Aberrationen in der DNA-Kopienzahl sind häufige genetische Veränderungen bei malignen Lymphomerkrankungen. Zugewinne sowie Deletionen stellen dabei Mechanismen zur Onkogen-Aktivierung sowie Tumorsuppressorgen-Inaktivierung dar und tragen somit zur Pathogenese der Erkrankung bei. Array-CGH und SNP-Array sind Messplattformen, die die genomweite Bestimmung von Kopienzahlaberrationen in einem Experiment ermöglichen. Die bei der Analyse entstehenden Datensätze sind komplex und erfordern automatische Methoden zur Unterstützung der Analyse und Interpretation der Messergebnisse. In dieser Promotionsarbeit wurden Methoden entwickelt, welche die Analyse von Array-CGH- und SNP-Array-Messungen ermöglichen. Diese Methoden wurden für die Auswertung umfangreicher Datensätze von malignen Non-Hodgkin-Lymphomen verwendet. Dabei wurden Lymphome der Entitäten Burkitt-Lymphom, diffus großzelliges B-Zell-Lymphom, Mantelzelllymphom, primäres ZNS-Lymphom und peripheres T-Zell-Lymphom – nicht anderweitig spezifiziert – analysiert. Für die untersuchten Lymphom-Entitäten konnten hierbei zahlreiche neue rekurrente Kopienzahlaberrationen sowie uniparentale Disomien gezeigt werden, die neue Einblicke in die Pathogenese der jeweiligen Erkrankungen erlauben. Darüber hinaus erfolgte ein Vergleich beider Messplattformen anhand eines Datensatzes mit gepaarten Array-CGH- und SNP-Array-Daten. Für die eingesetzten Plattformen (2800k-BAC-Array vs. Affymetrix 250k-Sty-SNP-Array) konnte eine circa zwölffach höhere effektive Auflösung der SNP-Array-Plattform gezeigt werden. Die wesentlichen Ergebnisse dieser Arbeit sind in sieben Publikationen eingeflossen.:Inhaltsverzeichnis Abkürzungsverzeichnis Tabellenverzeichnis Abbildungsverzeichnis 1. Einführung 1.1 Biologischer Hintergrund 1.1.1 Aberrationen der DNA-Kopienzahl und Tumorentstehung 1.1.2 Lymphome 1.2 Motivation und Rationale für die Arbeit 1.3 Array-CGH Analyse 1.4 SNP-Array-Analyse 1.5 Vergleich von Array-CGH und SNP-Array-Analyse 1.6 Assoziationen von DNA-Kopienzahlaberrationen mit RNA-Expression, Lymphomentität sowie klinischen und phänotypischen Faktoren 2.Publikationen 2.1 Publikation 1: “Development and implementation of an analysis tool for array-based comparative genomic hybridization” Methods Inf Med. 2007;46(5):608-13 2.2 Publikation 2: “Recurrent loss of the Y chromosome and homozygous deletions within the pseudoautosomal region 1: association with male predominance in mantle cell lymphoma” Haematologica. 2008 Jun;93(6):949-50 2.3 Publikation 3: “GeneChip analyses point to novel pathogenetic mechanisms in mantle cell lymphoma” Br J Haematol. 2009 Feb;144(3):317-31 2.4 Publikation 4: “Chromosomal imbalances and partial uniparental disomies in primary central nervous system lymphoma.” Leukemia. 2009 Oct;23(10):1875-84 2.5 Publikation 5: “High resolution SNP array genomic profiling of peripheral T cell lymphomas, not otherwise specified, identifies a subgroup with chromosomal aberrations affecting the REL locus” Br J Haematol. 2010 Feb;148(3):402-12 2.6 Publikation 6: “Detection of genomic aberrations in molecularly defined Burkitt\''s lymphoma by array-based, high resolution, single nucleotide polymorphism analysis” Haematologica. 2010 Dec;95(12):2047-55 2.7 Publikation 7: “Patient age at diagnosis is associated with the molecular characteristics of diffuse large B-cell lymphoma” Blood. 2012 Feb 23;119(8):1882-7 2.8 Kennzeichnung des Eigenanteils für alle eingeschlossenen Publikationen 3. Diskussion und Ausblick 4. Zusammenfassung 5. Referenzen 6. Eigene Publikationen 7. Erklärung 8. Danksagung 9. Curriculum vitae

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Thorough investigation of the glycine conjugation pathway has been neglected over the last 30 years. Environmental factors, nutrition, and the chronic use of medications are increasing the exposure of humans to benzoate and drugs that are metabolized to acyl-CoA intermediates. Glycine conjugation of mitochondrial acyl-CoAs, catalysed by glycine N-acyltransferase (GLYAT, E.C. 2.3.1.13), is an important metabolic pathway responsible for maintaining adequate levels of free coenzyme A (CoASH). However, because of the small number of pharmaceutical drugs that are conjugated to glycine, the pathway has not yet been characterised in detail. Therefore, one of the objectives of this thesis was to develop a better understanding of glycine conjugation and its role in metabolism. In humans and animals a number of endogenous and xenobiotic organic acids are conjugated