Biosensors have proven to be a powerful tool for detecting diverse targets, such as proteins, DNA, and small molecules representing disease biomarkers, toxins, drugs and their metabolites, environmental pollutants, agrichemicals, and antibiotics with high sensitivity and specificity. The major objective of the research described in this dissertation was to develop low cost, low sample volume, highly sensitive and specific AuNP-based colorimetric sensor platforms for the detection of DNA and small molecules. With this in mind, we propose an instrument-free approach in chapter three for the detection of NADH with a sensor constructed on a paper substrate, based on the target-induced inhibition of AuNP dissolution. The successful detection of this important molecule opens the door to numerous possibilities for dehydrogenase characterization, because NAD+/NADH are essential cofactors for more than 300 dehydrogenase enzymes. To further increase the sensitivity of our hybridization-based assay for DNA detection, we developed an enzyme-assisted target recycling (EATR) strategy in chapter four and have applied such an EATR-based colorimetric assay to detect single-nucleotide mismatches in a target DNA with DNA-functionalized AuNPs. This assay is based on the principle that nuclease enzymes recognize probe–target complexes, cleaving only the probe strand. This results in target release, enabling subsequent binding to and cleavage of another probe molecule. When the probe is conjugated onto AuNPs, complete cleavage from the AuNP surface produces a detectable signal in high ionic strength environments as the nanoparticles undergo aggregation. With such enzyme-assisted amplification, target detection can occur with a very low nM detection limit within 15 minutes. The extent of DNA loading on the AuNP surface plays an important role in the efficiency of DNA hybridization and aptamer-target assembly. Many studies have shown that high surface-coverage is associated with steric hindrance, electrostatic repulsive interactions and elevated surface salt concentration, whereas low surface-coverage can result in nonspecific binding of oligonucleotides to the particle surface. In chapter five, we investigated DNA surface coverage effects, and apply this optimization in conjunction with a highly-specific aptamer to develop a sensitive colorimetric sensor for rapid cocaine detection based on the inhibition of nuclease enzyme activity.

Secessionist movements have been found historically on both sides of the political spectrum, and sometimes have tried to remain apolitical completely, but because of the rise of partisan politics, secessionism has inevitably become politicized. Variations in Western European secessionism can be noticed, and as such, explanations put forward may be deemed insufficient, or incomplete. In my thesis I tested the hypothesis that secessionism varied on the political spectrum because it has been consolidated against ideological Others (in Scotland against Thatcher’s Conservatives between 1974 and 1990). I tested this methodologically through process tracing and theoretically by looking at the consolidation of the Scottish National Party through reactive nationalism. Specifically I analyzed the nationalist discourse used to justify ideological positioning in the 1970s and 1980s in propaganda materials and archival documents, and if and how this ideological choice was reflected or interpreted in newspapers (for opinions on how this consolidation was perceived by the electorate).

Archaeological and genetic evidence have long supported the notion that the Indian subcontinent played an important role in the dispersal of modern humans out of Africa. In the present study, two Dravidian populations, namely Lingayat (N=101) and Vokkaliga (N=102) were examined using high-resolution analyses of Y-chromosome single nucleotide polymorphism (Y-SNP) and their associated seventeen short tandem repeat (STR) loci. The results revealed a prevalence of the major indigenous Indian Y-haplogroups (H, L, F* and R2), which collectively accounted for three-fourths of the Lingayat and Vokkaliga paternal gene pool. In addition, the presence of ancient lineages such as F*-M213, H*-M69 and C*-M216 suggested that modern humans reached India very early after their migration out of Africa. Finally, high haplotype diversity values at 17 Y-STR loci for Lingayat (0.9981) and Vokkaliga (0.9901) populations as well as the absence of shared haplotypes between them emphasized the importance of independent databases for forensic casework.

