Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-09-04T21:36:01Z No. of bitstreams: 1 2017 - Odair Scatolin Rossafa Garcia.pdf: 1812000 bytes, checksum: 37d6d5a375daf6bc6b0ef40d4687a7f7 (MD5) / Made available in DSpace on 2018-09-04T21:36:01Z (GMT). No. of bitstreams: 1 2017 - Odair Scatolin Rossafa Garcia.pdf: 1812000 bytes, checksum: 37d6d5a375daf6bc6b0ef40d4687a7f7 (MD5) Previous issue date: 2017-05-26 / Goat meat has lower levels of fat than those found in other types of meat such as beef, pork, sheep and deer, but the lack of selection criteria for slaughter, storage and commercialization of meat leads to a low level because of the lack of standardization of the product presenting unpleasant sensory characteristics. A study of polymorphism, variation in gene expression and the association of these variations with the desired phenotype allows to broaden the understanding of the physiological processes, which helps in the strategies aimed at improving the characteristic of interest, resulting in the expected final phenotype. The objective of the present study was to evaluate polymorphisms and gene expression among some of the most promising genotypes of pleiotropic genes, comparing the polymorphism and expression among groups of animals with greater and lesser weight at slaughter to verify if there is any relation with the weight difference Or softness of the flesh. For this purpose, genotypes of 40 goats from the Saanen and Alpina breeds for the growth hormone (GH) gene, diacylglycerol acyltransferase 1 gene (DGAT1), myostatin gene (MSTN), growth factor gene Similar to insulin 1 (IGF1), fatty acid carrier protein (FATP1) gene, nuclear factor 1 (NF1) gene, gamma peroxisome proliferator activated receptor (PPAR?) gene. After analyzing the association of the different genotypes with the slaughter weight (AP), carcass weight (PC) and meat softness expressed in shear force (FC), some genes were selected for the analysis of expression and association with them Variables. The GH, NF1 and PPAR genes were not evaluated for expression, the first for not having presented a good result for the efficiency analysis of the other two primers due to the lack of substantial data for the preparation of the primers. For the softness test, previously performed in another study by the same team, the longissimus lumborum muscle was used. Polymerase chain reaction (PCR) technique was used for the genotyping and later, for some genes, the digestion of the fragments amplified by restriction enzymes, a technique known as PCR-RFLP. Gene expression was conducted using the Real Time PCR technique (qPCR) and the meat tenderness phenotype was analyzed in a texturometer. The data were statistically related using the SAS GLM procedure. The UNIVARIATE procedure was used to verify the normality of residues of expression of the genes under study (expressed as 2-?Ct) and softness data. The averages were compared by the Tukey test and the Pearson correlations tested by the t test. The polymorphism already described in the GH was also detected in the population studied in the present study, the genotype heterozygous AB presented a mean 2.78kg at slaughter weight more than the AA individuals, for the MSTN the individuals with heterozygous M1M2 genotype presented higher scores for weight at slaughter, while for the IGF1 gene the heterozygous AB animals present less tender meat. The group with lower weight at slaughter showed higher expression of the DGAT1 and FATP genes, which may reflect a higher deposition of fat in the carcass and greater softness, in comparison with the group of higher weight. / A carne caprina apresenta teores de gordura abaixo dos encontrados em outros tipos de carne como a de bovino, su?no, ovino e veado. Entretanto, a falta de crit?rio de sele??o para o abate, estocagem e comercializa??o da carne, acaba por gerar um baixo n?vel de consumo, devido ? falta de padroniza??o do