Cette étude soulève de nombreuses questions sur les Palestiniens vivant au sein de la diaspora en se concentrant sur les Palestiniens Chrétiens. Elle traite de sujets majeurs concernant les migrations, la diaspora, l'identité et les réseaux ; quatre concepts interdépendants mais qui ne peuvent être analysés de façon isolée les uns des autres. La majorité des migrations palestiniennes ont commencé pa rdes migrations forcées pour des raisons politiques ou économiques avant de devenir des migrations transnationales.Bien que des politiques d'absorption des migrants par les pays de la diaspora aient été mises en place, ceux-ci ont conservé leur identité, grâce aux réseaux religieux, familiaux,nationaux et palestiniens. La création de clubs de villages et de villes, de clubs familiaux, d’églises arabes, entre autres,ont relié les migrants les uns aux autres et ont également mis en lien la diaspora et le pays d'origine.Comprenant des réseaux sociaux, économiques et charitables, les réseaux transnationaux ont affirmé les relations des migrants avec leur pays d'origine comme un élément principal. Toutefois, la proportion de migrants palestiniens pouvant franchir les frontières de leur pays d'origine reste faible. Ceci confirme le fait que les Palestiniens à l'étranger constituent une vraie diaspora. Les Palestiniens ont vécu différentes expériences de migration et de diaspora dans les pays arabes voisins et dans les pays éloignés étrangers (non-arabes). Le concept de diaspora a été redéfini à partir de notre terrain palestinien.L'étude présente différents modèles géographiques de familles palestiniennes dans la diaspora / This study raises many questions and issues on Palestinians living in the diaspora through focusing on the segment of Palestinian Christians. This study discusses major issues on the level of migrations, diaspora, identity and networks; four interrelated concepts that could not be examined or understood in isolation from each other. The majority of Palestinian migrations started as forced emigrations for political or economic reasons before becoming transnationa lmigrations. This shift was accompanied by another shift in the legal statuses of this transient segment of Palestinians who obtained new nationalities.As a result of the adoption of migrants' absorption policies by countries of diaspora, migrants have preserved their identities, which ranged from religious, to familial, to nationalist and to Palestinian. The establishment of village and city clubs, Arab churches and family divans (Diwans) among others have connected migrants to one another and also connected the diaspora to the homeland .Ranging from social, to economic, to charitable, transnational networks have affirmed emigrants' relations with their country of origin as a main element. However, the proportion of Palestinian emigrants could cross borders to their country of origin is small. This is confirm the fact that Palestinians abroad constitute a real diaspora .Palestinians have gone through different experiences of migration and diaspora in neighboring Arab countries and remote foreign (non-Arab) countries; the concept of Diaspora has been redefined in a manner that fits the Palestinian case. The study presents different geographic patterns of Palestinian families in the diaspora.

Abstract Meniere’s disease (MD) is an inner ear disorder characterized by vertigo, tinnitus and sensorineural hearing impairment. An inherited form of the disease is called familial Meniere’s disease (FMD). The aim of this thesis was to describe the clinical and genetic features of Finnish FMD and to study its prevalence in Finland. In addition genetic factors previously associated with MD were studied in Finnish MD patients. A total of 38 Meniere-families were analysed in this study. In most of the families the mode of inheritance was found to be autosomal dominant. Meniere-like symptoms such as tinnitus or vertigo were common in these families even in individuals without a full triad of MD. Familial patients were affected earlier, suffered from longer spells of vertigo and had more autoimmune diseases compared to sporadic MD patients. The prevalence of FMD was studied among the patients treated in the Oulu University Hospital and Kainuu Central Hospital during the years 2005-2010. A family history of MD was probable in 23.4% of the cases, but only 9.3% could be confirmed, as it was not possible to gain information from deceased generations. Six candidate genes previously associated with MD were screened for mutations in Finnish MD patients. Two possibly adverse variations were observed in the KCNE1 gene in two