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The aim of this diploma thesis was association analysis of single nucleotide polymorphism of IGFBP-2 and MC4R genes in a selected group of Czech Large White pigs with indicators of pork quality. The surveyed traits were pH value, color of meat, electrical conductivity, drip loss, contents of intramuscular fat and fatty acids. The polymorphisms were detected by the PCR and RFLP procedures. The allele and genotype frequencies were calculated and results were statistically evaluated using SAS program. In the case of the IGFBP-2 gene was discovered, that the observed set of animals is marked out by the higher percentage of allele B (99,5 %), the relative frequency of the BB genotype was 99 %, AB 1 % and genotype AA has not been found. For this reason, an association analysis could not be performed. The MC4R gene showed a higher frequency of the G allele (74.52%), with the relative frequency of AA genotypes being 2.88%, AG 45.19% and GG 51.92%. On the basis of the association analysis, statistically significant differences were found in the content of the linoleic acid at significance level of P ≤ 0,05 between genotypes AG×GG and to the drip loss where was the difference between the genotypes AA×GG. No statistically significant differences were found for other meat quality indicators, not even value close to significant difference (P ≤ 0,1).

The study presents evaluation of pig genetic markers (MHS and MC4R) associated with performance traits in the Lithuanian White pig breed. The study presents a direct approach the testing and explanation of the quantitative part of the trait and QTL with marker based on estimated breeding values in the Lithuanian White population. Information about the strategies for association analysis and improvement can be applied for further characterization of the Lithuanian White population. The use of breeding values instead of single measurements reduces the bias in the recorded performance traits, therefore, the results obtained by using the marker for the Lithuanian White population gives animal breeders the opportunities for realization of a short-term goal in their selection criteria.

Mutations in the melanocortin 4 receptor (MC4R) are the most frequent cause of monogenetic forms of human obesity. Despite its importance, the MC4R signaling pathways and transcriptional regulation that underly the melanocortin pathway are far from being fully understood. The transcription factor Nescient Helix Loop Helix 2 (Nhlh2), is known to influence the melanocortin pathway. It regulates the transcription of genes by binding to the E-Box binding sites present in the promoter region. Here in this dissertation, Nhlh2's role as a transcriptional regulator of Mc4r and the effects of deletion of Nhlh2 on peripheral energy expenditure, glucose homeostasis and fatty acid oxidation are reported. To investigate the transcriptional mechanisms of Mc4r and the involvement of Nhlh2, gene expression analysis, DNA-protein binding, transactivation assays, and SiRNA induction were used. We show that Nhlh2 regulates the transcription of Mc4r by binding to the three E-Boxes present on the promoter at -553, -361 and +47. Further, SiRNA knockdown of Nhlh2 in the N29/2 cell line depresses Mc4r expression which suggests the requirement of Nhlh2 for Mc4r transcription. Development of adult onset obesity in the absence of evident hyperphagia questions the ability of mice which lacks Nhlh2 (N2KO) to utilize energy substrate efficiently. To test the effect of deletion of Nhlh2 in N2KO, body composition analysis, tissue specific characterization, fatty acid oxidation and glucose and insulin homeostasis were assessed. N2KO mice have a higher fat content than WT at the age of 12 weeks. There are architectural differences in adipose tissue of N2KO. White adipose tissue (WAT) shows infiltration of macrophages, and increased mRNA and serum levels of interleukin 6 which suggests the presence of a systemic inflammatory state in the N2KO mice. Sympathetic nervous system tone is reduced in both brown adipose tissue (BAT) and WAT, as evidenced by gene expression analysis, and this may be because of overall reduced melanocortinergic tone in N2KO mice. N2KO mice have an impaired glucose tolerance on the basis of their late glucose clearance on glucose (non-significant) and insulin (significant) challenges. Fatty acid oxidation (FAO) is higher in red fibers of skeletal muscle, and the respiratory exchange ratio (RER) is lower in N2KO, which is indicative of using fat as a preferential energy source. Increased expression of genes involved in the lipid metabolism in skeletal muscle and liver supports the RER and FAO, and are indicative of high turnover of lipids in N2KO. Findings from these studies implicate Nhlh2 as a transcriptional regulator of Mc4r which has a direct relevance to the ever increasing epidemic of obesity. Characterization of N2KO mice sheds light on the adult onset obesity phenotype. Knowledge gained from these findings will help us understand the monogenetic form of obesity more completely and could lead to the design of improved pharmacological therapies that target Nhlh2 or Mc4r or modify physical activity behavior. / Ph. D.