Les cellules gliales qui forment les segments de myéline du système nerveux périphérique (SNP) sont appelées cellules de Schwann. Elles assurent aux nerfs un soutien fonctionnel et permettent une conduction rapide et efficace de l'influx nerveux. Cette fonction requiert une communication réciproque entre les neurones et les cellules gliales qui les entourent. Une perturbation de cette interaction entraine le plus souvent une situation pathologique comme les neuropathies périphériques dont la plus connue est la maladie de Charcot-Marie-Tooth. Cependant, les mécanismes qui conduisent à ces pathologies sont encore peu connus et leur compréhension demande au préalable l'élucidation des mécanismes physiologiques qui contrôlent le développement du SNP. Ce travail a consisté en l'analyse de nouvelles fonctions des gènes ndrg4 et elmo1, dans le développement du système nerveux périphérique, chez le poisson zèbre. Nous avons montré que ndrg4 (n-myc downstream regulated gene) est un facteur neuronal qui régule le développement de la myéline périphérique en contrôlant le regroupement des canaux sodiques aux nœuds de Ranvier et l'expression de la mbp. Ndrg4 semble moduler l'échange vésiculaire entre les axones et les cellules de Schwann, en contrôlant l'expression de certaines protéines de relargage vésiculaire comme SNAP25, membre du complexe SNARE.Ce travail décrit par ailleurs une nouvelle fonction de elmo1 (engulfment and cell motility 1) dans le développement du SNP du poisson zèbre, où il favorise la survie des neurones dans lesquels il est exprimé. Nous avons montré qu'elmo1 protège les neurones de l'apoptose et que cette fonction est contrôlée par la voie nétrine1/unc5b en amont de Rac1. De ce fait, elmo1 est requis pour le développement du ganglion de la ligne latérale postérieure et des axones qui en émergent pour donner un système nerveux fonctionnel. Ainsi, ces travaux contribuent à une meilleure connaissance du développement du SNP et élucident pour la première fois une nouvelle voie de signalisation requise pour la survie des neurones dans le SNP. / The glial cells that form myelin segments in the peripheral nervous system (PNS) are called Schwann cells (SCs). They provide functional support for nerves and allow a fast and efficient conduction of the action potentials. This requires a bilateral communication between axons and the associated glial cells. Disruption of this interaction often leads to peripheral neuropathies e.g. Charcot-Marie-Tooth disease. However, the mechanisms underlying these diseases remain poorly known and their understanding requires, at first, the elucidation of the physiological mechanisms responsible for PNS development. This work consists of a functional analysis of two genes, ndrg4 and elmo1, in the PNS development, using the zebrafish model. We showed that the neuronal factor ndrg4 (n-myc downstream regulated gene 4) regulates nodes of Ranvier organization and mbp expression along the Posterior Lateral Line nerve (PLLn). This is achieved, most likely, by the ability of ndrg4 to modulate vesicular exchange between axons and SCs. Indeed, the expression of some key proteins involved in vesicle docking and release such as SNAP25, a member of the SNARE complex, are significantly dependent on ndrg4.Moreover, this work describes a novel role for neuronal elmo1 (engulfment and cell motility 1) in PNS development by promoting neuronal survival within the PLL ganglion. We showed that elmo1 has protective role against apoptosis and that its function is controlled by the netrin1/unc5b signalling upstream of Rac1 and independently of macrophages role in apoptotic clearance. Therefore, elmo1 is required for the proper development of the PLL ganglion and the axonal development of the PLLn. Thus, this study further contributes to our understanding of PNS development and unravels a novel molecular pathway required for neuronal survival in the PNS.

<div><div><div><p>Impacting individual’s social and physical well-being, psychiatric disorders have been a substantial burden on public health. As such disorders are frequently observed aggregating in families, we can expect a large involvement of heritable components underlying their etiologies. Therefore, studying the genetic architecture and basis is one of the most important aims toward developing effective treatments for psychiatric disorders. The overall objective of this dissertation is to contribute to understanding the genetics of psychiatric disorders. Analyzing summary statistics from genomewide association studies (GWAS) of psychiatric disorders, we mainly present results of two projects. In the first one, we evaluated commonalities and distinctions in genetic risk of four highly comorbid childhood onset neuropsychiatric disorders: attention deficit/hyperactivity disorder (ADHD), autism spectrum disorder (ASD), obsessive-compulsive disorder (OCD) and Tourette’s syndrome (TS). Through systematic analysis of genetic architecture and correlation, we confirmed exitance of genetic components shared across ADHD, ASD and TS, as well as OCD and TS. Subsequently, we identified those components at variant, gene, and tissue specificity levels through meta-analyses. Our results pointed toward possible involvement of hypothalamus-pituitary-adrenal (HPA) axis, a human stress response system, in the etiology of these childhood onset disorders. The second project includes the proposition of a novel framework for general GWAS summary statistics-based analyses. Instead of regular odds ratio and standard errors archived in the summary statistics, we proposed a recounstruction approach to rewrite the results in terms of single nucleotide polymorphisms (SNP) allelic and genotypic frequencies. We also put forward three applications built-upon the proposed framework, and evaluated the performance on both synthetic data and real GWAS results of psychiatric disorders for each of them. Through these three applications, we demonstrated that this framework can broaden the scope of GWAS summary statistics-based analyses and unify various of analyses pipelines. We hope our work can serve as a stepping-stone for future researchers aiming at understanding and utilizing GWAS results of complex psychiatric disorders.</p></div></div></div>

