The ubiquity of circadian systems has allowed their characterization in a broad range of model systems, which has greatly improved knowledge of how these systems are organized and the vast range of cellular and organismal processes under circadian control. Most of the advances, however, have come in describing the central oscillators of these systems, and, in some cases, the input pathways used to coordinate these oscillators to external time. Very little progress has been made in understanding the output pathways that allow circadian systems to regulate the breadth of processes shown to be clock-controlled. A genetic selection was designed to obtain mutations in genes involved in circadianregulated expression of the Neurospora crassa ccg-1 and ccg-2 genes. Some, but not all, of the strains obtained display altered regulation of more than one ccg as well as an ‘Easlike’ appearance on solid media, and altered circadian period on race tubes. The data suggest a model in which output from the clock to these two genes is through a single, bifurcated pathway. The cloning of the gene mutated (rrg-1) in one of the strains from the above selection led to the first molecular description of a circadian output pathway in Neurospora, the HOG MAP kinase pathway. The HOG pathway has been previously described with regard to its role in the osmotic-stress response. The discovery of the involvement of rrg-1 in circadian regulation of ccg-1 and ccg-2 led to the discovery of regulation of the HOG pathway by the circadian clock. The data indicate that osmotic stress information and time-of-day information are transduced through the HOG pathway and implicate a role for the clock in preparing the organism for daily occurrences of hyperosmotic stress associated with sun exposure. The genetic selection, and the description of the HOG pathway with regard to circadian output, provide a basis for further characterization of circadian output in Neurospora. The ubiquity of MAP kinase pathways, such as the HOG pathway, and the observed similarities in the mechanisms of circadian clock function across multiple phyla, indicate that these findings may well be applicable to other model systems.

O presente trabalho teve como objetivo avaliar novilhos resultantes da seleção genética de diferentes linhagens da raça Canchim (5/8 Charolês, 3/8 Zebu), recriados a pasto e terminados em confinamento, em relação ao consumo alimentar, desempenho, emissão de metano entérico, características de carcaça e cortes cárneos. O experimento foi desenvolvido na Empresa Brasileira de Pesquisa Agropecuária, unidade Pecuária Sudeste, São Carlos/SP. Foram utilizados 46 novilhos da raça Canchim (15 meses, 291 kg de peso vivo), 13 pertencentes a linhagem antiga, 20 a linhagem nova e 13 provenientes do acasalamento entre estas linhagens. A linhagem antiga é mantida como rebanho fechado desde 1953 e foi formada com base em 53 touros Charoleses importados da França, já a linhagem nova teve origem a partir de 1990 com a utilização de touros Charoleses de diferentes origens (Argentina, Brasil, Estados Unidos, França, Inglaterra), e a partir de 1998, com o acasalamento entre as duas linhagens formou-se a linhagem cruzada. Durante a recria a pasto (165 dias), os animais foram alojados em uma área de pastagem (Panicum maximum cv. Tanzânia), onde o manejo adotado foi o de pastejo rotacionado com lotação variável, para as medidas de desempenho foram executadas pesagens a cada 33 dias, e a mensuração da emissão de metano entérico através da técnica do gás traçador hexafluoreto de enxofre realizada em 12 animais da linhagem antiga e 12 da nova. No período de terminação em confinamento, os animais foram dispostos em 4 baias e receberam alimentação ad libitum durante 105 dias, o ganho de peso foi medido a cada 28 dias e para a mensuração de emissão de metano os mesmos animais avaliados a pasto, tiveram acesso ao sistema automatizado GreenFeed®. No momento do abate, foram coletadas informações sobre o peso de carcaça quente, e após 24 horas de resfriamento em câmara fria, o peso da carcaça fria, do traseiro especial, dianteiro e ponta de agulha e na desossa os pesos individuais dos cortes cárneos comerciais. A linhagem nova mostrou-se superior a antiga no período de recria nas variáveis de ganho médio diário, conversão e eficiência alimentar, não se diferenciando dos cruzados. Durante a terminação em confinamento, foi observado o mesmo em relação ao ganho médio diário, já em relação ao consumo, a linhagem nova e os cruzados apresentaram maiores ingestões de alimento comparados à linhagem antiga, enquanto a conversão alimentar dos animais cruzados apresentou maior valor comparados às das linhagens. Na recria a pasto, para se obter maiores ganhos de peso, melhor conversão alimentar e eficiência alimentar, devem ser utilizados animais resultantes de processos de seleção genética. A seleção genética para características de desempenho produtivo não garante animais com menores emissões de metano em condições de pastejo. Nos animais em fase de terminação em confinamento, o maior ganho de peso, melhor conversão alimentar e melhor acabamento de carcaça são obtidos através da utilização de animais resultantes de processos de seleção genética. O mesmo tipo de seleção para características de desempenho produtivo em confinamento leva a maiores consumos diários de alimento e consequentemente a maiores emissões de metano diárias. / The objective of this study was to evaluate steers resulting from the genetic selection of different Canchim (5/8 Charolais, 3/8 Zebu) lineages, rearing to pasture and finished in feedlot, in relation to feed intake, performance, enteric methane emission, carcass traits and retail cuts. The experiment was carried out at the Brazilian Agricultural Research Corporation (Embrapa), Southeast Livestock, São Carlos / SP. A total of 46 Canchim steers (15 months, 291 kg live weight) were used, 13 from the ancient lineage, 20 from the new lineage and 13 from the mating between these lineages. The ancient lineage is kept as a closed herd since 1953 and was formed by 53 Charolais bulls imported from France. The new lineage originated from 1990 with the use of Charolais bulls from different origins (Argentina, Brazil, the United States, France, England), and