<p> The mtDNA control region of the Brown-headed Cowbird (Molothrus ater) was sequenced and comparisons made at the inter- and intraspecific level. Comparison of the control region with that of another Passerine, Darwin's Finch (Geospiza scandens), revealed a high degree of both gross and fine scale structural similarity. At the nucleotide level, this comparison confirmed the presence of a hypervariable domain which evolves at rate approximately 5 times faster than coding mtDNA as well as a relatively conserved central domain which evolves at rate comparable to coding mtDNA. Both species displayed the typical avian mtDNA gene organisation previously described by Desjardins and Morais (1990, 1991) and Quinn and Wilson (in press). However, the most notable structural feature in common was the apparent deletion of the entire left hypervariable domain (CR1). At a finer scale, Conserved Sequence Block (CSB1) was perfectly conserved between cowbird and finch and Conserved Sequence Block 2 (CSB2) was 78% similar. The hypervariable right domain showed the largest degree of sequence divergence between species, 22.7%, while the central domain and phe-tRNA showed much less divergence, 6.47 and 4.41% respectively. At an intraspecific level, in 524 bases of sequence from 31 nestling cowbirds from a population at Delta, Manitoba, only 3 variable sites were detected which defined a total of 4 haplotypes. The average percent sequence divergence for this population was 0.27%. This level of variation within the cowbird population is low compared to other vertebrate populations. This relative lack of variation is largely attributable to the loss of the left hypervariable domain (CR1). The loss of CR1 will limit the control region's usefulness for high resolution population level studies but may make it a useful marker for phylogenetic studies within the class Aves.</p> / Thesis / Master of Science (MSc)

A deficiência de CD40 Ligante (CD40L) ou síndrome de Hiper-IgM ligada ao X (X-HIGM) é considerada uma imunodeficiência primária combinada de células T e B. O CD40L é expresso na superfície de linfócitos T ativados e interage com o CD40 expresso na superfície de linfócitos B, macrófagos, células dendríticas, células endoteliais e neutrófilos. A interação CD40L-CD40 transmite sinais que induzem ativação, diferenciação e proliferação celular. Nosso objetivo foi analisar as alterações genético-moleculares da molécula CD40L que acometeram indivíduos de 5 famílias brasileiras, ocasionando X-HIGM. Genotipamos 25 indivíduos, sendo 6 pacientes com X-HIGM, 13 parentes relacionados heterozigotos e 6 homozigotos sadios. Dentre os pacientes com X-HIGM dois eram de origem caucasóide e 4 eram mestiços. A idade dos pacientes variou de 2 a 20 anos e o quadro clínico de infecções de repetição teve início em média nos primeiros 4 meses de vida. As principais infecções recorrentes manifestadas pelos pacientes foram pneumonia e otite. O paciente TB apresentou blastomicose, observação original nesta imunodeficiência. A análise genético-molecular foi heterogênea. No paciente TB foi detectado um defeito de splicing levando a deleção do exon 3 (r.345_402del do gene CD40L (CD40LG) no paciente FS uma nova substituição missense g.11856 G>C (c.476 G>C, pW140C), no paciente KC uma substituição nonsense g.11855 G>A (c.475G>A, p. W140X), e nos pacientes CH, FE e VIC uma deleção g. 3074_3077delTAGA, levando a alteração no processamento do RNA. A fenotipagem dos leucócitos demonstrou que a contagem de linfócitos T auxiliares (CD3+CD4+), linfócitos citotóxicos (CD3+CD8+), linfócitos B (CD19+CD40+) e linfócitos T ativados (CD3+CD69+) dos pacientes foram similares aos controles sadios. Contudo, foi observada uma redução significativa nos níveis de expressão de CD40L na superfície de linfócitos CD3+ e CD4+ dos pacientes. A análise dos linfócitos T por microscopia confocal revelou que as células dos homozigotos com expressão residual do CD40L em sua superfície também apresentam redução na densidade da expressão da molécula CD3, sugerindo a necessidade da integridade molecular do CD40L para a expressão normal do CD3. Concluímos que mutações no CD40L que levam à síndrome de X-HIGM são heterogêneas e a análise genético-molecular permitiu um diagnóstico preciso tornando possível o aconselhamento genético e a triagem dos recém-nascidos das famílias avaliadas. / CD40-Ligand (CD40L) deficiency or X linked Hyper-IgM syndrome (X-HIGM) is considered a T and B cell combined primary immunodeficiency. CD40L is expressed on the cell surface of activated T lymphocytes and interacts with CD40, expressed