Myotonic dystrophy type 2 (DM2) is caused by the extreme expansion (from < 30 repeats in normal individuals to ~ 11,000 for the full mutation in certain patients) of the repeating tetranucleotide CCTG•CAGG sequence in the intron of the zinc finger protein 9 (ZNF9) gene. The genetic instabilities of the CCTG•CAGG repeats were investigated to evaluate the molecular mechanisms responsible for these massive expansions. The effects of replication, recombination, repair and transcription on the genetic instabilities have been investigated in COS-7 cells and E. coli model systems. A replication assay was established in COS-7 cells wherein the CCTG•CAGG repeats cloned proximal to the SV40 origin of replication resulted in expansions and deletions in a length and orientation-specific manner, whereas the repeats cloned distal to the same origin were comparatively stable. These results fit with our data obtained from biochemical studies on synthetic oligonucleotides since these biochemical studies revealed that the d(CAGG)26 oligomer had a marked propensity to adopt a hairpin structure as opposed to its complementary d(CCTG)26 that lacked this capacity. Furthermore, a genetic assay in E. coli was used to monitor the intramolecular frequency of recombination. This assay revealed that the tetranucleotide repeats were indeed hot spots for recombination. Moreover, studies conducted in SOS-repair mutants showed that recombination frequencies were much lower in a SOS¯ strain as compared to a SOS+ strain. However, experiments conducted to ascertain the level of induction of the SOS response revealed that the SOS pathway was not stimulated in our studies. These results revealed that although breaks may occur within the repeats, the damage is most likely repaired without induction of the SOS response contrary to previous beliefs. Thus, a complex interplay of replication, recombination, and repair is likely responsible for the expansions observed in DM2.

Die Arbeit befasst sich mit Abschätzung der Häufigkeit der proximalen myotonen Myopahtie (PROMM/DM2) in der deutschen Bevölkerung. Zugrunde liegend sind Daten aus dem Institut für Humangenetik der Universität Würzburg von 1993 bis 2006, sowie Daten der deutschlandweiten Diagnostik anbietenden Zentren aus den Jahren 2005 und 2006. Die Auswertung der Daten der Humangenetik in Würzburg bestätigte die Vermutung, dass die myotone Dystrophie (DM1) und die proximale myotone Myopathie (PROMM/DM2) gleich häufig sind. Aus dem Wissen heraus, dass frühere Angaben für die Häufigkeit der DM1 nicht nur diese Erkrankung erfasst haben, sondern auch Fälle mit PROMM/DM2, kann man davon ausgehen, dass damalige Häufigkeitsangaben anteilig beide Erkrankungen gemeinsam erfasssen. Das Ergebniss spricht für eine Inzidenz von 2,75/100.000 Einwohnern in Deutschland. / The central question of this work was, to asses the frequency of proximal myotonic dystrophy (PROMM/DM2) in germanys population. Based on data of the years 1993 to 2006 from institute for human genetics at the university of wuerzburg and data from laboratories all over germany offering genetic testing for proximal myotonic myopathy, the assumption of having both, proximal myotonic myopathy and myotonic dytsrophy, the same frequency has been confirmed. We know that data refering to the frequency of myotonic dystrophy must also have includet cases of proximal myotonic dytsrophy. Proceeding that previous data includes both malaties and that they are both, same often, we can say that the incidence of proximal myotonic dystrophy must be 2,75/100.000 inhabitans.