produto apresentando caracter?sticas sensoriais desagrad?veis. Estudo de polimorfismo, varia??o na express?o g?nica e associa??o destas varia??es com o fen?tipo desejado permite ampliar a compreens?o sobre os processos fisiol?gicos, al?m de auxiliar programas de melhoramento gen?tico animal para a sele??o de animais com fen?tipos superiores para a caracter?stica de interesse. O objetivo do presente trabalho foi avaliar a associa??o de polimorfismos e express?o g?nica com a caracter?stica peso ao abate e verificar se h? rela??o entre a diferen?a de peso e a maciez da carne. Para este prop?sito foram identificados inicialmente os gen?tipos de cabritos das ra?as Saanen e Alpina para os seguintes genes: horm?nio do crescimento (GH), diacilglicerol aciltransferase 1 (DGAT1), miostatina (MSTN), fator de crescimento semelhante ? insulina 1 (IGF1), prote?na transportadora de ?cidos graxos (FATP1), fator nuclear 1 (NF1), receptor ativado por proliferadores de peroxissomas gama (PPAR?). Ap?s a an?lise de associa??o dos diferentes gen?tipos com o peso ao abate (PA), peso da carca?a (PC) e maciez da carne expressa em for?a de cisalhamento (FC), foram selecionados alguns genes para a an?lises de express?o e associa??o com as mesmas vari?veis. Os genes GH, NF1 e PPAR n?o foram avaliados quanto a express?o, o primeiro por n?o ter apresentado um bom resultado para as analise de efici?ncia dos primers os outros dois devido ? problemas no genoma refer?ncia para a confec??o dos primers. Para o teste de maciez foi utilizado o m?sculo longissimus lumborum. Para a genotipagem foi utilizada a t?cnica da rea??o em cadeia pela polimerase (PCR) e posteriormente, para alguns genes, digest?o dos fragmentos amplificados por enzimas de restri??o (PCR-RFLP). A express?o g?nica foi conduzida utilizando a t?cnica de PCR em Tempo Real (qPCR) e o fen?tipo de maciez da carne foi analisado em textur?metro. Os dados foram analisados estat?sticamente utilizando o procedimento GLM do SAS. O procedimento UNIVARIATE foi utilizado para verificar a normalidade dos res?duos da express?o dos genes em estudo (expressos com 2-?Ct) e dados de maciez. As m?dias foram comparadas pelo teste de Tukey e as correla??es de Pearson testadas pelo teste de t. O polimorfismo j? descrito no GH foi tamb?m detectado na popula??o estudada no presente trabalho, o gen?tipo heterozigoto AB apresentou m?dia 2,78kg a mais de peso ao abate do que os indiv?duos AA, para a MSTN os indiv?duos com gen?tipo heterozigoto M1M2 apresentaram maiores escores para peso ao abate, enquanto para o gene IGF1 os animais heterozigotos AB apresentam carne menos macia. O grupo com menor peso ao abate apresentou maior express?o dos genes DGAT1 e FATP, o que pode refletir maior deposi??o de gordura de gordura na carca?a e maior maciez, em compara??o com o grupo de maior peso.

Os primeiros 30 dias pós-nascimento do bezerro, chamado período neonatal, é caracterizado por grande desenvolvimento imunológico. O sistema imune começa a se desenvolver ainda no início da gestação, porém, após o nascimento, mesmo que morfologicamente desenvolvido, não se apresenta totalmente funcional pela ausência de estímulos antigênicos. Ademais as particularidades anatômicas e fisiológicas da placentação dos bovinos são um impediente à transferência de imunidade durante a gestação, assim sendo o feto se desenvolve sem a influência das imunoglobulinas maternas, ficando altamente dependente ao início da vida extrauterina da transferência de imunidade passiva via colostro. As características do sistema imune do bezerro em seu período neonatal fazem com que este seja muito susceptível a doenças. Conhecer o comportamento imunitário dos bezerros recém-nascidos pode auxiliar a diminuir a incidência de doenças e o custo desse animal, além de aumentar o seu bem-estar. Deste modo, buscou-se avaliar o padrão de resposta imunitária do neonato nos primeiros 30 dias de vida, por meio de avaliações: da atividade fagocítica e do metabolismo oxidativo de neutrófilos circulantes e imunoenotipagem de linfócitos T (CD3+) e suas subpopulações (CD4+) e CD8+)) por citometria de fluxo e a expressão gênica das citocinas IL-4, IL-10, IL-12 e IFN-ϒ por PCR em Tempo Real. O exame físico, o hemograma e avaliação de transferência de imunidade passiva foram considerados como critério de inclusão para garantir a sanidade dos animais durante o período experimental. Com base nos resultados obtidos nesta pesquisa para pode se concluir que: a atividade de fagocitose dos granulócitos foi constante nos 30 dias avaliados; a produção de espécies reativas de oxigênio por granulócitos foi observada nos ensaios basal e estimulado; e com comportamento semelhante, apresentando os dois ensaios, maiores porcentagens nos dias 1, 25 e 30 p.n.; os linfócitos CD3+) e suas subpopulações:CD4+), CD8+), estes apresentaram porcentagens semelhantes àquelas encontradas nos bovinos adultos; e a relação CD4+) /CD8+) aumentou aos 30 dias de vida, pelo aumento de CD4+). Não foi possível mensurar a expressão gênica das citocinas IL-4 e IFN-ϒ em nenhum dos momentos avaliados; no entanto, foi verificada a expressão gênica de citocinas IL-10 e IL-12, com uma inclinação para o perfil Th2 induzido pela expressão mais frequente de IL-10, observando-se influência da expressão gênica das citocinas IL-10 e IL-12 no leucograma, na atividade dos granulócitos, e nas subpopulações de linfócitos T. / The first 30 days post-birth of the calf, called the neonatal period, is characterized by large immunological development. The immune system begins to develop even at the beginning of gestation, but after birth, even if morphologically developed, it is not fully functional due to the absence of antigenic stimuli. In addition, the anatomical and physiological characteristics of bovine placentation are an impediment to the transfer of immunity during gestation, so the fetus develops without the influence of maternal immunoglobulins, being highly dependent at the beginning of the extrauterine life of the transference of passive immunity via colostrum. The characteristics of the immune system of the calf in its neonatal period make it very susceptible to diseases. Knowing the immune behavior of newborn calves can help reduce the incidence of diseases and the cost of this animal, and increase their well-being. The aim of this study was to evaluate the immune response pattern of the neonate in the first 30 days of life through evaluations of phagocytic activity and oxidative metabolism of circulating neutrophils and T lymphocyte (CD3 +) immuno-typing and its subpopulations (CD4 + and CD8 +) by flow cytometry and the gene expression of the IL-4, IL-10, IL-12 and IFN-ϒ cytokines by Real-Time PCR. Physical examination, blood count and passive immunity assessment were considered as inclusion criteria to guarantee the health of the animals during the experimental period. Based on the results obtained in this research it can be concluded that: granulocyte phagocytosis activity was constant in the 30 days evaluated; the production of reactive oxygen species by granulocytes was observed in the basal and stimulated assays; and with similar behavior, presenting the two trials, highest percentages on days 1, 25 and 30 p.n .; the CD3 + lymphocytes and their subpopulations: CD4 +, CD8 +, these presented percentages similar to those found in adult bovines; and the CD4 + / CD8 + ratio increased at 30 days of life, due to the increase in CD4 +. It was not possible to measure the gene expression of IL-4 and IFN-ϒ cytokines in any of the evaluated moments; However, IL-10 and IL-12 cytokine gene expression was observed, with an inclination to the Th2 profile induced by the more frequent expression of IL-10, with the influence of the gene expression of the cytokines IL-10 and IL- 12 on leukogram, granulocyte activity, and T lymphocyte subpopulations.

