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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Investigation of the Effects of Xenoestrogens on the Protein Levels of the Estrogen Receptors

Lang, Claudia Nicole January 2006 (has links)
There has been an increase in reports of male reproductive disorders that include male infertility and testicular cancer worldwide. It has been suggested that agents such as xenoestrogens could be responsible. Xenoestrogens are chemical compounds that mimic the action of estrogens by binding to the estrogen receptors (ERs). The response ofa testicular cell line to estrogenic pesticides was examined. The effect of estrogenic pesticides on the growth and protein levels of ERα and ERβ of mouse Sertoli cells was investigated. Pyrethroids are widely used insecticides due to their insecticidal potency and low mammalian toxicity. In this study, the estrogenicity ofpyrethroid chemicals were tested using the yeast estrogen screen (YES) assay. The toxic effects of the pyrethroid compounds cypermethrin, 3-(4-hydroxy-phenoxy)benzyl alcohol (metabolite of permethrin), and the commercial product (Ripcord Plus) were evaluated. The Sertoli cells were exposed to pyrethroids at concentrations of 0.36 nM and 36 µM (cypermethrin and Ripcord Plus), and 0.69 nM and 69 µM (metabolite) for 100 h. The expression of the ERs was analysed through the use of Enzyme Linked ImmunoSorbent Assay (ELISA) experiments. The most toxic pyrethroid was the metabolite, followed by Ripcord Plus then cypermethrin. Overall the exposure of the cells to cypermethrin (36 µM), Ripcord Plus (36 µM) and the metabolite (69 µM) caused a significant decrease (p<0.05) in ERα levels. In the cultures exposed to the metabolite (69 µM), there was also a significant increase in ERβ levels. There appears to be a relation between cell toxicity and an increase in ERβ levels, which supports the theory that ERβ promotes apoptosis. Pyrethroids are rapidly excreted from the body, and it is unknown if there is accumulation in the male testes. Male fertility could be affected through molecular mechanisms involving the ERs, should cells in the male testes be exposed to these pyrethroids at physiologically relevant concentrations.
2

Gut microbiota dynamics in the weaner pig in response to experimental Escherichia coli challenge and dietary manipulation

Pollock, Jolinda January 2017 (has links)
The weaning transition period in pigs is linked to increased vulnerability to enteric disorders, which is partly attributed to destabilisation of the gut microbiota. Post-weaning colibacillosis is an economically important disease of the small intestine, which is most commonly caused by enterotoxigenic Escherichia coli (ETEC) strains. This disease has been variably linked to a diarrhoeal phenotype and decreased growth rate under clinical or sub-clinical conditions, and has been associated with shifts in particular bacterial populations using culturing methods. The emergence of next-generation sequencing technologies such as 16S rRNA gene metabarcoding now allows higher resolution study of complex microbial communities, without being reliant on the ability to culture fastidious micro-organisms. As part of this project, a 16S rRNA gene metabarcoding method was developed and validated to allow qualitative and quantitative measurement of gut microbiota shifts. A series of experimental ETEC challenge trials were carried out to monitor temporal faecal microbiota dynamics (Chapter 2), to further understand ETEC adhesion and shedding dynamics (Chapter 3) and to study potential changes in both ileal and faecal microbiota populations in response to dietary protein manipulation (Chapter 4). The effects of experimental treatments on pig health and performance were also measured as part of each experiment. Temporal shifts in ileal and faecal microbiota structure and stability were observed over the post-weaning period, as well as shifts in relative abundances of particular bacterial phylotypes (P < 0.05) (Chapter’s 2 and 4). ETEC challenge had no effects on faecal microbiota composition, pig health and performance when comparing to samples obtained from sham-challenged pigs (P > 0.05). However, when taking ETEC shedding level into account, variations in both microbiota structure and stability were observed at specific time points (P < 0.05) (Chapter 2). After a single-dose ETEC challenge, ETEC adhesion in the ileum and faecal shedding were evident up to 4 and 6 days post-challenge, respectively (Chapter 3). Changes in ileal microbiota structure and stability were observed in response to ETEC challenge (P < 0.05), with no changes exerted at faecal level (P > 0.05). Additionally, different dietary protein levels were linked to changes in ileal microbiota structure, stability and phylotype relative abundances (P < 0.05). Interestingly, significant differences in ileal microbiota structure were evident in samples obtained from ETEC-challenged pigs fed the low and high protein diets, with the pigs fed the high protein diet having significantly less stable ileal communities at population level (P < 0.05) (Chapter 4). The treatments had no effect on host performance (P > 0.05), but faecal consistency scores were higher in pigs fed the high protein diet (P < 0.05). In conclusion, both ETEC challenge and manipulation of dietary protein level had profound effects on ileal microbiota composition and faecal microbial communities were variable according to ETEC shedding status. These findings have implications for the development of alternative management strategies for enteric diseases in weaner pigs.
3