Single nucleotide polymorphism (SNP) arrays are important tools widely used for genotyping and copy number estimation. This technology utilizes the specific affinity of fragmented DNA for binding to surface-attached oligonucleotide DNA probes. This thesis contemplates the variability of the probe signals of Affymetrix GeneChip SNP arrays as a function of the probe sequence to identify relevant sequence motifs which potentially cause systematic biases of genotyping and copy number estimates.

Eine Vielzahl von Krankheiten und Phänotypen im Menschen werden durch Mutationen in G-Protein-gekoppelten Rezeptoren (GPCR) verursacht. Die Klasse der Adhäsions-GPCR (aGPCR) ist bisher wenig erforscht, wird aber mit diversen Funktionen in der Immunität, Nervenentwicklung, Embryonalentwicklung und im Tumorwachstum in Zusammenhang gebracht. Ein Vertreter der Gruppe V der aGPCR ist der GPR133 (ADGRD1). Einige genomweite Assoziationsstudien konnten den gpr133-Lokus mit Veränderungen im Metabolismus, in der Körpergröße und der Herzfrequenz verknüpfen. Für diesen aGPCR wurde bereits die Signaltransduktion über Gαs und Gαi gezeigt, sodass eine funktionelle Charakterisierung des GPR133 und dessen Varianten über cAMP-, cAMP response element- (CRE) und CRE binding protein- (CREB) Assays in vitro möglich ist. Systematische Untersuchungen der Struktur des GPR133 konnten die gebundene agonistische „Stachelsequenz“ aufzeigen. Dies legt den Grundstein für die funktionelle Untersuchung von Mutationen im GPR133. Eine Analyse von mehr als 1000 sequenzierten humanen Genomen ergab über 9000 Einzelnukleotidpolymorphismen (SNP) im gpr133-Gen. Ungefähr 2,4 % der SNP liegen in kodierenden Genabschnitten und resultieren in 129 nicht-synonymen SNP (nsSNP) an 119 Aminosäurepositionen. Die funktionelle Relevanz dieser Missense-Varianten war unbekannt. Tiefergehende Analysen konnten nsSNP identifizieren, die zu einem vollständigen bzw. partiellen Funktionsverlust (A448D, Q600stop, C632fs [frame shift], A761E, N795K) oder zu einer erhöhten Basalaktivität (F383S, D453N) führen. Ein Vergleich der aGPCR Subklassen basierend auf diversen Orthologsequenzen konnte zudem stark konservierte Bereiche aufzeigen, deren Änderungen durch nsSNP im GPR133 in Funktionsänderungen münden. Das große im Menschen vorhandene funktionelle Spektrum von GPR133-Varianten könnte für klinisch relevante Phänotypen verantwortlich sein, auch wenn die bisher erfassten heterozygoten Individuen lebensfähig sind.