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O diabetes mellitus tipo 2 (DM2), doenÃa multifatorial, heterogÃnica, resulta de suscetibilidade genÃtica associada a fatores ambientais, especialmente sedentarismo e dieta rica em gorduras saturadas, e a obesidade. Caracteriza-se por resistÃncia à insulina (RI) e pela diminuiÃÃo da secreÃÃo desse hormÃnio. InflamaÃÃo moderada e crÃnica no tecido adiposo branco disfuncional, denominada âmeta-inflamaÃÃoâ, parece ser o elo entre obesidade, RI e DM2. Papel de adipocinas produzidas pelo tecido adiposo nessas afecÃÃes vem sendo investigado. Objetiva-se neste estudo verificar se suplementaÃÃo oral (SO) de mistura de Ãleos (MXO) com relaÃÃo de Ãmega 9 / Ãmega 6 (&#969;9/&#969;6) elevada e de Ãmega 6 / Ãmega 3 (&#969;6/&#969;3) baixa, de diferentes fontes de &#969;3, interfere em adipocinas plasmÃticas de camundongos com DM2. Depois de alimentados ad libitum com dieta da AIN-93G atà ficarem adultos, camundongos Swiss (CSW) machos receberam, por onze semanas, dieta AIN-93HA, hiperlipÃdica adaptada, para induÃÃo de DM2, confirmado em 90% deles. Mantida a dieta AIN-93HA, os CSW com DM2 receberam, em grupos, por sete dias, SO com MXO: GA: H&#8322;O (controle nulo); GB: MXO [&#969;9:&#969;3 0,4:1;&#969;6:&#969;3 8:1 (controle neutro)]; GC: MXO [&#969;9:&#969;3 3,7:1; &#969;6:&#969;3-ALA 1,4:1]; GD: MXO [&#969;9:&#969;3 3,7:1;&#969;6:&#969;3-EPA+DHA de peixe 1,4:1]; GE: MXO [&#969;9:&#969;3 3,7:1; &#969;6:&#969;3-DHA de algas 1,4:1]. Por imunoensaios, realizou-se dosagem plasmÃtica de insulina e de adipocinas, fator de necrose tumoralâalfa (TNF-&#945;); interleucina-6 (IL-6); interleucina-1 beta (IL-1&#946;); fator ativador de monÃcitos (MCP-1); resistina (RES); leptina (LEP); inibidor do fator ativador de plasminogÃnio 1 (PAI-1) e adiponectina (AdipoQ). Constatou-se diferenÃa estatÃstica significante de adipocinas do grupo GE (&#969;3-DHA de algas), em relaÃÃo aos outros grupos, com aumento de IL-6 em relaÃÃo ao GC e GD; diminuiÃÃo de LEP em relaÃÃo ao GA; aumento de TNF-&#945; em relaÃÃo aos grupos GB, GC e GD; e diminuiÃÃo de AdipoQ em relaÃÃo ao GB; assim como de RES entre os grupos GC (&#969;3-ALA) e GD (&#969;3-EPA+DHA). NÃo houve diferenÃa estatÃstica significante em nenhuma das variÃveis entre grupos controles. Continuidade de dieta rica em gordura saturada pode ter comprometido a eficÃcia da suplementaÃÃo de MXO ricos em &#969;3 e &#969;9. O âestado da arteâ demanda outros estudos para esclarecer o papel do DHA na âmeta-inflamaÃÃoâ. / Diabetes mellitus type 2 (DM2), a multifactorial disease, heterogenic results of associated genetic susceptibility to environmental factors, especially sedentary lifestyle and a diet rich in saturated fats, and obesity. It is characterized by insulin resistance (IR) and by decreasing the secretion of this hormone. Moderate and chronic inflammation in white adipose tissue dysfunctional, called "meta-inflammation," seems to be the link between obesity, IR and DM2. Role of adipokines produced by adipose tissue in these diseases has been investigated. Objective of this study was to verify whether oral supplementation (SO) of oil blend (MXO) compared with omega 9 / omega 6 (&#969;9/&#969;6) and high omega 6 / omega 3 (&#969;6/&#969;3) low, from different sources of &#969;3 interferes with adipokines plasma of mice with T2DM. After fed ad libitum with AIN-93G diet until they become adult Swiss mice (CSW) males received by eleven weeks AIN-93HA, hyperlipidic adapted to induce DM2 confirmed in 90% of them. Maintained the AIN-93HA, the CSW with T2DM were in groups of seven days, with MXO SO: GA: H &#8322; O (null control) GB: MXO [&#969;9: &#969;3 0.4:1; &#969;6: &#969;3 8: 1 (neutral control)]; GC: MXO [&#969;9: 3.7:1 &#969;3, &#969;6: &#969;3-ALA 1.4:1]; GD: MXO [&#969;9: 3.7:1 &#969;3, &#969;6: &#969;3-EPA + DHA from fish 1.4:1]; GE: MXO [&#969;9: &#969;3 3.7:1; &#969;6: &#969;3, DHA from algae 1.4:1]. Why immunoassays, held measurement of plasma insulin and adipokines, tumor necrosis factor-alpha (TNF-&#945;), interleukin-6 (IL-6), interleukin-1 beta (IL-1&#946;), monocyte activating factor ( MCP-1), resistin (RES), leptin (LEP), an inhibitor of plasminogen activator 1 (PAI-1) and adiponectin (ADIPOQ). It found a statistically significant difference of adipokines group GE (&#969;3-DHA from algae), compared to the other groups, with increased IL-6 compared to GC and GD, fewer LEP compared to GA; increase of TNF- &#945; in relation to groups GB, GC and GD, and ADIPOQ decrease compared to GB, as well as RES between GC (&#969;3-ALA) and GD (&#969;3-EPA + DHA). There was no statistically significant difference in any of the variables between control groups. Continuity diet high in saturated fat may have compromised the effectiveness of supplementation MXO rich in &#969;3 and &#969;9. The "state of the art" demand further studies to clarify the role of DHA in the "meta-inflamaÃÃoDM2, Adipokines, saturated fatty acids, omega fatty acids 3, 6 and 9 and" meta-inflammation

