Дисертації з теми "Genetic diease"

Mon doctorat porte sur l'étude de deux maladies génétiques récessives qui touchent le développement cérébral postnatal et résultent de variants dans des gènes impliquant l'appareil de Golgi. La première maladie étudiée, le syndrome de Dyggve-Melchior-Clausen (DMC) a conduit à impliquer en 2003 la première protéine golgienne, la DYMECLINE, dans une microcéphalie postnatale, et a ensuite mené au concept de Golgipathies neurodéveloppementales. Les atteintes neurodéveloppementales du DMC sont associées à une déficience intellectuelle et à des anomalies squelettiques spécifiques également postnatales (dysplasie spondylo-epi-métaphysaire) et résultent de la déficience du gène DYM codant pour la DYMECLINE dont l'absence induit un défaut de transport antérograde entre le réticulum endoplasmique et l'appareil de Golgi notamment dans les neurones. Un variant clinique du DMC sans microcéphalie ni déficience intellectuelle, mais dont les caractéristiques osseuses sont identiques, la dysplasie de Smith McCort (SMC), peut résulter soit de variants moins délétères du gène DYM (SMC1) soit de variants du gène codant la RAB-GTPase RAB33B (SMC2) suggérant une relation entre les deux protéines. Dans ce travail, j'ai montré que ces deux protéines co-localisent au cis-Golgi, interagissent physiquement, que la DYMECLINE est recrutée au Golgi par RAB33B et intervient dans le contrôle de l'autophagie dans des cellules non neuronales. Dans les neurones déficients pour Dym, les deux protéines sont en revanche faiblement co-localisées et l'autophagie n'est pas perturbée, mais des défauts de transport rétrograde de la membrane plasmique vers le Golgi ont été identifiés, associés à des anomalies de croissance dendritique et des défauts de maturation synaptique. La seconde partie de ma thèse porte sur l'identification d'un variant biallélique du gène GORASP1 chez un patient présentant un nouveau syndrome neurodéveloppemental caractérisé par des atteintes de la substance blanche, neurosensorielles, neuromusculaires et squelettiques. GORASP1 code pour la protéine golgienne GRASP65, connue pour son rôle dans la structure du Golgi, dans la glycosylation des protéines et dans le contrôle de l'entrée en mitose. Malgré ces fonctions apparemment essentielles et ubiquitaires, le gène n'a été impliqué dans aucune pathologie humaine. A partir à la fois des fibroblastes du patient et de cellules RPE où j'ai introduit par CRISPR/Cas9 une mutation imitant le variant du patient, j'ai montré que la protéine GRASP65 n'est plus présente dans les cellules mutées et j'ai identifié dans ces cellules des défauts de glycosylation et de mitose. Ces défauts n'empêchent cependant pas les cellules RPE de proliférer normalement. En étudiant un autre mutant généré par hasard dans les cellules RPE et qui s'est avéré produire une protéine tronquée en C-terminal mais stable, j'ai observé un phénotype de croissance cellulaire plus sévère que lorsque la protéine est totalement absente, suggérant des effets dominants négatifs de la protéine tronquée. En revanche ce mutant stable n'a pas montré de défauts de glycosylation. Cette étude a permis d'impliquer GRASP65 dans une maladie neurodéveloppementale et suggère qu'une absence totale de protéine est parfois moins délétère qu'une protéine tronquée stable
My PhD focuses on the study of two recessive genetic diseases that affect postnatal brain development and result from variants in genes involving the Golgi apparatus. The first disease studied, Dyggve-Melchior-Clausen syndrome (DMC), led in 2003 to the involvement of the first Golgi protein, DYMECLIN, in postnatal microcephaly, and subsequently to the concept of neurodevelopmental Golgipathies. In DMC, neurodevelopmental impairments are associated with intellectual disability and specific skeletal defects, of postnatal onset as well (spondylo-epi-metaphyseal dysplasia) and result from a deficiency in the DYM gene encoding DYMECLIN, this absence of which induces a defect in anterograde transport between the endoplasmic reticulum and the Golgi apparatus, notably in neurons. Smith McCort dysplasia (SMC), a clinical variant of DMC without microcephaly or intellectual deficiency, but with identical skeletal features, can result either from less deleterious variants of the DYM gene (SMC1) or from variants of the gene encoding the RAB-GTPase RAB33B (SMC2), suggesting a relationship between the two proteins. In this work, I have shown that these two proteins co-localize at the cis-Golgi, interact physically, that DYMECLIN is recruited to the Golgi by RAB33B and is involved in the control of autophagy in non-neuronal cells. However, in Dym-deficient neurons, the two proteins are weakly co-localized and autophagy is not disrupted, but defects in retrograde transport from the plasma membrane to the Golgi have been identified, associated with abnormalities in dendritic growth and defects in synaptic maturation. The second part of my thesis concerns the identification of a biallelic variant in the GORASP1 gene in a patient with a new neurodevelopmental syndrome characterized by white matter, neurosensory, neuromuscular and skeletal abnormalities. GORASP1 encodes the Golgi protein GRASP65, known for its role in Golgi structure, protein glycosylation and control of mitosis entry. Despite these apparently essential and ubiquitous functions, the gene has not been implicated in any human pathology so far. Using both patient's fibroblasts and RPE cells in which I introduced by CRISPR/Cas9 a mutation mimicking the patient's variant, I showed that the GRASP65 protein is no longer present in mutated cells, and identified glycosylation and mitosis defects in these cells. However, these defects do not prevent RPE cells from proliferating normally. Studying another mutant generated incidentally in RPE cells, which turned out to produce a C-terminally truncated but stable protein, I observed a more severe cell growth phenotype than when the protein is totally absent, suggesting dominant negative effects of the truncated protein. In contrast, this stable mutant showed no glycosylation defects. This study implicates GRASP65 in a neurodevelopmental disease and suggests that a total absence of the protein is sometimes less deleterious than a stable truncated protein