patients but in none of the controls. The role of these variations in MD is still unclear and needs further study. The association of MD to the five other genes could not be confirmed, nor was Finnish FMD linked to a previously suggested locus on chromosome 12. / Tiivistelmä Menieren tauti on sisäkorvan sairaus, jolle on tyypillistä huimaus, korvien soiminen ja kuulon heikkeneminen. Tauti voi esiintyä myös perinnöllisenä. Tutkimustyön tavoitteena oli selvittää perinnöllisyyden osuutta Menieren taudissa, kuvata suomalaisen perinnöllisen Menieren taudin tyypilliset piirteet ja tutkia suomalaisessa aineistossa aikaisemmin tautiin yhdistettyjä perinnöllisiä tekijöitä. Tutkimuksessa analysoitiin 38 sukua, joissa Menieren tautia esiintyi perinnöllisenä. Suurimmassa osassa tapauksista periytyminen tapahtui vallitsevasti. Suvuissa esiintyi paljon Meniere-tyypistä oirehdintaa, kuten tinnitusta ja huimausta, ilman Menieren taudin koko taudinkuvaa. Meniere-suvuissa potilaat sairastuivat keskimääräistä aikaisemmin, kärsivät pidemmistä huimauskohtauksista ja sairastivat enemmän autoimmuunitauteja. Perinnöllisen Menieren taudin yleisyyttä tutkittiin Kainuun keskussairaalassa ja Oulun yliopistollisessa sairaalassa vuosina 2005−2010 hoidettujen potilaiden keskuudessa. Potilaista 23,4 %:lla Menieren taudin sukuhistoria oli positiivinen; kuitenkin vain 9,3 % pystyttiin vahvistamaan, sillä tietojen kerääminen edesmenneiltä sukupolvilta ei ollut mahdollista. Kuuden Menieren tautiin aikaisemmin yhdistetyn geenin merkitystä tutkittiin suomalaisessa aineistossa mutaatio- ja ehdokasgeenianalyysillä. KCNE1-geenistä löydettiin kaksi mahdollisesti proteiinia vaurioittavaa sekvenssinvaihtelua, joita ei havaittu kontrollihenkilöillä. Muutosten merkitys Menieren taudin synnyssä jäi kuitenkin epävarmaksi ja vaatii jatkotutkimuksia. Muiden geenien yhteyttä sairauteen ei pystytty vahvistamaan. Suomalainen Menieren tauti ei myöskään kytkeytynyt aikaisemmin ehdotettuun lokukseen kromosomissa 12.

Abstract This study utilized normal and defective epithelial cell cultures and epidermal skin samples to examine intra- and intercellular calcium signaling. The main interests of this thesis were Hailey-Hailey disease (HHD), Darier disease (DD), neurofibromatosis 1 (NF1) and transitional cell carcinoma (TCC). HHD and DD diseases are rare autosomal dominant skin disorders characterized by dissociation of epidermal keratinocytes (acantholysis) at the suprabasal layer of the epidermis. HHD and DD diseases are caused by mutations in the genes encoding the calcium pumps in the Golgi apparatus (hSPCA1) and endoplasmic reticulum (SERCA2b), respectively. Due to these mutations calcium uptake into the Golgi apparatus or ER is diminished, which is believed to cause abnormal cell junction protein processing and dissociation of keratinocytes. This study utilized electron probe microanalysis (EPMA) and demonstrated for the first time that lesional areas of HHD and DD and non-lesional areas of DD epidermis display abnormally low calcium content in the basal cell layer. Furthermore, ATP mediated calcium signaling was impaired in cultured HHD and DD keratinocytes and epidermal ATP receptor localization was disrupted. In conclusion, these results suggest that the low calcium content in the basal cell layer is the reason for suprabasal ruptures in HHD but not necessarily in DD lesions, and that abnormal ATP receptor localization contributes to the calcium signaling defects. NF1 deficient keratinocytes display abnormally low resting cytosolic calcium levels and it has been suggested that the calcium concentration in the lumen of the endoplasmic reticulum is decreased. This study demonstrated that NF1 keratinocytes rely mostly on ATP mediated calcium signaling while normal keratinocytes rely mostly on gap junctional intercellular communication (GJIC). Studies with TCC cells have demonstrated that gap junctions participate in intercellular calcium wave propagation. This thesis demonstrated that the ATP mediated pathway was also operational in calcium wave propagation in normal uroepithelial and TCC cell cultures. Furthermore, impaired calcium wave propagation in the TCC cell culture could be improved through PKC α/βI –isoenzyme inhibition with Gö6976. Gö6976 treatment increased connexin 26 clustering at plasma membrane but did not alter expression level of the protein. This thesis contains a wide repertoire of calcium detection techniques including a new cutting-edge technology for elemental calcium detection of epidermal samples. These techniques can be used for molecular specific analysis of calcium signaling in epithelial cells.