Evaluation of serum C-reactive protein levels as a predictor of outcome in puppies infected with parvovirus

McClure, Vanessa 25 June 2013 (has links)
Canine Parvovirus remains a leading cause of enteritis in dogs in South Africa and many other countries despite the wide availability of effective vaccines. The virus does not affect all dogs equally and the course of the disease depends on the age, immune status and breed of the puppies as well as the viral dose, route of exposure and the virulence of the strain. Although aggressive supportive treatment can be successful, the treatment and convalescent periods may be prolonged and consequently expensive and the mortality rate relatively high, causing many clients to forego treatment and elect for euthanasia of their pet. Acute phase proteins (APP) are proteins that change in concentration by at least 25% in animals subjected to external or internal inflammatory challenges, such as infection, inflammation or surgical trauma. Increased concentrations are associated with poor outcome in certain diseases. C-reactive protein (CRP) is the most sensitive APP in dogs. Its normal physiological concentration is low but increases rapidly with inflammation or tissue destruction. Due to the fact that CRP has a relatively short half life in serum (6-8 hours) and a high response in diseased animals, it can be used as a valid measure of a systemic response to an initiating stimulus at the time of blood sampling. By taking serial measurements, objective information about the extent of the ongoing lesions in the patient can be obtained and therefore may be used as a prognostic indicator. The objective of this prospective observational study was to evaluate the association of serum CRP concentrations in puppies suffering from canine parvoviral enteritis with morbidity and mortality, and to determine the usefulness of CRP to predict duration of hospitalisation time. Seventy-nine client owned puppies naturally infected with canine parvovirus were included. Parvovirus infection was diagnosed on electron microscopic examination of faeces from the puppies. CRP was measured using an automated human C-Reactive Protein Turbidimetric Immunoassay (TIA), which has been validated for use in dogs. Serum CRP measurements were performed at admission, twice daily for the first 48 hours, then once daily until death or discharge. There was a positive association between odds of mortality and CRP concentration on admission, as well as 12 and 24 hours after admission (P=0.04,P=0.005 and P=0.003, respectively). Survival time was negatively associated with CRP concentration at 12 and 24 hours after admission (P=0.002and P=0.001, respectively). Among the survivors, length of hospitalisation was positively associated with CRP concentration at 12, 24 and 36 hours after admission (P=0.012, P=0.001 and P=0.002, respectively). Utility for CRP concentration to correctly differentiate between survivors and non-survivors at 24 hours after admission had a sensitivity and specificity of 78.7% and 86.7% respectively. Although serum CRP concentration is associated with outcome in puppies infected with canine parvovirus, when used alone it did not prove to be a good predictor of survival. / Dissertation (MMedVet)--University of Pretoria, 2012. / Companion Animal Clinical Studies / unrestricted
4

Investigation of the Effects of Xenoestrogens on the Protein Levels of the Estrogen Receptors

Lang, Claudia Nicole January 2006 (has links)
There has been an increase in reports of male reproductive disorders that include male infertility and testicular cancer worldwide. It has been suggested that agents such as xenoestrogens could be responsible. Xenoestrogens are chemical compounds that mimic the action of estrogens by binding to the estrogen receptors (ERs). The response ofa testicular cell line to estrogenic pesticides was examined. The effect of estrogenic pesticides on the growth and protein levels of ERα and ERβ of mouse Sertoli cells was investigated. Pyrethroids are widely used insecticides due to their insecticidal potency and low mammalian toxicity. In this study, the estrogenicity ofpyrethroid chemicals were tested using the yeast estrogen screen (YES) assay. The toxic effects of the pyrethroid compounds cypermethrin, 3-(4-hydroxy-phenoxy)benzyl alcohol (metabolite of permethrin), and the commercial product (Ripcord Plus) were evaluated. The Sertoli cells were exposed to pyrethroids at concentrations of 0.36 nM and 36 µM (cypermethrin and Ripcord Plus), and 0.69 nM and 69 µM (metabolite) for 100 h. The expression of the ERs was analysed through the use of Enzyme Linked ImmunoSorbent Assay (ELISA) experiments. The most toxic pyrethroid was the metabolite, followed by Ripcord Plus then cypermethrin. Overall the exposure of the cells to cypermethrin (36 µM), Ripcord Plus (36 µM) and the metabolite (69 µM) caused a significant decrease (p<0.05) in ERα levels. In the cultures exposed to the metabolite (69 µM), there was also a significant increase in ERβ levels. There appears to be a relation between cell toxicity and an increase in ERβ levels, which supports the theory that ERβ promotes apoptosis. Pyrethroids are rapidly excreted from the body, and it is unknown if there is accumulation in the male testes. Male fertility could be affected through molecular mechanisms involving the ERs, should cells in the male testes be exposed to these pyrethroids at physiologically relevant concentrations.
5