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
O estudo foi conduzido para avaliar o efeito de duas proporções de concentrado, 70 e 80%, com duas fontes energéticas, milho e farelo de gérmen de milho, e volumoso à base de silagem de milho, sob o consumo de nutrientes, desempenho, características de carcaça, não componentes de carcaça e qualidade da carne de 28 novilhas Nelore confinadas, com aproximadamente 14 meses de idade e peso corporal médio de 200 kg, objetivando o abate. O delineamento experimental utilizado foi o inteiramente casualizado segundo esquema fatorial 2 x 2 ( 2 proporções de concentrado x 2 fontes energéticas) totalizando 4 tratamentos e 7 repetições, sendo as médias comparadas pelo teste de Tukey a 5%. Não houve interação da proporção de concentrado x fonte energética da dieta sobre o consumo de MS. Houve efeito significativo da interação proporção de concentrado x fonte energética da dieta para os consumos de proteína bruta (PB) e fibra em detergente neutro (FDN), sendo que a maior proporção de concentrado, associada ao milho, possibilitou maior consumo diário de PB (1,17 kg/dia) e FDN (4,05 kg/dia). Observou-se valor médio de consumo de fibra em detergente ácido superior para as dietas com 80% de concentrado, tanto para a dieta com milho quanto com farelo de gérmen de milho. Houve efeito significativo da fonte energética da dieta sobre o peso final dos animais, sendo que os animais alimentados com as dietas contendo milho apresentaram valor médio de peso final (326,14 kg) superior aos alimentados com as dietas contendo farelo de gérmen de milho (292,07 kg). A associação de altas proporções de concentrado, com milho como fonte energética, proporciona resultados satisfatórios de ganho em peso e desempenho de novilhas Nelore, já a substituição total do milho pelo farelo de gérmen de milho, associado à altas proporções de concentrado ricos em fibra...
The objective of this study was to evaluate the effect of two proportions of concentrate, 70 and 80%, with two energy sources, corn and corn germ meal, and roughage basis of corn silage on the intake of nutrients, performance, characteristics of a carcass, non components of carcass and meat quality of confined 28 Nellore heifers. The experimental design was a completely randomized design in factorial scheme 2 x 2 (2 proportions of concentrate x 2 energy sources) 4 replications and 7 treatments, and the averages has been compared by Tukey 5% test.. There was no interaction proportion of concentrate x energy source on the intake of dry matter. There was a significant effect of interaction proportion of concentrate x energy source intake for crude protein (CP) and neutral detergent fiber (NDF), suggesting that a higher proportion of concentrate, with the corn, allowed greater intake of CP (1.17 kg / day) and NDF (4,05 kg / day). It was observed average intake of acid detergent fiber than for diets with 80% concentrate, with both the diet with corn as the corn germ meal. There was a significant effect of dietary energy source on the final weight of animals, and the animals fed the diets with corn had average final weight (326.14 kg), above the fed with diets with corn germ meal (292.07 kg). The association of high proportions of concentrate with corn as an energy source, provided satisfactory results in performance of Nellore heifers, since the replacement of the corn germ meal, associated with high proportions of concentrate rich in fiber, not was recommended for young Nellore heifers. There was a significant effect the energy source of the diet on the weight of empty body, and the animals that received the diets with corn showed the average value of empty body weight (309.44 kg) than the diets of with corn germ meal (276.42 kg). The carcass yield, the thickness... (Complete abstract click electronic access below)

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É crescente o número de estudos que demonstram a importância de micronutrientes e compostos bioativos presentes nos alimentos na prevenção de diversas doenças degenerativas crônicas. Entretanto vários estudos moleculares epidemiológicos têm demonstrado que além de fatores ambientais, como a dieta, essas doenças degenerativas podem ser modulada por genes envolvidos no biometabolismo de xenobióticos, metabolismo do carbono e no reparo de DNA. Portanto, o presente estudo avaliou a possível influência do padrão alimentar e dos polimorfismos dos genes GSTP1, CYP1A1, XRCC1 e MTHFR sobre os níveis de danos oxidativos no DNA, uracilas incorporadas no DNA e eficiência do sistema de reparo de DNA em dois grupos de indivíduos residentes em Botucatu com diferentes padrões alimentares. Grupo I (GI): 87 indivíduos com alimentação rica em produtos orgânicos, grãos integrais, frutas e vegetais, e baixa ingestão de produtos industrializados; Grupo II (GII): 97 indivíduos com alimentação rica em produtos industrializados e pobres em frutas e vegetais. A quantificação do nível de danos oxidativos no DNA, uracilas incorporadas ao DNA e a eficiência do sistema reparo de DNA em linfócitos de sangue periférico, foi analisada utilizando-se o Teste do Ensaio Cometa. Os polimorfismos dos genes GSTP1, CYP1A1, XRCC1 e MTHFR foram analisados por realtime PCR. Também foi realizada a análise dos níveis de luteína, criptoxantina, -caroteno, -caroteno, licopeno, retinol e -tocoferol no plasma, pela técnica de cromatografia líquida de alta pressão (HPLC). Os indivíduos do GI apresentaram menores níveis de danos oxidativos no DNA e menores níveis de dano no DNA induzidos pela H2O2 quando comparados aos indivíduos do GII. Quanto aos subgrupos de micronutrientes: Indivíduos do subgrupo percentil 75 para todos os micronutrientes tiveram maior nível de danos no DNA do que os indivíduos...
Not available

