Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2007.

Circulating DNA is fragments of DNA which can be found in the blood of healthy as well as diseased individuals. Higher levels of these nucleic acid molecules can be found in diseased and pregnant individuals in contrast to healthy controls. The origin of circulating DNA has not been elucidated, but release of DNA after apoptosis or necrosis or active release by living cells has been hypothesized. It was concluded in this study that apoptosis or necrosis may only be a minor source of circulating DNA and that release of DNA by living cells might play a major role in the origin, while disturbance of the equilibrium between release by living cells and clearance mechanisms may cause the rise in the levels of circulating DNA observed in different conditions. Before circulating DNA can be analyzed, it has to be isolated from the blood. A number of different preanalytical conditions can have an impact on the quantity and quality of circulating DNA that can be isolated. Furthermore, the choice of isolation and quantification method may also influence the results obtained. Quantitative analysis of circulating DNA was done by real-time PCR analysis of the &Globin gene and the DNA levels obtained for healthy controls and cancer patients correlated with levels reported in the literature. Characterization of total circulating DNA may be beneficial in diagnosis and prognosis and may also contribute to determining the source and function of circulating DNA. In order for characterization to take place a method to clone total circulating DNA was developed and standardized and thirty-five clones were obtained and analyzed. It was found that the sequences contain a large amount of Alu repeats and the significance of this has not been determined yet. This is a first step towards future studies. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2007

Circulating DNA is fragments of DNA which can be found in the blood of healthy as well as diseased individuals. Higher levels of these nucleic acid molecules can be found in diseased and pregnant individuals in contrast to healthy controls. The origin of circulating DNA has not been elucidated, but release of DNA after apoptosis or necrosis or active release by living cells has been hypothesized. It was concluded in this study that apoptosis or necrosis may only be a minor source of circulating DNA and that release of DNA by living cells might play a major role in the origin, while disturbance of the equilibrium between release by living cells and clearance mechanisms may cause the rise in the levels of circulating DNA observed in different conditions. Before circulating DNA can be analyzed, it has to be isolated from the blood. A number of different preanalytical conditions can have an impact on the quantity and quality of circulating DNA that can be isolated. Furthermore, the choice of isolation and quantification method may also influence the results obtained. Quantitative analysis of circulating DNA was done by real-time PCR analysis of the &Globin gene and the DNA levels obtained for healthy controls and cancer patients correlated with levels reported in the literature. Characterization of total circulating DNA may be beneficial in diagnosis and prognosis and may also contribute to determining the source and function of circulating DNA. In order for characterization to take place a method to clone total circulating DNA was developed and standardized and thirty-five clones were obtained and analyzed. It was found that the sequences contain a large amount of Alu repeats and the significance of this has not been determined yet. This is a first step towards future studies. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2007

Le cancer broncho-pulmonaire du non-fumeur est considéré comme une entité à part du fait de ses particularités épidémiologiques. Il est en outre un excellent modèle pour l'étude des facteurs de risque de cancer bronchique autres que le tabagisme actif. Il n'existe que très peu de données non-asiatiques concernant cette entité d'intérêt. Le bio-observatoire national des cancers bronchiques de non-fumeurs (BioCAST I IFCT-1002) est une étude épidémiologique multicentrique prospective. Son objectif principal est de décrire une population de patient strictement non-fumeur (moins de 100 cigarettes au cours de la vie) atteint de cancer bronchique, notamment sur le plan de leur profil moléculaire somatique et de leur exposition aux facteurs de risque. Les objectifs secondaires étaient d'étudier si l'exposition aux différents facteurs de risque pouvait influencer le profil moléculaire ; et d'utiliser cette cohorte particulière (grande fréquence et diversité de mutations somatiques attendue) afin de développer un test multiplex pour le diagnostic non-invasif du profil moléculaire somatique tumoral à partir d'ADN circulant. Au total, 384 patients non-fumeurs atteints de cancer broncho-pulmonaire ont été inclus dans cette cohorte. Deux-tiers d'entre eux étaient exposés au tabagisme passif, et il s'agissait essentiellement d'une exposition domestique touchant les femmes. Inversement, 35% des hommes étaient exposés de manière certaine à au moins un cancérogène professionnel, contre 8% des femmes. Au total, 72% des patients présentait une anomalie moléculaire, essentiellement au niveau de l'EGFR (51% de l'ensemble de la cohorte). Le genre, ou l'exposition à différents facteurs de risque (tabagisme passif, exposition professionnelle, exposition hormonale chez les femmes) n'affectait pas de manière significative et cliniquement pertinente le profil mutationnel, avec les limites liée à de faibles effectifs dans certains groupes et aux expositions multiples. Seule l'exposition professionnelle à l'amiante et / ou à la silice semble avoir pour effet de diminuer la fréquence des mutations de l'EGFR / Lung cancer in never smokers (LCINS) is considered as a separate entity given its epidemiological specificities. It is also a very interesting template to assess alternative risk factors for lung cancers than tobacco smoking. However, there is very little non-Asian data about this particular topic. The BioCAST / IFCT1002 study is a prospective, nationwide, and multi-centric epidemiological study. Its main objective was to describe a French population of lung cancers in lifelong never smokers (less than 100 cigarette during all lifetime); with a special focus on molecular somatic profile and risk-factors exposure. Secondary objectives were to assess the interaction between risk-factor exposure and molecular profile; and to use this particular cohort to develop a multiplex test for non-invasive diagnosis of tumor mutations in circulating free DNA. Overall, 384 patients were recruited in the cohort. Two-third were exposed to passive smoking (mainly women and in domestic setting). By contrast, 35% of men were definitely exposed to occupational carcinogens versus 8% of women. Finally, 72% were found with a somatic mutation, mainly in the EGFR gene (51% of the whole population). Gender or exposure to risk factors such as passive smoking, occupational exposure, or hormonal status in women, were not significantly associated with a specific and/or clinically meaningful molecular profile in tumor. These findings should be interpreted with caution given that some subgroups were small and/or with many simultaneous exposures. However, exposure to asbestos and/or silica was significantly associated to a decreased risk for EGFR mutation. On the pilot study (n=106), circulating free DNA was associated with tumor burden. The multiplex diagnosis (12 amplicons on 5 genes) by next-generation sequencing was feasible and gave encouraging results in stage 4 patients (67% sensitivity, 73% concordance rate). LCINS is an interesting entity for the study of non-tobacco-related cancer risk factors; or to optimize liquid biopsy strategy

Type 1 diabetes mellitus (T1D) is an organ-specific autoimmune disease characterised by autoimmune destruction of insulin-producing beta cells in the islets of Langerhans. It is a long-term process initiated months or even years prior to the clinical onset. The main role in the pathogenesis is played by T lymphocytes but other cell types are involved as well. The presence of autoantibodies in the circulation is typical even before the disease onset. Nowadays, intensive research is focused on finding individuals at risk and developing an effective prevention. During my postgraduate studies I was involved mainly in the research of T1D prediction and prevention. We investigated the relationship of established autoimmune markers - autoantibodies - and the cellular reactivity to GAD65 and IA2 autoantigens. We discovered that the reaction to autoantigens is very individual and it is influenced by the patient's autoantibody profile. These results could be relevant in planning antigen-specific immunointervention studies and improving their efficacy. We also made an attempt to improve specificity and sensitivity of a beta cell destruction marker (specifically demethylated DNA), which would enable better understanding of the beta cell decline and identification of individuals at risk of T1D development. In...