on the surface of B lymphocytes, macrophages, dendritic cells, endothelial cells, and neutrophils. The CD40L-CD40 interaction induces activation, differentiation, and cell proliferation. Our aim was to analyze the molecular-genetic alterations of CD40L molecule affecting individuals of 5 brazilian families, leading to X-HIGM. We genotyped 25 individuals, whom 6 were X-HIGM patients, 13 were heterozygote related patients, and 6 were healthy homozygotes. Within the patients with X-HIGM, two of them were of caucasoid origin and four were mestiços. The patients age ranged from 2 to 20 years and their recurrent infections started in average during their first 4 months of life. The main recurrent infections were pneumonia and otitis. The patient TB presented blastomycosis, a unique observation in this immunodeficiency. The molecular-genetic analysis revealed heterogeneity. TB patient presented a splicing defect causing a deletion of exon 3 (r.345_402del) of CD40L gene (CD40LG). Patient FS presented a new missense mutation g.11856 G>C (c.476 G>C, pW140C). Patient KC presented a nonsense substitution g.11855 G>A (c.475G>A, p. W140X). Patients CH, VIC, and FE presented a deletion g. 3074_3077delTAGA, causing an alteration on RNA processing. The leukocytes fenotyping demonstrated that T helper lymphocytes (CD3+CD4+), T cytotoxic lymphocytes (CD3+CD8+), B lymphocytes (CD19+CD40+), and T activated (CD3+CD69+) cell counts of patients were similar to healthy controls. However it was observed a significant reduction of CD40L expression on cell surface patients CD3+ and CD4+ lymphocytes. The T lymphocyte confocal microscopy analysis revealed that homozygotes with residual expression of CD40L in their surface also presented a reduction on the density of CD3 molecule expression, suggesting the need of molecular integrity of CD40L for normal CD3 expression. We conclude that mutations on CD40L leading to X-HIGM syndrome are heterogeneous and the molecular-genetic analysis allowed a precise diagnosis making possible the genetic counseling and newborn screening of the involved families.

A deficiência de CD40 Ligante (CD40L) ou síndrome de Hiper-IgM ligada ao X (X-HIGM) é considerada uma imunodeficiência primária combinada de células T e B. O CD40L é expresso na superfície de linfócitos T ativados e interage com o CD40 expresso na superfície de linfócitos B, macrófagos, células dendríticas, células endoteliais e neutrófilos. A interação CD40L-CD40 transmite sinais que induzem ativação, diferenciação e proliferação celular. Nosso objetivo foi analisar as alterações genético-moleculares da molécula CD40L que acometeram indivíduos de 5 famílias brasileiras, ocasionando X-HIGM. Genotipamos 25 indivíduos, sendo 6 pacientes com X-HIGM, 13 parentes relacionados heterozigotos e 6 homozigotos sadios. Dentre os pacientes com X-HIGM dois eram de origem caucasóide e 4 eram mestiços. A idade dos pacientes variou de 2 a 20 anos e o quadro clínico de infecções de repetição teve início em média nos primeiros 4 meses de vida. As principais infecções recorrentes manifestadas pelos pacientes foram pneumonia e otite. O paciente TB apresentou blastomicose, observação original nesta imunodeficiência. A análise genético-molecular foi heterogênea. No paciente TB foi detectado um defeito de splicing levando a deleção do exon 3 (r.345_402del do gene CD40L (CD40LG) no paciente FS uma nova substituição missense g.11856 G>C (c.476 G>C, pW140C), no paciente KC uma substituição nonsense g.11855 G>A (c.475G>A, p. W140X), e nos pacientes CH, FE e VIC uma deleção g. 3074_3077delTAGA, levando a alteração no processamento do RNA. A fenotipagem dos leucócitos demonstrou que a contagem de linfócitos T auxiliares (CD3+CD4+), linfócitos citotóxicos (CD3+CD8+), linfócitos B (CD19+CD40+) e linfócitos T ativados (CD3+CD69+) dos pacientes foram similares aos controles sadios. Contudo, foi observada uma redução significativa nos níveis de expressão de CD40L na superfície de linfócitos CD3+ e CD4+ dos pacientes. A análise dos linfócitos T por microscopia confocal revelou que as células dos homozigotos com expressão residual do CD40L em sua superfície também apresentam redução na densidade da expressão da molécula CD3, sugerindo a necessidade da integridade molecular do CD40L para a expressão normal do CD3. Concluímos que mutações no CD40L que levam à síndrome de X-HIGM são heterogêneas e a análise genético-molecular permitiu um diagnóstico preciso tornando possível o aconselhamento genético e a triagem dos recém-nascidos das famílias avaliadas. / CD40-Ligand (CD40L) deficiency or X