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Worldwide, alcohol is the third leading risk factor for disease burden, while its harmful use leads to 2.5 million deaths every year. Alcohol dependence (AD) is a complex disease, with devastating effects on individuals, families, and society. It is estimated that 76.3 million people worldwide have suffered from alcohol use disorders (AUD), including alcohol abuse and AD. Alcohol withdrawal or alcohol withdrawal symptom (AWS) refers to a cluster of symptoms that may occur when a heavy drinker suddenly stops or significantly reduces their alcohol intake. These symptoms can start as early as 2 h after the last drink, persist for weeks, and range from mild anxiety and shakiness to severe complications, such as seizures and delirium tremens. Family, twin, and adoption studies have indicated that genetic and environmental factors and their interactions contribute to the development of AD and related phenotypes, with a heritability coefficient of more than 0.5 for AD. Whole-genome linkage and candidate gene association studies have successfully identified several chromosome regions and genes that are related to AD and AWS. Furthermore, gene expression analysis, epigenetic studies, and genome-wide association studies (GWAS) have provided regions and loci for AWS. This chapter reviews the recent findings in genetic studies of AWS.

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In den letzten Jahren gab es rasche Weiterentwicklungen auf dem Gebiet der Nukleinsäure-Erkennung. Von microRNA-Quantifizierung zur Untersuchung von Zelltods, --Teilung und -Regulation bis zur Bewertung genetischer Variabilität in Hinblick auf Krankheitsentstehung und -Behandlung: Die Analyse von Nukleinsäuren wird in der zukünftigen Medizin eine zentrale Rolle zukommen. Vor allem die Erkennung von SNPs als Hauptquelle der genetischen Vielfalt, aber aus Analysesicht auch eine der herausforderndsten Mutationen, stellt in dieser Hinsicht einen wesentlichen Aspekt dar. Methoden zur SNP-Erkennung müssen nicht nur sensibel, selektiv und stabil, sondern auch vielfältig sein und eine der wachsenden Analyseanzahl gerecht werdende hohe Verarbeitungsmenge bieten. Im Rahmendieser Arbeit wurde ein chemisches Prüfverfahren zur Erkennung von Nukleinsäuren und Einzelnukleotid-Polymorphismen (SNPs) entwickelt. Das Reaktionssystem zur Nukleinsäuren- Erkennung beruht hierbeiauf der Interaktion zweier modifizierter Peptid-Nukleinsäure (PNS) Oligonukleotiden. Das Erste beinhaltet einen C-terminalen Thioester (Donor-Sonde), die zweite einen N-terminalen Cysteinyl-Rest (Akzeptor-Sonde). Zusätzlich ist die Donor-Sonde durch einenmakrocyclischen Metall Chelatkomplex aus 1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraessigsäure(DOTA) mit einem gebundenen lanthanoid-tag funktionalisiert. In die Akzeptor-Sonde wurde, zurReinigung mit magnetischen Streptavidin Partikeln, Biotin integriert. Der Ziel-DNA-Strang bringt beideSonden in räumliche Nähe zueinander und ermöglicht so eine chemische Reaktion. Das so gewonneneLigationsprodukt beinhaltet den Lanthanoid-Tag und Biotin, über welches das Produkt gereinigt wird,bevor die Detektion mittels ICP-MS erfolgt. Die Lanthanoid Konzentration dient als Indikator desLigationsprodukts welches wiederum den Reporter des Ziel-DNS-Strangs darstellt. Die, mithilfe diesesSystems erreichte, methodische Nachweisgrenze lag bei 29 pM mit einem RSD von 6,8% bei 50 pM(n=5). Zur Erkennung von SNPs wurde das Experiment mit einer Kombination zweier-Sets PNS Sonden mit unterschiedlichen Lanthanoid Tags durchgeführt. Das erste Set zielte auf die SNP beinhaltende Sequenz (Reportersystem) ab, während das zweite an eine benachbarte Sequenz (Kontrollsystem) binden sollte. Zur Erkennung der SNP wurden die Signale bei der Lanthanoide wurden ins Verhältnis gesetzt. Mithilfe dieses Verfahrens konnte durch Messung von sechs Lanthaniden bei einer Konzentration von 5 nM erfolgreich simultan zwischen den Allelen dreier SNPs unterschieden werden. / The field of nucleic acid detection has evolved swiftly in recent years. From quantification of micro RNA for the study of cell death, proliferation, and regulation, to the assessment of the influence of genetic variability towards disease development and treatment, the analysis of nucleic acids will play a central role in future medicine. In that regard, the detection of SNPs, as the primary source of genetic variability and the most challenging mutation from the analytical point of view, will be at the forefront of the discussion. Methods for the detection of SNPs not only require sensitivity, selectivity and robustness, but they should also allow multiplexing and offer high throughput in order to face the growing analysis demand In this work an assay for the detection of nucleic acids and single nucleotide polymorphisms (SNPs) was developed. The reaction system for the detection of nucleic acids is based on the interaction between two modified peptide nucleic acid (PNA) oligonucleotides. The first incorporated a C-terminal thioester (donor probe), and the second one a N-terminal cysteinyl residue (acceptor probe). In addition, the donor probe is functionalized with a metal-tag, which consist of a macrocyclic metal chelate complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) with a chelated lanthanoide. A biotin tag for purification by streptavidin magnetic particles was incorporated in the acceptor probe. The target DNA strand brings together the reporter probes allowing the chemical reaction. The resulting ligation product contains the metal-tag and the biotin, which is used to purify the product before measurement in the ICP-MS system. The lanthanoid concentration is used as an indicator of the ligation product, which at the same time serves as reporter of the target template. The methodological limit of detection achieved with this system was 29 pM with RSD of 6.8% at 50 pM (n=5). Detection of SNPs was performed using a combination of two sets of PNA probes labeled with different lanthanoid metal tags. The first probe set targeted the sequence where the SNP was present (reporter probe system), while the second set of probes was designed to bind to a neighboring sequence (control probe system). The signals of both lanthanides were used to establish a ratio that allowed the detection of the SNP. This assay was successfully used to simultaneously differentiate between alleles of 3 SNPs by measuring six lanthanoids at 5 nM concentration.