Numerous studies have shown that respirable particles contribute to adverse human health outcomes including discomfort in irritated airways, increased asthma attacks, irregular heartbeat, non-fatal heart attacks, and even death. Particle emissions from diesel vehicles are a major source of airborne particles in urban areas. In response to energy security and global climate regulations, the use of biodiesel as an alternative fuel for petrodiesel has significantly increased in recent years. Particle emissions from diesel engines are highly dependent on fuel composition and, as such, the increased use of biodiesel in diesel vehicles may potentially change the concentration, size, and composition of particles in respirable air. One indicator used to evaluate the potential health risk of these particles to humans is particle diameter (Dp). Ultrafine particles (UFPs, Dp Current research in automotive emissions primarily focuses on particle emissions measured on a total particle mass (PM) basis from heavy-duty diesel vehicles. The nation's light-duty diesel fleet is, however, increasing; and because the mass of a UFP is much less than that of larger particles, the total PM metric is not sufficient for characterization of UFP emissions. As such, this research focuses on light-duty diesel engine transient UFP emissions, measured by particle number (PN), from petrodiesel, biodiesel, and blends thereof. The research objectives were to determine: 1) the difference in UFP emissions between petrodiesel and blends of waste vegetable oil-based biodiesel (WVO), 2) the differences between UFP emissions from blends of WVO and soybean oil-based biodiesel (SOY), and 3) the feasibility of using genetic programming (GP) to select the primary engine operating parameters needed to predict UFP emissions from different blends of biodiesel. The results of this research are significant in that: 1) Total UFP number emission rates (ERs) exhibited a non-monotonic increasing trend relative to biodiesel content of the fuel for both WVO and SOY that is contrary to the majority of prior studies and suggests that certain intermediate biodiesel bends may produce lower UFP emissions than lower and higher blends, 2) The data collected corroborate reports in the literature that fuel consumption of diesel engines equipped with pump-line-nozzle fuel injection systems can increase with biodiesel content of the fuel without operational changes, 3) WVO biodiesel blends reduced the overall mean diameter of the particle distribution relative to petrodiesel more so than SOY biodiesel blends, and 4) Feature selection using genetic programming (GP) suggests that the primary model inputs needed to predict total UFP emissions are exhaust manifold temperature, intake manifold air temperature, mass air flow, and the percentage of biodiesel in the fuel; These are different than inputs typically used for emissions modeling such as engine speed, throttle position, and torque suggesting that UFP emissions modeling could be improved by using other commonly measured engine operating parameters.

The current modern commercial pig is an animal that has evolved through deliberate breeding programs, controlled environmental influences and nutrition to yield a highly efficient feed converter and fertile animal. The objective of this study was to measure the growth performances and carcass characteristics of entire male grower – finisher pigs which were subjected to different seasonal variations, nutrient dense diets, feeding regimes and group situations under South African circumstances. Period 1 was conducted in the winter from 6 June 2008 to 13 August 2008 and Period 2 in the summer from 3 October 2008 to 10 December 2008. The sire lines that were selected for the experiment had the same genetic breeding values (Topigs Selection Index value), of which two sire lines were the same in both Period 1 and Period 2 and one of two different sires lines was used either in Period 1 or Period 2. The animals were fed two different rations, a high (FH) and a low ration (FL), with the low ration’s specifications being 95 % of the high ration. The animals were randomly allocated three different feed regimes throughout the trial; restricted single feeding (RSF), ad libitum single feeding (ASF) and ad libitum group feeding (AGF). In the winter animals had a greater growth response compared to the summer, with end weight and average daily gain being significant (P<0.05) higher. A significant (P<0.05) improvement in average daily gain, feed efficiency and protein deposition rate were observed when animals were fed a higher energy and protein content in their diet, especially during summer. A significant (P<0.05) improved feed conversion was observed for restricted animals, but end weight, average daily gain and average protein deposition rates were significant (P<0.05) lower compared to ad libitum group and individually fed animals. In conclusion; the impact of decreasing the nutrient density of the diet for growing pigs through incremental changes in diet composition had a variable impact on overall growth performance and carcass quality. Feeding the high energy and protein ration improved growth performance during summer, but also in the initial stages of growth when feed intake capacity was limited. The objective when formulating diets should be to provide the essential amino acids and energy in amounts needed to support maximal and efficient growth. Using growth models estimated optimal feed intake curves will not deliver optimal results. Only when measuring and calculating the actual feed intake and protein deposition rates optimal performance levels will be reached. Measurements of feed intake and growth performance data derived from pigs penned individually should be adjusted before they can be applied to commercial situations or research conditions in which pigs are penned in groups.
Dissertation (MSc(Agric))--University of Pretoria, 2011.
Animal and Wildlife Sciences
unrestricted

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Universidade Estadual Paulista (UNESP)
Para avaliar os efeitos da suplementação dietética de maltodextrina em substituição a lactose para leitões dos 21 aos 51 dias de idade, foram utilizados 120 animais, distribuídos em um delineamento experimental de blocos ao acaso com arranjo fatorial 2x4 (duas dietas: simples e complexa e níveis de maltodextrina substituindo a lactose: 0,0%; 33,5%; 66,5% e 100,0%). Foram avaliados o consumo diário de ração (CDR), o ganho diário de peso (GDP) e a conversão alimentar (CA) em dois períodos pósdesmame (0-15 dias e 0-30 dias). Não houve interação entre os níveis de maltodextrina e as dietas para as variáveis de desempenho. Os leitões que consumiram as dietas complexas apresentaram maior GDP (P = 0,016) no período de 0 a 15 dias e melhor CA nos períodos de 0 a 15 dias (P = 0,010) e de 0 a 30 dias (P = 0,020) pós-desmame. Não foram observados efeitos dos níveis de maltodextrina sobre o CDR e GDP nos dois períodos e na CA no período de 0 a 15 dias. O aumento do nível de maltodextrina na dieta dos leitões determinou efeito quadrático na CA no período de 0 a 30 dias (Ŷ = 1,530000 + 0,002287X + 0,000022X2; R2 = 0,64). As rações contendo somente maltodextrina em sua composição proporcionaram melhor resultado econômico. A maltodextrina pode substituir totalmente a lactose nas dietas pré-iniciais e iniciais, simples ou complexas, sem afetar o desempenho de leitões dos 21 aos 51 dias de idade.
One hundred and twenty piglets were used to evaluate the effects of dietary supplementation of maltodextrin as a replacement of lactose for weaned piglets from 21 to 51 days of age, in a randomized complete block design, according to a 2x4 factorial (two diet types: simple or complex; and levels of maltodextrin as a replacement of lactose: 0.0%, 33.5%, 66.5% and 100,0%). The daily feed intake (DFI), daily weight gain (DWG) and feed conversion (FC) were measured. No interaction between maltodextrin levels and diet type was observed. The pigs fed complex diets showed higher DWG from 0 to 15 days (P = 0.016) and better FC from 0 to 15 days (P = 0.010) and from 0 to 30 days (P = 0.020) post-weaning period. No effect of maltodextrin levels on DWG or DFI was observed from 0 to 15 days or from 0 to 30 days; on FC from 0 to 15 days. Maltodextrin showed a quadratic effect on FC from 0 to 30 days (Ŷ = 1.53000 + 0.002287X + 0.000022X2; R2 = 0.64). Diets containing only maltodextrin provided the best economical results. The maltodextrin can fully replace lactose in simple or complex pre-starter or starter diets without affecting performance weanling pigs from 21 and 51 days of age.