from 1998 the crossed lineage was formed with the mating between the two lineages. During grazing (165 days), the animals were housed in an pasture area (Panicum maximum cv. Tanzania), where the management used was rotational grazing with variable stocking, for the performance measures were performed weighing every 33 days, and the measurement of the enteric methane emission through the sulfur hexafluoride tracer gas technique was performed in 12 animals of the ancient and 12 of the new lineage. In feedlot the animals were housed in 4 pens and fed ad libitum for 105 days. The weight gain was measured every 28 days and as for the measurement of methane emission the same animals evaluated on pasture had access to the GreenFeed® automated system. Information on the hot carcass weight was collected at the time of slaughter and after 24 hours of cooling in the cold chamber, the weights of the cold carcass, of the special hind, fore and spare ribs, and the individual weights of the retail cuts in boning. The new lineage was superior to the ancient one in the rearing period in the variables of average daily gain, conversion and feed efficiency, not differing from the animals crossed. During feedlot, it was observed the same in relation to the average daily gain, already in relation to intake, the new lineage and the crossbreeds presented higher feed intakes compared to the ancient lineage while the feed conversion of the crossed animals presented higher value compared with the new and ancient lineages. In pasture recreating, in order to obtain greater weight gains, better feed conversion and feed efficiency, animals resulting from genetic selection processes must be used. Genetic selection for performance traits does not guarantee animals with lower methane emissions under grazing conditions. For feedlot animals, the highest weight gain, better feed conversion and better carcass finishing are obtained through the use of animals resulting from genetic selection processes. The same type of selection for traits of performance in feedlot leads to higher daily feed intake and consequently to higher daily methane emissions.

O presente trabalho teve como objetivo avaliar novilhos resultantes da seleção genética de diferentes linhagens da raça Canchim (5/8 Charolês, 3/8 Zebu), recriados a pasto e terminados em confinamento, em relação ao consumo alimentar, desempenho, emissão de metano entérico, características de carcaça e cortes cárneos. O experimento foi desenvolvido na Empresa Brasileira de Pesquisa Agropecuária, unidade Pecuária Sudeste, São Carlos/SP. Foram utilizados 46 novilhos da raça Canchim (15 meses, 291 kg de peso vivo), 13 pertencentes a linhagem antiga, 20 a linhagem nova e 13 provenientes do acasalamento entre estas linhagens. A linhagem antiga é mantida como rebanho fechado desde 1953 e foi formada com base em 53 touros Charoleses importados da França, já a linhagem nova teve origem a partir de 1990 com a utilização de touros Charoleses de diferentes origens (Argentina, Brasil, Estados Unidos, França, Inglaterra), e a partir de 1998, com o acasalamento entre as duas linhagens formou-se a linhagem cruzada. Durante a recria a pasto (165 dias), os animais foram alojados em uma área de pastagem (Panicum maximum cv. Tanzânia), onde o manejo adotado foi o de pastejo rotacionado com lotação variável, para as medidas de desempenho foram executadas pesagens a cada 33 dias, e a mensuração da emissão de metano entérico através da técnica do gás traçador hexafluoreto de enxofre realizada em 12 animais da linhagem antiga e 12 da nova. No período de terminação em confinamento, os animais foram dispostos em 4 baias e receberam alimentação ad libitum durante 105 dias, o ganho de peso foi medido a cada 28 dias e para a mensuração de emissão de metano os mesmos animais avaliados a pasto, tiveram acesso ao sistema automatizado GreenFeed®. No momento do abate, foram coletadas informações sobre o peso de carcaça quente, e após 24 horas de resfriamento em câmara fria, o peso da carcaça fria, do traseiro especial, dianteiro e ponta de agulha e na desossa os pesos individuais dos cortes cárneos comerciais. A linhagem nova mostrou-se superior a antiga no período de recria nas variáveis de ganho médio diário, conversão e eficiência alimentar, não se diferenciando dos cruzados. Durante a terminação em confinamento, foi observado o mesmo em relação ao ganho médio diário, já em relação ao consumo, a linhagem nova e os cruzados apresentaram maiores ingestões de alimento comparados à linhagem antiga, enquanto a conversão alimentar dos animais cruzados apresentou maior valor comparados às das linhagens. Na recria a pasto, para se obter maiores ganhos de peso, melhor conversão alimentar e eficiência alimentar, devem ser utilizados animais resultantes de processos de seleção genética. A seleção genética para características de desempenho produtivo não garante animais com menores emissões de metano em condições de pastejo. Nos animais em fase de terminação em confinamento, o maior ganho de peso, melhor conversão alimentar e melhor acabamento de carcaça são obtidos através da utilização de animais resultantes de processos de seleção genética. O mesmo tipo de seleção para características de desempenho produtivo em confinamento leva a maiores consumos diários de alimento e consequentemente a maiores emissões de metano diárias. / The objective of this study was to evaluate steers resulting from the genetic selection of different Canchim (5/8 Charolais, 3/8 Zebu) lineages, rearing to pasture and finished in feedlot, in relation to feed intake, performance, enteric methane emission, carcass traits and retail cuts. The experiment was carried out at the Brazilian Agricultural Research Corporation (Embrapa), Southeast Livestock, São Carlos / SP. A total of 46 Canchim steers (15 months, 291 kg live weight) were used, 13 from the ancient lineage, 20 from the new lineage and 13 from the mating between these lineages. The ancient lineage is kept as a closed herd since 1953 and was formed by 53 Charolais bulls imported from France. The new lineage originated from 1990 with the use of Charolais bulls from different origins (Argentina, Brazil, the United States, France, England), and from 1998 the crossed lineage