Novel approach to cancer therapeutics using comparative cancer biology

Revi, Bhindu January 2018 (has links)
Developing personalized cancer therapies based on cancer genomics methodologies forms the basis for future cancer therapeutics. A genomics platform was developed based on canine cancer to produce a proof-of-concept for personalized genomics led therapeutic choices but also developing personalized therapeutics for canine cancer patients themselves. The platform identified the genetic state of a canine cancer patient within two drugable pathways; p53 and HSP90/IRF1. The former gene was wild-type p53 thus directing the use of p53 activating molecules. The latter mutations in both HSP90 and IRF1 suggested an investigation into HSP90 and interferon signalling molecules as drug leads. Drugs that target both of these pathways were subsequently used to measure drug effects in cell line models but also to identify novel biomarkers of drug responses. My study focused on the effect of the HSP90-inhibitor Ganetespib had on its client proteins, particularly IRF-1. Briefly my results indicated the following:(i) Ganetespib downregulated IRF-1 protein levels in A375 cell lines and this attenuation was not mediated by either MDM2 or CHIP (E3 ligase). IFNγ- induced IRF-1 was also observed to be downregulated when Ganetespib was used in combination therapy.(ii) Insitu proximity ligation assay showed induced HSC70 upregulation upon HSP90 inhibition by Ganetespib and HSC70/MDM2 complexes were seen to be stabilized compared to the usage of MDM2/p53 inhibitor-nutlin. I hypothesize that MDM2/HSC70 complex might chaperon IRF-1 into lysosome for degradation via chaperon mediated autophagy pathway. (iii) My results also indicate that Ganetespib can downregulate IFN γ- induced PDL-1 expression in melanoma cell lines. Pre-sensitizing the cells with Ganetespib prior to the addition of IFNγ could attenuate PDL-1 to basal levels. (iv) My results also showed that the downregulation of PDL-1 by Ganetespib is an IRF-1 dependent mechanism. Therefore, my results suggest that HSP90 represents an important emerging target for cancer therapy because its inactivation results in the simultaneous blockade of multiple signalling pathways and can also sensitize tumor cells to other anticancer agents. Targeting HSP90 could also help to disrupt PD1/PDL- 1 interaction and activate immune system to recognise tumor cells. I conclude that HSP90 and IRF-1 play a critical role in types II interferon pathways and these findings establish a novel basis for the design of future Ganetespib-based combinatorial approaches to improve patient outcomes in this disease. These approaches finally demonstrate that cancer genomics can stratify choice of cancer drugs used on patients but also provide evidence that cancer patient samples can be used for the specific stratification of cancer drug choice based on cancer genomics data.
6

Utilização do conceito de proteína ideal para perus de corte fêmea: desempenho, retenção de nitrogênio corporal e excreção de nitrogênio / Using the concepto of ideal protein for turkeys cutting female: performance, nitrogen retention and excretion of body nitrogen