The aim of this thesis was to evaluate the level of reading comprehension in children at risk of developing grammar difficulties. The main goal consisted of assessing the performance and children's success in two experimental groups (with impaired speech development and the familial risk of dyslexia) and one control group (individuals with typical speech development) at the Test of Reading Comprehension based on the YARC diagnostic scale (Hulme et al., 2009). Additionally, we also observed the performance of children throughout a longer period of time. Consequently, it enabled us to compare the performance of children in the original test with the results of the Test of Reading and Comprehension by M. Caravolas and J. Volín (2005), which was administered two years later. Further analysis reveal that the individuals with the impaired speech development performed significantly worse than the children with familial risk of dyslexia whose performance was very much on the same level as the ones with typical speech development. Additionally, we found out that the performance of children in terms of comprehension does not change significantly during their development. The results of this study may be beneficial both for specialists and parents who may obtain a better understanding of their children's...

Magister Artium - MA / Adolescent substance abuse is a widely researched area both internationally and nationally. It has been known to affect many problems which are prevalent in most low socioeconomic communities such as crime, school truancy and family fragmentation. It is of particular concern in low socioeconomic communities within the Cape Flats District which continues to be plagued with social ills such as gangsterism, adolescent criminal behaviour, and high rates of school dropout. The problem of adolescent substance abuse cannot be explored in isolation, but rather, in conjunction with all other spheres which it affects such as family relationships, dynamics and functioning. Parents fulfil an important role in managing their adolescent's addiction problem. These parents often feel helpless, hopeless, guilty, and angry, and are inclined to blame themselves for their child's delinquent behaviour. In some cases spouses blame one another for their being too permissive or too stern. However, there is a dearth in research of parents' lived experiences and the ways in which they attribute meaning to their situations. The aim of this study was to explore parents' lived experiences of their adolescent's substance abuse. More specifically the study explored parents' perceptions of the ways in which one family member's substance abuse affects the dynamics and the functioning of the family. At a theoretical level, this study aligned with Bowen's Family Systems theory, as it allowed the researcher to explore the ways in which family roles, dynamics and functioning are affected by a relative's substance addiction. The current study was conducted within the qualitative methodological framework, as the aim was to gain an in-depth understanding of parent's lived experiences of managing their adolescent's substance abuse. Furthermore, this study was positioned within the phenomenological epistemological framework as it aligns well with the aims of this study, which is to acquire an understanding of parents' lived experiences of their adolescent's substance abuse.

L'objectif de cette thèse est d'explorer la stigmatisation perçue liée à la maladie d'Alzheimer (MA) parmi une population d'aidants familiaux (enfants adultes ou conjoints) accompagnant leurs proches (conjoints ou parents) vivant avec une probable MA. Parmi les patients, non seulement, il est examiné les sources de stigmatisation envers les personnes vivant avec une probable MA associées aux réactions émotionnelles et comportementales d'affiliation ou de distance sociale de l'entourage. Mais encore il est observé les facteurs de stigmatisation qui prédisent une variation de leur qualité de vie. Parmi, leurs aidants familiaux, il est étudié les facteurs de stigmatisation qui prédisent une variation de la symptomatologie dépressive et du fardeau de soins. Dans cette relation, il est étudié le rôle modérateur du soutien social. Nos résultats indiquent parmi les patients, que la fréquence des symptômes comportementaux liés à la dépression prédisent les émotions négatives et les comportements de distance sociale de l'entourage. Aussi, nous observons que la qualité de vie du patient varie selon son lieu de résidence. Au domicile, il est observé davantage de comportements de distance sociale venant de l'entourage. Parmi les proches aidants, il est constaté que soutien social modère les effets entre les émotions négatives, les comportements de distance sociale de l'entourage envers le patient, et la symptomatologie, le fardeau de soins des aidants. Mais que le soutien social exacerbe les effets entre les émotions positives de l'entourage et le fardeau de soins. Cette thèse permet d'objectiver la stigmatisation liée à la maladie d'Alzheimer parmi les patients et