to glycine. Glycine conjugation has generally been assumed to be a detoxification mechanism, increasing the water solubility of organic acids in order to facilitate urinary excretion. However, recently it was proposed that the role of the amino acid conjugations, including glycine conjugation, is to regulate systemic levels of amino acids that are also utilised as neurotransmitters in the central nervous systems of animals. The glycine deportation hypothesis was based on the observation that, compared to glucuronidation, glycine conjugation does not significantly increase the water solubility of aromatic acids. A thorough review of the literature for this thesis showed that the major role of glycine conjugation, however, is to dispose of the end products of phenylpropionate metabolism. The review also introduced the new perspective that mitochondrial glycine conjugation prevents the accumulation of benzoate in the mitochondrial matrix by forming hippuric acid a less lipophilic conjugate that can be more readily transported out of the mitochondria. Although organic anion transporters can export benzoate from the matrix, this process would likely be futile because benzoic acid can simply diffuse back into the matrix. Hippurate, however, is significantly less lipophilic and therefore less capable of diffusing into the matrix. It is therefore not the transport out of the mitochondrial matrix that is facilitated by glycine conjugation, but rather the ability of the glycine conjugates to re-enter the matrix that is decreased. Lastly, glycine conjugation of benzoate also exacerbates the dietary deficiency of glycine in humans. Because the resulting shortage of glycine can negatively influence brain neurochemistry and the synthesis of collagen, nucleic acids, porphyrins, and other important metabolites, the risks of using benzoate as a preservative should not be underestimated. To date, no defect of the glycine conjugation pathway has been reported and this, together with the fact that GLYAT plays an important role in hepatic metabolism, suggests that this pathway is essential for survival. GLYAT activity affects mitochondrial ATP production, glycine availability, CoASH availability and the toxicity of various organic acids. Therefore, variation in the glycine conjugation pathway could influence liver cancer, musculoskeletal development and mitochondrial energy metabolism. Significant interindividual variation exists in glycine conjugation capacity. The molecular basis for this variability is not known. The main aim of this thesis was to investigate and characterise the genetic variation in the coding region of the GLYAT gene. This was accomplished by firstly, investigating the influence of non-synonymous single nucleotide polymorphisms (SNPs) on the enzyme activity of a recombinant human GLYAT and secondly, by analysing the level of genetic variation in the coding region of the GLYAT gene using existing worldwide population data. To investigate the influence of non-synonymous SNPs in the GLYAT gene on the enzyme activity, a recombinant human GLYAT was prepared, and characterised. Site-directed mutagenesis was used to generate six variants of the enzyme (K16N; S17T; R131H; N156S; F168L; R199C). The variants were expressed, purified, and enzymatically characterised. The enzyme activities of the K16N, S17T and R131H variants were similar to that of the wild-type, whereas the N156S variant was more active, the F168L variant less active, and the R199C variant was inactive. The results showed that SNP variations in the human GLYAT gene can influence the kinetic properties of the enzyme. The genetic variation data of the human GLYAT open reading frame (ORF) available on public databases was investigated by formulating the hypothesis that due to the essential nature of the glycine conjugation pathway, the genetic variation in the ORF of the GLYAT gene should be low and that deleterious alleles will be found at low frequencies. Data from the i) 1000 Genome Project, ii) the HapMap Project, and iii) the Khoi-San/Bantu Sequencing Project was downloaded from available databases. Sequence data of the coding region of a small cohort of South African Afrikaner Caucasian individuals was also generated and included in the analyses. In the GLYAT ORF of the 1537 individuals analysed, only two haplotypes (S156 and T17S156) out of 14 haplotypes were identified in all populations as having the highest haplotype frequencies (70% and 20% respectively). The S156C199 and S156H131 haplotypes, which have a deleterious effect on the enzyme activity of a recombinant human GLYAT, were detected at very low frequencies. The results of this study indicated that the GLYAT ORF is remarkably conserved, which supports the hypothesis that the glycine conjugation pathway is an essential detoxification pathway. The findings presented in this thesis highlight the importance that future investigations should determine the in vivo capacity of the glycine conjugation pathway for the detoxification of benzoate and other xenobiotics. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2015