A sequência de eventos bacterianos que ocorre durante a colonização do trato gastrointestinal pode afetar o futuro da saúde do hospedeiro, particularmente no que diz respeito à regulação do sistema imunológico. Um entendimento claro do processo de colonização do intestino humano neonatal nos países em desenvolvimento está faltando, porque os poucos estudos disponíveis foram, em sua maioria, realizados utilizando técnicas de cultura. O objetivo deste estudo foi detectar e quantificar as bactérias dos gêneros Bifidobacterium, Lactobacillus, Eubacterium e Lactococcus, importantes componentes anaeróbios da microbiota intestinal usando PCR em tempo real. O grupo de estudo foi composto por 10 crianças, acompanhadas durante o primeiro ano de vida, vivendo em baixas condições sócio-econômicas em São Paulo, Brasil. Amostras de fezes foram avaliadas em períodos de 24 horas, 7 dias, 30 dias, 3 meses, 6 meses e 1 ano. Durante o primeiro ano de vida, há um aumento da quantidade de Bifidobacterium spp., quando comparada com as outras bactérias anaeróbias estudadas, com médias variando de 8,27x1010 a 2,51x1012 número de cópias de DNA/g de fezes. Lactobacillus spp. também foi encontrado em todos os pontos de tempo estudado, com médias variando de 4,03x108 a 1,46x1010 número de cópias de DNA/g de fezes. Lactococcus spp. foi o gênero bacteriano encontrado em quantidades menores. As contagens máximas desses gêneros foram encontradas entre o terceiro e sexto mês de vida. Embora o gênero Eubacterium seja descrito como um dos principais membros da microbiota intestinal, este foi encontrado em amostras de apenas duas crianças. A inclusão de dieta sólida e a mudança do tipo de amamentação influenciam a composição da microbiota. No entanto, não se pode estabelecer um padrão para a presença destes micro-organismos ao longo dos meses, mostrando que a microbiota é única e está sujeita a interferências ambientais. / The sequence of bacterial events that occurs during the colonization of the gastrointestinal tract may affect the future health of the host, particularly with respect to the regulation of the naive immune system. A clear understanding of the colonization process of the human neonatal gut in developing countries is lacking because the few available studies were mostly performed using culture techniques. The aim of this study was to detect and quantify the bacterial genera Bifidobacterium, Eubacterium, Lactobacillus and Lactococcus, important anaerobic components of the intestinal microbiota using real-time PCR. The study group comprised 10 children followed during the first year of life, living in low socio-economic conditions in São Paulo, Brazil. Fecal samples were evaluated at times of 24 hours, 7 days, 30 days, 3 months, 6 months and 1 year. During the first year of life, there is an increased amount of Bifidobacterium spp. compared to others studied anaerobic bacteria, with averages ranging from 8,27x1010 to 2,51x1012 DNA copy number/g of feces. Lacotbacillus was also found in all studied time point, with averages ranging from 4,03x108 to 1,46x1010 DNA copy number/g of feces. Bacterial genus Lactococcus is found in smaller quantities. The maximum counts of these genera were found between the third to sixth month of life. Although the genus Eubacterium is described as one of the leading members of the intestinal microbiota, this was found in samples of only 2 children. The inclusion of solid diet and change of type of feeding influences the composition of the microbiota, however, could not set a standard for the presence of these micro-organisms over the months, showing that the microbiota is unique and is subject to environmental interference.

Mixed Linkage Glucan (MLG, or (1,3;1,4)-ß-D glucan) is a component of cell walls for major cereal crops and is significant to food and beverage industries. To better understand genetic factors affecting MLG content in oats, this study investigates the presence of glucosidases likely to participate in MLG production. A glucosidase showing co-expression with CslF6—the primary gene responsible for MLG synthesis—could indicate a hand in MLG production by association. Reference genes for expression analysis as well as glucosidase candidates were first selected using in silico methods. In both cases, barley was used as model species because it has abundant public bioinformatic resources for in silico data mining, and it generates large amounts of MLG, like oats. Actin, malate dehydrogenase, and elongation factor 2, were validated in oat and barley as top reference genes. They were then used to compare the expression activity of the top glucosidase candidate gene, ßglux, with CslF6. ßglux was found to have increased activity with CslF6 during caryopsis development. It is a strong candidate for future transgenic experiments regarding its effect on MLG production.