was formed with the mating between the two lineages. During grazing (165 days), the animals were housed in an pasture area (Panicum maximum cv. Tanzania), where the management used was rotational grazing with variable stocking, for the performance measures were performed weighing every 33 days, and the measurement of the enteric methane emission through the sulfur hexafluoride tracer gas technique was performed in 12 animals of the ancient and 12 of the new lineage. In feedlot the animals were housed in 4 pens and fed ad libitum for 105 days. The weight gain was measured every 28 days and as for the measurement of methane emission the same animals evaluated on pasture had access to the GreenFeed® automated system. Information on the hot carcass weight was collected at the time of slaughter and after 24 hours of cooling in the cold chamber, the weights of the cold carcass, of the special hind, fore and spare ribs, and the individual weights of the retail cuts in boning. The new lineage was superior to the ancient one in the rearing period in the variables of average daily gain, conversion and feed efficiency, not differing from the animals crossed. During feedlot, it was observed the same in relation to the average daily gain, already in relation to intake, the new lineage and the crossbreeds presented higher feed intakes compared to the ancient lineage while the feed conversion of the crossed animals presented higher value compared with the new and ancient lineages. In pasture recreating, in order to obtain greater weight gains, better feed conversion and feed efficiency, animals resulting from genetic selection processes must be used. Genetic selection for performance traits does not guarantee animals with lower methane emissions under grazing conditions. For feedlot animals, the highest weight gain, better feed conversion and better carcass finishing are obtained through the use of animals resulting from genetic selection processes. The same type of selection for traits of performance in feedlot leads to higher daily feed intake and consequently to higher daily methane emissions.

L'amélioration de l'efficacité alimentaire est un enjeu économique et environnemental important pour la production porcine. L'aliment est le premier poste de dépenses en élevage porcin, et le restera vraisemblablement au vu de l'évolution du coût des matières premières. Historiquement, l'efficacité alimentaire a été améliorée en sélectionnant, avec succès, pour une diminution de l'indice de consommation (IC), qui représente l'efficacité de conversion de l'aliment en gain de poids. Dans un contexte d'évolution des objectifs de sélection, de nouvelles stratégies d'amélioration de l'efficacité alimentaire sont recherchées. Ce travail de thèse propose d'en explorer certaines en utilisant des données enregistrées en stations publiques de contrôle de performance entre 2000 et 2009 dans les quatre races porcines en sélection collective en France, deux races maternelles (Large White femelle et Landrace) et deux races paternelles (Large White mâle et Piétrain). L'approche privilégiée s'appuie sur la consommation moyenne journalière résiduelle (CMJR), qui est un critère d'efficacité alimentaire indépendant des performances des animaux, afin d'estimer ses relations génétiques avec les performances de production, ainsi qu'avec les quantités d'azote et de phosphore excrétées. Dans un premier temps, l'étude d'une population expérimentale Large White a montré que les corrélations génétiques entre sexes pour la CMJR étaient proches de 1, permettant par la suite de comparer les estimations de paramètres obtenues sur des femelles Piétrain à celles obtenues sur les mâles castrés des trois autres races. Nous montrons que ce caractère a une héritabilité (h²) comprise entre 0,21 et 0,33 selon les races, qu'il est un peu moins héritable que l'IC (h² entre 0,30 et 0,40), que ces deux caractères présentent des corrélations génétiques favorables (entre 0,52 et 0,85), et que si la CMJR est par définition phénotypiquement indépendante des caractères de croissance et de composition corporelle, elle l'est aussi au niveau génétique (corrélations inférieures à 0,16 en valeur absolue quelle que soit la race). En revanche, la CMJR, comme l'IC, a des relations génétiques antagonistes avec les caractères de qualité de la viande. Cependant, nous montrons que la CMJR, contrairement à l'IC, n'est pas affectée par le génotype halothane en ségrégation chez le Piétrain. Enfin, des corrélations génétiques très élevées avec l'IC (0,97 et plus), et plus modérées avec la CMJR (entre 0,38 et 0,86) ont été trouvées pour l'excrétion d'azote et de phosphore, suggérant la réduction attendue des rejets en réponse à la sélection sur l'efficacité alimentaire. Ces corrélations étaient globalement plus élevées dans les races paternelles que dans les races maternelles. Enfin, l'étude des cinétiques de croissance et d'ingestion des animaux en fonction de leur niveau d'efficacité alimentaire a montré qu'une sélection pour des animaux plus efficaces ne peut être réalisée qu'en adaptant la formulation des aliments, notamment leur concentration en acides aminés, de façon à permettre la pleine expression de leur potentiel génétique. / Improvement of feed efficiency is a main economic and environmental goal for pig production. Feed is the first component of cost production for pig farmers, and this will remain the case due to the evolution of feedstuffs cost. Historically, feed efficiency has been improved through selection, with success, for a decrease of feed conversion ratio (FCR) representing efficiency of feed conversion into body weight gain. In a context of evolving breeding objectives, new strategies of feed efficiency improvement are investigated. This PhD work has studied some of them using data collected in the French central test stations between 2000 and 2009 for the four main pigs breeds used in collective selection, i.e. two dam breeds (Large White dam line and Landrace) and two sire breeds (Large White sire line and Piétrain). The main approach retained here deals with residual feed intake (RFI), which is a criterion of feed efficiency independent from animal's performance level, in order to estimate its genetic relationships with usual production