtle but important ways. Although the articulation of the pedagogic implications of this insight would be beyond the remit of this Master Thesis, a conceptual foundation will have been laid here upon which a new edifice for further research at the doctoral level can be built.

The Olfactory Epithelium (OE) is a specialized epithelial tissue inside the nasal cavity that is involved in the smell sensation. The OE maintains neuroregeneration, i.e. producing new olfactory sensory neurons, throughout the adult life via neural stem cell self-renewal, proliferation, neuronal differentiation and maturation. The neural stem cell niche regulates stem cell self-renewal and proliferation, and consists of stem cells, blood vessels and multiple extracellular matrix proteins (ECMs). ECMs regulate stem cell adhesion, proliferation, differentiation and migration via integrins. One of the main mediators of intracellular integrin signaling is the Focal Adhesion Kinase (FAK). Our previous studies found that FAK inhibition increased cell proliferation in adult mouse olfactory epithelium (OE) via up-regulation of Ciliary Neurotrophic Factor (CNTF). Now we continue to test whether FAK inhibition increases neuroregeneration through CNTF in the adult mouse OE using BrdU-chase pulse method. Adult male and female C57BL/6, CNTF wildtype and CNTF knockout (lack the CNTF gene) mice were systemically injected with PBS or FAK inhibitor (FAK14) for 3 days. During these 3 days, BrdU was injected into mice 4 h following PBS or FAK on each day. BrdU acts as a thymidine analog and is incorporated into DNA during DNA syntheses. Using immunohistochemistry with anti-BrdU antibody, BrdU+ cells can be visualized in the tissue. The BrdU+ cells are the ones who are replicating during the time frame when BrdU was given. 20 days after last BrdU injection, we fixed the mice via cardiac perfusion. The whole heads of mice was decalcified with EDTA and then frozen cross head sections including OE were cut using cryostat and mounted onto slides. The OE sections were then stained with anti-BrdU antibody followed by FITC-conjugated secondary antibody. The BrdU+ cells in the OE were counted in three sections (both left and right sides) per mouse and normalized to linear length of OE basement membrane. The results of the experiment showed that FAK 14 significantly increased BrdU+ stem cells and olfactory sensory neurons in the OE of C57BL/6 and CNTF wildtype mice but not knockout mice, indicating that FAK inhibition promotes olfactory stem cell self-renewal and neuroregeneration via CNTF. Collectively, this data indicates that FAK normally inhibits OE neuroregeneration by inhibiting CNTF expression and identifies the OE is a good model to study neuroregenerative mechanisms in the CNS.

The construction and operation of hydroelectric projects consist of multiple activities in a single watershed, which can generate significant impacts on the surrounding biophysical environment and on the health and well-being of local communities. The impacts of those activities may be insignificant individually, yet together may have an important cumulative effect. The impacts of hydroelectric development on human health and well-being have been widely documented. Current practices of cumulative effects assessment (CEA), however, as conducted under project-based environmental assessment (EA), often fail to address the deeper issues of human health and social well-being. This thesis was developed to examine how health effects, including cumulative health effects, are considered within regulatory EA practices in the hydroelectric sector in Manitoba. This was achieved by reviewing the EAs of three recent hydroelectric projects –Wuskwatim Generating Station, Bipole III Transmission Project, and Keeyask Hydroelectric Generating Station – located in the Nelson River watershed in northern Manitoba. Results indicate that the consideration of human health issues in EA has gradually improved over time; however, the assessment of health impacts was invariably limited to physical health components and often lacked due consideration of broader social health issues. The inadequacy of the practice of health impact assessment (HIA) was also evident by the lack of health-related indicators and the poor consistency in the use of indicators across projects and over time for measuring and predicting changes in the health conditions of the communities due to project development. An in-depth analysis regarding the assessment of cumulative health effects was carried out in the CEA of the most recent hydroelectric development – the Keeyask project. The findings show that cumulative health effects were not adequately considered in each of the basic components of CEA – scoping, retrospective analysis, prospective analysis, and management measures. Improving the consideration of health in EA requires paying more attention to broader range of health determinants, including both biophysical and social determinants and their interconnectedness in EA. Moreover, there is a need to improve greater consistency in the use of health indicators across projects and over time. It can be assisted by developing standardized terms of reference (ToR) for project proponents to ensure the consideration and monitoring of those indicators used for development projects built within the same geographic region and affecting the same communities and environments. Approaching cumulative health effects in a more regional and strategic framework of CEA, beyond the scale of individual projects, is likely to provide the best mechanism to understand and monitor the cumulative impacts of project development on the health and well-being of the affected communities.