João Guilherme Ferreira 19 June 2012 (has links)
Avaliaram-se diferentes níveis de proteína bruta para perus de corte fêmeas, utilizando-se 576 aves de zero a 84 dias (Experimento I), 550 aves de 28 a 56 dias (Experimento II) e 550 aves dos 57 aos 84 dias de idade (Experimento III). O delineamento experimental foi inteiramente casualizado com seis tratamentos e seis repetições. Nos três experimentos, as dietas dos tratamentos eram isoenergéticas, sendo à base de milho e farelo de soja, a suplementação dos aminoácidos foram realizadas, quando necessário. No Experimento I, a fase inicial recebeu tratamentos correspondentes aos níveis 23, 24, 25, 26, 27 e 28% de PB, em dietas com 3020 kcal de EM/kg. Nas duas fases seguintes (fase de ganho compensatório) as dietas foram de 26 e 23% de PB e com 3100 e 3250 de EM/kg para as fases de crescimento e final, respectivamente. No Experimento II, os tratamentos corresponderam aos níveis de 21, 22, 23, 24, 25 e 26% de PB, em dietas com 3100 de EM/kg. No Experimento III, os níveis para os tratamentos foram: 17, 18, 19, 20, 21 e 22% de PB, com 3250 de EM/kg. As variáveis avaliadas foram ganho de peso, consumo de ração, conversão alimentar, composição e deposição de nutrientes corporais, e na fase final (do primeiro e terceiro experimento) as características e rendimento de cortes na carcaça. Na fase inicial, do Experimento I, os níveis de PB dietéticos influenciaram o consumo de ração, consumo de PB e excreção de nitrogênio, constatando-se resposta (P<0,001) linear crescente. Observou-se efeito (P<0,01) quadrático para peso vivo e ganho de peso. Dos componentes químicos, expressos em porcentagem na carcaça, houve resposta (P<0,001) quadrática para todas as variáveis. Nas fases posteriores não houve resposta para nenhuma variável, observando-se um efeito de ganho compensatório, possibilitando a redução protéica na fase inicial. No Experimento II, houve resposta linear crescente para as variáveis de peso vivo, consumo de ração, ganho de peso, deposição de proteína e água. As variáveis restantes não apresentaram resposta significativa (P>0,05), o nível de 26% foi estabelecido como melhor nível para desempenho e deposição de nutrientes nesta fase. Foram encontradas respostas no desempenho, excreção de nitrogênio e composição química, no experimento III, apenas a viabilidade não foi influenciada pelo nível de PB, todas as outras variáveis obtiveram resposta linear decrescente (P<0,05). Para as variáveis de rendimento de abate, houve resposta linear para o rendimento de carcaça (P<0,01) e quadrática para rendimento de peito e peito sem pele e osso, permitindo uma redução para 17% de PB na fase final. Essas informações obtidas nos três experimentos confirmam que a redução de PB pode ser feita sem prejudicar o desempenho e a deposição protéica, reduzindo a excreção de nitrogênio. / Was evaluated at different levels of crude protein for female turkeys, using 576 birds from zero to 84 days (Experiment I), 550 birds 28-56 days (Experiment II) and from 57 to 550 birds 84 days (Experiment III). The experimental design was completely randomized with six treatments and six repetitions. In three experiments, the diets were isocaloric treatments, being based on corn and soybean meal supplementation of amino acids were supplemented when necessary. In Experiment I, the initial treatment received corresponding to levels 23, 24, 25, 26, 27 and 28% CP in diets with 3020 kcal / kg. In the next two phases (phase of compensatory growth) diets were 26 and 23% CP and 3100 and 3250 / kg for the grower and finisher phases, respectively. In Experiment II, the treatments were to levels 21, 22, 23, 24, 25 and 26% CP in diets with 3,100 / kg. In Experiment III, levels for the treatments were: 17, 18, 19, 20, 21 and 22% CP with 3,250 / kg. The parameters evaluated were weight gain, feed intake, feed conversion, composition and body nutrient deposition, and the final stage (the first and third experiment) the characteristics and yield of the carcass. In the initial phase of Experiment I, the levels of dietary CP influenced feed intake, CP intake and nitrogen excretion, we noted the response (P <0.001) increased linearly. Effect was observed (P <0.01) quadratic for body weight and weight gain. Of chemical components, expressed in percentage in the carcass was no response (P <0.001) quadratic for all variables. In later stages there was no answer for any variable, observing an effect of compensatory growth, leading to a protein in the initial phase. In Experiment II, there was a linear correlation for the variables of body weight, feed intake, weight gain, protein deposition and water. The remaining variables showed no significant response (P> 0.05), the level of 26% was established as a better standard for performance and nutrient deposition at this stage. Responses were found in performance, nitrogen excretion and composition in experiment III, only the viability was not affected by CP level, all other variables had decreased linearly (P <0.05). Variables of income for the slaughter, linear response to the carcass (P <0.01) and quadratic for breast meat yield and breast without skin and bone, allowing a reduction to 17% CP in the finals. This information obtained from the three experiments confirm that the reduction of CP can be done without sacrificing performance and protein deposition, reducing nitrogen excretion.
7

Utilização do conceito de proteína ideal para perus de corte fêmea: desempenho, retenção de nitrogênio corporal e excreção de nitrogênio / Using the concepto of ideal protein for turkeys cutting female: performance, nitrogen retention and excretion of body nitrogen

