The fidelity of DNA replication prevents mutations that may lead to cancer predisposition or neurodegenerative diseases. One mechanism that enhances DNA replication fidelity is DNA mismatch repair, which corrects mismatches and small insertion/deletion loops that have escaped polymerase proofreading. In all eukaryotes and most prokaryotes, MutL (a key mismatch repair protein) has an intrinsic endonuclease activity that nicks the newly synthesized strand and recruits downstream factors to remove and correct errors. It has been proposed that ATP binding promotes a series of conformational changes that induce structural order within MutL and stimulates its endonuclease activity. The C-terminal domain of MutL, which harbors the endonuclease site, does not bind to DNA. This has prevented the molecular characterization of its endonuclease activity. In this thesis, we first show that MutL in B. subtilis exhibits asymmetric conformations similar to yeast and human MutL homologs. We also devise a novel approach to bypass the binding defect of the C-terminal domain by using fusion proteins. We find that these fusions bind to DNA specifically and, in the presence of the processivity clamp, can nick DNA. One of these fusion proteins in particular stimulates the nicking activity much more efficiently than the C-terminal domain alone. This work lays the foundation for the mechanistic characterization of the MutL endonuclease and provides a method to stabilize transient protein-DNA interactions. / Thesis / Master of Science (MSc)

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DNA mismatch repair corrects base-base mismatches and small insertion/deletion loops generated during normal DNA replication. If left unrepaired, these errors become permanent mutations and can lead to increased susceptibility to cancer. In most prokaryotes and all eukaryotes, the mismatch repair protein MutL is a sequence-unspecific endonuclease that plays an essential role in the strand discrimination step of this pathway. Prokaryotic MutL forms homodimers with two endonuclease sites, whereas eukaryotic MutL homologs form heterodimers with a single active site. To elucidate the mechanistic differences between prokaryotic and eukaryotic MutL, we tested whether both endonuclease sites are necessary for prokaryotic MutL nicking activity. MutL interaction with the processivity clamp is required to stimulate endonuclease activity. Therefore, we also tested whether both subunits of the MutL dimer needed to interact with the processivity clamp. To this end, we engineered a system to independently manipulate each protomer of the homodimer. We demonstrated that prokaryotic MutL is regulated by the processivity clamp to act in a similar manner to eukaryotic MutL with only one functional site contributing to the endonuclease activity. We also devised a strategy to stabilize the transient interactions between MutL, the β-clamp, and DNA through disulfide bridge crosslinking and heterobifunctional crosslinking. Stabilizing transient protein-protein and protein-DNA interactions will help optimize future structural studies in obtaining the ternary complex for mechanistic insights to the MutL endonuclease activity and regulation imposed by the β-clamp. / Thesis / Master of Science (MSc)

In humans, multi enzymatic processes are involved in maintaining DNA stability and cellular homeostasis. Cells undergo several episodes to survive and protect itself in daily basis. Accumulation of DNA errors and breaks are repaired by dynamic machinery, such as mismatch repair (MMR), replication-related process. In presented diploma thesis, we report the studied MMR pathway and its involvement in malignancy of epithelial ovarian cancer (EOC). Our working hypothesis postulated that core genes of MMR, such as MLH1 and MSH2 are down-regulated in malignant cells. Cells therefore become incapable to repair accumulating DNA damage, undergo apoptosis or most likely uncontrolled proliferation. Above mentioned genes may also be silenced in cancer patients at transcription, translation or epigenetic levels. Our aims were to clarify and to investigate the importance of MMR based on mRNA transcription, protein stability and promoter hypermethylation on a set of major MMR genes, particularly MLH1, MSH2, PMS1, MLH3, MSH6, MSH3, and PMS2. In our study, we analysed samples from 63 epithelial ovarian cancer patients and 12 non-malignant reference tissues using RT-qPCR, MS-HRM, and Western Blotting methods. Consequently, our results show down-regulation of all MMR genes except for MSH2 (up-regulated) in tumor...