leurs proches aidants. Les résultats obtenus justifieraient l'élaboration d'actions de communication centrées sur la nécessité du soutien social auprès de l'ensemble des personnes affectées par la maladie d'Alzheimer. / This thesis aims at exploring the perceived stigmatization towards persons possibly leaving with Alzheimer’s disease among the population of the family caregivers – grown-up children and spouses. Not only do we have observed sources of stigmatizations aimed at patients possibly leaving with Alzheimer’s disease, in relation with emotional reactions and either affiliation behavior, or social distancing from the family and social circle, but we have also observed factors of stigmatization leading to variations in patients’ quality of life. Amongst family caregivers, we have studied factors of stigmatization leading to changes in the associated depressive symptoms and the increased caregiver burden. With respect to the relationship induced, we have given attention to the moderating role of social support. Our results show that, among patients, the negative emotions and the social distancing behavior from social circle can be linked to the frequency of behavioral symptoms related to the depression. With respect to this observation, we show an variation in the patients’ quality of life according to the places they live in. At home, we have noted increased social distancing behavior from the social circle. Amongst the relatives caregivers, we have also noticed that social support has a moderating influence on the effects of negative emotions, the social distancing behavior from the social circle towards the patient, the symptoms and the burden of care felt by the caregivers. However, the social support exacerbates the relation between positive emotions among the relatives and the burden of care. This thesis gives us the opportunity to objectify the stigmatization process with respect to Alzheimer’s disease amongst patients and their relatives caregivers. Our results could open the way to specific communications promoting the necessity for social support in favor of the entire population concerned by Alzheimer’s disease.

Ce travail a pour objectif d’étudier, selon une approche psychosociale, les facteurs de vulnérabilité associés au risque de malnutrition au sein du binôme personne âgée démente/aidant familial. Cette étude transversale a été réalisée dans le cadre d’une intervention psychosociale effectuée à domicile et proposée par un Centre Local d’Information et de Coordination (CLIC) en milieu rural. Les caractéristiques socio-démographiques et des mesures sur la santé psychologique et physique ont été recueillies auprès d’un échantillon composé de 56 binômes. Nos résultats montrent que le risque de malnutrition des personnes âgées démentes serait plus particulièrement expliqué par leur dépendance dans les activités de base de la vie quotidienne et par le propre risque de malnutrition des aidants familiaux. Ce dernier serait lui-même expliqué par la dépression, leur niveau de dépendance, et la sévérité de l’apathie de leur proche dément. Après avoir défini des profils nutritionnels de binômes et les facteurs de vulnérabilité qui y sont associés, la description de leur vécu et de leurs interactions autour de l’alimentation permet de mieux comprendre les problématiques psychologiques en jeu. Ces résultats sont discutés en fonction de la dimension psychosociale de l’alimentation, entre dépendance comme facteur de vulnérabilité et interdépendance comme fonction d’affirmation et de maintien du lien au sein du binôme. Finalement, ces éléments de réponses ainsi que les limites identifiées nous amènent à proposer des perspectives de recherche et de prise en charge / This work aims to study psychosocial factors associated with risk of malnutrition in the dyad demented elderly/ family caregiver. This cross-sectional study comprising 56 community-dwelling demented elderly and 56 family caregivers was performed in a French gerontological institution providing psychosocial interventions. The data collected included their socio-demographic characteristics and measures of their psychological and physical health. Our results show that the risk of malnutrition of demented elderly would be particularly explained by their dependence in activities of daily life and the own risk of malnutrition of family caregivers. This one would be explained by their dependence, depression, and the severity of apathy of demented elderly. Nutritional profiles of caregiving dyads and vulnerability factors associated are presented. According to these profiles, description of their emotional experiences and their interactions concerning feeding-related activities is interesting to better understand the psychological issues. These results are discussed according to psychosocial dimension of feeding, between dependence as a vulnerability factor and interdependence as a function to affirm and preserve the link within caregiving dyad. Finally, the limits of this work and its implications for both clinical and research are argued.