Thorough investigation of the glycine conjugation pathway has been neglected over the last 30 years. Environmental factors, nutrition, and the chronic use of medications are increasing the exposure of humans to benzoate and drugs that are metabolized to acyl-CoA intermediates. Glycine conjugation of mitochondrial acyl-CoAs, catalysed by glycine N-acyltransferase (GLYAT, E.C. 2.3.1.13), is an important metabolic pathway responsible for maintaining adequate levels of free coenzyme A (CoASH). However, because of the small number of pharmaceutical drugs that are conjugated to glycine, the pathway has not yet been characterised in detail. Therefore, one of the objectives of this thesis was to develop a better understanding of glycine conjugation and its role in metabolism. In humans and animals a number of endogenous and xenobiotic organic acids are conjugated to glycine. Glycine conjugation has generally been assumed to be a detoxification mechanism, increasing the water solubility of organic acids in order to facilitate urinary excretion. However, recently it was proposed that the role of the amino acid conjugations, including glycine conjugation, is to regulate systemic levels of amino acids that are also utilised as neurotransmitters in the central nervous systems of animals. The glycine deportation hypothesis was based on the observation that, compared to glucuronidation, glycine conjugation does not significantly increase the water solubility of aromatic acids. A thorough review of the literature for this thesis showed that the major role of glycine conjugation, however, is to dispose of the end products of phenylpropionate metabolism. The review also introduced the new perspective that mitochondrial glycine conjugation prevents the accumulation of benzoate in the mitochondrial matrix by forming hippuric acid a less lipophilic conjugate that can be more readily transported out of the mitochondria. Although organic anion transporters can export benzoate from the matrix, this process would likely be futile because benzoic acid can simply diffuse back into the matrix. Hippurate, however, is significantly less lipophilic and therefore less capable of diffusing into the matrix. It is therefore not the transport out of the mitochondrial matrix that is facilitated by glycine conjugation, but rather the ability of the glycine conjugates to re-enter the matrix that is decreased. Lastly, glycine conjugation of benzoate also exacerbates the dietary deficiency of glycine in humans. Because the resulting shortage of glycine can negatively influence brain neurochemistry and the synthesis of collagen, nucleic acids, porphyrins, and other important metabolites, the risks of using benzoate as a preservative should not be underestimated. To date, no defect of the glycine conjugation pathway has been reported and this, together with the fact that GLYAT plays an important role in hepatic metabolism, suggests that this pathway is essential for survival. GLYAT activity affects mitochondrial ATP production, glycine availability, CoASH availability and the toxicity of various organic acids. Therefore, variation in the glycine conjugation pathway could influence liver cancer, musculoskeletal development and mitochondrial energy metabolism. Significant interindividual variation exists in glycine conjugation capacity. The molecular basis for this variability is not known. The main aim of this thesis was to investigate and characterise the genetic variation in the coding region of the GLYAT gene. This was accomplished by firstly, investigating the influence of non-synonymous single nucleotide polymorphisms (SNPs) on the enzyme activity of a recombinant human GLYAT and secondly, by analysing the level of genetic variation in the coding region of the GLYAT gene using existing worldwide population data. To investigate the influence of non-synonymous SNPs in the GLYAT gene