When applied to drug discovery, modern computational systems can provide insight into the highly complex systems underlying drug activity and predict compounds or targets of interest. Many tools have been developed for computer aided drug discovery (CADD), focusing on small molecule ligands, protein targets, or both. The aim of this thesis is the improvement of CADD tools for describing small molecule properties and application of CADD to several stages of drug discovery regarding two targets for the treatment of obesity and related diseases: the neuropeptide Y4 receptor (Y4R) and the melanocortin-4 receptor (MC4R). In the first chapter, the major categories of CADD are outlined, including descriptions for many of the popular tools and examples where these tools have directly contributed to the discovery of new drugs. Following the introduction, several improvements for encoding stereochemistry and signed property distribution are introduced and tested in scenarios meant to simulate applications in virtual high-throughput screening. Y4R and MC4R are both class A G-protein coupled receptors (GPCRs) with endogenous peptide ligands that play critical roles in the signaling of satiety and energy metabolism. So far, no structures from either receptor family have been experimentally elucidated. CADD was combined with high-throughput screening (HTS) to discover the first small molecule positive allosteric modulators (PAMs) of Y4R. Secondly, CADD techniques were used to model the interaction of Y4R and pancreatic polypeptide based on experimental results that elucidate specific binding contacts. Similar SB-CADD approaches were used to model the interaction of MC4R with its high affinity peptide agonist α-MSH. Due to its role in monogenic forms of obesity, these models were used to predict which residues directly participate in binding and correlate mutated residues with their potential role in the binding site.

When applied to drug discovery, modern computational systems can provide insight into the highly complex systems underlying drug activity and predict compounds or targets of interest. Many tools have been developed for computer aided drug discovery (CADD), focusing on small molecule ligands, protein targets, or both. The aim of this thesis is the improvement of CADD tools for describing small molecule properties and application of CADD to several stages of drug discovery regarding two targets for the treatment of obesity and related diseases: the neuropeptide Y4 receptor (Y4R) and the melanocortin-4 receptor (MC4R). In the first chapter, the major categories of CADD are outlined, including descriptions for many of the popular tools and examples where these tools have directly contributed to the discovery of new drugs. Following the introduction, several improvements for encoding stereochemistry and signed property distribution are introduced and tested in scenarios meant to simulate applications in virtual high-throughput screening. Y4R and MC4R are both class A G-protein coupled receptors (GPCRs) with endogenous peptide ligands that play critical roles in the signaling of satiety and energy metabolism. So far, no structures from either receptor family have been experimentally elucidated. CADD was combined with high-throughput screening (HTS) to discover the first small molecule positive allosteric modulators (PAMs) of Y4R. Secondly, CADD techniques were used to model the interaction of Y4R and pancreatic polypeptide based on experimental results that elucidate specific binding contacts. Similar SB-CADD approaches were used to model the interaction of MC4R with its high affinity peptide agonist α-MSH. Due to its role in monogenic forms of obesity, these models were used to predict which residues directly participate in binding and correlate mutated residues with their potential role in the binding site.

Obesity is a frequent metabolic disease that causes many other health and socioeconomic complications. Obesity arises due to excessive energy intake and decrease in energy expenditure, which is a conseqence of contemporary lifestyle. Moreover, obesity has a strong genetic component. Common obesity is polygenic, multifactorial disease, in which individual genes interact with each other and with environmental factors. Genome-wide association studies, conducted between 2006-09, led to the discovery of dozens of gene loci that predispose individuals to obesity. The strongest signals were registered for polymorphisms in FTO (fat mass and obesity-associated) and near a gene MC4R (melanocortin 4 receptor). However, the contributions of these variations on the phenotype of obesity are very small, therefore, it is necessary to validate the results of such robust studies. It is very important to uncover the effects of genetic variants for understanding the molecular mechanisms of energy metabolism. The studies presented in this thesis refer about the impact of polymorphisms in selected genes on anthropometric and metabolic parameters of the patients of the Institute of Endocrinology and of healthy volunteers who underwent functional tests. Our cohort includes a representative sample of Czech children (COPAT...