linked Hyper-IgM syndrome (X-HIGM) is considered a T and B cell combined primary immunodeficiency. CD40L is expressed on the cell surface of activated T lymphocytes and interacts with CD40, expressed on the surface of B lymphocytes, macrophages, dendritic cells, endothelial cells, and neutrophils. The CD40L-CD40 interaction induces activation, differentiation, and cell proliferation. Our aim was to analyze the molecular-genetic alterations of CD40L molecule affecting individuals of 5 brazilian families, leading to X-HIGM. We genotyped 25 individuals, whom 6 were X-HIGM patients, 13 were heterozygote related patients, and 6 were healthy homozygotes. Within the patients with X-HIGM, two of them were of caucasoid origin and four were mestiços. The patients age ranged from 2 to 20 years and their recurrent infections started in average during their first 4 months of life. The main recurrent infections were pneumonia and otitis. The patient TB presented blastomycosis, a unique observation in this immunodeficiency. The molecular-genetic analysis revealed heterogeneity. TB patient presented a splicing defect causing a deletion of exon 3 (r.345_402del) of CD40L gene (CD40LG). Patient FS presented a new missense mutation g.11856 G>C (c.476 G>C, pW140C). Patient KC presented a nonsense substitution g.11855 G>A (c.475G>A, p. W140X). Patients CH, VIC, and FE presented a deletion g. 3074_3077delTAGA, causing an alteration on RNA processing. The leukocytes fenotyping demonstrated that T helper lymphocytes (CD3+CD4+), T cytotoxic lymphocytes (CD3+CD8+), B lymphocytes (CD19+CD40+), and T activated (CD3+CD69+) cell counts of patients were similar to healthy controls. However it was observed a significant reduction of CD40L expression on cell surface patients CD3+ and CD4+ lymphocytes. The T lymphocyte confocal microscopy analysis revealed that homozygotes with residual expression of CD40L in their surface also presented a reduction on the density of CD3 molecule expression, suggesting the need of molecular integrity of CD40L for normal CD3 expression. We conclude that mutations on CD40L leading to X-HIGM syndrome are heterogeneous and the molecular-genetic analysis allowed a precise diagnosis making possible the genetic counseling and newborn screening of the involved families.

The growing obesity epidemic in America carries with it numerous health risks, including diabetes, increased serum lipid levels, and excess fat accumulation in the liver. If these conditions persist or become exacerbated, they may lead to the development of cardiovascular disease, the current leading cause of death among Americans, or to nonalcoholic fatty liver disease (NAFLD) which can progress to hepatocellular carcinoma (HCC), one of the deadliest forms of cancer. Better understanding of the genes involved in these diseases can lead to improved identification of at-risk individuals and treatment strategies. Our lab previously identified zinc fingers and homeoboxes 2 (Zhx2) as a regulator of hepatic gene expression. The BALB/cJ mouse strain has a hypomorphic mutation in the Zhx2 gene, causing a 95% reduction in Zhx2 protein expression. The near ablation of Zhx2 in BALB/cJ mice confers protection from cardiovascular disease when fed a high fat diet, yet these mice show increased hepatic lipid accumulation and liver damage. Microarray data indicates Zhx2 may be involved in the regulation of numerous genes involved in lipid metabolism. Recent GWAS studies indicate ZHX2 may contribute to the risk of cardiovascular disease and liver damage in humans as well. In this dissertation, I characterize the role of Zhx2 expression in the liver and how it affects the risk of both cardiovascular disease and liver damage. I generated liver-specific Zhx2 knockout mice and confirmed Zhx2 regulates several novel targets that could contribute to the fatty liver phenotype seen in BALB/cJ mice. Further studies revealed that hepatic Zhx2 expression is necessary for proper sex-specific expression of several Cyptochrome P450 (CYP) genes and could contribute to gender differences in disease susceptibility. Lastly, I performed studies into the functional role of the Zhx2 target gene Elovl3. A mouse model of HCC revealed that Elovl3 is completely repressed in HCC tumors. Cell viability and cell cycle assays indicate that Elovl3 expression slows cell proliferation and may be important for proper cell cycle checkpoints. Together, these data indicate that Zhx2 and/or its targets could be clinically relevant in the detection, prevention, or treatment of cardiovascular disease, fatty liver, and HCC.