on the enzyme activity, a recombinant human GLYAT was prepared, and characterised. Site-directed mutagenesis was used to generate six variants of the enzyme (K16N; S17T; R131H; N156S; F168L; R199C). The variants were expressed, purified, and enzymatically characterised. The enzyme activities of the K16N, S17T and R131H variants were similar to that of the wild-type, whereas the N156S variant was more active, the F168L variant less active, and the R199C variant was inactive. The results showed that SNP variations in the human GLYAT gene can influence the kinetic properties of the enzyme. The genetic variation data of the human GLYAT open reading frame (ORF) available on public databases was investigated by formulating the hypothesis that due to the essential nature of the glycine conjugation pathway, the genetic variation in the ORF of the GLYAT gene should be low and that deleterious alleles will be found at low frequencies. Data from the i) 1000 Genome Project, ii) the HapMap Project, and iii) the Khoi-San/Bantu Sequencing Project was downloaded from available databases. Sequence data of the coding region of a small cohort of South African Afrikaner Caucasian individuals was also generated and included in the analyses. In the GLYAT ORF of the 1537 individuals analysed, only two haplotypes (S156 and T17S156) out of 14 haplotypes were identified in all populations as having the highest haplotype frequencies (70% and 20% respectively). The S156C199 and S156H131 haplotypes, which have a deleterious effect on the enzyme activity of a recombinant human GLYAT, were detected at very low frequencies. The results of this study indicated that the GLYAT ORF is remarkably conserved, which supports the hypothesis that the glycine conjugation pathway is an essential detoxification pathway. The findings presented in this thesis highlight the importance that future investigations should determine the in vivo capacity of the glycine conjugation pathway for the detoxification of benzoate and other xenobiotics. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2015

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Reproduction and robustness traits are integral in ensuring sustainable, efficient and profitable sheep farming. Increases in genetic gain of reproduction and robustness traits are however, hampered by low heritability coupled with the difficulty in quantification of these traits for traditional selective breeding strategies. The aim of the current study was therefore to identify genomic regions underlying variation in reproduction traits and elucidate quantitative trait loci (QTL) and/or genes associated with reproductive traits. The Elsenburg Merino flock has been divergently selected for the ability to raise multiple offspring and has resulted in a High and a Low line that differ markedly with regard to reproductive output and other robustness traits. The flock thus served as an ideal platform to identify genomic regions subject to selection for reproductive traits. To pinpoint genomic regions subject to selection, a whole-genome genotyping platform, the OvineSNP50 chip, was selected to determine the genotype of more than 50 000 SNPs spread evenly across the ovine genome. The utility of the OvineSNP50 chip was determined for the Elsenburg Merino flock as well as additional South African Merino samples and three other important South African sheep breeds, the Blackheaded Dorper, South African Mutton Merino (SAMM) and the Namaqua Afrikaner. Although genotyping analysis of the Elsenburg Merino flock indicated some signs of poor genotype quality, the overall utility of the genotype data were successfully demonstrated for the South African Merino and the other two commercial breeds, the Dorper and SAMM. Genotyping results of the Namaqua Afrikaner and possibly other indigenous African breeds may be influenced by SNP ascertainment bias due to the limited number of indigenous African breeds used during SNP discovery. Analysis of pedigree, phenotypic records and SNP genotype data of the Elsenburg Merino cohort used in the current study, confirmed that the lines are phenotypically as well as genetically distinct. Numerous putative genomic regions subject to selection were identified by either an FST outlier approach or a genomic scan for regions of homozygosity (ROH) in the High and Low lines. Although annotated genes with putative roles in reproduction were identified, the exact mechanism of involvement with variation in reproduction traits could