Ferreira, João Guilherme 19 June 2012 (has links)
Avaliaram-se diferentes níveis de proteína bruta para perus de corte fêmeas, utilizando-se 576 aves de zero a 84 dias (Experimento I), 550 aves de 28 a 56 dias (Experimento II) e 550 aves dos 57 aos 84 dias de idade (Experimento III). O delineamento experimental foi inteiramente casualizado com seis tratamentos e seis repetições. Nos três experimentos, as dietas dos tratamentos eram isoenergéticas, sendo à base de milho e farelo de soja, a suplementação dos aminoácidos foram realizadas, quando necessário. No Experimento I, a fase inicial recebeu tratamentos correspondentes aos níveis 23, 24, 25, 26, 27 e 28% de PB, em dietas com 3020 kcal de EM/kg. Nas duas fases seguintes (fase de ganho compensatório) as dietas foram de 26 e 23% de PB e com 3100 e 3250 de EM/kg para as fases de crescimento e final, respectivamente. No Experimento II, os tratamentos corresponderam aos níveis de 21, 22, 23, 24, 25 e 26% de PB, em dietas com 3100 de EM/kg. No Experimento III, os níveis para os tratamentos foram: 17, 18, 19, 20, 21 e 22% de PB, com 3250 de EM/kg. As variáveis avaliadas foram ganho de peso, consumo de ração, conversão alimentar, composição e deposição de nutrientes corporais, e na fase final (do primeiro e terceiro experimento) as características e rendimento de cortes na carcaça. Na fase inicial, do Experimento I, os níveis de PB dietéticos influenciaram o consumo de ração, consumo de PB e excreção de nitrogênio, constatando-se resposta (P<0,001) linear crescente. Observou-se efeito (P<0,01) quadrático para peso vivo e ganho de peso. Dos componentes químicos, expressos em porcentagem na carcaça, houve resposta (P<0,001) quadrática para todas as variáveis. Nas fases posteriores não houve resposta para nenhuma variável, observando-se um efeito de ganho compensatório, possibilitando a redução protéica na fase inicial. No Experimento II, houve resposta linear crescente para as variáveis de peso vivo, consumo de ração, ganho de peso, deposição de proteína e água. As variáveis restantes não apresentaram resposta significativa (P>0,05), o nível de 26% foi estabelecido como melhor nível para desempenho e deposição de nutrientes nesta fase. Foram encontradas respostas no desempenho, excreção de nitrogênio e composição química, no experimento III, apenas a viabilidade não foi influenciada pelo nível de PB, todas as outras variáveis obtiveram resposta linear decrescente (P<0,05). Para as variáveis de rendimento de abate, houve resposta linear para o rendimento de carcaça (P<0,01) e quadrática para rendimento de peito e peito sem pele e osso, permitindo uma redução para 17% de PB na fase final. Essas informações obtidas nos três experimentos confirmam que a redução de PB pode ser feita sem prejudicar o desempenho e a deposição protéica, reduzindo a excreção de nitrogênio. / Was evaluated at different levels of crude protein for female turkeys, using 576 birds from zero to 84 days (Experiment I), 550 birds 28-56 days (Experiment II) and from 57 to 550 birds 84 days (Experiment III). The experimental design was completely randomized with six treatments and six repetitions. In three experiments, the diets were isocaloric treatments, being based on corn and soybean meal supplementation of amino acids were supplemented when necessary. In Experiment I, the initial treatment received corresponding to levels 23, 24, 25, 26, 27 and 28% CP in diets with 3020 kcal / kg. In the next two phases (phase of compensatory growth) diets were 26 and 23% CP and 3100 and 3250 / kg for the grower and finisher phases, respectively. In Experiment II, the treatments were to levels 21, 22, 23, 24, 25 and 26% CP in diets with 3,100 / kg. In Experiment III, levels for the treatments were: 17, 18, 19, 20, 21 and 22% CP with 3,250 / kg. The parameters evaluated were weight gain, feed intake, feed conversion, composition and body nutrient deposition, and the final stage (the first and third experiment) the characteristics and yield of the carcass. In the initial phase of Experiment I, the levels of dietary CP influenced feed intake, CP intake and nitrogen excretion, we noted the response (P <0.001) increased linearly. Effect was observed (P <0.01) quadratic for body weight and weight gain. Of chemical components, expressed in percentage in the carcass was no response (P <0.001) quadratic for all variables. In later stages there was no answer for any variable, observing an effect of compensatory growth, leading to a protein in the initial phase. In Experiment II, there was a linear correlation for the variables of body weight, feed intake, weight gain, protein deposition and water. The remaining variables showed no significant response (P> 0.05), the level of 26% was established as a better standard for performance and nutrient deposition at this stage. Responses were found in performance, nitrogen excretion and composition in experiment III, only the viability was not affected by CP level, all other variables had decreased linearly (P <0.05). Variables of income for the slaughter, linear response to the carcass (P <0.01) and quadratic for breast meat yield and breast without skin and bone, allowing a reduction to 17% CP in the finals. This information obtained from the three experiments confirm that the reduction of CP can be done without sacrificing performance and protein deposition, reducing nitrogen excretion.
8

A Loss of the Fragile X mental retardation protein alters the spatial and temporal expression of glutamate receptors in the mouse brain