Numerous epidemiology studies suggest a correlation between exposures to polychlorinated biphenyls (PCBs) and the development and severity of type 2 diabetes (T2D); however, mechanisms remain largely unknown. Previous studies demonstrated that PCBs that are ligands of the aryl hydrocarbon receptor (AhR) promote the expression of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), that are linked to insulin resistance in adipocytes. To explore potential mechanisms linking PCB exposures to diabetes, we developed a mouse model of glucose and insulin intolerance induced by acute and chronic exposures to PCB-77. We hypothesized that PCB ligands of AhR result in adipocyte-specific elevations in TNF-α and dysregulated glucose homeostasis. Results demonstrated that PCB77 resulted in rapid and sustained glucose and insulin intolerance in low fat (LF)-fed mice, and that these effects were associated with adipose-specific elevations in TNF-α. When mice were made obese from consumption of a high fat (HF) diet, effects of PCB77 were lost presumably due to concentration of the toxin in adipose lipids. However, upon weight loss, mice exposed to PCB77 exhibit impaired glucose homeostasis. These results suggest that lipophilic PCBs redistribute from adipose lipids with weight loss and mitigate beneficial effects to improve glucose homeostasis. To define the role of adipocyte AhR in PCB-induced diabetes, we created a mouse 3 model of adipocyte AhR deficiency using the Cre/LoxP system. Adipocyte-AhR deficiency conferred protection from the development of PCB-77-induced impairments in glucose and insulin tolerance in obese mice undergoing weight loss. Unexpectedly, adipocyte-AhR deficient mice fed the HF diet exhibited adipocyte hypertrophy, increased adipose mass and elevated body weight. These results suggest that (1) adipocyte AhRs are responsible for effects of PCB77 to impair glucose homeostasis during weight loss and (2) adipocyte AhRs respond to the HF diet to regulate adipose mass and body weight. We used resveratrol as a putative AhR antagonist to determine if the polyphenol confers protection against PCB-77-induced diabetes. Resveratrol abolished acute effects of PCB77 to impair glucose and insulin tolerance in LF-fed mice. Notably, PCB77 administration abolished insulin-induced phosphorylation of Akt in adipose tissue and these effects were abolished by resveratrol. Resveratrol also abolished marked suppressions in glucose uptake in adipocytes exposed to PCB77. These studies suggest the adipocyte AhR plays a potentially significant role in the development of diabetes and obesity, and that resveratrol may represent a novel therapeutic for PCB exposed populations.

A pesquisa de linhagens com duplicação cromossômica, como a linhagem A de Aspergillus nidulans, teve oseu início no final da década de 1970. Durante este período foram isolados da linhagem A, diversos variantes deteriorados, que foram caracterizados genética e citologicamente. Neste trabalho de pesquisa, as analises genéticas demonstraram que os determinantes de deterioração ou segmentos de inserção de V5, V101, V102, V103 e V104 estão localizados nos grupos de ligação VIII, III, IV, VII e I respectivamente. As análises citogenéticas revelaram diversas alterações no ciclo celular e migração nucleares nas fases iniciais de desenvolvimento. A duplicação cromossômica da linhagem A e os variantes deteriorados foram investigados a nível molecular, por técnica de PCR. Os resultados mostraram que o segmento de inserção consiste de um provável Elemento de Transposição, denominado de MATE, o qual é característico do fungo Aspergillus nidulans. Os segmentos de inserção analisados apresentam características típicas de MATE, como o motivo "Spe" que é encontrado por toda seqüência dos Elementos MATE. / The research with chromosome duplication strains, as strain A of Aspergillus nidulans, began during the 70's, with isolation of several deteriorates variants of strain A and characterization by genetic and cytological analysis. In this work the genetic analysis has demonstrated that the deterioration determinant or insertion sequence in V5, V101, V102, V103 and V104 deteriorates variants are located in the linkages groups VIII, III, IV, VII and I, respectively. The cytological analyses have demonstrated changes in cellular cycle and nuclear migration in initial phases of development. The chromosome duplication of strain A and the deteriorated variants were investigated by PCR with designed primers to mobile elements, what have resulted in the identification of the transposable element MATE, mainly by great similarity with "Spe" motif sequence that is described as essential in activity of these elements.