Orientadora: Jane Maria Bertocco Ezequiel
Banca: Alexandre Amstalden Moraes Sampaio
Banca: Antonio Ferriani Branco
Resumo: O estudo foi conduzido para avaliar o efeito de duas proporções de concentrado, 70 e 80%, com duas fontes energéticas, milho e farelo de gérmen de milho, e volumoso à base de silagem de milho, sob o consumo de nutrientes, desempenho, características de carcaça, não componentes de carcaça e qualidade da carne de 28 novilhas Nelore confinadas, com aproximadamente 14 meses de idade e peso corporal médio de 200 kg, objetivando o abate. O delineamento experimental utilizado foi o inteiramente casualizado segundo esquema fatorial 2 x 2 ( 2 proporções de concentrado x 2 fontes energéticas) totalizando 4 tratamentos e 7 repetições, sendo as médias comparadas pelo teste de Tukey a 5%. Não houve interação da proporção de concentrado x fonte energética da dieta sobre o consumo de MS. Houve efeito significativo da interação proporção de concentrado x fonte energética da dieta para os consumos de proteína bruta (PB) e fibra em detergente neutro (FDN), sendo que a maior proporção de concentrado, associada ao milho, possibilitou maior consumo diário de PB (1,17 kg/dia) e FDN (4,05 kg/dia). Observou-se valor médio de consumo de fibra em detergente ácido superior para as dietas com 80% de concentrado, tanto para a dieta com milho quanto com farelo de gérmen de milho. Houve efeito significativo da fonte energética da dieta sobre o peso final dos animais, sendo que os animais alimentados com as dietas contendo milho apresentaram valor médio de peso final (326,14 kg) superior aos alimentados com as dietas contendo farelo de gérmen de milho (292,07 kg). A associação de altas proporções de concentrado, com milho como fonte energética, proporciona resultados satisfatórios de ganho em peso e desempenho de novilhas Nelore, já a substituição total do milho pelo farelo de gérmen de milho, associado à altas proporções de concentrado ricos em fibra... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The objective of this study was to evaluate the effect of two proportions of concentrate, 70 and 80%, with two energy sources, corn and corn germ meal, and roughage basis of corn silage on the intake of nutrients, performance, characteristics of a carcass, non components of carcass and meat quality of confined 28 Nellore heifers. The experimental design was a completely randomized design in factorial scheme 2 x 2 (2 proportions of concentrate x 2 energy sources) 4 replications and 7 treatments, and the averages has been compared by Tukey 5% test.. There was no interaction proportion of concentrate x energy source on the intake of dry matter. There was a significant effect of interaction proportion of concentrate x energy source intake for crude protein (CP) and neutral detergent fiber (NDF), suggesting that a higher proportion of concentrate, with the corn, allowed greater intake of CP (1.17 kg / day) and NDF (4,05 kg / day). It was observed average intake of acid detergent fiber than for diets with 80% concentrate, with both the diet with corn as the corn germ meal. There was a significant effect of dietary energy source on the final weight of animals, and the animals fed the diets with corn had average final weight (326.14 kg), above the fed with diets with corn germ meal (292.07 kg). The association of high proportions of concentrate with corn as an energy source, provided satisfactory results in performance of Nellore heifers, since the replacement of the corn germ meal, associated with high proportions of concentrate rich in fiber, not was recommended for young Nellore heifers. There was a significant effect the energy source of the diet on the weight of empty body, and the animals that received the diets with corn showed the average value of empty body weight (309.44 kg) than the diets of with corn germ meal (276.42 kg). The carcass yield, the thickness... (Complete abstract click electronic access below)
Mestre

It is important to understand the underlying plant defense mechanisms in order to establish best strategies to reduce losses due to diseases in cereals. The current available information is mostly on model organisms and their plant-pathogen interactions. However, this study is focused on the identification of genes involved in the resistance mechanism of one of the most devastating diseases of wheat, yellow rust. The strategy undertaken was to use differential display method (DD) together with microarray technology, on yellow rust differential lines of wheat (Avocet-Yr1 and Avocet-Yr10) infected with the virulent and avirulent Puccinia striiformis f. sp. tritici races (Pst: PST17, PST45, 169E136 and 232E137) together with appropriate control infections. DD primer combinations of ninety allowed the detection of fourteen differentially expressed genes which were also confirmed by real-time QRT-PCR analysis. All of but one were found to be novel sequences in wheat genome. Among those, two very important genes were identified as full ORF including 5&rsquo
and 3&rsquo
end untranslated regions (UTR)
namely cyclophilin like protein (putative antifungal activity) and ubiquitin conjugating enzyme (E2). The sequence homology analysis of the cloned gene fragments reveled that the genes detected have roles in ubiquitinylation, programmed cell death (apoptosis), putative antifungal activities, disease resistance, pathogen related responses, including a few with no known function. In addition to DD analysis, using wheat Affymetrix &ldquo
GeneChip&rdquo
, we identified 93 differentially expressed ESTs of wheat in response to avirulent pathogen attack. We also investigated the differential expression profiles of wheat leaves during the virulent infections and determined 75 differentially regulated ESTs. 1Selected ESTs were further analyzed using QRT-PCR analysis and 15 were confirmed to be differentially regulated. For the further characterization of the identified genes, such as determination of their putative roles in disease response, functional studies have to be performed. For this purpose, BSMV (Barley Stripe Mosaic Virus) mediated virus induced gene silencing (VIGS) method is optimized in this thesis for wheat. We have successfully managed to silence the endogenous PDS gene (Phytoene desaturase) of wheat which can be used as a positive control for the monitoring of silencing of the genes we have identified. Our results show that BSMV mediated VIGS can be used efficiently and effectively to silence wheat genes that we identified through differential display and microarray analysis and can be used to study the functions of those genes