not be determined for all regions and genes. Putative ROH overlapped with QTL for several reproduction, milk, production and parasite resistance traits, and sheds some light on the possible function of these regions. The overlap between QTL for production and parasite resistance with putative ROH may indicate that several, seemingly unrelated traits add to the net-reproduction and may have been indirectly selected in the Elsenburg Merino flock. A SNP genotyping panel based solely on reproduction traits may therefore be ineffective to capture the variation in all traits influencing reproduction and robustness traits. A holistic selection strategy taking several important traits, such as robustness, reproduction and production into account may as such be a more effective strategy to breed animals with the ability to produce and reproduce more efficiently and thereby ensure profitable and sustainable sheep farming in South Africa. / AFRIKAANSE OPSOMMING: Reproduksie- en gehardheids-eienskappe is noodsaaklik om volhoubare, doeltreffende en winsgewende skaapboerdery te verseker. ‘n Toename in genetiese vordering in reproduksie- en gehardheids-eienskappe word egter bemoeilik deur lae oorerflikhede tesame met die probleme in kwantifisering van hierdie eienskappe vir tradisionele selektiewe diereteelt strategieë. Die doel van die huidige studie was dus om gebiede in die genoom onderliggend tot variasie in reproduksie-eienskappe te identifiseer en die rol van verwante kwantitatiewe eienskap loki (KEL) en/of gene met reproduktiewe eienskappe te bepaal. Die Elsenburg Merinokudde is uiteenlopend geselekteer vir die vermoë om meerlinge groot te maak en het gelei tot 'n Hoë en 'n Lae lyn wat merkbaar verskil ten opsigte van reproduksie-uitsette en ander gehardheids-eienskappe. Die kudde het dus gedien as 'n ideale platform om genomiese areas onderhewig aan seleksie vir reproduksie-eienskappe te identifiseer. Om vas te stel waar genomiese areas onderhewig aan seleksie gevind kan word, is ‘n heel-genoom genotiperingsplatform, die OvineSNP50 skyfie, gekies om die genotipes van meer as 50 000 enkel nukleotied polimorfismes (ENPs) eweredig versprei oor die skaap genoom, te bepaal. Die nut van die OvineSNP50 skyfie is bepaal vir die Elsenburg Merinokudde sowel as addisionele Suid-Afrikaanse Merinos en drie ander belangrike Suid-Afrikaanse skaaprasse, die Swartkop Dorper, Suid-Afrikaanse Vleismerino (SAVM) en die Namakwa Afrikaner. Hoewel genotipe resultate van die Elsenburg Merino kudde sommige tekens van swak genotipe gehalte getoon het, kon die algehele nut van die genotipering resultate vir die Suid-Afrikaanse Merino en die ander twee kommersiële rasse, die Dorper en SAVM, bevestig word. Genotipering resultate van die Namakwa Afrikaner en moontlik ook ander inheemse Afrika rasse kan deur ENP vasstellingspartydigheid beïnvloed word as gevolg van die beperkte aantal inheemse Afrika rasse gebruik tydens ENP ontdekking. Ontleding van stamboom inligting, fenotipe rekords en ENP genotipe data van die Elsenburg Merino-kohort gebruik in die huidige studie, het bevestig dat die lyne fenotipies asook geneties verskil. Talle vermeende genomiese areas onderhewig aan seleksie is geïdentifiseer deur 'n FST uitskieter benadering of deur ‘n genomiese skandering vir gebiede van homogositeit (GVH) in die Hoë en Lae lyne. Hoewel geannoteerde gene met potensiële rolle in reproduksie geïdentifiseer is, kan die presiese meganisme van betrokkenheid by variasie in reproduksie-eienskappe nie bevestig word vir al die gebiede en gene nie. Vermeende GVH oorvleuel met KEL vir 'n paar reproduksie-, melk-, produksie- en parasietweerstand-eienskappe, en werp daarom lig op die moontlike funksie van hierdie gebiede. Die oorvleueling tussen KEL vir produksie en parasietweerstand met vermeende GVH kan daarop dui dat 'n hele paar, skynbaar onverwante, eienskappe bydrae tot net-reproduksie, wat indirek geselekteer mag wees in die Elsenburg Merino-kudde. ‘n ENP genotiperingspaneel uitsluitlik gebaseer op reproduksie-eienskappe mag daarom onvoldoende wees om die variasie in alle eienskappe wat betrekking het op reproduksie- en gehardheids-eienskappe, in te sluit. ‘n Holistiese seleksie strategie wat verskeie belangrike eienskappe, soos gehardheid, reproduksie en produksie in ag neem, mag ‘n meer effektiewe strategie wees om diere te teel met die vermoë om in 'n meer doeltreffende manier te produseer en reproduseer en om daardeur winsgewende en volhoubare skaapboerdery in Suid-Afrika te verseker.