Ziel dieser Arbeit war es Kandidatengene, die mit der Entwicklung von Adipositas assoziiert sind, auf eine veränderte Kopienzahl zu untersuchen. Zwar fanden wir in den von uns ausgewählten Genen keine Veränderungen der Kopienzahl, es gelang uns jedoch der Nachweis eines Nukleotid-Polymorphismus in SIM1 (rs3734354). Für diesen konnten wir erstmals eine Assoziation mit Parametern der Adipositas in einer Kinderkohorte zeigen. Weiterführend untersuchten wir die Effekte von Typ-2-Diabetes-assoziierten SNPs auf Glukosehomöostase und Insulinmetabolismus. Wir fanden eine Assoziation von rs2877716 (ADCY5) mit einer verminderten Insulinsekretion und von rs17271305 (VPS13C) mit einem erhöhten Nüchternglukosespiegel. Zusammenfassend unterstreichen die Effekte von SNPs wie VPS13C und ADCY5 die Komplexität der bisher unvollständig verstandenen Interaktionen von Alter, Genotyp und Phänotyp auf die Entwicklung des Typ-2-Diabetes. Für die Entwicklung der Adipositas scheinen Veränderungen der Kopienzahl in den untersuchten Genen bei übergewichtigen Kindern eine untergeordnete Rolle zu spielen. Weiterführende Arbeiten sind zur Erklärung der Heritabilität beider Krankheitsbilder und der komplexen Interaktionen von Phänotyp und Genotyp notwendig.:1 Bibliographische Beschreibung 2 Einführung 2.1 Bedeutung und Interaktion von Diabetes und Adipositas 2.2 Überblick Genetik Adipositas und Typ-2-Diabetes: Epidemiologie, Monogen vs. Polygen und Methodik 2.3 Der Leptin-Melanocortin Kreislauf 2.4 Überblick über Gene des Leptin-Melanocortin Kreislaufs (Leptin, POMC, MCR, SIM1) 2.5 Von Adipositas zu Diabetes 2.6 Charakteristik der Typ-2-Diabetes Kandidatengene (GIPR, ADCY5, GCKR, VPS13C) 2.7 Ziele der Arbeit 3 Publikation 1 'Copy number variations in “classical” obesity candidate genes are not frequently associated with severe early-onset obesity in children' 4 Publikation 2 'Effects of Genetic Variants in ADCY5, GIPR, GCKR and VPS13C on Early Impairment of Glucose and Insulin Metabolism in Children' 5 Zusammenfassung der Arbeit 6 Literaturverzeichnis 7 Anlagen 7.1 Supplementary Methods, Publikation 1 7.2 Supplementary Methods, Publikation 2 7.3 Erklärung über die eigenständige Abfassung der Arbeit 7.4 Erklärung über den wissenschaftlichen Beitrag des Promovenden an der ausgewählten Publikation 7.5 Lebenslauf 7.6 Publikationsverzeichnis 7.7 Danksagung

Introduction: Diabetes mellitus is a polygene disease and on its manifestation have influence also enviromental factors. We have studied the role of extrapituitary prolactin (PRL) and toll-like receptors (TLR) 2 and 4 in the etiopathogenesis of autoimmune diabetes. PRL is mainly produced by hypophysis, but in small concentrations also in the periphery, where it participates in the immune reactions. Therefore, we investigated the influence of the levels of monocytic PRL mRNA on the development of diabetes, and also the influence of G allele of the -1149 G/T polymorphism in the extrapituitary promotor, which has already been associated with other autoimmune diseases. TLRs are receptors of the immune cells that recognize patogenes entering into the body. They play an important role in the iniciation of the immune response. We aimed to find out their function in the pathogenesis of the autoimmune diabetes by the detection of their mRNA levels and protein levels expressed on the cell surface of the monocytes. Material and methods: In this study we included 30 T1D and 21 LADA patients. Three control groups consisted of 23 T2D patients, 23 patients with a nondiabetic disease (neDM) and 60 healthy blood donors (TO). Blood samples have been taken from the individuals. From these blood samples we isolated...

Background: Hypertension affects 33% of Americans while type 2 diabetes and Alzheimer's disease (AD) affect 10% of Americans, respectively. Ryanodine receptor 3 gene (RYR3) codes for the RYR which functions to release stored endoplasmic reticulum calcium ions (Ca2+) to increase intracellular Ca2+ concentration. Increasing studies demonstrate that altered levels of intracellular Ca2+ affect cardiac contraction, insulin secretion, and neurodegeneration. In this study, we investigated associations of the RYR3 genetic variants with hypertension, AD, and diabetes. Methods: Family data sets were used to explore association of RYR3 polymorphisms with risk and age at onset (AAO) of hypertension, diabetes, and AD. Results: Family-based association tests using generalized estimating equations (FBAT-GEE) showed several unique or shared disease-1 associated variants in the RYR3 gene. Three single nuclear polymorphisms (SNPs; rs2033610, rs2596164, and rs2278317) are significantly associated with risk for hypertension, diabetes, and AD. Two SNPs (rs4780174 and rs7498093) are significantly associated with AAO of the 3 diseases. Conclusions: RYR3 variants are associated with hypertension, diabetes, and AD. Replication of these results of this gene in these 3 complex traits may help to better understand the genetic basis of calcium-signaling gene, RYR3 in association with risk and AAO of these diseases.