In order for an economy to survive and thrive it requires resources. Water is a resource that not only the economy is dependent on but also ecological and human communities. The deteriorated state of South African rivers suggests the intensive use of the country’s freshwater reserves by the population and industry. Such a source of freshwater is the Wonderfonteinspruit. It flows through an area that requires water for gold mining that has taken place in the area for more than 120 years. Furthermore, the Wonderfonteinspruit runs past communities such as Bekkersdal and eventually forms part of the source waters for the Boskop Dam, the main drinking water reservoir for Potchefstroom. Literature suggests that the Wonderfonteinspruit is impacted by anthropogenic activities, in particular impacts associated with both historical and current gold mining activities. The Wonderfonteinspruit has its origin in the Tudor Dam in Krugersdorp (now Mogale City), and then flows into Donaldson Dam from where it is piped in a 32 km long pipeline, before its confluence with the Mooi River which subsequently flows into the Boskop Dam. The study area specifically focuses on the Upper Wonderfonteinspruit from just downstream of the Donaldson Dam to just upstream of the dam. The study area was selected due to the close proximity of the Donaldson Dam to the community of Bekkersdal which formed the second part of the investigation for this thesis. Bekkersdal is primarily a mining community that has historically faced issues with sufficient land provision, housing, unemployment and service delivery. It is located in the Gauteng Province and falls under the jurisdiction of Westonaria Local Municipality. Recent protests by community members have occurred due to the lack of service delivery and inappropriate development of infrastructure with regards to water services. Due to the close proximity of Bekkersdal to the Wonderfonteinspruit (as it is situated on the border of the Donaldson Dam) the community provided an ideal study area to explore the use of the river by the community. In order to determine the relationship between the Wonderfonteinspruit and the community of Bekkersdal the study comprised two parts: during the first part of the study, the ecological state of the Wonderfonteinspruit was determined through the evaluation of the quality of water, sediment and biota within the river; while in the second part an assessment of Bekkersdal (both formal and informal sections) was undertaken through the use of questionnaires in order to determine past, current and future water use of both municipal water and water sourced from the Wonderfonteinspruit. The final outcomes of both the environmental and social assessments were then compared with national and international standards. Water quality assessment of the Wonderfonteinspruit was done by assessing the following: - in situ water quality parameters (such as pH, total dissolved solids and dissolved oxygen) - metal and ionic composition analysis of water samples using inductively coupled plasma mass spectrophotometry (ICP-MS) - nutrient loads using a spectrophotometer and Spectroquant® test kits - bacteriological quality by determining presence of total coliforms and faecal coliforms through the growth of bacterial cultures on M-ENDO and m-FC agar plates - the Physico-chemical Driver Assessment Index (PAI) was applied according to DWAF 2008 - statistical relevance between sites and results through principal component analysis (PCA) Finally, these results, where applicable, were compared to both national and international standards for human and ecological use. The results indicated that the water quality levels exceeded the guideline values of national and international standards for the following uses: drinking water, certain industrial activities, watering of certain livestock and crop types as well as aquaculture. It was also found that the water quality was acceptable for certain activities (e.g. recreation) only if precautions and further analysis are performed. The guideline values of national water quality standards for ecological status were also exceeded, while the PAI results indicated that the ecological category (EC) for the Wonderfonteinspruit is a D which indicates that the state of the water quality in terms of the ecology is fair. The sediment quality of the Wonderfonteinspruit was determined by ICP-MS. The metal composition of the sediment was compared to that of other rivers and the following indices were applied: enrichment factor (EF), contamination factor (CF), pollution load index (PLI) and geo-accumulation index (Igeo). It was found that the sediment composition is comparable to that