traits, and also with nitrogen and phosphorus quantities excreted by the animals. In a first step, a study of an experimental Large White population showed that genetic correlations between the three sexes for RFI were very close to 1 for RFI. This result allowed the comparisons of the results obtained on Piétrain females with those obtained on castrated males in the three other breeds. We showed that RFI had an heritability (h²) ranging between 0.21 and 0.33 depending on the breed, slightly less heritable than FCR (h² between 0.30 and 0.40) and with a favourable genetic correlation between them (between 0.52 and 0.85). The RFI is, by definition, phenotypically independent from growth rate and carcass composition traits, and this was also the case at the genetic level (genetic correlations lower than 0.16 in absolute values whatever the breed). However, RFI, like FCR, did not display favourable genetic correlations with meat quality traits. Contrary to FCR, RFI was shown to be unaffected by the halothane genotype segregating in the Piétrain breed. Very high genetic correlations with FCR (0.97 and higher), and more moderate genetic correlations with RFI (between 0.38 and 0.86) were found for nitrogen and phosphorus excreted quantities, which indicates that the reduction of excreted quantities is expected in response to selection for a better feed efficiency. These genetic correlations were globally higher in sire lines than in dam lines. Finally, the study of growth and feed intake curves of animals classified according to their feed efficiency level (either RFI or FCR) suggested that breeding for more efficient animals could not be conducted without an adaptation of feed formulation (e.g. for amino acids concentration), in order to allow the full expression of their genetic potential.

Os índices de seleção foram inicialmente propostos para a seleção simultânea de diversos caracteres e, por requerem a obtenção de estimativas de variâncias e covariâncias, são mais apropriados para programas de seleção recorrente. Posteriormente, desenvolveram-se outros índices, que por não requererem o conhecimento de tais estimativas, podem ser aplicados tanto em programas de seleção recorrente, como para a seleção de genótipos já fixados, como clones, linhagens e híbridos. Recentemente, Garcia (1998) desenvolveu um índice específico para a seleção de genótipos fixados, que se baseia no cálculo da distância de cada genótipo a um ideótipo. As principais vantagens são que este índice permite a aplicação de testes de comparações de médias, bem como a identificação de genótipos com performances aquém daquela exigida para os cultivares comerciais para um dado caráter. O objetivo deste trabalho foi avaliar a eficiência do índice de Garcia (1998) para a seleção de linhagens de soja na fase de pré-cultivares. O material experimental consistiu de 88 linhagens (Geração F7), derivadas pelo método S.S.D. e oriundas do cruzamento entre os genitores BR-80-14853 x PI-123439, e três testemunhas comerciais: IAC-12, IAC-Foscarin-31 e IAS-5. Os experimentos foram conduzidos no delineamento em blocos casualizados em seis anos agrícolas, com diferentes números de repetições por ano: 3 (1995/6), 4 (1996/7), 6 (1998/9), 6 (1999/0), 10 (2000/1) e 10 (2001/2). Os caracteres avaliados foram: produção de grãos (PG: g/parcela) altura das plantas na maturação (AM: cm/planta), e acamamento (AC: notas de 1 a 5). No cálculo das distâncias utilizaram-se a Distância Euclidiana e a Distância de Mahalanobis. Os resultados obtidos mostraram que os índices baseados tanto na Distância Euclidiana quanto na de Mahalanobis foram eficientes para a discriminação dos genótipos, embora os melhores resultados tenham sido obtidos com a Distância de Mahalanobis. Devido a isso, sugere-se o uso da Distância de Mahalanobis para identificar e selecionar pré-cultivares de soja e também, como proposto por Garcia (1998), que esta seleção seja acompanhada da avaliação de caracteres individuais, para evitar que genótipos promissores sejam descartados por apresentarem pequenos defeitos em caracteres de importância secundária. / Selection indices were first proposed for the selection of several traits at the same time, and because of the requirement of variances and covariances estimates, they are useful for recurrent selection programs. Later on, other indices were proposed, which do not require the knowledge of these estimates, and thus are useful for both recurrent selection programs and for the selection of fixed genotypes, such as clones, inbred lines and hybrids. Recently, Garcia (1998) proposed a new index for the selection of fixed genotypes which is based on the distance of each genotype to and ideotype. The main advantages of this index is that multiple comparison tests can be done and also the identification of genotypes that perform below than those required by commercial cultivars for a given trait. The objective of this work was to evaluate the efficiency of Garcia (1998)´s index for the selection of pre-cultivars in soybeans. Experimental material comprised 88 lines (F7 generation) derived by the S.S.D. method from the cross between the BR-80-14853 and PI-123439 parents, and three cultivars as checks: IAC-12, IAC-Foscarin-31 and IAS-5. The evaluation trials were carried out in randomized block designs across six growing seasons, with different number of replications each: 3 (1995/6), 4 (1996/7), 6 (1998/9), 6 (1999/0), 10 (2000/1) and 10 (2001/2). The following traits were evaluated: grain yield (PG: g/plot), plant height at maturity (AM: cm/plant) and lodging (AC: scale: 1 to 5). For the calculation of the distance of each genotype to the ideotype, the Euclidean and Mahalanobis methods were used. General results have shown that the index based on both Euclidean and Mahalanobis distances were efficient to discriminate the genotypes, but the best results were reached by using Mahalanobis distance. Consequently, in order to identify and select fixed genotypes of soybeans we suggest the use of Mahalanobis distance, and as proposed by Garcia (1998), that the selection be based on the index and also on the evaluation of each trait, in order to avoid the discard of superior genotypes showing small problems in traits of minor importance.