A cachaça é uma bebida típica brasileira produzida a partir da destilação do caldo de canade- açúcar fermentado principalmente por Saccharomyces cerevisiae. Grande parte da produção nacional é artesanal, e não há uma preocupação por parte dos produtores quanto ao controle microbiológico da fermentação. Este trabalho objetivou caracterizar a comunidade bacteriana contaminante do fermento utilizado na produção de cachaça. Foram coletadas quatro amostras em um alambique artesanal. A primeira (NA) foi coletada um ano anterior às outras três e utilizada como controle. As restantes foram coletadas ao final do primeiro dia de fermentação (NP), após quinze (NS) e trinta dias (NT) utilizando o mesmo fermento. Um total de 587 seqüências de 16S rDNA foram analisadas, sendo 81 da amostra NA, 177 da amostra NP, 159 da amostra NS e 170 da amostra NT. As análises das seqüências revelaram a presença de 17 gêneros e 27 espécies, além de 27 bactérias não conhecidas. Cento e setenta unidades taxonômicas operacionais (UTOs) foram identificadas utilizando o software DOTUR com uma distância evolucionária de 0.03. Quarenta e três UTOs foram identificadas na amostra controle (NA), 38 na NP, 57 na amostra NS e 38 na terceira amostra (NT). Das 170 UTOs identificadas, apenas dezessete foram identificadas duas vezes em diferentes amostras e somente uma, identificada como Lactobacillus hilgardii, foi identificada três vezes, evidenciando uma grande dinâmica populacional bacteriana durante o processo fermentativo. Análises estatísticas utilizando o software S-LIBSHUFF indicaram diferenças significativas na composição bacteriana entre amostras. Foram encontradas espécies/gêneros ainda não descritas na literatura em fermentos de cachaça, como Weissella cibaria, Leuconostoc citreum e algumas espécies de Lactobacillus. Além destas, várias bactérias não conhecidas também foram identificadas. Os resultados revelaram que a comunidade de bactérias contaminantes do processo fermentativo é muito mais complexa do que se conhecia, com características heterofermentativas e produção de congêneres que provavelmente refletem na qualidade da bebida. Este é o primeiro relato da utilização do método de seqüenciamento do gene 16S rDNA para determinar contaminantes bacterianos em fermentados de cana-de-açúcar para produção de cachaça. / Cachaça is a typical Brazilian spirit made from sugar cane fermented mainly by Saccharomyces cerevisiae. The production is mostly artisanal, and there is no microbiological control during the fermentation process. The objective of this study was to assess the bacterial community associated with the ferment used in the production of cachaça. Four ferment samples were collected from an artisanal still. The first one (NA), was collected one year previously to the other three and constituted a control reference. The remaining three samples were collected at the end of the first day of fermentation (NP), and fifteen (NS) and thirty days (NT) after using this same ferment. A total of 587 16S rDNA sequences were analyzed, being 81 sequences from the NA sample, 177 from NP, 159 from NS, and 170 from the NT sample. Sequence analyses revealed the presence of 17 genus and 27 species plus 27 unknown species. One hundred and seventy operational taxonomic units (OTUs) were identified using the DOTUR software using a cut-off evolutionary distance of 0.03. Forty three were identified in the control (NA) sample, 38 in the NP, 57 in NS, and 38 in the third (NT) sample. Of the 170 OTUs, only seventeen were detected twice in different samples and only one, identified as Lactobacillus hilgardii, was identified thrice, indicating a dynamic nature of the bacterial community during the fermentation process. Statistical analysis using the software S-LIBSHUFF indicated significant differences in the bacterial composition among samples. Our analyses also allowed to identify several bacteria not yet described as contaminants of the cachaça ferment, such as Weissella cibaria, Leuconostoc citreum and some Lactobacillus species. In addition, several unknown bacteria were also detected. The results revealed a much larger bacterial contaminant community present in the fermentative process of cachaça than previously reported with heterofermentative ability to produce secondary compounds which probably influence the quality of the final beverage. This is the first report on the utilization of the 16S rDNA sequencing method to assess the bacterial diversity of sugar cane fermentation for the production of cachaça.