of other rivers impacted by gold mining and that uranium, cobalt and nickel enriched the sediment according to the indices. Biotic indicators that were assessed included fish, diatoms and invertebrates. The fish health assessment index (HAI) was applied to fish caught in the Donaldson Dam. The muscle tissue was also removed and its metal concentration was determined by ICP-MS. Thereafter, the edibility of the fish muscle tissue was determined and the following indices were applied: condition factor (CF), hepatosomatic index (HSI), gonadosomatic index (GSI) and spleen somatic index (SSI). The diatom community composition was assessed by applying the Biological Diatom Index (BDI), Specific Pollution Sensitivity Index (SPI) and the percentage pollution tolerant valves (%PTV). The Macroinvertebrate Response Assessment Index (MIRAI) was applied in order to determine the state of the macroinvertebrate community. The fish assessment indicated that arsenic contamination may negatively impact the health of consumers. Diatom indices indicated that the EC of the Wonderfonteinspruit is a D/E which indicates poor water quality; likewise, this is supported by the MIRAI results as the EC for MIRAI was a D which indicates that the river is largely modified. The investigation into the water use of the Bekkersdal community, with a special focus on the use of the Wonderfonteinspruit, was achieved through the use of questionnaires that were distributed in both formal and informal sections in Bekkersdal. The research forms part of a larger Integrative Multidisciplinary study and was given ethical clearance under the NRF Community Engagement Project (see Ethical Clearance: no. FH-BE-2013-0014. The National Research Fund (NRF) provided the funding for the research, the views expressed is that of the author and not those of the NRF. The aim of the questionnaire was to determine the following aspects in terms of the community of Bekkersdal: - Demographic details, such as language preference, employment status and age distribution. - Current water use practices . - Use of the Wonderfonteinspruit. - Future water use of the Wonderfonteinspruit. - Water quality perceptions of the Wonderfonteinspruit. - Field notes that included any relevant observations of the fieldworkers. The unemployment rate of the Bekkersdal community was found to be high (78.20%) and 86.40% of the residents are South African citizens. The community relies heavily on municipal provision of sources of water with 100% of the respondents indicating that it is their primary source of water. However, several issues were identified in terms of municipal water supply in the community. Some 10.14% of the residents indicated that they make regular use of the Wonderfonteinspruit (in particular the Donaldson Dam) most often for drinking water, laundry and washing of cars, etc. Regarding the state of the Wonderfonteinspruit, the overall viewpoint of the Bekkersdal community was that it is largely polluted with sewage, litter and mining waste. However, some 87.80% of the residents expressed their willingness to participate in environmental clean-up initiatives in their area. The link between the ecological state of the Wonderfonteinspruit and human health and wellbeing was explored through the use of spider diagrams where rank scores were assigned to both index results and human water quality use categories. These were compared and it was found that ecological indicators are more sensitive than human water quality use scores and therefore can aid in acting as early detection indicators of possible negative impacts on human health and wellbeing. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

In order for an economy to survive and thrive it requires resources. Water is a resource that not only the economy is dependent on but also ecological and human communities. The deteriorated state of South African rivers suggests the intensive use of the country’s freshwater reserves by the population and industry. Such a source of freshwater is the Wonderfonteinspruit. It flows through an area that requires water for gold mining that has taken place in the area for more than 120 years. Furthermore, the Wonderfonteinspruit runs past communities such as Bekkersdal and eventually forms part of the source waters for the Boskop Dam, the main drinking water reservoir for Potchefstroom. Literature suggests that the Wonderfonteinspruit is impacted by anthropogenic activities, in particular impacts associated with both historical and current gold mining activities. The Wonderfonteinspruit has its origin in the Tudor Dam in Krugersdorp (now Mogale City), and then flows into Donaldson Dam from where it is piped in