Many cancers have long been known to display an absolute requirement for the amino acid methionine (L-Met). Studies have shown that in the absence of L-Met, sensitive neoplasms experience cell cycle arrest and perish. Without the metabolic deviations that characterize L-Met auxotrophs, normal cells are able to grow on precursors such as homocysteine and tolerate periods of L-Met starvation. The differential requirement for this amino acid between normal and tumor cells has been exploited through enzymatic serum degradation of L-Met by a bacterial methionine-γ-lyase (MGL). Though MGL was able to deplete L-Met to therapeutically useful levels in animal models and exert a significant cytotoxic effect on malignant cell lines in vitro and on tumor xenografts in vivo, the clinical implementation of this enzyme is hampered by its short serum half-life and potential for catastrophic immune response. In the chapters that follow, we describe the engineering of a novel human methionine degrading enzyme (hMGL) that overcomes the limitations of the bacterial therapeutic. We have shown that hMGL is capable of degrading methionine at a therapeutically useful rate and inducing extensive cell killing in a variety of neoplasms. This enzyme is expected to have low immunogenicity in patients and a high therapeutic index. We have developed a high throughput screen for methionine degrading activity that we can utilize to further engineer the enzyme based on the results of additional preclinical development. We have found that hMGL is also capable of degrading cystine to operate as a dual amino acid depletion treatment that is expected to be more potent than methionine depletion alone. Due to the wide array of neoplasms sensitive to methionine and cystine starvation, the engineered enzyme holds a great deal of promise as a unique and powerful cancer therapeutic. / text

Beyond their physiological roles, nuclear receptors have been exploited for their ability to act as intracellular sensors of small molecules. Accordingly, yeast two- and three-hybrid systems have been developed, exploiting them to control reporter gene expression. These systems may be used to identify nuclear receptor ligand interaction, or for protein engineering applications, particularly of the nuclear receptor ligand binding domain. In this work, the use of estrogen receptors as sensors for enzyme catalysis is explored, where expression of a reporter gene is induced in the presence of the product from an enzymatic reaction. This system, which we have called enzyme-activated growth, has applications for the engineering of biocatalysts. Biocatalytic routes are currently being explored in industrial applications since they often have financial and environmental benefits over traditional heterogeneous catalysis. Enzyme-activated growth is designed to serve as a system to select for engineered enzymes capable of catalyzing the desired reaction. For this work, a new yeast two-hybrid strain has been developed and characterized to allow for detection of both agonist and antagonist compounds. To increase the sensitivity of this assay, a variant of the estrogen receptor was created through random mutation, which responded to ligand concentrations an order of magnitude lower than the wild type receptor. The five mutations identified in the best variant were previously unknown in the literature and the roles of each of these are investigated, as is the mechanism by which they alter ligand sensitivity. As a proof-of-principle, the enzymatic production of genistein, an estrogenic metabolite from plants, using the enzyme isoflavone synthase, as well as the production of estrogen from testosterone, is explored. Synthesis of genistein from the starting material naringenin in vivo was detected in the yeast two-hybrid strain; however, attempts at pairing this with estrogen receptor activation and cell growth were met with limited success. Lastly, targeting the estrogen receptor with a series of novel anti-cancer therapeutics is explored. These compounds were designed to both bind and (in)activate the estrogen receptor while inhibiting histone deacetylase activity. The (anti-)estrogenic properties were analyzed as well as their potency as histone deacetylase inhibitors. These properties were compared to their anti-proliferative effects against various cancerous and healthy cell lines to determine their potential as selective anti-cancer therapeutics.