Two trials were conducted using 48 infant pigs of both sexes. Pigs were taken from the sow when three to seven days old and placed in wire-bottomed metal cages by pairs. Animals in the first trial received synthetic diets containing 100 or 1000 international units of ergocalciferol and 1000 or 4000 international units of retinyl acetate or four or sixteen milligrams of beta carotene per kilogram of feed. Baby pigs in the second trial received the same synthetic diet plus 1000 international units of ergocalciferol (D2) or cholecalciferol (D3) and 2000 international units of vitamin A or eight milligrams of beta carotene per kilogram of feed. Neither growth nor feed efficiency were influenced by diet, but in Trial II, pigs receiving vitamin D2 showed higher gains per kilogram of diet than pigs fed vitamin D3. The weight and density of ribs and femurs did not show any differences due to diet. Different sources and levels of vitamins A and D did not effect strength characteristics of femurs. However, trends indicated 1000 IU of vitamin D in Trial I and vitamin D2 in Trial II slightly increased the bone ash in comparison with beta carotene and in Trial II retinyl acetate with vitamin D2 resulted in the maximum amount of mineral deposition in bone. Liver as a percent of body weight was higher in pigs treated with beta carotene than in those fed retinyl acetate either with vitamin D2 or D3. Serum total sulfur was increased in pigs receiving vitamin D3 in Trial II. Inorganic sulfur in Trial I was lowered significantly by high levels of vitamin D and either low beta carotene, low retinyl acetate, or high beta carotene. There were not significant effects of level and source of vitamin A or D on uronic acid or total sulfur concentration in cartilage. However, on the high level of dietary vitamin D, uronic acid in cartilage was slightly increased (Trial I). Trends indicated that beta carotene increased and high levels of vitamin D lowered total sulfur in cartilage (Trial I).

Vitamin D has been connected with increased intramyocellular lipid (IMCL) mitochondrial function in skeletal muscle. It is also shown to prevent lipotoxicity in several tissues, but this has not yet been examined in skeletal muscle. Perilipin 2 (PLIN2), a lipid droplet protein upregulated with vitamin D treatment, is integral to managing IMCL capacity and lipid oxidation in skeletal muscle. Increased lipid storage and oxidation is associated with increased tolerance to a hyperlipidic environment and resistance to lipotoxicity. Therefore, I hypothesized that vitamin D increases β-oxidation and lipid turnover though a PLIN2 mediated mechanism, thereby preventing lipotoxicity. This hypothesis was divided into two specific aims: 1) Characterize the effect of vitamin D and PLIN2 on lipid turnover and β-oxidation in mature myotubes, and 2) Determine the role of vitamin D and PLIN2 in regulating key markers of lipotoxicity. To address these aims, cells were treated with or without vitamin D, palmitate, and PLIN2 siRNA in an eight group, 2x2x2 design. Key experiments included quantitative real time polymerase chain reaction for markers of lipid accumulation, lipolysis, and lipotoxicity; Seahorse oxygen consumption assay; 14C-palmitate oxidation assay; and analyses of lipid accumulation and profile. Failure of the palmitate treatment to produce a reliable model for lipotoxicity resulted in negative data for Aim 2 of this dissertation and a focus on vitamin D and PLIN2 knockdown treatments as a four group, 2x2 model. Aim 1 showed that vitamin D reliably increases markers of lipolysis and lipid accumulation. Most of these markers were in turn decreased after PLIN2 knockdown, and DGAT2 exhibited an interaction effect between the two treatments. Contrary to our hypothesis and some published research, PLIN2 knockdown did not prevent lipid accumulation. Vitamin D increased oxygen consumption, especially consumption driven by mitochondrial complex II. PLIN2 knockdown decreased oxygen consumption and demonstrated an interaction effect specific to mitochondrial complex II. Data in this dissertation show that vitamin D increases mitochondrial function, and these effects are at least in part accomplished through a PLIN2 mediated mechanism. However, this work lacks the data required to make specific claims regarding β-oxidation and lipid turnover. This research is some of the first to show that PLIN2 knockdown carries negative impacts for skeletal muscle mitochondria and makes valuable contributions to general knowledge of how vitamin D and lipid storage impact muscle health and function. This ultimately provides additional evidence to advocate for vitamin D supplementation as a means of improving musculoskeletal health and function. Future research should investigate how vitamin D and PLIN2 impact markers of lipotoxicity in skeletal muscle.