a 32 km long pipeline, before its confluence with the Mooi River which subsequently flows into the Boskop Dam. The study area specifically focuses on the Upper Wonderfonteinspruit from just downstream of the Donaldson Dam to just upstream of the dam. The study area was selected due to the close proximity of the Donaldson Dam to the community of Bekkersdal which formed the second part of the investigation for this thesis. Bekkersdal is primarily a mining community that has historically faced issues with sufficient land provision, housing, unemployment and service delivery. It is located in the Gauteng Province and falls under the jurisdiction of Westonaria Local Municipality. Recent protests by community members have occurred due to the lack of service delivery and inappropriate development of infrastructure with regards to water services. Due to the close proximity of Bekkersdal to the Wonderfonteinspruit (as it is situated on the border of the Donaldson Dam) the community provided an ideal study area to explore the use of the river by the community. In order to determine the relationship between the Wonderfonteinspruit and the community of Bekkersdal the study comprised two parts: during the first part of the study, the ecological state of the Wonderfonteinspruit was determined through the evaluation of the quality of water, sediment and biota within the river; while in the second part an assessment of Bekkersdal (both formal and informal sections) was undertaken through the use of questionnaires in order to determine past, current and future water use of both municipal water and water sourced from the Wonderfonteinspruit. The final outcomes of both the environmental and social assessments were then compared with national and international standards. Water quality assessment of the Wonderfonteinspruit was done by assessing the following: - in situ water quality parameters (such as pH, total dissolved solids and dissolved oxygen) - metal and ionic composition analysis of water samples using inductively coupled plasma mass spectrophotometry (ICP-MS) - nutrient loads using a spectrophotometer and Spectroquant® test kits - bacteriological quality by determining presence of total coliforms and faecal coliforms through the growth of bacterial cultures on M-ENDO and m-FC agar plates - the Physico-chemical Driver Assessment Index (PAI) was applied according to DWAF 2008 - statistical relevance between sites and results through principal component analysis (PCA) Finally, these results, where applicable, were compared to both national and international standards for human and ecological use. The results indicated that the water quality levels exceeded the guideline values of national and international standards for the following uses: drinking water, certain industrial activities, watering of certain livestock and crop types as well as aquaculture. It was also found that the water quality was acceptable for certain activities (e.g. recreation) only if precautions and further analysis are performed. The guideline values of national water quality standards for ecological status were also exceeded, while the PAI results indicated that the ecological category (EC) for the Wonderfonteinspruit is a D which indicates that the state of the water quality in terms of the ecology is fair. The sediment quality of the Wonderfonteinspruit was determined by ICP-MS. The metal composition of the sediment was compared to that of other rivers and the following indices were applied: enrichment factor (EF), contamination factor (CF), pollution load index (PLI) and geo-accumulation index (Igeo). It was found that the sediment composition is comparable to that of other rivers impacted by gold mining and that uranium, cobalt and nickel enriched the sediment according to the indices. Biotic indicators that were assessed included fish, diatoms and invertebrates. The fish health assessment index (HAI) was applied to fish caught in the Donaldson Dam. The muscle tissue was also removed and its metal concentration was determined by ICP-MS. Thereafter, the edibility of the fish muscle tissue was determined and the following indices were applied: condition factor (CF), hepatosomatic index (HSI), gonadosomatic index (GSI) and spleen somatic index (SSI). The diatom community composition was assessed by applying the Biological Diatom Index (BDI), Specific Pollution Sensitivity Index (SPI) and the percentage pollution tolerant valves (%PTV). The Macroinvertebrate Response Assessment Index (MIRAI) was applied in order to determine the state of the macroinvertebrate community. The fish assessment indicated that arsenic contamination may negatively impact the health of