L'analyse longitudinale de la fécondité montre que les valeurs élevées de l’ICF pendant unepremière période (1960-1980) résultent de l'adoption d'un calendrier plus précoce des femmes néesen 1940 et au-delà. Aussi, son effondrement au cours d’une seconde période (1980-2000) est dû à uncalendrier fécond plus mature des femmes nées à partir de 1960. L’augmentation de l’ICF despremières années de 2000 est due au phénomène de récupération des naissances à traversl’augmentation des taux de fécondité à des âges supérieurs à 30 ans. Cette récupération estcependant incomplète car la descendance finale des générations s’est nettement réduite au fil dutemps. En tenant compte de la mortalité, aucune de générations examinées ne s’est complètementreproduite. L'analyse de la fécondité longitudinale selon le rang biologique de naissance de l’enfantmontre un âge moyen à la maternité de plus en plus élevé au premier enfant et l'augmentationsignificative de l’infécondité définitive pour les femmes nées depuis la fin des années 1960. Plus de lamoitié des femmes nées entre 1940 et le début des années 1960 ont obtenu 2 enfants. Le modèlestandard de la famille de deux enfants semble donc apparaître un plus tôt en Grèce que dans d’autrespays européens. Le découplage de la fécondité de la nuptialité, observée dans les pays occidentauxn’a pas encore été confirmée pour la Grèce. Le début de la crise économique a coïncidé avec ladiminution de la fécondité transversale. Le faible recul temporel ne nous permet pas de savoir si cetteréduction aura un impact sur la descendance finale des générations. / The longitudinal analysis of fertility shows that the low values of the period TF from 1980 to 2000was the result of the postponement of births, as women who born after 1960 were putting offparenthood to later ages which depressed period fertility rates. The increase of period TF in the firstdecade of 2000 is due to fertility “recuperation”, through the increase in fertility rates at ages over 30years old. The recuperation is incomplete and the cohort fertility has significantly reduced over timeAnalysis of cohort fertility by biological birth order shows a mean age of childbearing in first childincreasingly high and a significant increase of childlessness. The 20-25% of woman born from1970 to1975 in Greece will remain childlessness. The reduction of complete fertility in generations is largelydue to the fact that more and more women reaching the age of 49 years old without having achildren. Also the family size is reduced over the generations, two child family becoming the norm.The parity progression ratios reduced at all birth orders and especially a2 and a3. The low percentageof births outside marriage in Greece (6,7% 2013) revealed the important role of marriage inchildbearing. Data from the censuses (1991,2001,2011) show that unmarried women over 49 yearsold, had on average a total fertility between 0,05 to 0,15 children/women and a childlessness ratebetween 85 and 95%. The examination of a series of economic indicators like GDP andunemployment rate alongside with period TF reveals the strong correlation between the twophenomena. The short time series (2009-2012) does not allow us to know whether this reduction ofperiod fertility will have an impact on the cohort fertility.

Introdução: A Hipercolesterolemia Familiar (HF) é uma doença genética caracterizada clinicamente por elevados níveis de lipoproteína de baixa densidade (LDL-C) na corrente sanguínea desde a infância. Indivíduos que apresentam HF podem desenvolver doença aterosclerótica ainda em idade jovem. Os principais preditores de risco no desenvolvimento da doença cardiovascular (DCV) nesses indivíduos após entrarem em um programa de rastreamento genético não são conhecidos na nossa população. Além disso, a HF é subdiagnosticada e subtratada mundialmente e o rastreamento genético em cascata dos familiares tem sido mundialmente avaliado como o método diagnóstico mais custo. Contudo, a efetividade do rastreamento genético em cascata é dependente dos critérios clínicos de entrada do primeiro indivíduo da família e não há um consenso de qual critério apresenta a melhor acurácia para detecção de uma mutação. Objetivos: Identificar os fatores determinantes para ocorrência de eventos cardiovasculares (CV) em todos os indivíduos da coorte e avaliar o critério clínico para detecção de uma variante genética patogênica para HF, no primeiro indivíduo da família, após serem inseridos em um programa de rastreamento genético em cascata.Métodos: Estudo de coorte prospectiva aberta dos pacientes que foram inseridos no programa de rastreamento genético em cascata para HF. A população do estudo é definida como caso índice (CI), o primeiro da família