Humans are continuously challenged by harmful endogenous and xenobiotic substances. Detoxification is the ability to neutralise and remove these substances from the body. Glycine N-acyltransferase, EC 2.3.1.13 (GLYAT) is a key enzyme in detoxification. GLYAT catalyses an amino acid (glycine) conjugation reaction in phase II of detoxification. It is expected that, similar to what has been observed in the Cytochrome P450 enzymes, variations within the GLYAT gene may lead to altered enzyme activity that may affect the efficacy of detoxification. The aim of this study was to identify genetic variations within the GLYAT gene of a cohort of individuals whose GLYAT activity has been biochemically characterized. Biochemical profiles of phase I and II detoxification of a number of individuals was screened to select those with possible aberrant GLYAT activity. Eighteen selected individuals agreed to participate in the study. The 23.21 kb GLYAT gene of the participants was amplified in four fragments and sent for pyrosequencing (Roche GS FLX titanium) at Inqaba Biotec. The results were analysed with the Lasergene software package from DNAStar (Madison, Wisconsin, USA). A total of 94 variations were identified from the Next Generation Sequencing data. Of these three found in the exons were known variations and four variations located in the exons were novel. A total of 62 known and 25 novel variations were identified in the introns of the GLYAT gene. Sanger sequencing verified 70.29% (68 in total) of the variation, which included 12 novel variations, of which one is located in exon six. Real-time quantitative PCR (qPCR) experiments were conducted and the data analysed using CopyCaller software to identify copy number variations within the cohort. It was found that participant 17 may have multiple copies of parts of the 3-terminal end of the gene (exons five and six), which might have an effect on GLYAT activity. Variations could possibly affect GLYAT activity, but the data was inconclusive and must be confirmed. Some of the variations could possibly affect GLYAT activity, but no correlation could be made between the variations identified during this study and the cohort’s detoxification ability. Further studies needs to be conducted to establish the effect of the variations in combination with one another on GLYAT activity. If some of these variations affect GLYAT activity such data might shed some light on variations observed between the glycine conjugation ability of individuals. Such information could eventually be of value in treatment of inborn errors of metabolism. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2014

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).

Skeletal muscles in broilers are generally characterised by pathological muscle damage, indicated by greater plasma creatine kinase (CK) activity, higher incidence of haemorrhages, lighter and less coloured breast muscles, compared with layers and traditional breeds of chicken. Muscle damage is further exacerbated by exposure to stressful conditions such as high ambient temperatures which results in a further decrease in the quality of broiler meat and leads to the production of pale, soft and exudative (PSE) meat. This growing incidence of poor quality poultry meat is causing substantial losses to the meat industry. However, in contrast to pork the genetics of poor muscle and meat quality in chicken is unknown. The present project was conducted to identify the underlying genetics of this low quality meat by using heat-stress as a tool to amplify muscle damage and expression of the relevant genes. Whole-genome expression studies in broiler and layer breast muscles were conducted before and after heat-stress and some phenotypic data were also recorded. From the gene expression studies, 2213 differentially expressed genes (P<0.05) were found. About 700 of these genes had no gene ontology (GO) terms associated with them for biological process or function. The significant gene set was analysed in BioLayout Express and interesting clusters of the genes, based on their positive correlation with each other, were selected for further investigation. Genes were grouped together in 6 different categories or clusters, on the basis of their expression pattern. The genes in the selected clusters were analysed in Ingenuity Pathway Analysis (IPA) software, for each category separately, and relevant biological pathways and networks for those genes were studied. Similarly, the genes filtered out by BioLayout Express at a Pearson threshold of 0.80 were also analysed in IPA separately and interesting pathways and networks were selected. From