consumers. Diatom indices indicated that the EC of the Wonderfonteinspruit is a D/E which indicates poor water quality; likewise, this is supported by the MIRAI results as the EC for MIRAI was a D which indicates that the river is largely modified. The investigation into the water use of the Bekkersdal community, with a special focus on the use of the Wonderfonteinspruit, was achieved through the use of questionnaires that were distributed in both formal and informal sections in Bekkersdal. The research forms part of a larger Integrative Multidisciplinary study and was given ethical clearance under the NRF Community Engagement Project (see Ethical Clearance: no. FH-BE-2013-0014. The National Research Fund (NRF) provided the funding for the research, the views expressed is that of the author and not those of the NRF. The aim of the questionnaire was to determine the following aspects in terms of the community of Bekkersdal: - Demographic details, such as language preference, employment status and age distribution. - Current water use practices . - Use of the Wonderfonteinspruit. - Future water use of the Wonderfonteinspruit. - Water quality perceptions of the Wonderfonteinspruit. - Field notes that included any relevant observations of the fieldworkers. The unemployment rate of the Bekkersdal community was found to be high (78.20%) and 86.40% of the residents are South African citizens. The community relies heavily on municipal provision of sources of water with 100% of the respondents indicating that it is their primary source of water. However, several issues were identified in terms of municipal water supply in the community. Some 10.14% of the residents indicated that they make regular use of the Wonderfonteinspruit (in particular the Donaldson Dam) most often for drinking water, laundry and washing of cars, etc. Regarding the state of the Wonderfonteinspruit, the overall viewpoint of the Bekkersdal community was that it is largely polluted with sewage, litter and mining waste. However, some 87.80% of the residents expressed their willingness to participate in environmental clean-up initiatives in their area. The link between the ecological state of the Wonderfonteinspruit and human health and wellbeing was explored through the use of spider diagrams where rank scores were assigned to both index results and human water quality use categories. These were compared and it was found that ecological indicators are more sensitive than human water quality use scores and therefore can aid in acting as early detection indicators of possible negative impacts on human health and wellbeing. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Malgré l'arsenal antifongique existant aujourd'hui, les mycoses superficielles sont en constante progression de par le monde et notamment dans le bassin caribéen. Nous nous sommes focalisés sur les pathologies qui posent, en Guadeloupe, de nombreux problèmes de résistance ou de rechute vis-à-vis des antifongiques actuels, à savoir : les dermatophyties, le Pityriasis versicolor (Malassezia sp.), les candidoses et les scytalidioses. Suite à ce constat, nous nous sommes tournés vers la flore guadeloupéenne où ont été sélectionnées dix plantes sur des critères ethnobotaniques, chimiotaxonomiques ou encore d'observations naturalistes avec un double objectif : trouver de nouveaux extraits antifongiques d'une part et, d'autre part, étudier leur composition et vérifier leur innocuité. Le screening antifongique in vitro des extraits c-hexane, EtOH et EtOH/eau (1:1, v/v) a été réalisé vis-à-vis de 4 dermatophytes, 1 Malassezia sp., 5 Candida spp. et 1 Scytalidium sp. Les extraits ont également été testés vis-à-vis d'un autre pathogène, Pneumocystis jirovecii responsable de la pneumocystose pulmonaire. Quatre plantes : Bursera simaruba, Cedrela odorata, Enterolobium cyclocarpum et Pluchea carolinensis ont été retenues afin de définir leurs cytotoxicités puis de procéder à l'isolement des composés responsables de leur activité antifongique par bioguidage. Cedrela odorata a montré une activité significative vis-à-vis de Pneumocystis jirovecii due en partie à la (+)-catéchine. Concernant les mycoses superficielles, Bursera simaruba et Cedrela odorata présentent une activité due à une synergie de composés non identifiés par bioguidage alors que Pluchea carolinensis et Enterolobium cyclocarpum doivent respectivement leurs activités à des flavonoïdes sulfatés et à des saponosides triterpéniques. Faisant suite à cette étude phytochimique et pharmacologique, la formulation des extraits sous forme de gels et vernis a été développée. Ainsi, cette étude permet d'apporter une réponse originale et efficace aux pathologies ciblées.