Bermudagrass (Cynodon spp.) breeding and cultivar development is hampered by limited information regarding its genetic and phenotypic diversity. A germplasm collection of 206 bermudagrass accessions from 29 countries was genotyped with high-throughput genotyping-by-sequencing technique. Genomic diversity in this diverse germplasm panel was assessed with multifaceted approaches including population structure, phylogenetic analysis, principal component analysis, and genetic diversity parameters. This study revealed substantial genetic variation in the Cynodon accessions, demonstrating the potential of this germplasm panel for further genetic studies and cultivar development in breeding programs. Another critical issue in turfgrass breeding is the lack of information regarding the genetic architecture of traits. Four agronomic traits leaf length, leaf width, internode distance and stem diameter were evaluated in a germplasm panel of common bermudagrass accessions. Then genome-wide association study was performed to dissect the genetic basis of the traits.

The innate immune system in vertebrates is considered to lack specific memory. To investigate innate immune system based immunological protection mediated by cells that are not part of the acquired immune system the Tübingen recombination activation gene1 (rag1)t26683 mutant (MT) zebrafish was chosen. Molecular analysis demonstrated MT zebrafish kidney cells expressed Non-specific Cytotoxic cell receptor protein-1 (NCCRP-1) and Natural Killer cell (NK) lysin but lacked T cell receptor (TCR) and immunoglobulin (Ig) VH1, VH2, VH3 and VH4 expression. Differential counts of peripheral blood leukocytes indicated that MT fish had decreased lymphocyte populations (34.7%) compared to rag1+/+ wild-type (WT) fish (70.5%), and increased granulocyte populations (34.7%) compared to WT (17.6%). Further, endocytic functions of phagocytes from MT fish were compared to WT fish. No significant differences in the selective and non-selective mechanisms of uptake in phagocytes were observed between MT and WT zebrafish. For the first time it was shown that zebrafish phagocytes utilize macropinocytosis and Ca2+ dependant endocytosis mechanisms for antigen uptake. These characterization studies suggest that MT zebrafish provide a unique model for investigating innate immune responses because fully functional innate defenses are present without the influence of lymphocytes and lymphocyte associated acquired immune responses. To conduct such large scale investigations the first ongoing rag1t26683 mutant zebrafish breeding colony was established. To meet special husbandry needs of immunodeficient MT zebrafish, standard rearing protocols were advanced and the information was made available to the zebrafish community at: http://www.cvm.msstate.edu/zebrafish/index.html. Multiple trials were conducted to evaluate the potential for memory of the innate immune system. Significant reduction in mortality was observed in MT vaccinated zebrafish upon secondary exposure to Edwardsiella ictaluri when compared to unvaccinated, MT fish. This documents for the first time, that MT zebrafish, lacking an acquired immune system, are able to mount a protective immune response to Edwardsiella ictaluri and generate protection upon a repeated encounter to the same pathogen. The observed protection is long lasting and mediated by the innate immune system, but a specific mechanism is not yet defined.