Die Regulation der Nahrungsaufnahme erfolgt zentral im Hypothalamus wo eine Vielzahl von G-Protein-gekoppelten Rezeptoren exprimiert werden die an der Gewichtsregulation beteiligt sind. Periphere hormonelle Signale aktivieren ihre korrespondierenden Rezeptoren im Nucleus arcuatus (ARC) oder im Nucleus paraventricularis (PVN) und modifizieren dadurch sowohl das anorexigene System, z.B. über die Stimulation des Melanocortin-4-Rezeptors (MC4R) im PVN, als auch das orexigene System mit dem Neuropeptide Y (NPY) sowie dem Agouti-related Protein (AgRP). Im Zuge einer systematischen Interaktionsstudie wurden verschiedene GPCRs, die entweder mit dem MC3R oder dem MC4R auf dem gleichen Neuron koexprimiert werden und nachweißlich die Appetit- und Gewichtregulation beeinflussen, untersucht. Basierend auf den Ergebnissen von Sandwich-ELISA und FRET- (Fluoreszenz-Energie-Transfer)Studien konnte eine Interaktion des MC3R mit dem Growth hormone secretagogues Rezeptor (GHSR) bestimmt werden, die beide auf den NPY/AgRP-Neuronen des ARC lokalisiert sind. Der MC3R gehört zu den Gαs bindenden Rezeptoren wohingegen GHSR über den Gαq vermittelten Signaltransduktionsweg signalisiert. Es konnte eine Erhöhung der induzierten cAMP-Spiegel infolge der Stimulation des MC3R sowohl mit α-, als auch β- und γ-MSH für die Koexpression von MC3R mit GHSR im Vergleich zum MC3R Homodimer ermittelt werden. Die Charakterisierung des neuen Signalisierungsverhaltens des Heterodimers unter der Verwendung verschiedener Inhibitoren zeigte eine Aktivierung von Gαi in Gegenwart der endogenen Agonisten beider Rezeptoren. Die Beobachtung unterschiedlicher Regulationsmuster nach der Kostimulation des Heterodimers in Abhängigkeit von α- oder γ-MSH jeweils in Anwesenheit von Ghrelin verweist auf komplexe Interaktionsmechanismen zwischen dem Melanocortin- und dem Ghrelin-Rezeptor innerhalb der hypothalamischen Gewichtsregulation. / Food intake is centrally regulated in hypothalamic nuclei where many GPCRs are expressed which are known to be involved in weight regulation.Peripheral hormonal signals activate their corresponding receptors in the arcuate nucleus (ARC) or paraventricular nucleus (PVN) and modulate the orexigenic (appetite-supressing) pathway mediated by stimulation of the melanocortin-4-receptor (MC4R) as well as the anorexigenic (appetite-stimulating) pathway including neuropeptide Y (NPY) and agouti-related protein (AgRP). In a systematic approach we investigated the interaction of a selective number of GPCRs which are co-expressed on the same neurons like MC3R or MC4R and know to play an essential role in hypothalamic weight regulation. Based on the results of a sandwich ELISA and fluorescence resonance energy transfer (FRET) approach we report the interaction of the MC3R and the growth hormone secretagogue receptor (GHSR) which are co-expressed on arcuate NPY/AgRP neurons. It is known that MC3R couple to the Gαs whereas GHSR couple to the Gαq signaling pathway. However, here the co-expression of MC3R and GHSR reveal a profoundly increase cAMP-accumulation after melanocortin (α-, β- and γ-MSH) challenge, that is higher compared to MC3R activation alone. In-depth characterization of the new signaling properties of the MC3R/GHSR heterodimer by different inhibitors revealed the activation of Gαi in the presents of both endogene agonists. The observation of different regulatory pattern after co-stimulation of the heterodimer depending on the endogenouse ligands (α- or γ-MSH) of MC3R reflect complex functional interaction mechanisms between melanocortin and ghrelin receptors within the hypothalamic signaling pathways of weight regulation.