In this thesis we have addressed three different although related topics. First, we studied the post-mortem mutation damage rate of contaminated DNA sequences in ancient human remains focusing on the development of strategies to avoid pre-laboratory derived contaminations. We proposed a guideline to control them consisting in typing every single person involved on the manipulation of the remains, especially when they have not been excavated and washed under controlled conditions. Second, we successfully develop a non-invasive technique to sequence ancient remains but preserving it from the destruction. And third, we sequenced ancient human remains from different evolutionary times (from Paleolithic to post-Neolithic) to make inferences about the peopling of Western Europe focusing mainly in the Iberia peninsula. We found that there is a long term genetic continuity at least since the Neolithic. The only clear genetic discontinuity found is that involving two different human species, H. sapiens and H. neanderthalensis.
En la presente tesis hemos tratado tres temas diferentes aunque muy relacionados. Primero, hemos estudiado la tasa de mutación post-mortem de secuencias de ADN contaminante en restos humanos antiguos centrándonos en el desarrollo de estrategias para evitar que las muestras se contaminen antes de llegar al laboratorio. Proponemos una guía que consiste en el tipado genético de cada persona implicada en la manipulación de los restos, especialmente cuando estos han sido excavados y lavados bajo condiciones no controladas. Segundo, hemos desarrollado una técnica no invasiva para secuenciar DNA de restos humanos antiguos pero sin destruirlos. Y por ultimo, hemos secuenciado restos humanos antiguos pertenecientes a diferentes periodos evolutivos (desde el Paleolitico hasta el post-Neolitico) que nos han permitido hacer inferencias sobre el poblamiento Europeo centrándonos básicamente en la Península Ibérica. Hemos encontrado que ha habido una continuidad genética desde el Neolítico. La única clara discontinuidad genética encontrada es entre dos especies distintas: H. Sapiens y H.neanderthalensis.

As enzimas lipolíticas vêm atraindo atenção no mercado global devido ao enorme potencial biotecnológico, como: na formulação de detergentes; na indústria de couro; produção de cosméticos, fármacos, aromas, biodiesel, etc. O objetivo deste trabalho foi prospectar genes codificadores de enzimas lipolíticas em biblioteca metagenômica de um consórcio microbiano degradador de óleo diesel. A seleção foi feita pela atividade lipolítica através do cultivo dos clones em placa de petri e a avaliação foi pela observação de halo ao redor da colônia, sendo positiva para 30 clones dentre os quais dois se destacaram. Estes dois clones foram selecionados e subclonados. Os DNAs das sub-bibliotecas foram sequenciados, gerando um contig completo para cada clone. Através do ORF Finder foi identificado cinco ORFs de esterase/lipase, dentre as quais uma alcançou 58% de identidade com uma bactéria não cultivável. As árvores filogenéticas indicam que duas ORFs são similares à família IV das enzimas lipolíticas, enquanto que as outras três ORFs à família V.
Lipolytic enzymes have been attracting global market attention because they show enormous biotechnological potential. The present work was done as an attempt to find genes which codify lipolytic enzymes in a metagenomic library composed of diesel oil degradation microbe consortia. Clones were selected according to lipolytic activity and were then evaluated after cultivation in Petri dishes by observation of halo formation around the colonies. 30 clones produced halo formations and were identified as positives, two of which showed prominent results. These two were then selected and sub cloned. DNA from the sub libraries was sequenced, generating a complete contig for each clone. Using the ORF Finder five esterase/lipase ORFs were identified, with one of these attaining 58% of identity to a non cultivatable bacteria species. Assessment of the cladograms showed that two ORFs were similar to lipolytic enzyme family IV, while the other three ORFs were similar to family V.

Orientador: Dirlei Antonio Berto
Banca: Jacinta Diva Gomes Ferrugem
Banca: José Roberto Sartori
Banca: Silvia Maria Alves Gomes
Banca: Valdomiro Shigueru Miyada
Resumo: Para avaliar os efeitos da suplementação dietética de maltodextrina em substituição a lactose para leitões dos 21 aos 51 dias de idade, foram utilizados 120 animais, distribuídos em um delineamento experimental de blocos ao acaso com arranjo fatorial 2x4 (duas dietas: simples e complexa e níveis de maltodextrina substituindo a lactose: 0,0%; 33,5%; 66,5% e 100,0%). Foram avaliados o consumo diário de ração (CDR), o ganho diário de peso (GDP) e a conversão alimentar (CA) em dois períodos pósdesmame (0-15 dias e 0-30 dias). Não houve interação entre os níveis de maltodextrina e as dietas para as variáveis de desempenho. Os leitões que consumiram as dietas complexas apresentaram maior GDP (P = 0,016) no período de 0 a 15 dias e melhor CA nos períodos de 0 a 15 dias (P = 0,010) e de 0 a 30 dias (P = 0,020) pós-desmame. Não foram observados efeitos dos níveis de maltodextrina sobre o CDR e GDP nos dois períodos e na CA no período de 0 a 15 dias. O aumento do nível de maltodextrina na dieta dos leitões determinou efeito quadrático na CA no período de 0 a 30 dias (Ŷ = 1,530000 + 0,002287X + 0,000022X2; R2 = 0,64). As rações contendo somente maltodextrina em sua composição proporcionaram melhor resultado econômico. A maltodextrina pode substituir totalmente a lactose nas dietas pré-iniciais e iniciais, simples ou complexas, sem afetar o desempenho de leitões dos 21 aos 51 dias de idade.
Abstract: One hundred and twenty piglets were used to evaluate the effects of dietary supplementation of maltodextrin as a replacement of lactose for weaned piglets from 21 to 51 days of age, in a randomized complete block design, according to a 2x4 factorial (two diet types: simple or complex; and levels of maltodextrin as a replacement of lactose: 0.0%, 33.5%, 66.5% and 100,0%). The daily feed intake (DFI), daily weight gain (DWG) and feed conversion (FC) were measured. No interaction between maltodextrin levels and diet type was observed. The pigs fed complex diets showed higher DWG from 0 to 15 days (P = 0.016) and better FC from 0 to 15 days (P = 0.010) and from 0 to 30 days (P = 0.020) post-weaning period. No effect of maltodextrin levels on DWG or DFI was observed from 0 to 15 days or from 0 to 30 days; on FC from 0 to 15 days. Maltodextrin showed a quadratic effect on FC from 0 to 30 days (Ŷ = 1.53000 + 0.002287X + 0.000022X2; R2 = 0.64). Diets containing only maltodextrin provided the best economical results. The maltodextrin can fully replace lactose in simple or complex pre-starter or starter diets without affecting performance weanling pigs from 21 and 51 days of age.
Doutor