the pathways and networks analyses of these genes, it was discovered that genes involved in inflammatory, cell death, oxidative stress and tissue damage related functions were up-regulated in control broilers compared with control and similar to heat-stressed layers. After exposure to heat-stress the expression levels of these genes were further increased in broilers. These results led us to develop the hypothesis that breast muscles in broilers are under stress-related damage even under the normal rearing conditions. This hypothesis was tested by rearing the broilers birds at normal/conventional and comparatively low ambient temperature and its effects on breast muscle quality and meat quality were studied. Significant improvement of breast muscle redness was observed. Additionally substantial numerical improvements for other meat and muscle quality traits like breast muscle lightness and histopathology were observed. From the key positions of interesting significant pathways and networks, candidate genes were selected for further investigation. In total, 25 candidate genes were selected for SNP genotyping: 19 genes were selected from the interesting pathways and networks and 6 genes were selected on the basis of their GO terms. For each gene 4-5 SNPs were selected, where possible, that were present in exons and promoter regions of the candidate genes. The selected SNPs were genotyped for muscle and meat quality traits in 34 breeds of chicken and significant causative SNPs for each trait including plasma CK activity, pHi and pHu for breast muscles, colour (L*, a*, and b*) traits for breast and thigh muscles were found. These SNPs were responsible for explaining a moderate to high (15-55%) percentage of phenotypic variance for these traits. To our knowledge this is the first study in which gene-expression in chicken breast muscle was conducted in response to heat-stress and additionally, for the first time, a set of novel SNPs for all of these traits were identified. Some of the significant causative SNPs were lying in the protein coding sequences and some were present in the promoter regions of the candidate genes.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Pour améliorer la compétitivité de l'industrie porcine, le porc canadien doit être attrayant et présenter un avantage unique qui permettrait à cette industrie de se démarquer des autres produits alimentaires. La carnosine pourrait offrir un tel avantage et ainsi aider l'industrie porcine à obtenir une plus grande part de marché, tout en modifiant les perceptions négatives liées à la consommation de viande. La carnosine fut, en 1900, le premier peptide jamais isolé à partir de matériel biologique. Chez l'homme, certaines propriétés curatives ont été associées à la consommation de carnosine. Ces propriétés peuvent être expliquées, en partie, par la capacité de la carnosine à inhiber la glycation non enzymatique des protéines et leurs agrégations au cours du vieillissement. Mais son potentiel d'améliorer la qualité de la viande de porc est tout aussi intéressant à développer. En effet, la carnosine musculaire agirait comme tampon de pH, ce qui permettrait de ralentir l’acidification dans le muscle squelettique. La carnosine présente également des propriétés antioxydantes, ce qui peut aussi contribuer à améliorer la qualité de la viande. Ce projet de maîtrise fait partie d’un programme de recherche plus large ayant pour objectif d'augmenter la teneur en carnosine musculaire chez le porc en croissance afin de différencier et de donner une valeur ajoutée au porc canadien. Dans cette étude, nous avons supposé qu’un contenu élevé en carnosine musculaire serait associé à de meilleurs paramètres de qualité de la viande chez le porc en croissance et que l'expression des gènes liés au métabolisme de la carnosine serait modulée chez les porcs ayant différents niveaux de carnosine musculaire, de même que dans les muscles de porcs de différentes races. En second lieu, nous avons supposé que des polymorphismes d'un seul nucléotide (SNP), observés sur les gènes liés au métabolisme de la carnosine, pourraient aussi affecter le dépôt de carnosine musculaire chez le porc. Un total de 282 porcs de race pure a été utilisé pour ce projet, incluant 85 Duroc, 92 Landrace et 105 Yorkshire, lesquels provenaient de 16 producteurs différents à travers le Canada. Ces porcs sont entrés à la station de Deschambault (Québec, Canada) entre 10 à 16 jours d’âge, ont tous été élevés dans des conditions similaires et abattus à 120 kg de poids vif. Les carcasses ont été suivies individuellement à l’abattoir pour permettre l’échantillonnage du muscle