The kynurenine pathway produces NAD+ from L-tryptophan. Metabolites known as the kynurenines are produced within the pathway. The effects of the kynurenines have been associated with a number of diseases including cancer, Alzheimer’s disease, Huntington’s disease, and acute pancreatitis. Kynurenine monooxygenase (KMO) is an enzyme that catalyses the conversion of L-kynurenine to 3-hydroxy-L-kynurenine, the downstream product of which is the neurotoxic quinolinic acid. L-kynurenine is positioned at a branching point within the pathway. Metabolism via KMO leads to quinolinic acid production whereas conversion via kynurenine aminotransferase (KAT) produces the neuroprotective kynurenic acid. Inhibition of KMO leads to an increase in kynurenic acid concentration. This has also been shown to ameliorate the symptoms of neurological diseases in a number of animal models as well as to protect against multiple organ dysfunction caused by acute pancreatitis in rodent models. These findings present KMO as a promising drug target. Due to the hydrophobic nature of human KMO (hKMO) it has been necessary to utilise other forms of KMO as models. Past studies have produced crystal structures of a truncated Saccharomyces cerevisiae KMO and of Pseudomonas fluorescens KMO (PfKMO). Previous work in this research group has resulted in the structure of variants of PfKMO bound to either inhibitor molecules or substrate. These structures identified residues involved in substrate binding and the presence of a highly mobile section of the C-terminus, giving rise to open and closed conformations. It was surmised the movement of the C-terminus was dependent upon the presence of substrate and an interactive network between the C-terminus and the rest of the protein. Using improved crystallising conditions high-resolution structures of PfKMO have been produced that allow for further study of residues involved in substrate binding and the interactive network within the C-terminus. The mutants R84K and Y404F showed severely decreased enzyme activity. Crystal structures of these proteins showed disrupted interactions between substrate and active site. These findings underline the importance of residues R84 and Y404 in substrate binding. An H320F mutation gives an analogous active site to hKMO. Crystallographic and kinetic study of this mutant proved very similar to PfKMO, supporting the use of PfKMO as a model for hKMO. Throughout the work each structure had a P21221 space group with two molecules in the asymmetric unit. The presence of an open and closed molecule within each structure, including substrate-free molecules refuted the connection between C-terminus and substrate. R386K and E372T mutations were separately introduced in order to interrupt the interactive network. The presence of both open and closed conformations in the structures of R386K and E372T refutes the necessity for the interactive network in C-terminus movement. The data analysed throughout the project suggest simple mobility and thermal motion as the cause of the movement of the C-terminus. This work, in conjunction with kinetic data from the thesis of Helen Bell, presents structural data to characterise the role of binding residues within the active site of KMO as well as the mechanistic role of the C-terminus. It also highlights the importance of certain binding residues and countered the previously held hypotheses surrounding the significance of the C-terminus. The mechanistic role of the C-terminus therefore remains unclear and requires further study.