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Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq)
Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (Capes)
Funda??o de Amparo ? Pesquisa do Estado de Minas Gerais (FAPEMIG)
Esta pesquisa foi realizada com o objetivo de avaliar o efeito da intera??o gen?tipo x n?veis de lisina digest?vel das dietas sobre caracter?sticas morfom?tricas e de desempenho de til?pias do Nilo, variedade GIFT, em fase de crescimento. O experimento foi constitu?do de duas fases, sendo que na primeira as til?pias foram alimentadas dos 90 aos 150 dias de idade com dietas contendo percentuais de lisina iguais a 1,43, 1,53, 1,63, 1,73 e 1,83 e na segunda fase, dos 151 aos 255 dias, os percentuais de lisina das dietas foram iguais a 1,28, 1,38, 1,48, 1,58 e 1,68. As til?pias foram pesadas dos 90 aos 255 dias de idade com intervalos de aproximadamente 30 dias, foram calculados o ganho de peso total e o ganho de peso di?rio e, posteriormente, as idades foram ajustadas para 90 e 150 dias na primeira fase, e 185, 220 e 255 dias na segunda fase. As medidas de altura, largura, comprimento de cabe?a, comprimento padr?o e comprimento total das til?pias foram coletadas aos 150 dias de idade. A partir desses dados, foram calculadas as rela??es cabe?a/comprimento padr?o, comprimento padr?o/comprimento total e largura/altura. Modelos de regress?o aleat?ria, por meio do modelo animal uni caracter?stica, foram utilizados para avaliar a sensibilidades dos valores gen?ticos aos n?veis de lisina digest?vel das dietas, plotando-se as normas de rea??o de 25 peixes amostrados aleatoriamente para as diferentes caracter?sticas para demonstrar o padr?o de comportamento dos valores gen?ticos das caracter?sticas ao longo do gradiente ambiental. Foram testados modelos com homogeneidade (1 classe) e heterogeneidade de vari?ncia residual (2, 3 e 4 classes). Modelos com heterogeneidade de vari?ncia residual foram mais adequados para avalia??o gen?tica da maioria das caracter?sticas das til?pias na primeira fase e modelos com homogeneidade de vari?ncia residual para a maioria das caracter?sticas de desempenho das til?pias avaliadas na segunda fase. Os valores gen?ticos das til?pias, em ambas as fases, variaram entre os n?veis de lisina estudados para a maioria das caracter?sticas, indicando a presen?a de intera??o gen?tipo x ambiente. No geral, as herdabilidades e vari?ncias gen?ticas aditivas variaram de baixas a altas magnitudes. As correla??es de Spearman, assim como as normas de rea??o, indicaram reordenamento dos valores gen?ticos ao longo do gradiente ambiental, sugerindo a presen?a de intera??o gen?tipo x ambiente. A sele??o para desempenho e medidas morfom?tricas de til?pias GIFT deve ser realizada no n?vel de lisina da dieta com a qual as til?pias ser?o alimentadas.
Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Zootecnia, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2017.
This research was carried out with the objective of evaluating the effect of genotype x levels of digestible lysine of the diets on morphometric and performance characteristics of Nile tilapia, GIFT variety, in the growing phase. The experiment consisted of two phases, and in the first the tilapia were fed from 90 to 150 days of age diets containing lysine percentages equal to 1.43, 1.53, 1.63, 1.73 and 1.83. In the second phase, from 151 to 255 days, the lysine percentages of the diets were 1.28, 1.38, 1.48, 1.58 and 1.68. Tilapia were weighed from 90 to 255 days of age at intervals of approximately 30 days. Total weight gain and daily weight gain were calculated and, subsequently, were adjusted for 90 and 150 days in the first phase, and 185 , 220 and 255 days in the second phase. The measurements of height, width, head length, standard length and total length of tilapias were collected at 150 days of age. From these data, the head / standard length ratios, standard length / total length and width / height ratios were calculated. Single trait random regression modes were used in the analyses and the reaction norm plots of 25 randomly sampled fishes were used to demonstrate the sensitivity of breeding values to dietary digestible lysine level. Models with homogeneity (1 class) and heterogeneity of residual variance (2, 3 and 4 classes) were tested. Models considering heterogeneity of residual variance were more adequate for genetic evaluation of most of the characteristics of tilapia in the first phase and models considering homogeneity of residual variance for most of the performance characteristics of tilapia evaluated in the second phase. The breeding values of tilapia, in both phases, varied among the lysine levels studied for most of the traits, indicating the presence of genotype x environment interaction. In general, heritabilities and additive genetic variances varied from low to high magnitude. Spearman correlations, as well as the reaction norms, suggested rearrangement of breeding values along the environmental gradient, again indicating the presence of genotype x environment interaction. The selection for performance and morphometric traits of tilapia GIFT strain should be performed at the lysine level of the diet with which the tilapia will be fed.