longissimus thoracis immédiatement après la mort de l’animal et de prendre les mesures de qualité de la viande, 24 h postmortem. Les valeurs de qualité de la viande et de carnosine musculaire étaient disponibles au début de ce projet de maîtrise (projet de recherche antérieur). Dans un premier temps, les niveaux d’expression génique de gènes du métabolisme de la carnosine ont été mesurés par la méthode de PCR en temps réel. Les gènes sélectionnés incluent des enzymes (ABAT, 4-aminobutyrate aminotransferase; CARNS1, carnosine synthase 1; CNDP1, CNDP dipeptidase 1; CNDP2, CNDP dipeptidase 2; HDC, histidine decarboxylase) et des transporteurs (SLC6A6, solute carrier family 6, member 6; SLC15A1, solute carrier family 15, member 1; SLC15A2, solute carrier family 15, member 2; SLC15A3, solute carrier family 15, member 3; SLC15A4, solute carrier family 15, member 4; SLC36A1, solute carrier family 36, member 1) reliés au métabolisme de la carnosine. Les gènes ABAT, CDNP1, HDC, SLC15A1 et SLC15A2 ont été abandonnés puisque leurs transcrits étaient indétectables ou présents à de très faibles quantités. En ce qui concerne les autres gènes, l’analyse des résultats a révélé un effet de race pour l’expression génique de CARNS1, CNDP2 et SLC36A1, les valeurs les plus élevées étant observées chez les porcs de race Duroc, lesquels présentent également des niveaux plus élevés en carnosine musculaire. Pour chaque race, les animaux ont été regroupés en trois catégories selon leur teneur en carnosine musculaire (Bas, Moyen et Élevé). Pour les Duroc, l'abondance en ARNm de la CARNS1 est plus élevée pour le groupe Bas que pour les groupes Moyen et Élevé en carnosine. Ce résultat laisse croire à un possible rétrocontrôle de la carnosine musculaire sur la transcription de la CARNS1, l’enzyme responsable de sa synthèse. Pour les gènes SLC15A3 et SLC15A4, nous observons des niveaux d’ARNm plus élevés chez les animaux à haute teneur en carnosine. Ainsi, des niveaux plus élevés de carnosine musculaire pourraient entrainer une augmentation de la transcription de SLC15A3 et SLC15A4, deux gènes impliqués dans le transport de la carnosine. Dans un deuxième temps, l’effet de la quantité de carnosine musculaire sur différents caractères de qualité de la viande a aussi été étudié. Ces résultats nous démontrent que les porcs ayant des niveaux de carnosine élevés, présentent également une meilleure qualité de la viande, tel que démontré par un pH à 24h plus élevé, une meilleure capacité de rétention d’eau et une diminution des indices de couleur b* (couleur jaune) et L* (luminance).Enfin, une recherche de polymorphismes d’ADN a aussi été effectuée sur quatre gènes cibles (CARNS1, SLC6A6, SLC15A3 et SLC15A4) ayant un fort potentiel d’affecter les niveaux de carnosine musculaire. Les séquences codantes et 3’UTR de chacun de ces gènes ont tout d’abord été séquencées sur une population restreinte de 28 Duroc, 27 Landrace et 30 Yorkshire, incluant des porcs présentant de faibles et de hauts niveaux de carnosine musculaire. Un total de 27 SNPs ont été identifiés pour ces quatre gènes. De ce nombre, quatre SNPs furent sélectionnés pour effectuer le génotypage de la population entière (n = 590). De ces SNPs, deux entrainent un changement d’acide aminé dans le gène SLC15A4 (SNP c.658A>G : Ile220Val; SNP c.818G>A : Ser273Asn)) et deux autres SNPs dans le gène SLC15A3 se retrouvent sur un site reconnu par un micro-ARN (c.*35C>T and c.*52C>T). Des analyses d’association ont ensuite été effectuées afin de déterminer l’effet des différents SNPs et diplotypes sur les caractères de qualité de la viande et sur les niveaux de carnosine et d’ansérine (analogue méthylé de la carnosine). Nos résultats démontrent qu’une sélection en faveur du génotype c.658AA ou du diplotype AA/GG pour le gène SLC15A4 résulterait en une augmentation de carnosine musculaire et une amélioration de certains paramètres de qualité de la viande tels que la couleur, la rétention d’eau et le pH à 24 h. Collectivement ces résultats montrent que les porcs présentant de hauts niveaux de carnosine musculaire présentent également une meilleure qualité de la viande. De plus, les niveaux d’expression de certains gènes du métabolisme de la carnosine sont modulés selon la race de porc (influence génétique possible) et aussi selon le contenu de carnosine musculaire. Enfin, une amélioration générale de la qualité de la viande serait possible par la sélection d’allèles favorables du gène SLC15A4. Cependant, les fréquences observées pour les allèles mineures du SNP c.658A>G (i.e. Duroc, 0.01; Landrace, 0.09) suggèrent une amélioration génétique limitée chez les Duroc et les Landrace.