L’hypertension artérielle pulmonaire (HTAP) est définie par une pression artérielle moyenne >25 mmHg associée à une pression capillaire ≤ 15 mmHg. L’augmentation des résistances artérielles pulmonaires dans l’HTAP est associée à des phénomènes de vasoconstriction, de remodelage pariétal et de thrombose au niveau des artères pulmonaires de petit calibre. Plusieurs données expérimentales montrent que l’hypoxie est susceptible d’induire ces mêmes changements au niveau du lit artériel pulmonaire. De plus, d’après les petites cohortes étudiées dans la littérature, une hypoxémie nocturne (HN) serait fréquente dans l’HTAP pouvant se rencontrer chez presque 77% des malades. Les mécanismes de cette HN sont mal élucidés, en rapport avec une respiration périodique de type Cheyne Stokes (CS) pour certains auteurs, ou avec des apnées-hypopnées obstructives (AHO) du sommeil pour d’autres.Nous avons voulu ainsi explorer le sommeil des malades porteurs d’HTAP à la recherche de ces anomalies du sommeil et dans le but de déterminer leur prévalence et leurs mécanismes physiopathologiques. Quarante six malades hospitalisés dans le service de pneumologie de l’Hôpital Antoine Béclère, centre de référence national pour l’HTAP, ont bénéficié d’une nuit de polysomnographie (CIDELEC) avec une mesure de la capnographie transcutanée (TOSCA). Il s’agissait d’une population homogène comprenant 29 malades porteurs d’une HTAP idiopathique (HTAPI) et de 17 malades porteurs d’un coeur pulmonaire chronique postembolique (CPCPE), n’ayant pas d’anomalies fonctionnelles respiratoires sévères (VEMS etCPT ≥ 60 % de la théorique), ni d’obésité sévère (IMC < 35 Kg/m²), et stables depuis au moins 3mois, sous traitement spécifique optimal pour l’HTAP. Ces malades étaient pour la plupart en classe fonctionnelle II de la NYHA, parcouraient > 400m au test de marche de 6 minutes, etavaient un index cardiaque moyen mesuré sur le cathétérisme cardiaque droit dans les normes(3,2 ± 0,6 L/min/m²).La majorité (38/46 soit 82,6%) avaient une HN définie par un temps de sommeil passé avec uneSpO2 < 90%, > 60min et/ou un index de désaturation ≥ 20/h. Ces patients passaient 48.9 ± 35.9%de leur temps de sommeil avec une SpO2 < 90%. Le mécanisme le plus fréquemment rencontré(76% des malades désaturateurs) correspond à une hétérogénéité ventilation/perfusion (VA/Q)isolée ou associée à des AHO du sommeil. La prévalence des apnées-hypopnées (AH) était très élevée avec un index d’apnées-hypopnées (IAH) ≥ 5/h chez 89% des malades et un IAH moyende 24.9 ± 22.1/ h. La majorité de ces évènements était d’origine obstructive, seulement 4malades présentaient des AH de mécanisme central dont 3 avaient une respiration périodique de type CS. Un seul cas d’hypoventilation alvéolaire nocturne associé à un IAH obstructif modéré a été identifié, avec une désaturation nocturne prolongée concomitante d’une majoration significative de la capnie transcutanée de plus que 30 mmHg. Aucun facteur clinique ni hémodynamique n’a pu être identifié comme prédictif de la survenue de l’HN malgré certaines corrélations notées entre les paramètres de désaturation nocturne et la PaO2 diurne d’une part, et l’obstruction des petites voies aériennes d’autre part.Nous avons pu conclure que l’HN est fréquente dans l’HTAPI et dans le CPCPE, en rapport avec un déséquilibre VA/Q et/ou des AHO du sommeil. Reste à préciser dans des études ultérieures si la correction de cette HN aura des effets bénéfiques pour les patients en termes d’amélioration fonctionnelle, hémodynamique ou de réponse au traitement. / Precapillary pulmonary hypertension (PH) is defined by a mean pulmonary pressure > 25 mmHg associated with a normal wedge pressure (≤ 15 mmHg). Increase in vascular resistances in PH is due to vasoconstriction, vascular wall remodelling and thromboses of small pulmonary arterioles. Hypoxia is known to cause similar changes in pulmonary vasculature. Although some cohorts studies have shown that nocturnal hypoxemia (NH) is frequent in PH, accounting for up to 77% of cases, the mechanisms of this NH were not well established, being associated to periodic respiration like the Cheyne Stokes respiration (CS) by some authors, and to obstructive apneas-hypopneas (OAH) by others.The aim of our study was to search for sleep-related breathing disorders in PH, to determine their prevalence and their mechanisms. Fourty six patients hospitalized in the pulmonary department of Antoine Béclère Hospital, which is the national referral center for PH in France, underwent a one night polysomnography (CIDELEC) with a transcutaneous capnography (TOSCA). Our population was homogeneous with 29 patients having idiopathic pulmonary arterial hypertension (IPAH) and 17 patients having chronic thrombo-embolic PH (CTEPH). Patients had no severe functional limitation (FEV1 and TLC ≥ 60 % of predicted), nor severe obesity (BMI < 35 Kg/m²), and they were in a steady state with optimal PH treatment for at least three months. The majority of patients were in NYHA functional class II, had a 6 minutes walking distance > 400m and a mean cardiac index measured on right heart catheterization within normal ranges (3,2 ± 0,6 L/min/m²).Thirty eight out of the 46 patients (82,6%) had a NH as defined by a sleeping time spent with a SpO2 < 90%, > 60min and/or an oxygen desaturation index ≥ 20/h. These patients spent 48.9 ± 35.9% of their sleeping time with a SpO2 < 90%. The most frequent mechanism underlying theses abnormalities (76% of desaturators patients) was due to ventilation/perfusion (VA/Q) heterogeneity, isolated or associated to OAH. Apneas-hypopneas (AH) were frequent in PH patients with an AH index (AHI) ≥ 5/h in 89% of cases, and a mean AHI of 24.9 ± 22.1/ h. Most of these respiratory events were obstructive, only four patients had central AH with a CS pattern in 3 out them. A single case of alveolar hypoventilation associated with a moderate AHI was identified and was caracterized by a sustained nocturnal desaturation associated with a significant increase in transcutaneous CO2 pressure ( > 30 mmHg). No clinical nor hemodynamic factor was found to be predicting for NH, although minor correlations were found between nocturnal desaturation parameters and PaO2, and nocturnal desaturation parameters and small airways obstruction.We conclude that NH is frequent in IPAH and CTEPH, due to VA/Q mismatch and/or OAH. Future studies are needed to determine the impact of the correction of this NH on PH patients regarding their NYHA functionnal class, their hemodynamic parameters and their responsiveness to PH specific treatment.