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Financiadora de Estudos e Projetos
Demands for seafood are growing in the whole world and the fish farming industries are in frank development over the last decade. However, stressful conditions, which favor higher incidence of diseases and impair growth in fishes, are usually found in fish farming. Therefore, several studies are focused in the improvement of management to reduce stress and keep the animals healthy, reducing losses in farm production. The use of immunostimulants in the diets, such as vitamin E, has been suggested as an alternative to improve fish resistance to diseases under stressful conditions. Pacu, Piaractus mesopotamicus, is one of the main species from the Brazilian aquatic fauna and is of great importance in fish farming. Hence, the objective of this study was to evaluate the metabolic responses and innate immunity of pacu fed with vitamin E supplemented diets and challenged to temperature variation. The experiment was conducted as a totally randomized design in a factorial scheme 3x3x2, with three levels of dietary vitamin E (0, 100 and 300 mg/kg), three sampling spans (0, 24 and 72h) and two temperature conditions (controlled and variable), with four replicates (tanks) per treatment. The main responses observed to cope with stress were 1) elevation of plasma cortisol in the animals subjected to temperature variation, independent of the concentration of vitamin E in the diet, 2) hyperglycemia only in the animals subjected to temperature variations and fed with not supplemented diets and 3) hyperlactemia in the fish subjected to temperature variation and fed with not supplemented and supplemented diets with 300 mg of vitamin E. Animals fed with diets supplemented with 100 mg of vitamin E and subjected to temperature variation depicted the highest percentage of granular leukocytes, the highest concentration of serum lysozyme and the highest activity of alternative complement pathway. There upon, supplementation with 100 mg of vitamin E seems to be the best one for this species under the studied conditions.
A demanda mundial por produtos pesqueiros é crescente e a piscicultura teve na última década desenvolvimento emergente. Porém, situações estressantes, que favorecem maior incidência de doenças e prejudicam o crescimento dos peixes, são encontradas nas práticas diárias da piscicultura. Assim, tem-se buscado cada vez mais o conhecimento de boas práticas de manejo, a fim de reduzir o estresse e manter os animais saudáveis, reduzindo as perdas na produção. O uso de imunoestimulantes, como a vitamina E, na dieta tem sido apontado como uma alternativa para melhorar a resistência dos peixes a doenças em situações de estresse. O pacu, Piaractus mesopotamicus, é uma das principais espécies da fauna aquática brasileira e possui grande importância para a piscicultura comercial. Assim, o objetivo desse trabalho foi avaliar algumas respostas metabólicas e de imunidade inata em pacus alimentados com dietas suplementadas com vitamina E e submetidos à variação de temperatura. O experimento foi realizado em um delineamento inteiramente casualizado, em esquema fatorial 3x3x2, sendo três níveis de vitamina E na ração (0, 100 e 300 mg/kg), três horários de coleta (0, 24 e 72h) e duas condições de temperatura (controlada e variável), com quatro repetições (aquários) por tratamento. As principais respostas ao estresse observadas foram: 1) elevação do cortisol plasmático nos animais submetidos à variação de temperatura, independente da concentração de vitamina E na dieta, 2) hiperglicemia apenas nos animais submetidos à variação de temperatura e alimentados com dieta não suplementada, 3) hiperlactemia nos animais submetidos à variação de temperatura e alimentados com dieta não suplementada e suplementada com 300 mg de vitamina E e 4) os animais alimentados com dieta suplementada com 100 mg de vitamina E e submetidos à variação de temperatura apresentaram a maior porcentagem de leucócitos granulares, a maior concentração de lisozima sérica e a maior atividade da via alternativa do sistema complemento. Diante disso, a suplementação da ração com 100 mg de vitamina E mostrou-se a melhor para a espécie nas condições experimentais do presente trabalho.