Heavily-selected livestock production traits rarely come without compromise; altered physiology arising from intensive selection often gives rise to concern of a welfare trade-off. A particularly clear example of welfare challenge caused by genetic selection in chickens is the ‘broiler-breeder paradox’, wherein breeding populations of broiler-type birds selected for fast growth are feed-restricted in order to reduce growth and maintain reproductive viability at sexual maturity. In order to better-inform management and breeding strategies for alleviating reproductive problems resulting from genetic selection for growth, it is essential to develop a better understanding of the physiological processes underpinning growth. Whereas the molecular mechanisms governing energy balance in mammals have been relatively welldescribed, analogous avian systems have not received as much research attention and remain somewhat poorly understood. The broad aim of this doctoral project was to contribute to understanding of avian energy balance, particularly in the context of selection for high growth. Using an advanced broiler-layer intercross chicken line (AIL), high- and low-growth haplotypes at the locus encoding the cholecystokinin A receptor (CCKAR), underlying the most significant QTL for growth in chickens, were characterised. Of over 300 variations detected, a select panel spaced across the CCKAR locus were tested for prediction of bodyweight in a diverse cohort of chicken populations. One intronic SNP was found to be significant (p < 0.05) and proximal to transcription factor binding sites. The effect of this locus on gross bodyweight remained significant into the 20th AIL generation (~20% at 10wk, p < 0.05). In this otherwise effectively genetically homogeneous population, several specific physiological traits were predicted by CCKAR haplotype alone, yielding some clues as to the significance of perturbed cholecystokinin (CCK) signalling in broiler strains. While birds with high-growth CCKAR haplotype (HG) did not appear to consume more, feed conversion efficiency (FCE) was improved, at least for males, compared to low-growth (LG) (p < 0.05). Visceral organ anatomies were morphologically disparate, with HG individuals exhibiting ~1/3 less gallbladder mass (p < 0.01), and ~10% shorter GI tract (p < 0.01) and metatarsal bone (p < 0.05). Further gaps in knowledge of the expression of peripheral satiety hormones in chicken are addressed in this thesis. Tissue distributions for expression of CCK, gastrin, pancreatic polypeptide (PPY) and peptide YY (PYY), were mapped and their respective dynamic responses to nutritive state examined. CCK was found to be most highly expressed in the brain, whereas PYY, PPY and gastrin were far more abundant in distinct regions of the periphery. Interestingly, peripheral CCK was not responsive to short-term (< 10h) satiety in experimental populations where PYY and gastrin were. PYY expression was found to be greatest in the pancreas and consistently upregulated within hours after feeding (p < 0.01), whereas gastrin expression was confined to the gastric antrum and paradoxically highest in fasting birds (p < 0.01). PPY expression is strictly limited to the pancreas and appears dependent on longerterm energy state. These results highlight similarities and differences to mammalian systems; notably, the avian pancreas seems to fulfil an exceptional role as a site of signal integration, perhaps unsurprising considering its disproportionate size compared to mammals. Indeed, pancreatic PYY appears to act as a primary peripheral short-term satiety hormone in birds. This body of work contributes to the understanding of avian energy balance and growth. An invaluable foundation for future research is formed by the identification of the major locations of production, and basic nutrient-responsive trends, for several peripheral avian hormones. Information on the growth role of CCKAR is consolidated and expanded upon, demonstrating a clear genetic contribution to maintenance organ morphology and overall growth. Such knowledge can be used to reliably assess and advise on selection and management of chickens to stem welfare concerns without compromising production. Comparisons between avian and other vertebrate endocrine systems make for interesting insight into the adaptive role of energy homeostatic mechanisms in divergent evolution of mammals and non-mammalian vertebrates. In some aspects, birds might better represent the ancestral phenotype from which each vertebrate clade arose.

A esquistossomose acomete 207 milhões de pessoas, com mais de 200 mil mortes anuais. Seu principal agente etiológico é o helminto Schistosoma e o principal modelo experimental, o camundongo. Linhagens de camundongos selecionadas geneticamente para susceptibilidade (TS) e resistência (TR) a tolerância imunológica constituem bons modelos para o estudo da resposta imunológica específica e inespecífica nas infecções. O objetivo deste trabalho foi caracterizar a infecção experimental por S. mansoni nestes camundongos, evidenciando a imunopatologia por diversos parâmetros na fase aguda da infecção. TR e TS não diferiram quanto a penetração de cercárias, recuperação de vermes adultos, fecundidade/produtividade de ovos das fêmeas de S. mansoni, mas predominaram ovos mortos em TS. Quanto maior o número de casais, maior a probabilidade de troca de casais e regressão sexual da fêmea, além de pequena redução da produtividade de ovos. Análise ultraestrutural dos parasitos machos recuperados de TS apresentaram tubérculos edemaciados, espinhos encurtados e em menor densidade que os parasitos dos TR. O tegumento dos parasitos recuperados de TS apresentou-se desorganizado, intensamente vacuolizado e com tendência a se desprender da superfície e espinhos internalizados e células vitelínicas desorganizadas. TS desenvolveram granulomas hepáticos grandes, com fibras radiais