Workers whose credentials and skills do not meet or exceed the required competencies for their jobs have been of interest to scholars investigating the transition from school to jobs. To understand how such mismatch arises in the transitional period, some scholars emphasize that the labor market cannot keep up with the pace of educational expansion. Thus, many highly educated workers do not find jobs that fit their schooling and skill level. Others locate the source of mismatch in the inability of education to produce enough workers with the desired skill levels in the labor market. By focusing on this mismatch, this dissertation aims to provide a better understanding of the relationship between education and work. In particular, this study examines data covering the past two decades to see how the number of workers with skill and educational mismatch has changed and how educational expansion and transformations in the labor market have contributed to the change. The results indicate that workers with such mismatch have generally increased over the past two decades, but educational expansion has minimally contributed to this change. Rather, it is more likely caused by business cycles or job characteristics. The study also explores how the practices applied to select suitable workers in the hiring process affects workers’ job matching. This study suggests that workers are classified into various types depending on strategies by which employers use to determine workers’ degree of fit. Subsequently, their earnings and job satisfaction vary according to workers’ membership in these types of groups.

Graded InGaN buffers were employed to relax the strain arising from the lattice and thermal mismatch in GaN/InGaN epilayers grown on sapphire. An enhanced strain relaxation was observed in GaN grown on a stack of five InGaN layers, each 200 nm thick with the In content increased in each layer, and with an intermediate thin GaN layer, 10 nm thick inserted between the InGaN layers, as compared to the conventional two-step growth of GaN epilayer on sapphire. The function of the intermediate layer is to progressively relax the strain and to annihilate the dislocations that build up in the InGaN layer. If the InGaN layers were graded too rapidly, more dislocations will be generated. This increases the probability of the dislocations getting entangled and thereby impeding the motion of the dislocations to relax the strain in the InGaN layer. The optimum growth conditions of the intermediate layer play a major role in promoting the suppression and filling of the V-pits in the GaN cap layer, and were empirically found to be a thin 10 nm GaN grown at 750 0°C and annealed at 1000 0°C. / Singapore-MIT Alliance (SMA)