Pig industry is required to reduce its environmental footprint, in order to satisfy the increasing demand of eco-friendly products. Moreover, the producers need to cut down with the feeding costs, by improving the pigs’ feed efficiency. Despite the rich literature, knowledge about the effect of some feeding strategies on feeding behaviour, performance, and quality of meat and meat products seems still controversial or scarce. With this background, the aims of this Ph.D. project were: i) to assess the influence of a reduction in dietary indispensable amino acids (AA) on feed intake, growth performance, N excretion and carcass and meat quality traits of fast growing pigs fed according to an ad libitum or a restricted feeding regime; ii) to study the feeding behaviour of group-housed pigs fed individually from single-space feeders and subjected to feed restriction and a reduction in the dietary indispensable AA content; iii) to explore the influence of feeding behaviour on growth performance, carcass and meat characteristics of pigs; iv) to investigate the influence of low-protein and AA diets on the characteristics of San Daniele like dry-cured hams obtained from two genetic groups of pigs with different lean growth potential. In the 1st Chapter, 96 barrows were individually fed through automatic feeding stations, according to an ad libitum or restricted feeding regime and to a conventional or low-AA diet. Feed restriction decreased feed intake, average daily gain, carcass weight, backfat depth, but increased gain:feed ratio. The low-AA diets increased feed intake, carcass weight and the intramuscular fat content, with no effects on the feed efficiency and the estimated Pr. Nitrogen excretion was reduced by feed restriction and by the reduction of the dietary AA content. In the 2nd Chapter, the data recorded by the automatic feeding stations were used to study the effects of the feeding regime and the dietary AA restriction on the feeding patterns of the pigs of the previous contribution. Feed restriction decreased daily feed intake, the number of visits and the time spent feeding, but increased feed consumption per visit and feeding rate. The low-AA diets increased daily feed intake, tended to increase feeding rate and interacted with feeding regime for the number and duration of feeding visits. The pigs were able to adapt their feeding patterns to compensate for a reduction in feed allowance or nutrient restriction. In the 3rd Chapter the phenotypic correlations among feeding patterns, growth performances and carcass traits were studied. The records of each pig were classified into feeding rate tertiles. Pigs eating faster showed greater final body weights, average daily gains, estimated protein gains, estimated lipid retention, carcass weights, weights of lean cuts, weights of fat cuts, proportions of fat in the carcass, and lower proportions of carcass lean cuts than pigs eating slowly. Manipulating the eating rate, through management or genetic strategies, could affect growth performances and carcass quality, with little influence on feed efficiency. The 4th Chapter investigated the influence of diets lowered in protein and AA contents on the quality of 40 San Daniele like dry-cured hams produced by pigs of two genetic groups (Danbred and Anas) characterized by different potential for lean growth, eating conventional or low-protein diets. The Danbred fresh hams were heavier, but showed greater seasoning losses and thinner fat cover than Anas hams. Dry-cured hams from Danbred had higher protein content than the Anas ones. Dietary protein restriction had small influence on dry-cured ham quality. Due to its positive effects on sustainability of dry-cured ham chain by decreasing pig farm nitrogen excretion and feeding costs, the use of low-protein diets seems an advisable strategy for the feeding of traditional PDO heavy pigs.
Il settore suinicolo deve oggi soddisfare la domanda di prodotti eco-friendly, ridurre i costi di produzione e migliorare l’efficienza alimentare dei maiali attraverso l’utilizzo di nuove strategie alimentari. Gli obiettivi di questo progetto di dottorato sono: i) valutare l’influenza di una riduzione del contenuto di amminoacidi della dieta su consumo alimentare, prestazioni e qulità della carne di maiali alimentati secondo un regime alimentare ad libitum o razionato; ii) studiare il comportamento alimentare dei maiali sottoposti ad una restrizione alimentare e ad una riduzione del contenuto di amminoacidi essenziali dell’alimento; iii) conoscere l’influenza del comportamento alimentare sulle prestazioni e le caratteristiche della carcassa; iv) esaminare gli effetti di una riduzione del contenuto di proteina e amminoacidi sulla qualità di prosciutti crudi ottenuti da due linee genetiche caratterizzate da diversi potenziali di crescita magra e stagionati come prosciutti San Daniele DOP. Nel I contributo, 96 maiali sono stati alimentati attraverso un regime alimentare ad libitum o una leggera restrizione alimentare utilizzando diete convenzionali o con basso contenuto di amminoacidi essenziali (LAA). La restrizione alimentare ha ridotto i consumi di alimento, gli accrescimenti, i pesi delle carcasse e gli spessori del grasso, ma ha aumentato l’efficienza alimentare. La dieta LAA ha aumentato i consumi, i pesi e il contenuto di grasso delle carcasse, senza influenzare l’efficienza alimentare e la crescita proteica. I due trattamenti alimentari hanno ridotto l’escrezione di azoto. Nel II contributo, si sono valutati gli effetti del regime alimentare e della dieta LAA sul comportamento alimentare dei maiali. La restrizione alimentare ha ridotto l’ingestione di alimento, il numero di visite alla mangiatoia e il tempo utilizzato per mangiare, ma ha aumentato il consumo per visita, e la velocità d’ingestione. La dieta LAA ha aumentato i consumi alimentari, e ha provocato un leggero aumento della velocità d’ingestione. I maiali sono stati in grado di adattare il loro comportamento alimentare per superare i limiti dovuti alla restrizione alimentare e alla riduzione di nutrienti del mangime. Nel III capitolo, si sono studiate le relazioni tra comportamento alimentare, prestazioni e caratteristiche della carcassa. I dati di ciascun maiale sono stati classificati secondo tre classi di velocità d’ingestione. I maiali che mangiavano più rapidamente avevano maggiori pesi, accrescimenti proteici e lipidici, pesi delle carcasse, dei tagli magri e dei tagli grassi, con una più elevata proporzione di tagli grassi, rispetto ai maiali più lenti. Manipolare il comportamento alimentare attraverso strategie manageriali e alimentari può avere effetti favorevoli sulle prestazioni produttive e la qualità della carcassa, senza alterare l’efficienza alimentare dei maiali. Nel IV capitolo della tesi si è valutato l’effetto di una riduzione del contenuto di proteina e amminoacidi essenziali della dieta sulle caratteristiche di qualità di 40 prosciutti crudi lavorati come prosciutti San Daniele DOP e ottenuti da due linee genetiche con un diverso potenziale per la crescita magra alimentate con diete convenzionali o a basso tenore proteico. Le cosce rifilate dei Danbred erano più pesanti, ma mostravano maggiori perdite di stagionatura e minori spessori del grasso di copertura rispetto a quelle ottenute degli Anas. Inoltre, i prosciutti dei Danbred hanno avuto un maggior contenuto di proteina. Invece, la riduzione del contenuto proteico e amminoacidico della dieta ha influenzato poco le caratteristiche di qualità dei prosciutti crudi. Dunque, dati gli effetti favorevoli alla riduzione delle escrezioni di azoto e dei costi di alimentazione, l’uso di diete a basso tenore proteico può essere un’efficace soluzione per l’alimentazione del suino pesante italiano.

Thesis (M.S.)--University of Wisconsin--Madison, 2001.
Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 149-154).

Thesis (M.S.)--University of Wisconsin--Madison, 2004.
Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 154-159).

This thesis deals with the application of BEAS-2B cell line for micronucleus assay in genetic toxicology. It is divided into two main parts: a) theoretical introduction to the analysis and search for suitable models for testing the impact of air pollution and manufactured nanoparticles, b) practical part that describes the results of micronuclei induction by polycyclic aromatic hydrocarbons (PAHs), extractable organic matter (EOM) from diesel exhaust particles obtained from emissions of three types of fuel and engineered nanoparticles. BEAS-2B cell line is a nonmalignant human model of lung epithelium which seems to be suitable for micronucleus assay. This assay is commonly used for determining the genotoxicity of various substances to wide variety of cell cultures and also in human studies. In this thesis, the following substances were tested: benzo[a]pyrene, 3-nitrobenzanthrone and 1-nitropyrene as carcinogenic PAHs commonly found in polluted air; EOMs from exhaust particles of 100 % diesel fuel, a blend of diesel fuel and 30 % of biodiesel, 100 % biodiesel and two types of engineered nanoparticles (TiO2 and Ag). The cells were treated with the compounds for 28, 48 and 72 hours. The results confirm the suitability of BEAS-2B cell line as a model for testing the genotoxicity of substances under...