e predomínio do estágio exsudativo-produtivo com características de fase produtiva (EP/P), enquanto camundongos TR desenvolveram granulomas menores, com fibras concêntricas e predomínio de granulomas exsudativo-produtivos. TS desenvolveu hepatomegalia mais acentuada na fase aguda da infecção e exacerbada esplenomegalia na fase crônica. A aspartato aminotransferase mais elevada nos TR foi coerente com a acentuada histólise nos granulomas iniciais dos TR. É possível que a histólise menor em TS tenha contribuído para sua intensa hepatomegalia na fase aguda. Leucócitos totais séricos aumentaram em TS, nas fases aguda e crônica, mas não em TR. TS apresentaram anemia durante a fase crônica da infecção, possivelmente devido ao desvio na hematopoiese medular para a produção de leucócitos ou apoptose das hemácias. A mieloperoxidase neutrofílica hepática e no íleo foi maior em TS e a peroxidase de eosinófilos foi mais elevada no íleo do TS. Ambas as linhagens produziram IFN-&#947;, mas os níveis funcionais de IFN-&#947; foram diferentes nas duas linhagens em cultura de células. É possível que a imunopatologia hepática grave na linhagem TS possa estar relacionada aos altos títulos IFN-&#947;. TS produziu IL-10 em maior quantidade, entretanto esta citocina não foi capaz de regular o crescimento exacerbado dos granulomas hepáticos. Altos títulos de IL-4 na linhagem TS também são coerentes com a exacerbação dos granulomas, pois, como a IL-13, a IL-4 induz síntese de colágeno e está relacionada ao desenvolvimento da fibrose no granuloma esquistossomótico. Observamos redução do percentual relativo de células T CD4+ hepáticas de animais infectados em ambas as linhagens e redução percentual nas subpopulações de linfócitos B na medula óssea (precursores, linfócitos B imaturos, maduros e plasmócitos) mais acentuada em TS que em TR, possivelmente devido a extensa mobilização de B imaturos induzida pela inflamação ou desvio da hematopoiese para síntese de granulócitos em TS. Quantitativamente, TR não alterou suas subpopulações de linfócitos B. TS e TR são bons modelos para estudo da resposta imunológica na infecção esquistossomótica experimental. Novos estudos são necessários para confirmar nossas propostas e compreender os mecanismos envolvidos na diferença da resposta imunológica dessas linhagens na relação schistosoma-hospedeiro. / Schistosomiasis affects 207 million people, with more than 200 000 deaths annually. Its main etiological agent is the helminth Schistosoma and the main experimental model, the mouse. Strains of mice genetically selected for susceptibility (TS) and resistance (TR) immunological tolerance are good models for the study of specific and nonspecific immune response in infections. The objective of this study was to characterize the experimental infection with S. mansoni in these mice, demonstrating the immunopathology of several parameters in the acute phase of infection. TR and TS did not differ in the penetration of cercariae, adult worms recovery, fecundity/ productivity of eggs from females of S. mansoni, but dead eggs prevailed in TS. The greater the number of couples, the more probability of changing couples and female sexual regression, and slight reduction of the eggs productivity. Ultrastructural analysis of parasites recovered from TS males had swollen tubercles, shortened spines and lower density of them than the parasites of TR. The tegument of the parasites recovered from TS appeared disorganized, intensely vacuolated and with a tendency to detach from the surface and internalized spines and disorganized vitelline cells. TS developed large hepatic granulomas with radial fibers and predominance of exudative-productive stage with characteristics of productive stage (EP/P), while TR mice developed smaller granulomas, with concentric fibers and predominance of exudative-productive granulomas. TS developed hepatomegaly more pronounced in the acute phase of infection and exacerbated splenomegaly in chronic phase. The aspartate aminotransferase was higher in TR mice consistent with the marked histolysis in initials TR granulomas. It is possible that the lower histolysis in TS mice has contributed to its severe hepatomegaly in acute phase. Serum total leukocytes increased in TS acute and chronic phases, but not in TR. TS had anemia during the chronic phase of infection, possibly due to deviation in bone marrow hematopoiesis for the production of leukocytes or apoptosis of red blood cells. The neutrophil myeloperoxidase from liver and ileum were higher in TS and the eosinophil peroxidase was higher in the TS ileum. Both strains produced IFN-&#947;, but functional levels of IFN-&#947; were different in the two strains in cell culture. It is possible that severe liver immunopathology in TS strain may to be related to high IFN-&#947; titers. TS produced IL-10 in larger quantities, however this cytokine was not able to regulate the overgrowth of hepatic granulomas. High levels of IL-4 in TS strain are also consistent with the exacerbation of granulomas, because as IL-13, IL-4 induces collagen synthesis and is related to the development of fibrosis in schistosomal granuloma. We observed reduction in the relative percentage of TCD4 + liver cells of infected animals in both strains and percentage reduction in subpopulations of B lymphocytes in bone marrow (precursors, immature and mature B lymphocytes, plasma cells) stronger in TS than TR, possibly due to extensive mobilization of immature B cells induced by inflammation or hematopoiesis deviation for synthesis of granulocytes in TS. Quantitatively, TR did not change their subpopulations of B lymphocytes. TS and TR are good models for studying the immune response in experimental schistosome infection. Further studies are needed to confirm our proposals and to understand the mechanisms underlying the difference in immune response of these strains in the relationship schistosoma-host.