Abstract The main objective of the present follow-up study was to investigate auditory processing by using auditory event related potentials (ERPs), and language development to determine whether a correlation exists between auditory ERPs and language development. Auditory processing was investigated in very low birth weight (VLBW) preterm children and matched controls at mean ages of 4 and 6 years to determine whether there are differences in ERPs between VLBW preterm children and controls. Language development was measured at the mean ages of 2, 4 and 6 years to investigate the developmental course of language learning and to determine whether a relationship exists between ERPs, especially mismatch negativity (MMN), and language development. Auditory ERPs were also measured in children with AS (mean age 9;1 years) and matched controls to assess whether differences can be found between these two groups of children. Language development in children with AS was not investigated for this study. VLBW preterm children exhibited difficulties in the auditory processing at the level of obligatory ERPs, MMN, late MMN (lMMN) and behavioural tests. Both language comprehension and production were deficient in the preterm group compared to their controls. Lexical development was the most prominent phenomenon differentiating preterm children from their controls. MMN and lMMN amplitudes were attenuated most in children with naming difficulty at the ages of 4 and 6 years. Weak or totally missing MMN at the age of 4 years was mainly found in children with naming difficulties. Children with AS also displayed abnormalities in auditory processing, as indexed by delayed MMN latency. MMN was most delayed in the right hemisphere and specifically for tones. In conclusion: VLBW preterm children and children with AS exhibited difficulties in auditory processing. MMN correlated well with language development in preterm children. Therefore, auditory ERPs, especially MMN, should be used in combination with language measures to identify the children at a risk for deficient auditory processing and language delays.

Beim akuten ischämischen Schlaganfall ist die Größe der initialen DWI-Läsion (Diffusions-gewichtete Sequenz) in der zerebralen MRT zur Abschätzung der Prognose des Patienten umstritten. Die Beobachtung des Verlaufs der DWI-Läsion in den ersten Tagen nach einem Schlaganfall scheint hierfür besser geeignet. Hierbei zeigt sich, dass ein Infarktwachstum eher mit einem schlechteren klinischen Ergebnis korreliert. Ein mögliches Infarktwachstum vorherzusagen ist deshalb von großem Interesse. Bei nicht-lakunären Infarkten, die durch Verschlüsse größerer Arterien verursacht werden, ist die Ausbreitung des Infarktkerns in zuvor minderperfundierte Areale (d.h. in die Penumbra), als Ursache eines Wachstums anerkannt. Die Penumbra wird als die Differenz zwischen DWI- und PWI-Läsionen (Perfusions-gewichtete Sequenz) angesehen, was als PWI/DWI-Mismatch bezeichnet wird. Unklar ist, ob dieses Mismatch-Konzept auch auf lakunäre Ischämien (DWI-Läsionen < 2 cm Ø) angewandt werden kann. Ziel dieser prospektiven Studie war, den Zusammenhang zwischen dem initialen DWI/PWI-Mismatch und der Infarktentwicklung im kurzfristigen Verlauf bei 100 konservativ behandelten Patienten zu untersuchen. Das Patientenkollektiv war hinsichtlich der Infarktsubtypen (lakunäre und nicht-lakunäre) als inhomogen einzustufen. Am Tag der Aufnahme und an Tag 7 wurden anhand eines MRT-Schlaganfallprotokolls die Volumina der DWI und PWI-Läsionen, die Mismatch-Volumina, das Infarktwachstum, das relative Mismatch und das relative Infarktwachstum untersucht. Das Infarktwachstum zeigte einen engen Zusammenhang mit dem initialen DWI/PWI-Mismatch. Dies traf vor allem dann zu, wenn man die relativen Werte betrachtete. Hieraus ergibt sich, dass ein großes initiales Mismatch ein späteres Infarktwachstum wahrscheinlicher macht. Für die Zukunft könnte man daher davon ausgehen, dass das relative Mismatch, bezogen auf die relative Infarktentwicklung, einen besseren prognostischen Wert aufweist, als die absoluten Werte.

This essay investigates the potential educational mismatch of immigrants compared to natives concerning the Swedish labor market. The data is collected from the European Social Survey between 2002-2014. Our results show that immigrants tend to be more overeducated than natives. Our results also indicate that more recent cohorts are more likely to be overeducated compared to cohorts that have lived for a longer time period in Sweden. Disparities in language and country specific skills but also if a person belong to an ethnic minority are reasons for why these mismatches occur.