Le rein est particulièrement exposé à de nombreux composés chimiques dont des médicaments, potentiellement néphrotoxiques. Parmi-eux, le Tacrolimus, un anti-calcineurine largement utilisé en transplantation rénale, est associé à l'apparition plus ou moins rapide de lésions histologique de toxicité conduisant à la fibrose rénale et, à terme, à la perte de fonction du greffon. Des systèmes enzymatiques et protéiques impliqués dans la prise en charge cellulaire des xénobiotiques, ainsi que des protéines aux propriétés anti-fibrosantes, telles que la cavéoline-1, lui permettent de se défendre contre ce type d'agression locale prolongée. La mesure de l'expression de 380 gènes impliqués dans la prise en charge des xénobiotiques dans des échantillons de tissu rénal humain sain nous a permis de confirmer que le rein possède un arsenal de défense important et complet. Ces différents systèmes de défense étant hautement polymorphes, le principal objectif de notre travail était de déterminer l'impact de certains polymorphismes génétiques sur la susceptibilité individuelle à la néphrotoxicité du Tacrolimus.Dans un premier temps, l'impact sur les paramètres pharmacocinétiques du Tacrolimus et le devenir du greffon de deux polymorphismes génétiques affectant les CYP3A5 et ABCB1, deux protéines participant au transport et au métabolisme du Tacrolimus, a été évalué dans une cohorte de 209 patients transplantés rénaux. Le génotypage a été réalisé à la fois sur l'ADN des receveurs et des donneurs. Les patients ont été suivis jusqu'à 2 ans après transplantation rénale. Cette étude a confirmé que les receveurs sous Tacrolimus (Prograf) porteurs d'au moins un allèle CYP3A5*1 fonctionnel nécessitent, quelque soit le moment de la greffe, des posologies de Tacrolimus plus élevées. Malgré ces doses, leur taux résiduel de Tacrolimus (C0) reste plus faible. Cependant, l'analyse de la distribution des rapports résiduelles/posologies de Tacrolimus montre qu'il existe une forte zone de chevauchement entre les deux populations *1/- vs *3/*3. Ainsi, certains patients ayant un CYP3A5 génétiquement déficitaire se comportent phénotypiquement comme des patients fonctionnels. D'autres polymorphismes génétiques sont probablement à l'origine de ce phénomène. En ce qui concerne le devenir du greffon, malgré son impact sur la pharmacocinétique du Tacrolimus, le polymorphisme génétique du CYP3A5 du donneur ou du receveur n'est statistiquement pas associé à la survenue de rejet, de retard de fonctionnement de greffon, ou n'a d'impact direct sur la fonction rénale (MDRD) et la survie du greffon. La mutation 3435C>T affectant le gène ABCB1 du donneur ou du receveur n'influence ni les paramètres pharmacocinétiques, ni le devenir clinique du greffon. Par ailleurs, le Tacrolimus est désormais disponible sous une forme à libération prolongée (Advagraf), permettant une prise unique journalière. Des pharmacocinétiques sur 24h ont été réalisées chez 32 patients (17 fonctionnels et 15 non fonctionnels pour le CYP3A5) avant conversion et quinze jours après conversion Prograf-Advagraf. Les résultats ont montré que, après conversion, l'exposition au Tacrolimus diminue de manière significative pour les patients CYP3A5 fonctionnelsEnfin, l'influence d'un polymorphisme génétique, rs4730751, affectant le gène CAV1 (codant la cavéoline-1, une protéine inhibitrice de la fibrose tissulaire) sur la survie du greffon rénal a récemment été rapporté. Nous avons confirmé dans une cohorte de 475 transplantés rénaux que les patients porteurs de ce polymorphisme génétique à l'état homozygote (ADN du greffon rénale) ont une altération de la fonction rénale significativement plus rapide.