Karenia brevis (Davis cf. Hansen & Moestrup = Gymnodinium breve) is the non-peridinin containing dinoflagellate responsible for many harmful algal blooms (red tides) in the Gulf of Mexico. These recurrent blooms can have significant negative ecological, economic, and human health impacts including fish kills, tainting of shellfish, poisoning of marine mammals, loss of tourism revenue due to beach closures, and respiratory distress and food poisoning in humans. A method for detection of Karenia brevis was developed based upon amplification of the mRNA for the plastid-encoded gene of the carbon fixing enzyme ribulose 1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) large subunit (rbcL). Using sequence information from a primer set targeting a 554-bp region of the Karenia rbcL gene, a small (91 bp amplicon) primer and probe set was created for TaqMan(registered trademark) real time RT-PCR of K. brevis rbcL. The primer/probe set is sensitive to as little as 0.1 fg of target transcript and as little as 1 pg of total cellular K. brevis RNA extract, corresponding to less than 1 cell reaction-1. The primer/probe set did not amplify rbcL transcript from any of the non-target algae tested. Bloom samples analyzed by this method have shown the assay to be a reliable method, with effective enumeration and a linear relationship showing good correlation to the cell counts by microscopy (r2= 0.8344). The assay has been shown to be robust and perform well even in non-ideal conditions, with pre-extraction RNA from unialgal culture stable at room temperature for up to 3 days and up to a month at -80 degrees C in Stratagene's lysis buffer. The transcription of the rbcL gene demonstrated minor variation throughout the diel period, however the variation was not linked to the diel cycle or to carbon fixation, which showed a distinct diel signal. Due to the relatively constant expression of the rbcL gene, the real-time RT-PCR assay developed should be able to reliably enumerate K. brevis populations in the natural environment, as long as the sample is placed in Stratagene's lysis buffer and processed within one or two days or frozen at -80 degrees C and processed within a month.

The incidence of Squamous Cell Carcinoma is growing in certain populations to the extent that it is now the most common skin lesion in young men and women in high ultraviolet exposure regions such as Queensland. In terms of incidence up to 45% of the Australian population over 40 years of age is thought to possess the precancerous Solar Keratosis lesion and with a small but significant chance of progression into SCC, understanding the genetic events that play a role in this process is essential. The major aims of this study were to analyse whole blood derived samples for DNA aberrations in genes associated with tumour development and cellular maintenance, with the ultimate aim of identifying genes associated with non-melanoma skin cancer development. This study had an explicit emphasis on the mitochondrial genome and nuclear genes that encode for subunits in the mitochondrial regulated energy transducing oxidative phosphorylation pathways. More specifically the first aim of this project was to analyse the NDUFA8, PTCH, NDUFAS, SMOH, SDHD, MMPI2, NDUFV1, EMSI, COXVIIc, and RASAI genes via non-specific fluorophoric Real-Time PCR for genetic aberrations in an affected Solar Keratosis and control cohort. The second aim was to analyse two specific genes, SDHD and MMPI2, for copy number aberrations via Dual-Labelled Probe Real-Time PCR in the same affected Solar Keratosis and control cohort. The third aim was to analyse Mitochondrial DNA Depletion syndrome (MDS) in a chemically exposed RAAF personnel cohort via Dual-Labelled Probe Real-Time PCR. The significance of these studies is in their contribution to the knowledge of the genetic pathways that are malformed in the progression and development of the pre-cancerous skin lesion Solar Keratosis. Furthermore, it would determine whether the genes analysed in this study exist in greater prevalence in the affected Solar Keratosis population compared to the control cohort. With regard to the MDS component, identifying the presence of this disease in these individuals was initially undertaken as part of a study to provide evidence in compensation claims. The diagnosis may assist in their medical therapy, insofar as some of them were now suffering from liver malfunctions and atypical male breast cancer. Another application of this effective and low cost method of diagnosing MDS is in populations with high HTV incidences. This