a ser identificado clinicamente e encaminhado para o teste genético, e os familiares, que são os parentes de 1º grau do CI em que foi encontrada uma alteração genética. Todos os indivíduos são inseridos na coorte no momento em que recebem o laudo genético (tempo zero, T0). Um ano depois do T0 é realizado o primeiro contato telefônico, ou seja, primeiro ano de seguimento (T1) Resultados: No T1, o total de 818 indivíduos foi incluído, sendo verificados 47 eventos CV, sendo 14 (29,7%) fatais. Para o CI, o único fator independente associado ao aumento do risco de eventos CV no T1 foi a presença de arco corneano (OR: 9,39; IC 95%: 2,46-35,82). Para os familiares com uma mutação positiva os fatores associados ao aumento do risco de eventos CV foram diabetes mellitus (OR: 7,97; IC 95%: 2,07-30,66) e consumo de tabaco (OR: 3,70; IC 95%: 1,09-12,50). Na análise do melhor critério clínico para detecção de uma mutação patogênica no CI os valores de LDL-C >= 230 mg/dL tiveram a melhor relação entre sensibilidade e especificidade. Na análise da curva ROC o escore Dutch Lipid Clinic Network (DLCN) apresentou melhor desempenho do que o LDL-C para identificar uma mutação, a área sob a curva ROC foi 0,744 (IC 95%: 0,704-0,784) e 0,730 (IC 95%: 0,687-0,774), respectivamente, p = 0, 014. Conclusão: Em um ano de seguimento essa coorte identificou uma alta incidência de eventos CV após a entrada em um programa de rastreamento genético em cascata e os preditores dos eventos CV diferem entre CI e familiares. Esses resultados podem contribuir para o desenvolvimento de ações preventivas nesse grupo altamente susceptível de indivíduos. Além disso, devido a importância da detecção da mutação para um diagnóstico definitivo de HF e a importância da cascata ser custo efetiva o estudo identificou que o critério único do LDL-C >= 230 mg/dl é viável para indicar o CI para o teste genético / Introduction: Familial Hypercholesterolemia (FH) is a genetic disease characterized clinically by high levels of low density lipoprotein (LDL-C) in the bloodstream since childhood. Individuals with FH can develop atherosclerotic disease at a young age. The main predictors of cardiovascular disease (CVD) risk in these individuals after entering a genetic screening program are not known in our population. In addition, FH is underdiagnosed and undertreated worldwide and cascaded genetic screening of family members has been evaluated globally as the most cost effective for the diagnosis of FH. However, the effectiveness of cascading genetic screening is dependent on the clinical entry criteria of the first individual in the family and there is no consensus as to which criterion shows the best accuracy for detecting a mutation. Objectives: To identify the determinant factors for cardiovascular (CV) events in all individuals in the cohort and to evaluate the clinical criteria for detecting a genetic variant pathogenic to FH in the first individual of the family after being inserted into a genetic screening program in cascade. Methods: Open prospective cohort study of patients who were enrolled in the cascade genetic screening program for FH. The study population is defined as index case (IC), the first of the family to be clinically identified and referred to the genetic test, and relatives, who are the first-degree relatives of the IC in which a genetic alteration was found. All individuals are inserted into the cohort at the moment they receive the genetic report (time zero, T0). The first follow-up telephone contact is made one year after T0 (first year of follow-up, T1). Results: In T1, a total of 818 subjects were included, and 47 CV events were verified, of which 14 (29.7%) were fatal. For IC, the only factor independently associated with the increased risk of CV events in T1 was the presence of a corneal arch (OR: 9.39; 95% CI: 2.46-35.82). For relatives with positive mutation, factors associated with increased risk of CV events were diabetes mellitus (OR: 7.97; 95% CI: 2.07-30.66) and tobacco consumption (OR: 3.70; 95% CI: 1.09-12.50). In the analysis of the best clinical criteria for the detection of a pathogenic mutation in the IC, the LDL-C values >= 230 mg/dL had the best relationship between sensitivity and specificity. In the ROC curve analysis, the Dutch Lipid Clinic Network (DLCN) score performed better than LDL-C to identify a mutation, the area under the ROC curve was 0.744 (95% CI: 0.704-0.784) and 0.730 (CI 95 %: 0.687-0.774), respectively, p = 0.014. Conclusion: At one year follow-up this cohort identified a high incidence of CV events following entry into a cascade genetic screening program and the predictors of CV events differ between IC and family members. These results may contribute to the development of preventive actions in this group highly susceptible to individuals. In addition, because of the importance of detecting the mutation for a definitive diagnosis of HF and the importance of the cascade being cost effective, the study identified that the single LDL-C criterion >= 230 mg / dl is feasible to indicate IC for the genetic test