La cellule cardiaque requiert un apport énergétique conséquent qui exige une production et un transfert énergétiques efficaces. Si la production de l'énergie dépend essentiellement des propriétés intrinsèques des mitochondries, il semblerait que l'efficacité du transfert d'énergie du site de production vers les sites consommateurs (ATPases) pourrait être liée à l'architecture spécifique du cardiomyocyte qui conduit à une organisation spatiale singulière des structures internes (mitochondries, réticulum sarcoplasmique, myofilaments). Pour comprendre ce qui lie la cytoarchitecture, la compartimentation cellulaire et la fonction contractile, il a été entrepris d'étudier l'architecture cellulaire et la signalisation énergétique de cardiomyocytes au cours du processus de maturation de la cytoarchitecture et dans un modèle présentant une désorganisation des structures intracellulaires. La première partie de ce travail, réalisée durant le développement postnatal de la souris, a permis de démontré qu'il existe une synchronisation parfaite entre la mise en place de la cytoarchitecture et la maturation fonctionnelle du transfert d'énergie par canalisation directe des nucléotides adényliques entre les mitochondries et les ATPases. Si cette étude apporte un élément qui tendrait à démontrer l'implication de l'architecture cellulaire dans l'efficacité des transferts d'énergie, elle a également mis en avant la maturation très précoce de l'énergétique cellulaire. La mitochondrie faisant partie intégrante de cette architecture et étant modelée par des mécanismes de fusion et de fission, la deuxième étape de ce travail de thèse a consisté à étudier l'implication de la morphologie mitochondriale dans l'énergétique du cardiomyocyte. Il a ainsi été montré que, chez la souris, la diminution d'expression de la protéine OPA1, impliquée dans la fusion mitochondriale, conduit à des perturbations de la morphologie mitochondriale qui n'affectent pas la fonction intrinsèque mitochondriale mais qui altèrent le système de canalisation directe entre les mitochondries et les ATPases des myofilaments. De manière générale, ces résultats démontrent clairement une dépendance des transferts d'énergie à l'architecture cellulaire spécifique de la cellule musculaire cardiaque.

Le cancer du sein est une cause importante de mortalité féminine en France et l'émergence de chimiorésistance, en particulier avec les taxanes, dont le paclitaxel (Taxol®), est une limite importante à l'emploi de ces molécules. Comme de nombreux anticancéreux, les taxanes ciblent les microtubules (MTs), des polymères de tubuline intervenant dans de nombreuses fonctions cellulaires. Ces derniers alternent entre des phases de croissance et de désassemblage, leur conférant ainsi un caractère dynamique. En se liant à la beta-tubuline, le Taxol stabilise les MTs et bloque la mitose, conduisant la cellule vers l'apoptose. La résistance au Taxol est un processus multifactoriel impliquant des mécanismes tels que la surexpression de pompes d'efflux (P-glycoprotéine), des mutations des gènes d'alpha- et de beta-tubuline ou une expression altérée de protéines associées aux MTs (MAPs) telles que MAP4 (stabilisatrice des MTs) ou la stathmine (dépolymérisante), contribuant ainsi à la restauration de la dynamique microtubulaire.Le projet repose sur l'emploi de la lignée tumorale mammaire MDA-MB 231 rendue résistante par paliers à 25nM de Taxol dans des conditions de blocage des pompes d'efflux. L'objectif étant d'explorer les variations protéiques de l'environnement des MTs associées au phénotype chimiorésistant, nous avons comparé le protéome d'extraits microtubulaires de cellules sensibles (Tv) et résistantes au Taxol (T8). Parmi les 112 protéines