Majaess, Namat-Maria 20 December 2012 (has links)
Fragile X Syndrome (FXS) is the leading cause of inherited intellectual disability. The disorder is caused by a trinucleotide expansion that silences the Fragile X Mental Retardation 1 (Fmr1) gene resulting in the loss of its protein product, the Fragile X Mental Retardation Protein (FMRP). FXS patients show broad clinical phenotypes including intellectual disability, as well as a number of cognitive and behavioral problems. The lack of FMRP is believed to be the direct cause of the deficits seen in FXS patients. FMRP is an RNA-binding protein that is expressed in the brain and testes. This protein is believed to form a messenger ribonucleoprotein complex with mRNAs in the nucleus and subsequently export them to polyribosomes in the cytoplasm, therefore influencing translation of its bound mRNAs. Importantly, FMRP has long been suspected to be involved in synaptic plasticity due to its ability to bind several mRNAs that encode for proteins important in synaptic plasticity. Such proteins include the GluN1, GluN2A and GluN2B subunits of the N-methyl-D- aspartate receptor (NMDAR). FMRP is expressed in the hippocampus, a region of the brain involved in learning and memory processes. Recently, impaired NMDAR functioning in the dentate gyrus (DG) subregion of the hippocampus has been observed in Fmr1 knockout (-/y) mice. This impairment also resulted in reduction in long-term potentiation (LTP) and long-term depression (LTD) of synaptic efficacy, two biological models of learning and memory. In the present study, I focused on the levels of the NMDAR GluN1, GluN2B and Glu2B subunits in order to determine the synaptic plasticity alterations seen in the DG of Fmr1-/y mice. Using Western blotting, I found that there is a decrease in the GluN1, GluN2A and GluN2B subunits in the DG of young adult Fmr1-/y mice, indicating that these mice have significantly lower amounts of total NMDARs. These results could explain the altered LTP and LTD seen in Fmr1-/y mice at the molecular level and might contribute to the intellectual impairments seen in these KO mice. NMDARs appear to be important in the development and maturation of synapses. The GluN2A and GluN2B subunits are developmentally regulated, where GluN2B is predominantly expressed early in development and GluN2A in the adult brain. A dysregulation of GluN2A and GluN2B subunits has been proposed to affect the maturation and formation of synapses. Intriguingly, FMRP is also believed to play a functional role in early brain development. Thus, this study also focused on the developmental expression of the GluN1, GluN2A and GluN2B subunits in the DG, Cornu Ammonis, prefrontal cortex and cerebellum of Fmr1-/y mice, all of which are brain regions implicated in FXS. We found that the developmental expression of these subunits is altered in Fmr1-/y mice in specific brain regions. Together, these results demonstrate that the loss of FMRP differentially affects GluN1, GluN2A and GluN2B subunit expression both developmentally and spatially, further implicating NMDARs in the pathophysiology of FXS. / Graduate
9

Desempenho e características de carcaça de juvenis de carpa capim (ctenopharyngodon idella) em resposta a níveis e fontes de proteína da dieta / Growth and carcass characteristics of grass carp (ctenopharyngodon idella) juveniles in response of dietary protein levels and sources