Thesis (MScMedSc (Biomedical Sciences))--University of Stellenbosch, 2010. / Thesis presented in partial fulfillment of the requirements for the degree Master of Science in Medical Biochemistry at the University of Stellenbosch. / Bibliography / ENGLISH ABSTRACT: Tuberculosis (TB) is an infectious disease which has plagued society for thousands of years. Despite public health programs, anti-TB drugs and a vaccine, the absolute numbers of people infected with TB each year continue to rise as populations grow. The high TB-burden areas are also plagued by other debilitating factors including HIV/AIDS infection, poverty and malnutrition. Nutrition has been implicated in TB susceptibility in a number of studies. While most are observational reports made during times of war, famine or natural disaster, multiple studies provide convincing evidence for poor nutritional status increasing the morbidity and mortality of TB. Numerous approaches are currently utilized in TB research, and there has been convincing evidence to support the role of host genetics in TB susceptibility. Based on previous linkage studies and a search of current literature, three genes were selected for this case-control study. Subsequently, variations located in cathepsin Z (CTSZ), melanocortin 3 receptor (MC3R) and melanocortin 4 receptor (MC4R) were genotyped in the South African Coloured (SAC) population to determine the existence of an association with TB disease. CTSZ is a lysosomal cysteine protease expressed in cells of the immune system. Interaction between this 303 amino acid protein and β2 integrin receptors lymphocyte function-associated antigen-1 (LFA-1) and macrophage antigen-1 (MAC-1) leads to altered lymphocyte proliferation. As a result, a single exonic variant in CTSZ, rs34069356, the same identified in a previous linkage study, showed strong evidence for association with TB susceptibility in cases (n = 410) and controls (n = 301) in the SAC population (p< 0.0001). MC3R and MC4R are two of 5 melanocortin receptors. MC3R has been found to be a key regulator in energy expenditure and host metabolism while activation of MC4R leads to a decrease in food intake. Activation of these two receptors is regulated by leptin, a hormone released by adipose tissue. A variant located upstream of the MC3R gene, rs6127698, also showed evidence of disease association with the less frequent allele, T, being under-represented in cases (n = 540) compared to controls (n = 541) (genotypic frequency, p = 0.0039), suggesting a possible resistance phenotype. Functional analysis of this variant revealed an increase in MC3R expression when stimulated with BCG, with individuals homozygous for the T allele exhibiting an even larger upregulation of MC3R expression than individuals homozygous for the G allele, though this difference was not statistically significant. A single haplotype in MC3R was found to be associated with TB susceptibility (p = 0.0008) and this association remained after permutation testing to correct for multiple testing (p = 0.0061) Three variants were selected for genotyping in MC4R and while none of these showed a statistically significant difference between cases (n = 510) and controls (n = 487), this gene should not be ruled out as both MC3R and MC4R have been found to work closely though not redundantly and double knockout experiments result in exacerbated obesity, suggesting that these proteins have a synergistic effect. The results of this study support both a role of host genetics and nutritional status in TB and strongly motivate further research in both of these fields. / AFRIKAANSE OPSOMMING: Tuberkulose (TB) is ‘n aansteeklike siekte wat reeds vir eeue die gesondheid van die publiek bedreig. Ten spyte van publieke gesondheidsprogramme en verskeie anti-TB medikasie middele, groei die aantal van mense wat hiermee ge-infekteer word steeds jaarliks. Dit is veral in areas waar TB steeds groei, waar ook ander neerdrukkende faktore soos HIV/Vigs, armoede en wanvoeding hoogty vier. Na aanleiding van verskeie verslae tydens oorloë, hongersnood en ander natuulike rampe is dit veral duidelik dat swak nutriënt inname morbiditeit en sterftes wat met TB gepaard gaan verhoog. Talle benaderings word tans gebruik in TB-navorsing, Bewyse is oortuigend om die rol van genetika van die gaheer met vatbaarheid vir TB te verbind. Op grond van vorige studies en die huidige literatuur, het ons drie gene gekies vir hierdie pasiënt-kontrole studie. Variante geleë in cathepsin Z (CTSZ), melanocortin 3 receptor (MC3R) en melanocortin 4 receptor (MC4R) is ge-genotipeer in die Suid-Afrikaanse Kleurling bevolking (SAK) (540 gevalle en 540 kontrole) om sodoende die assosiasie met TB te bepaal. CTSZ is ‘n lisosomale sisteïen protease wat uitgedruk word in immuunselle. Interaksie tussen hierdie 303 aminosuur protein en β2 integrin reseptore nl. LFA-1 en MAK-1 bring veranderde limfosiet proliferasie mee. ‘n Enkele eksoniese variant in CTSZ, rs34069356, dieselfde soos ge-identifiseer in ‘n vorige studie, verskaf sterk bewys vir assosiasie met TB vatbaarheid in gevalle (n = 410) en kontrole (n = 301) in die SAK bevolking. MC3R en MC4R is twee van 5 melanokortien reseptore. Daar is gevind dat MC3R 'n sleutelrol speel in die energie regulering van gasheer metabolisme, terwyl die aktivering van MC4R eindelik lei tot 'n afname in voedsel inname. Aktivering van hierdie twee reseptore word gereguleer deur Leptien, 'n hormoon wat vrygestel word deur adipose weefsel, ‘n Variant, stroomop geleë vanaf MC3R, rs6127698, is ook bewys om met TB ge-assosieer te wees, met die T-alleel meer seldsaam in gevalle (n = 540) as in kontroles (n = 541) wat dui op 'n moontlike weerstandsfenotipe. Funksionele analise van hierdie variant onthul 'n toename in MC3R uitdrukking wanneer gestimuleer met BCG, met individue homosigoties vir die T-alleel wat selfs groter opregulation veroorsaak wanneer vergelyk word met individue homosigoties vir die G allele. Hierdie resultaat was egter nie statisties beduidend nie. 'n Enkele haplotiepe in MC3R is ge-assosieer met TB vatbaarheid en die assosiasie is onveranderd nadat ‘n permutasie korreksie aangebring is (p = .0061). Voorts is drie variante gekies vir genotipering in MC4R en ten spyte daarvan dat nie een daarvan 'n statisties beduidende verskil getoon het tussen pasiënte (n = 510) en kontroles (n = 487) nie, behoort hierdie geen nie uitgesluit word nie, Die rede hiervoor is dat beide MC3R en MC4R verskeie kere gevind is om in samewerking ‘n rol te speel om vetsug te voorkom of te vererger. Die resultate van hierdie studie beaam beide 'n rol van gasheer genetika en voedingstatus in TB en motiveer veral verdere navorsing in beide van hierdie vakgebiede.

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