is due to the fact that the most common drug used to treat this disease can give rise to the expression of MDS, thus further complicating the health status of HIV infected individuals. The analysis of this research was accomplished via the Real-Time PCR technique, with a non-specific fluorophore component in addition to specific Dual-Labelled Probe components, to ascertain the general nature of any aberration identified in the sample cohort. This project also employed additional methods of analysis such as DHPLC and DNA sequencing to assist in determining the veracity of its aims, particularly in terms of the precise detection of genetic aberrations via Real-Time PCR. Patients exhibiting male breast cancer and liver malftinctions were also analysed via Dual-Labelled Probe RealTime PCR to ascertain the presence of Mitochondrial DNA Depletion syndrome, a disorder characterised by lactic acidosis, liver failure, seizures, and congestive heart failure. Determining the presence of this syndrome in these patients would assist in their medical treatment, and contribute to the analytical methods available to diagnose this syndrome, which is known to occur in HIV sufferers due to the nucleoside drugs used to combat the disease. Real-Time PCR can adequately gauge the integrity of a genetic area in terms of amplicon malformities (non-specific-fluorophoric) and DNA copy number aberrations (Dual-Labelled Probe) via fluorophore signal differentials compared to wild-type samples and housekeeper profiles. The results of the first component of this project, namely the analysis of five gene pairs by non-specific fluorophoric Real-Time PCR, highlighted that a significantly higher incidence of putative aberrants is evident in the affected population when compared to the control cohort. The genes analysed were NDUFA8, PTCH, NDUFA5, SMOH, SDHD, MMP 12, NDUFVI, EMS 1, COXVIIc, and RASA 1. These ten genes were subdivided into five pairs; one of the pair being a gene associated with the development of a non-melanotic skin cancer (NMSC), the other a gene encoding for a subunit of the Electron Transport Chain (ETC). Each of these pairs exists in close proximity to one another on a particular chromosomal locale. Differences were highlighted in the single gene triplicate run population. The ETC genes (NDUFA8, NDUFA5, SDHD, NIDUFVI, COXVIIc) exhibited 10 / 720 (1.37%) as being putative mutants in the control population, compared to 117 / 675 (17.3%) for the affected population (p value less than 0.0001). The NMSC gene analysis (PTCH, SMOH, MMPI2, EMSI, RASA1) produced a 16 / 720 (2.22%) ratio for the control population, with the affected population having an incidence of 97 / 675 (14.4 %) for putative mutants (p value less than 0.0001). The observance of putative aberrants in the NDUFVI (p less than 0.018), EMS1 (p less than 0.003), COXVTIc (p less than 0.001), and RASA I (p less than 0.009) genes in the affected Solar Keratosis (SK) population was significantly higher than that observed in the control population. The majority of aberrations detected via the non-specific fluorophoric Real-Time PCR technique were small nucleotide base insertions and deletions. The analysis of the SK affected and control cohort via Real-Time PCR proved a cost-effective and reliable method in identifying the presence of DNA aberrations such as non-instructional sites. The results of the second component extended the findings of the non-specific fluorophoric analysis. The SDHD and MMPI 2 genes were analysed for copy number aberrations via Dual-Labelled Probe Real-Time PCR for genetic aberrations the same affected and control Solar Keratosis cohort. It was found that 12 of 279 samples had identifiable copy-number aberrations in either the SDHD or MMPI2 gene (this means that a genetic section of either of these two genes is aberrantly amplified or deleted), with five of the samples exhibiting aberrations in both genes. The MMPI2 gene also had nine samples identified as possessing an intronic heterozygous base-pair substitution anomaly via DNA sequencing. The NDUFA8 gene had 12 samples identified as anomalous via the DHPLC technique, 11 of which were identified via non-specific fluorophoric Real-Time PCR, with the analysis performed to verify the accuracy of the Real-Time technique in identifying DNA aberrations. This study identified DNA aberrations in an affected Solar Keratosis and control cohort and ascertained several particular genomic abnomialities in the SDHD, MMPI2 and NDUFA8 genes, with an emphasis on copy-number aberrations and amplicon abnormalities. In