Veiverberg, Cátia Aline 06 February 2009 (has links)
Conselho Nacional de Desenvolvimento Científico e Tecnológico / This work was carried out to evaluate the performance and carcass characteristics of grass carp (Ctenopharyngodon idella) juveniles in response to dietary protein levels and sources. For this, two experiments were conducted: the first (80 days), evaluating four crude protein (CP) levels (22, 30, 36 and 44%) and the second (60 days), comparing protein sources in the diet: FCS (porcine meat meal - control); FC: canola meal; FG: sunflower meal and FCG: canola meal + sunflower meal. Both experiments were conducted in a water re-use system composed of 12 tanks (850 L), with three replicates per treatment. In experiment 1, 10 fish by tank (initial weight 153,0±1,5g) were fed 3% of body weight, twice daily, and the experiment 2 was provided ration (2% of biomass) in the morning and forage (Napier grass ad libitum) in the afternoon, to 15 fish by tank (initial weight 54,6±1,0g). Growth parameters (weight, specific growth rate, daily weight gain, relative weight gain and feed conversion ratio) and carcass (carcass and fillet yield, digestive somatic index, hepatic somatic index and visceral fat index, intestinal quotient, protein retention, protein efficiency rate and whole body and fillet protein and fat deposition) were evaluated. Moreover, the proximate composition (moisture, ash, fat and protein) in fillet and whole fish and blood parameters (glucose, total triglycerides, total cholesterol and total protein in both experiments and hematocrit in Experiment 1) were also evaluated. In experiment 2 was also determined the daily consumption of forage and the instrumental color. In experiment 1, linear positive effect of protein level for all growth variables was observed. However, the same effect was observed to whole body and fillet fat deposition, triglycerides and total cholesterol in serum, indicating that the protein from the diet was used as energy source. To feed conversion ratio and fat in whole fish, the effect was quadratic, with maximum response with 40.6 and 37.1% CP, respectively. Protein retention, protein efficiency ratio, protein deposition in whole body and fillet and hematocrit also showed linear positive effect, while the other parameters were not affected. In experiment 2, the growth parameters and the daily consumption of forage (1.24 to 2.11% of body weight) did not differ among the treatments. About proximate composition of whole fish, higher fat content and lower protein content, besides fillet ash, were obtained in the treatment FCG. The diet FCS presented the highest values of serum protein, triglycerides and total cholesterol. The fillet yield was higher in the treatments FCG and FC, while the digestive somatic index was higher in treatment FG and FCG. In the instrumental evaluation of color, the fillets from FCS and FCG diets showed higher value of L (brightness), differing only treatment FC. The other parameters did not differ among them. Based on this results, we can conclude that: the minimum protein level for maximum growth of grass carp in the growing phase, with practical diets, is 44%; the variation in dietary protein level promotes changes in metabolism of juvenile grass carp, reflected in hematological and carcass parameters; canola meal and sunflower meal can be used in diets for grass carp growing phase, when supplemented with limiting essential amino acids, without compromising growth. / Este trabalho foi conduzido com o objetivo de avaliar o desempenho produtivo e qualidade de pescado de juvenis de carpa capim (Ctenopharyngodon idella) em resposta a níveis e fontes de proteína da dieta. Para isso, foram conduzidos dois experimentos: o primeiro, com 80 dias, avaliando quatro níveis de proteína bruta (22, 30, 36 e 44%) e o segundo, com 60 dias, avaliando fontes protéicas na dieta, em combinação com farelo de soja: FCS: farinha de carne suína; FC: farelo de canola; FG: farelo de girassol e FCG: farelo de canola + farelo de girassol. Ambos os experimentos foram conduzidos em sistema de recirculação de água com temperatura controlada, composto de 12 unidades experimentais (850 L), com três repetições por tratamento. No experimento 1, 10 animais por unidade experimental (peso inicial 153,0 ± 18,2g) foram alimentados com ração (3% da biomassa) duas vezes ao dia. No experimento 2, foram utilizados 15 animais por unidade experimental (peso inicial 54,7 ± 7,8g), alimentados com ração (2% da biomassa) pela manhã e capim elefante (à vontade) à tarde. Foram avaliados os parâmetros de crescimento (peso, taxa de crescimento específico, ganho em peso diário e relativo e conversão alimentar aparente) e de carcaça (rendimento de carcaça e filé, índices digestivossomático, hepatossomático e de gordura visceral, quociente intestinal, coeficiente de retenção protéica e deposições de proteína e gordura corporal e no filé). Além disso, a composição centesimal (umidade, cinzas, gordura e proteína) no filé e no peixe inteiro e os parâmetros sangüíneos (glicose, triglicerídeos totais, colesterol total e proteínas totais nos dois experimentos e hematócrito no experimento 1) também foram avaliados. No experimento 2 também foi determinado o consumo diário de forragem e a medida instrumental da cor. No experimento 1, houve efeito linear positivo do nível de proteína para todas as variáveis de crescimento. Entretanto, o mesmo efeito foi observado para a deposição de gordura corporal e no filé, triglicerídeos totais e colesterol total no soro, indicando que a proteína proveniente da dieta estava sendo utilizada como fonte de energia. Para conversão alimentar aparente e gordura no peixe inteiro, o efeito foi quadrático, com ponto de máxima em 40,6% de PB e 37,1%, respectivamente. Coeficiente de retenção protéica, taxa de eficiência protéica, deposição de proteína corporal e no filé e hematócrito também apresentaram efeito linear positivo. No experimento 2, os parâmetros de crescimento não diferiram estatisticamente entre os tratamentos. O consumo de forragem variou entre 1,24 e 2,11% do PV por dia, não diferindo entre os tratamentos. Na composição centesimal do peixe inteiro, maior teor de gordura e menor teor de proteína foram obtidos no tratamento FCG, bem como para cinzas no filé. A dieta FCS foi a que apresentou maiores valores de proteínas, triglicerídeos e colesterol total circulantes. O rendimento de filé foi maior nos tratamentos FC e FCG, enquanto o índice digestivossomático foi maior nos tratamentos FG e FCG. Na avaliação instrumental da cor, os filés obtidos dos tratamentos FCS e FCG apresentaram maior valor de L (luminosidade), diferindo apenas do tratamento FC. Com base nos resultados obtidos, pode-se concluir que: o nível mínimo de proteína para o máximo crescimento da carpa capim na fase de recria, com dietas práticas, é de 44%; a variação do nível de proteína da dieta promove alterações no metabolismo dos juvenis de carpa capim, refletido nos parâmetros sangüíneos e de carcaça; farelo de canola e farelo de girassol podem ser utilizados em dietas para recria da carpa capim, quando for feita a suplementação com lisina e forragem, sem comprometer o crescimento.
10