statistiquement enrichies dans une des deux populations de MTs, on retrouve notamment une augmentation des tubulines beta III et IV, une augmentation et une diminution respectives des moteurs moléculaires kinésine-1 et dynéine ainsi que l'enrichissement de plusieurs septines (SEPT2, 8, 9 et 11) dans les fractions de MTs T8. Les septines sont des GTPases associées en complexes hétéro-oligomériques impliquées dans la cytocinèse et l'organisation du cytosquelette de MTs et d'actine. Le recrutement de ces protéines candidates sur les MTs des cellules T8 a été validé par Western blot (Froidevaux-Klipfel et al., 2011) et immunofluorescence. Dans la littérature, les septines sont décrites comme étant localisées soit au niveau de l'actine soit au niveau des MTs dans les cellules de mammifères. De façon intéressante, dans nos cellules MDA-MB 231, nous observons un recrutement partiel de SEPT2 sur les fibres d'actine dans les cellules Tv alors qu'elle colocalise avec les MTs des cellules T8. Bien que la dépolymérisation de l'actine dans les cellules Tv n'entraine aucun déplacement des septines vers les MTs, cette localisation différentielle des septines sur les MTs des cellules résistantes pourrait néanmoins participer au phénotype chimiorésistant.Par ailleurs, il a été décrit dans la littérature que SEPT2 se lie aux MTs polyglutamylés afin de faciliter le transport vésiculaire à la membrane de protéines impliquées dans la polarisation cellulaire. La polyglutamylation est une modification post-traductionnelle permettant la formation de chaines latérales d'un à plusieurs résidus glutamate sur les tubulines alpha ou beta, régulant ainsi les interactions entre MTs et MAPs. Nos résultats montrent que l'accumulation des septines sur le réseau de MTs des cellules T8 s'accompagne d'une augmentation de la polyglutamylation mais aussi de la tyrosination de la tubuline. De plus, des tests de viabilité cellulaire ont mis en évidence que l'inhibition partielle par RNAi des septines ainsi que des polyglutamylases et de la tubuline tyrosine ligase, comme la surexpression d'enzymes responsables de la déglutamylation de la tubuline, permettent de restaurer une certaine sensibilité au Taxol des cellules T8. Inversement, la surexpression de certaines enzymes responsables de la polyglutamylation et de la tyrosination de la tubuline dans les cellules Tv permet d'instaurer une résistance au Taxol des cellules sensibles.La compilation de nos résultats permet de proposer un nouveau mécanisme de résistance au Taxol des cellules cancéreuses mammaires MDA-MB 231 : une augmentation du niveau de tyrosination de l'alpha-tubuline serait à l'origine de l'allongement des chaînes polyglutamylées sur le MT entraînant une diminution de la liaison de la protéine stabilisatrice MAP4 ainsi que le recrutement des septines sur les MTs. Ces modifications favoriseraient alors le recrutement du facteur de sauvetage CLIP-170 et de kinésines dépolymérisantes telles que MCAK à l'extrémité en croissance du MT des cellules résistantes T8, permettant une certaine restauration de la dynamique microtubulaire et contribuant ainsi à l'apparition du phénotype chimiorésistant.Ces études sont indispensables pour établir les bases d'un nouveau mécanisme de résistance au Taxol impliquant les septines, ainsi qu'un lien de causalité avec la tyrosination et la polyglutamylation. Au-delà, la recherche de ces modulations clés pourra être réalisée sur des cancers mammaires sensibles et résistants au Taxol, issus de biopsies de patients. Ainsi, il sera possible à terme, de déterminer si les septines, la tyrosination et la polyglutamylation de la tubuline ont une véritable importance fonctionnelle dans la résistance de cancers du sein au Taxol.