the third component of this study, namely the analysis of Mitochondrial DNA Depletion syndrome (MDS) in a jet-fuel exposed RAAF personnel cohort via Dual-Labelled Probe Real-Time PCR, the results indicated that four of the seven patients were expressing MDS. Of the four patients who exhibited a reduction in mitochondrial copy-number the average decrease was of a four-fold level, or approximately a depletion of mitochondrial copies from 200 plus to ~ 54 (74 % reduction in MtDNA). The patients who contributed DNA for investigation into the presence of MDS were suffering from liver malfunction and atypical male breast cancer. The Dual-Labelled Probe technique proved a reliable and cost effective method in identifying the presence of MDS in these patients, with the DNA extracted from fresh white blood cells that had been isolated using the Ficoll-Hypaque method. The importance of this is that accurate levels of Mitochondrial DNA copy numbers can be ascertained in white blood cells as it removes the presence of platelets, which also contain mitochondria but no nucleus. The analysis of ETC and NMSC associated genes in addition to mitochondrial copy number integrity means that this study investigated two aspects of the carcinogenetic pathway i.e. abnormal energy regulation and the regulation of micromolecular and macromolecular cellular homeostatic mechanisms. The mechanism of programmed cell death or apoptosis is regulated by the mitochondria and the ability of a genetically damaged cell to evade the apoptotic process is directly linked to a cell becoming cancerous. It is only after the evasion of apoptosis and the replication of the damaged cells' DNA into daughter cells that neoplastic events can occur. Thus, this study contributed to the understanding of how neo-plastic lesions may develop and progress into invasive tumours. It additionally assisted in proving the effectiveness of the RealTime PCR technique in detecting DNA aberrations and mitochondrial copy number anomalies.

<p>ABSTRACT</p><p>A defect implantation process is the major reason for unexplained infertility. Estrogen andprogesterone are steroid hormones preparing the endometrium for implantation. They mediatetheir effect through their receptors: estrogen receptor alpha and beta and progesteronereceptor A and B, respectively. Leukemia inhibitory factor (LIF), which is also important forimplantation, mediates its effect through LIF receptor and the coreceptor, gp130, and is downregulated by suppressors of cytokine signaling 1. The aim of the study was to compare thelevels of the steroid hormone receptors and LIF related factors in the endometrium of twogroups of women with the diagnosis unexplained infertility: one that became pregnant afterassisted reproduction and one that did not become pregnant. Before treatment of thesewomen, endometrial mRNA was collected during the window of implantation in themenstrual cycle. The levels of specific mRNAs were measured with real-time PCR. Womenwho had become pregnant had a significantly higher level of steroid hormone receptors. Thus,these proteins seem to be important for a pregnancy and may be suitable as receptivitymarkers.</p>

<p>The field of forensic genetics is growing fast and the development and optimisation of more sensitive, faster and more discriminating forensic DNA analysis methods is highly important. In this thesis, an evaluation of the use of novel DNA technologies and the development of specific applications for use in forensic casework investigations are presented.</p><p>In order to maximise the use of valuable limited DNA samples, a fast and user-friendly Real-time PCR quantification assay, of nuclear and mitochondrial DNA copies, was developed. The system is based on the 5’ exonuclease detection assay and was evaluated and successfully used for quantification of a number of different evidence material types commonly found on crime scenes. Furthermore, a system is described that allows both nuclear DNA quantification and sex determination in limited samples, based on intercalation of the SYBR Green dye to double stranded DNA. </p><p>To enable highly sensitive DNA analysis, Pyrosequencing of short stretches of mitochondrial DNA was developed. The system covers both control region and coding region variation, thus providing increased discrimination power for mitochondrial DNA analysis. Finally, due to the lack of optimal assays for quantification of mitochondrial DNA mixture, an alternative use of the Pyrosequencing system was developed. This assay allows precise ratio quantification of mitochondrial DNA in samples showing contribution from more than one individual.</p><p>In conclusion, the development of optimised forensic DNA analysis methods in this thesis provides several novel quantification assays and increased knowledge of typical DNA amounts in various forensic samples. The new, fast and sensitive mitochondrial DNA Pyrosequencing assay was developed and has the potential for increased discrimination power.</p>