Consumption of endophyte-infected tall fescue results in a syndrome of negatively altered physiological systems, collectively known as fescue toxicosis. Another challenge to endophyte-infected tall fescue -based beef cattle operations is that the soils often are selenium (Se) poor, necessitating the need to provide supplemental Se. To test the general hypothesis that different forms of supplemental Se would ameliorate the negative effects of fescue toxicosis, predominately-Angus steers (BW = 183 ± 34 kg) were randomly selected from herds of fall-calving cows grazing an endophyte-infected tall fescue pasture and consuming vitamin-mineral mixes that contained 35 ppm Se as sodium selenite (ISe), SELPLEX (OSe), or an 1:1 blend of ISe and OSe (MIX). Steers were commonly weaned and depleted of Se for 98 d. Steers were assigned (n = 8 per treatment) to the same Se-form treatments upon which they were raised and subjected to summer-long common grazing of an endophyte-infected tall fescue pasture (0.51 ppm ergot alkaloids: ergovaline plus ergovalinine; 10.1 ha). Selenium treatments were administered by daily top-dressing 85 g of vitamin-mineral mix onto 0.23 kg soyhulls, using in-pasture Calan gates. The first project objective was to determine the effect of forms of supplemental Se on whole blood Se, serum prolactin, liver glutamine synthetase (GS) activity, carcass parameters, and growth performance (Experiment 1). In Experiment 1, whole blood Se increased for all treatments from day 0 to 22 and then did not change. Across periods, MIX and OSe steers had greater whole blood Se than ISe steer. Compared to ISe steers, MIX and OSe steers had more serum prolactin. Liver GS mRNA, protein content, and activity were greater in MIX and OSe steers than ISe steers. However, the ADG and carcass parameters were not affected by Se treatments. The second project objective was to determine the effect of forms of supplemental Se on serum clinical parameters of Experiment 1 steers (Experiment 2). In Experiment 2, across periods, MIX steers had more serum albumin than OSe, and ISe steers, respectively. Serum alkaline phosphatase (ALP) activity was greater in MIX and OSe steers. In addition, blood urea nitrogen (BUN), serum sodium, phosphorus, and magnesium concentration were affected by Se treatments. Partial correlation analysis revealed that serum albumin, BUN, and ALP activity were correlated with whole blood Se concentration. The third project objective was to evaluate the hepatic transcriptome profiles of Experiment 1 steers using microarray and targeted RT-PCR analyses (Experiment 3). In Experiment 3, bioinformatic analysis of microarray data indicated that hepatic glutamate/glutamine, proline, arginine, and citrulline metabolism was affected by different forms of supplemental Se. The mRNA expression of critical proteins involved in glutamate/glutamine (GLS2, GLUD1, GLUL), proline (PYCR1, ALDH18A1), and urea (ARG1, ARG2, OAT, NAGS, OTC, ORNT1) metabolism were differentially expressed by Se treatments. Collectively, we conclude that consumption of 3 mg Se/d as OSe or MIX forms of Se in vitamin-mineral mixes 1) increased whole blood Se content, an indicator of greater whole-body Se assimilation; 2) increased serum prolactin, albumin, and ALP, the reduction of which are hallmarks of fescue toxicosis; and 3) altered hepatic nitrogen metabolism, as indicated by changes in key enzymes of glutamate/glutamine, proline, and urea metabolism. However, 4) these positive effects on metabolic parameters were not accompanied by increased growth performance.