Type 2 diabetes mellitus (T2DM) has become a major health problem worldwide. The St. Vincent declaration emphasized the urgent need to improve the epidemiological knowledge of this disease in Europe. Within Europe, research on the link between socioeconomic position (SEP) and type 2 diabetes is scarce. The objective of this thesis was to conduct an extensive review of the current literature on socioeconomic inequalities in type 2 diabetes within European countries, while analyzing the relationship between, incidence, prevalence and mortality due to T2DM and SEP. In addition, we also analyzed trends on SEP inequalities in the prevalence of T2DM in Spain (1983-2006). Finally, we also assessed the appropriate use of health surveys with self-reported diagnosis in order to further analyze the relation between SEP and T2DM. Different sources of information were used throughout the study. The systematic review was completed using the PUBMED database while the empirical studies used data of two European projects, the EUROTHINE, SHARE and the Spanish National Health Survey (study of trends in SEP inequalities in T2DM) along with the Catalonia health surveys (study of validation). The thesis consists of 5 papers that attempt to respond to the different objectives. The studies included in this thesis suggest that socio-economic position (SEP) inequalities affect the incidence, prevalence and mortality by T2DM in Europe. These SEP inequalities are partly explained for body mass index, diet and physical activity. Moreover, these inequalities seemed to have remained constant or increased over time. Finally, health interview surveys with self-reported T2DM seems to be a good instrument to evaluate SEP inequalities in T2DM. / La Diabetis Mellitus Tipus 2 (DM2) ha esdevingut un dels principals problemes de salut a nivell mundial. La declaració de ST VINCENT emfatitzava la necessitat i la urgència de millorar-ne el coneixement epidemiològic a nivell Europeu. Els estudis a nivell europeu sobre les desigualtats per Posició Socioeconòmica (PSE) en la DM2 eren força escassos. L’objectiu d’aquesta tesi era fer una revisió extensa dels estudis publicats sobre desigualtats per PSE en la DM2 a Europa, així com analitzar la relació entre la incidència, la prevalença i la mortalitat per DM2 i la PSE. Un altre objectiu també era analitzar la tendència de les desigualtats per PSE en la prevalença de DM2 a Espanya (1983-2006). Finalment, com a objectiu també hi figurava el valorar l’ús adequat de les enquestes de salut amb auto - declaració de DM2 per tal d’avaluar les desigualtats per PSE en la DM2. Per tal de dur a terme els objectius es van emprar diferents fonts d’informació. Per tal de dur a terme la revisió sistemàtica es va emprar la base de dades de PUBMED mentre que pels estudis empírics es van utilitzar les dades de dos projectes europeus com són el projecte EUROTHINE i el SHARE i les enquestes nacionals de salut d’Espanya (per la tendència de diabetis) i de Catalunya (per la validació). La tesi consta de 5 articles que intenten donar resposta als diferents objectius. Els estudis inclosos en aquesta tesi suggereixen que existeixen desigualtats per posició socioeconòmica (SEP) en la DM2, tant en la incidència, en la prevalença com en la mortalitat a Europa. Aquestes desigualtats per PSE s’expliquen en part per l’índex de massa corporal, la dieta o l’activitat física. A més a més, aquestes desigualtats sembla que s’han mantingut constants o han crescut al llarg del temps. Finalment, s’ha vist que les enquestes de salut amb la pregunta d’auto-declaració de la diabetis són un bon instrument per avaluar les desigualtats per PSE en la DM2.