Malignant catarrhal fever (MCF) is a lymphoproliferative and inflammatory syndrome affecting primarily ruminant species. The disease, which is often fatal, is most often described as affecting bovids and cervids. No vaccines are available for prevention of MCFV infection. The primary method to control spread of disease is to prevent contact between carriers and clinically susceptible species. There is no known method to control infection of malignant catarrhal fever virus-white-tailed deer variant (MCFV-WTD), as the carrier animal of this virus is unknown. To determine the prevalence of malignant catarrhal fever viruses in Tennessee ruminant populations, blood and/or lymph node samples were collected from farms, animal processing and disposal facilities, and hunter check-in stations from 2006-2008 from several species of animals including cervids, cattle, and goats. Strain-specific real time PCR was developed to detect ovine herpesvirus-2 (OvHV-2), caprine herpesvirus-2 (CpHV-2), and MCFV-WTD DNA. MCFV DNA was detected in all species of ruminants sampled. Although disease related to infection with MCFV-WTD and CpHV-2 has not been reported in Tennessee cattle or cervid populations, MCFV-WTD DNA was detected in 3 percent of cervid samples, and MCFV-WTD and CpHV-2 DNA was detected in 27 and 3 percent respectively of cattle samples from animal disposal facilities that process dead or debilitated animals. One hunter harvested deer (n=781) and 25 cattle (n=165) tested from animal disposal facilities were positive for OvHV-2 DNA. This study demonstrated that healthy cattle and cervids can be infected with OvHV-2 and MCFV-WTD without apparent disease, and dead or debilitated cattle were infected with OvHV-2, MCFV-WTD and CpHV-2 at a higher percentage than healthy herd animals. Prevalence of CpHV-2 in Tennessee goat populations (7%) was significantly lower than reported in other goat populations (73%). Low prevalence of CpHV-2 in Tennessee goat populations likely explains why no evidence of infection was found in cervids tested, and the low prevalence of CpHV-2 infection in dead or debilitated cattle compared to prevalence of infection with OvHV-2 and MCFV-WTD. The discovery of infection in cattle with CpHV-2 and MCFV-WTD opens a new avenue of investigation into the pathology and virulence of MCFV’s in domestic cattle.

The field of forensic genetics is growing fast and the development and optimisation of more sensitive, faster and more discriminating forensic DNA analysis methods is highly important. In this thesis, an evaluation of the use of novel DNA technologies and the development of specific applications for use in forensic casework investigations are presented. In order to maximise the use of valuable limited DNA samples, a fast and user-friendly Real-time PCR quantification assay, of nuclear and mitochondrial DNA copies, was developed. The system is based on the 5’ exonuclease detection assay and was evaluated and successfully used for quantification of a number of different evidence material types commonly found on crime scenes. Furthermore, a system is described that allows both nuclear DNA quantification and sex determination in limited samples, based on intercalation of the SYBR Green dye to double stranded DNA. To enable highly sensitive DNA analysis, Pyrosequencing of short stretches of mitochondrial DNA was developed. The system covers both control region and coding region variation, thus providing increased discrimination power for mitochondrial DNA analysis. Finally, due to the lack of optimal assays for quantification of mitochondrial DNA mixture, an alternative use of the Pyrosequencing system was developed. This assay allows precise ratio quantification of mitochondrial DNA in samples showing contribution from more than one individual. In conclusion, the development of optimised forensic DNA analysis methods in this thesis provides several novel quantification assays and increased knowledge of typical DNA amounts in various forensic samples. The new, fast and sensitive mitochondrial DNA Pyrosequencing assay was developed and has the potential for increased discrimination power.