Global analysis of cellular protein dynamics by pulse-labeling and quanti tati ve mass spectrometry

Schwanhäußer, Björn 05 April 2011 (has links)
Der erste Teil der Arbeit beschreibt die Etablierung einer modifizierten Form des klassichen SILAC-Verfahrens, das in der quantitativen Massenspektrometrie zur Bestimmung von relativen Änderungen in Proteinmengen benutzt wird. Im sog. „pulsed SILAC (pSILAC)“ Verfahren werden Zellen im Zuge einer differentiellen Behandlung in Kulturmedien transferiert, die unterschiedlich Isotop-markierte Aminosäuren enthalten. Da hier die Quantifizierung auf dem Verhältnis der neusynthetisierten Proteinmengen beruht, können gezielt Unterschiede in der Proteinproduktion bestimmt werden. Mit Hilfe von pSILAC konnte im zweiten Teil der Arbeit erstmals quantitativ erfasst werden, welchen Einfluss microRNAs auf die Proteinsynthese ausüben. So konnte gezeigt werden, dass sowohl die Überexpression als auch die Repression einzelner microRNAs die Produktion hunderter Proteine beeinflussen kann. Außerdem konnten Genprodukte identifiziert werden, die ausschließlich translational reguliert werden. Die Messung von Proteinneusynthese ermöglichte auch die Bestimmung von Proteinumsatzraten, dargestellt im dritten Teil der Arbeit. Zusammen mit mRNA-Umsatzraten sowie Protein- und mRNA-Mengen bilden sie die Grundlage für eine dynamische Beschreibung zelluärer Genexpression. Durch den gleichzeitigen Einsatz des Nukleosidanalogons 4-Thiouridin (4sU) und von schweren Aminosäuren (SILAC) konnte eine metabolische Markierung neusynthetiserter mRNAs und Proteine in murinen Fibroblasten erreicht und damit eine Berechnung von Protein- und mRNA-Halbwertszeiten und absoluten Mengen für ca. 5,000 Gene ermöglicht werden. Während mRNA- und Proteinenmengen deutlich korrelierten, war zwischen mRNA- und Proteinhalbwertszeiten nur eine äußerste schwache Korrelation zu erkennen. Dennoch stehen mRNA- und Proteinumsatzraten nicht einem willkürlichen Zusammhang zu einander, da bestimmte Kombinationen von mRNA- und Proteinhalbwertszeiten eine Optimierung von Genen hinsichtlich ihrer biologischen Funktionen erkennen ließen. / The first part of the thesis describes the establishment of a modified version of the classic SILAC approach routinely used in quantitative mass spectrometry (MS) to assay relative changes in protein levels. In the newly-devised approach termed pulsed SILAC (pSILAC) differentially treated cells are transferred to culture medium supplemented with different versions of stable-isotope labeled heavy amino acids. As MS-based relative quantification is exclusively based on the newly-synthesized heavy protein amounts the method enables the detection of differences in protein production resulting from the treatment. The second part of the thesis shows the use of pSILAC to globally quantify the impact of microRNAs onto the proteome. Ectopic over-expression or knock-down of a single microRNA both affected protein production of hundreds of proteins. pSILAC identified several target genes as exclusively translationally regulated as changes in corresponding transcript levels were virtually absent. Measuring newly-synthesized protein amounts with heavy amino acids in a pulsed-labeling fashion has also been used to determine turnover rates of individual proteins, described in the third part of the present work. Along with transcript turnover as well as mRNA and protein levels they are essential for a dynamic description of gene expression. Simultaneous application of the nucleoside analogue 4-thiouridine (4sU) and heavy amino acids (SILAC) to metabolically label newly-produced mRNAs and proteins in mouse fibroblasts resulted in the calculation of mRNA and protein lifetimes and absolute levels for approximately 5,000 genes. While mRNA and protein levels were overall well correlated, a correlation between mRNA and protein half-lives was virtually absent. Yet this seemingly chaotic distribution of mRNA and protein half-lives was highly instructive since specific gene subsets have obviously evolved distinct combinations of half-lives that relate to their biological functions.

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