A esquistossomose acomete 207 milhões de pessoas, com mais de 200 mil mortes anuais. Seu principal agente etiológico é o helminto Schistosoma e o principal modelo experimental, o camundongo. Linhagens de camundongos selecionadas geneticamente para susceptibilidade (TS) e resistência (TR) a tolerância imunológica constituem bons modelos para o estudo da resposta imunológica específica e inespecífica nas infecções. O objetivo deste trabalho foi caracterizar a infecção experimental por S. mansoni nestes camundongos, evidenciando a imunopatologia por diversos parâmetros na fase aguda da infecção. TR e TS não diferiram quanto a penetração de cercárias, recuperação de vermes adultos, fecundidade/produtividade de ovos das fêmeas de S. mansoni, mas predominaram ovos mortos em TS. Quanto maior o número de casais, maior a probabilidade de troca de casais e regressão sexual da fêmea, além de pequena redução da produtividade de ovos. Análise ultraestrutural dos parasitos machos recuperados de TS apresentaram tubérculos edemaciados, espinhos encurtados e em menor densidade que os parasitos dos TR. O tegumento dos parasitos recuperados de TS apresentou-se desorganizado, intensamente vacuolizado e com tendência a se desprender da superfície e espinhos internalizados e células vitelínicas desorganizadas. TS desenvolveram granulomas hepáticos grandes, com fibras radiais e predomínio do estágio exsudativo-produtivo com características de fase produtiva (EP/P), enquanto camundongos TR desenvolveram granulomas menores, com fibras concêntricas e predomínio de granulomas exsudativo-produtivos. TS desenvolveu hepatomegalia mais acentuada na fase aguda da infecção e exacerbada esplenomegalia na fase crônica. A aspartato aminotransferase mais elevada nos TR foi coerente com a acentuada histólise nos granulomas iniciais dos TR. É possível que a histólise menor em TS tenha contribuído para sua intensa hepatomegalia na fase aguda. Leucócitos totais séricos aumentaram em TS, nas fases aguda e crônica, mas não em TR. TS apresentaram anemia durante a fase crônica da infecção, possivelmente devido ao desvio na hematopoiese medular para a produção de leucócitos ou apoptose das hemácias. A mieloperoxidase neutrofílica hepática e no íleo foi maior em TS e a peroxidase de eosinófilos foi mais elevada no íleo do TS. Ambas as linhagens produziram IFN-&#947;, mas os níveis funcionais de IFN-&#947; foram diferentes nas duas linhagens em cultura de células. É possível que a imunopatologia hepática grave na linhagem TS possa estar relacionada aos altos títulos IFN-&#947;. TS produziu IL-10 em maior quantidade, entretanto esta citocina não foi capaz de regular o crescimento exacerbado dos granulomas hepáticos. Altos títulos de IL-4 na linhagem TS também são coerentes com a exacerbação dos granulomas, pois, como a IL-13, a IL-4 induz síntese de colágeno e está relacionada ao desenvolvimento da fibrose no granuloma esquistossomótico. Observamos redução do percentual relativo de células T CD4+ hepáticas de animais infectados em ambas as linhagens e redução percentual nas subpopulações de linfócitos B na medula óssea (precursores, linfócitos B imaturos, maduros e plasmócitos) mais acentuada em TS que em TR, possivelmente devido a extensa mobilização de B imaturos induzida pela inflamação ou desvio da hematopoiese para síntese de granulócitos em TS. Quantitativamente, TR não alterou suas subpopulações de linfócitos B. TS e TR são bons modelos para estudo da resposta imunológica na infecção esquistossomótica experimental. Novos estudos são necessários para confirmar nossas propostas e compreender os mecanismos envolvidos na diferença da resposta imunológica dessas linhagens na relação schistosoma-hospedeiro. / Schistosomiasis affects 207 million people, with more than 200 000 deaths annually. Its main etiological agent is the helminth Schistosoma and the main experimental model, the mouse. Strains of mice genetically selected for susceptibility (TS) and resistance (TR) immunological tolerance are good models for the study of specific and nonspecific immune response in infections. The objective of this study was to characterize the experimental infection with S. mansoni in these mice, demonstrating the immunopathology of several parameters in the acute phase of infection. TR and TS did not differ in the penetration of cercariae, adult worms recovery, fecundity/ productivity of eggs from females of S. mansoni, but dead eggs prevailed in TS. The greater the number of couples, the more probability of changing couples and female sexual regression, and slight reduction of the eggs productivity. Ultrastructural analysis of parasites recovered from TS males had swollen tubercles, shortened spines and lower density of them than the parasites of TR. The tegument of the parasites recovered from TS appeared disorganized, intensely vacuolated and with a tendency to detach from the surface and internalized spines and disorganized vitelline cells. TS developed large hepatic granulomas with radial fibers and predominance of exudative-productive stage with characteristics of productive stage (EP/P), while TR mice developed smaller granulomas, with concentric fibers and predominance of exudative-productive granulomas. TS developed hepatomegaly more pronounced in the acute phase of infection and exacerbated splenomegaly in chronic phase. The aspartate aminotransferase was higher in TR mice consistent with the marked histolysis in initials TR granulomas. It is possible that the lower histolysis in TS mice has contributed to its severe hepatomegaly in acute phase. Serum total leukocytes increased in TS acute and chronic phases, but not in TR. TS had anemia during the chronic phase of infection, possibly due to deviation in bone marrow hematopoiesis for the production of leukocytes or apoptosis of red blood cells. The neutrophil myeloperoxidase from liver and ileum were higher in TS and the eosinophil peroxidase was higher in the TS ileum. Both strains produced IFN-&#947;, but functional levels of IFN-&#947; were different in the two strains in cell culture. It is possible that severe liver immunopathology in TS strain may to be related to high IFN-&#947; titers. TS produced IL-10 in larger quantities, however this cytokine was not able to regulate the overgrowth of hepatic granulomas. High levels of IL-4 in TS strain are also consistent with the exacerbation of granulomas, because as IL-13, IL-4 induces collagen synthesis and is related to the development of fibrosis in schistosomal granuloma. We observed reduction in the relative percentage of TCD4 + liver cells of infected animals in both strains and percentage reduction in subpopulations of B lymphocytes in bone marrow (precursors, immature and mature B lymphocytes, plasma cells) stronger in TS than TR, possibly due to extensive mobilization of immature B cells induced by inflammation or hematopoiesis deviation for synthesis of granulocytes in TS. Quantitatively, TR did not change their subpopulations of B lymphocytes. TS and TR are good models for studying the immune response in experimental schistosome infection. Further studies are needed to confirm our proposals and to understand the mechanisms underlying the difference in immune response of these strains in the relationship schistosoma-host.