La polyneuropathie amyloïde familiale à transthyrétine (FAP-TTR) est une maladie rare héréditaire à transmission autosomique dominante liée à la production de formes mutantes de la transthyrétine (TTR). Ces mutations sont à l’origine d’un changement de conformation de la protéine qui, in vivo, se présente sous forme de tétramère. Celui-ci est alors déstabilisé et évolue vers la formation de fibrilles amyloïdes qui s'accumulent au niveau du système nerveux autonome, des nerfs périphériques et des organes. Ces dépôts sont responsables de la pathologie. Dans le but d’évaluer l’efficacité des thérapies pouvant être mises en œuvre, nous avons développé des stratégies analytiques visant à concevoir un système « point of care » à usage hospitalier permettant de quantifier les formes mutante et native circulantes de la TTR. La méthodologie développée consiste à réaliser la séparation électrocinétique de fragments ciblés de la TTR, obtenus par digestion enzymatique de la protéine. Cette approche analytique a été développée en se focalisant sur une mutation fréquente en France : la TTR Thr49Ala où une thréonine est remplacée par une alanine en position 49. Au cours de cette thèse, deux types de microréacteurs enzymatiques ont été étudiés, i.e. (i) un lit fluidisé contenant des particules magnétiques fonctionnalisées par des molécules de trypsine et (ii) une puce monolithique à base de thiol-ène fonctionnalisée également par la trypsine. Le pouvoir catalytique de ces microsystèmes a été comparé en mesurant l’efficacité de digestion du BApNA (substrat modèle) et de la TTR à l’aide de méthodes analytiques telles que la spectrophotométrie d’absorption UV-Visible, l’électrophorèse capillaire couplée à une détection UV (EC-UV) et la chromatographie liquide couplée à la détection par spectrométrie de masse (UHPLC-SM). Les résultats obtenus ont montré que le microréacteur enzymatique monolithique à base de thiol-ène était le plus performant pour digérer la TTR. Par ailleurs, nous avons réalisé, au cours de cette étude, l’optimisation d’une méthode EC-UV, adaptée à l’analyse des digestats recueillis en sortie de microréacteur. Elle a permis de séparer et de quantifier les peptides d’intérêt pour déterminer le rapport de TTR mutante (Thr49Ala) sur TTR native. / Transthyretin familial amyloid polyneuropathy (TTR-FAP) is a hereditary rare disease with an autosomal dominant transmission, related to the production of mutant forms of transthyretin (TTR). These mutations lead to a conformational change of the protein whose in vivo form is a tetramer. As a consequence, this tetramer is destabilized and evolves towards the formation of amyloid fibrils that aggregate on the autonomic nervous system, peripheral nerves, and organs. These deposits are responsible for the pathology. In order to evaluate the efficiency of possible therapies, we developed analytical strategies aiming at designing a “point of care” system for hospital use, which would enable the quantification of circulating mutant and native forms of TTR. The methodology that we developed consists in undertaking the electrokinetic separation of targeted TTR fragments obtained through the enzymatic digestion of the protein. This analytical approach has been developed while focusing on a frequent mutation in France: the TTR Thr49Ala mutation in which a threonine is substituted by an alanine in position 49. In this thesis, two types of enzymatic microreactors have been studied, i.e. (i) a fluidized bed containing magnetic particles functionalized by trypsin molecules and (ii) a thiol-ene-based monolithic chip also functionalized by trypsin. The catalytic power of these microsystems has been compared by measuring the digestion efficiency of BApNA (model substrate) and TTR through analytical methods such as UV-visible absorption spectrophotometry, capillary electrophoresis with UV detection (CE-UV), and liquid chromatography with mass spectrometry detection (UHPLC-MS). The results showed that the thiol-ene-based monolithic enzymatic microreactor was the most efficient system to digest TTR. Besides, during this study, we undertook the optimization of a CE-UV method which is adapted to the analysis of digested sample collected directly out of the microreactor. This allowed us to isolate and quantify the peptides of interest to measure the ratio of mutant TTR (Thr49Ala) versus the wild one.