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Chickpea (Cicer arietinum L.) was recently introduced to the Canadian prairies, a region which has a short growing season in which crop maturation often occurs under cool and wet conditions. To improve the yield of chickpea, crop duration must closely match the available growing season. The objectives of this study were to: i) examine the days to flowering of diverse chickpea accessions grown in either long or short-days; ii) examine the days to flowering of selected chickpea accessions grown in a range of thermal regimes combined with either long or short days and to examine the interaction between photoperiod and day and night temperatures on crop duration; iii) determine the timing and duration of the photoperiod-sensitive phase in selected chickpea accessions, and vi) determine the genetic basis of the association between flowering time and reaction to ascochyta blight in chickpea. A wide variation was observed in chickpea accessions for their response to flowering under long (16/8 hours day /night) and short days (10/14 hours day/night). Earlier flowering was observed under long photoperiod regimes compared with the short photoperiod regimes. Variability was detected among chickpea accessions for their flowering responses when different temperatures were combined with different photoperiods. Earlier flowering was observed under long days (16/8 hours day/night) coupled with high to moderate temperature regimes (24/16 ºC and 20/12 ºC, day and night respectively) compared to short-days (10/14 hours day and night) and moderate to low temperature regimes (20/12 ºC and 16/8 ºC day and night, respectively). Those chickpea accessions such as ICC 6821 and ICCV 96029 which originated from the lower latitudes of Ethiopia and India, respectively, flowered earlier compared to accessions such as CDC Corinne and CDC Frontier which originated from the higher latitudes and cooler temperate environments of western Canada. Photoperiod sensitivity phases were detected in chickpea accessions adapted to the cold environments of western Canada, whereas no photoperiod sensitivity phase was identified in the extra-early flowering cultivar ICCV 96029. The duration of the photoperiod sensitive phase in the chickpea accessions was longer under short days compared to long days. Field and growth chamber evaluation of a chickpea RIL population (CP-RIL-1) revealed the presence of variability among the lines and the two parents for their days to flowering and level of resistance to ascochyta blight. Broad sense heritability across different site-years for days to flower 0.45 to 0.78, plant height 0.48 to 0.78, ascochyta blight resistance 0.14 to 0.68, days to maturity 0.26, photoperiod sensitivity 0.83 and nodes number of first flowering 0.37 to 0.75 were estimated. Days to flower and photoperiod sensitivity were significantly r = -0.21 to -0.58 (P ≤ 0.05 to 0.001) and -0.28 to -0.41 (P ≤ 0.01 to 0.001), respectively and negatively correlated with ascochyta blight resistance in the CP-RIL-1 population. A genetic linkage map consisting of eight linkage groups was developed using 349 SNP markers. Seven QTLs were identified for days to flowering under growth chamber and field conditions on chromosomes 3, 5, 6 and 8 each and 3 QTLs on chromosome 4. The total phenotypic variation explained by QTLs for days to flowering ranged from 7 to 44%. Two QTLs for days to maturity were identified on chromosomes 3 and 8. Three QTLs, one each on chromosomes 3, 4 and 5 were identified for photoperiod sensitivity. The total phenotypic variation explained by each QTL for photoperiod sensitivity ranged from 7 to 41%. A total of three QTL for node of first flowering, one on chromosomes 3 and 8 each, and two on chromosome 4 were identified. The two QTL on chromosome 4 explained total phenotypic variations of 11 and 32%, respectively. Ten QTLs distributed across all chromosomes, except chromosomes 2 and 5, were identified for ascochyta blight resistance. The phenotypic variability explained by each QTL for ascochyta blight resistance ranged from 7 to 17%. The molecular markers associated with these QTLs have potential for use in chickpea breeding.

Infertility affects 15% of reproductive-aged couples. The milieu surrounding the growing embryo is of outmost importance, and should be optimised during in vitro fertilisation (IVF). Many biological processes, such as angiogenesis, coagulation, and immune processes need to be well regulated for a pregnancy to occur and progress normally. Histidine-rich glycoprotein (HRG) is a plasma protein that regulates components of these systems by building complexes with various ligands. A single nucleotide polymorphism (SNP) in HRG, denoted HRG C633T, seem to be of importance for IVF treatment outcomes. The aim of this thesis was to further investigate the proposed human fertility effects of the HRG C633T SNP. According to the findings of this thesis, the HRG C633T genotype is associated with primary recurrent miscarriage. Male HRG C633T genotype is associated with semen characteristics in infertile men, and pregnancy rates following IVF. However, the distribution of the HRG C633T SNP does not differ between infertile and fertile couples. We further examined the role of the region surrounding the HRG C633T SNP for regulation of endometrial angiogenesis and human embryo development. The region affects primary endometrial endothelial cell migration, proliferation and tube-formation in vitro but does not appear to affect human embryo development. No effect of the HRG peptide was noted on the secretome of human embryos. However, early embryos secrete proteins into the surrounding culture media and the level of secretion of VEGF-A, IL-6, EMMPRIN and PlGF is greater in embryos of higher developmental stages. In conclusion, the HRG C633T genotype appears to play a role only if infertility is established. The region surrounding HRG C633T SNP is of relevance in vitro for regulation of human endometrial endothelial cell angiogenesis. To predict which embryos to transfer in IVF, we have highlighted a number of proteins of interest for further investigation.