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<p>Abstract</p><p>The trefoil factor family of secreted proteins contains three members; trefoil factor 1 or TFF1, trefoil factor 2 or TFF2, and trefoil factor 3 or TFF3. These three proteins share a conserved 42-43 amino acid domain containing 6 cysteine residues resulting in three disulfide bonds that holds the protein in a characteristic three-loop or "trefoil structure" known as the P domain. TFF1 is primarily localized to the stomach and secreted by the gastric mucosa while TFF2 and TFF3 are primarily localized to the colon and duodenum and secreted by the goblet cells. All three of these proteins play a protective role in the gastrointestinal tract where they are normally localized and have been identified as possible tumor suppressors, however, these proteins are also upregulated in cancer within tissues where they are not normally expressed including the breast, pancreas, prostate, and liver. The mechanisms by which two of these factors, TFF1 and TFF3, promote tumorigenesis remain largely undefined. In this dissertation we will attempt to elucidate these mechanisms as well as the regulation of these two proteins in both pancreatic and prostate cancer. Many of the underlying genetic and molecular mechanisms involved in the development of both pancreatic and prostate cancer remain largely unknown and as a result, therapeutic and diagnostic tools for treating these diseases are not as effective as they could be. By deciphering the role of TFF1 and TFF3 in these cancers, they could potentially serve as new therapeutic targets or biomarkers for treating both diseases.</p><p>Chapter 2 of this dissertation will examine the functional role of TFF1 promoting tumorigenesis in pancreatic and prostate cancer. We will show that TFF1 expression is critical for the viability of both pancreatic and prostate cancer cells and that reduction of TFF1 expression in these cells results in decreased tumorigenicity when implanted in immunocompromised mice. It will also be demonstrated that TFF1's function in promoting tumorigenicity is its ability to assist tumor cells overcome the tumor suppressive barrier of senescence. Thirdly, we show that the form of senescence that TFF1 assists in allowing the cells overcome is oncogene-induced senescence (OIS). Lastly, a cell cycle array identifies the potential downstream target p21CIP, a cyclin-dependent kinase inhibitor and OIS marker, whose expression is induced by loss of TFF1 expression.</p><p>In Chapter 3 of this work, we examine the role of another trefoil factor family member, TFF3, and its role in promoting prostate tumorigenesis. Just as with TFF1, it appears that TFF3 3 expression is critical for prostate cancer cell viability and tumorigenicity using the same experimental techniques used in Chapter 2. Using a genetically defined model of prostate cancer, a PI3-kinase-dependent regulatory mechanism of TFF3 emerges in this prostate cancer context. Using this system we begin to see a divergence in both regulation and function of TFF1 and TFF3 in prostate cancer. Finally, a mouse model expressing TFF3 was developed to monitor the histopthological changes associated with expression of this protein. Initial characterization of this model suggests a hyperplastic phenotype coinciding with TFF3 expression in the prostate.</p><p>The two studies in this dissertation establish a role of TFF1 and TFF3 in both prostate and pancreatic tumorigenesis and demonstrate that ablation of expression of both proteins is a potent inhibitor of tumorigenesis. With this knowledge, it is possible that TFF1 and TFF3 may become a potential therapeutic target or diagnostic marker for better treatment of prostate and pancreatic cancer.</p> / Dissertation

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Trefoil brooches are one of the most abundant types of Viking Age jewellery in Scandinavia. This thesis delves into the journey of one such brooch, known as 555783, discovered in Birka, Sweden. Through an archaeological object biography approach this thesis examines the brooch's construction, provenance, transportation, transformation, and deposition in an attempt to shed light on how this and other similar examples evolved in Scandinavian society compared to their cultural origins. Furthermore, this research aims to uncover broader patterns in the Viking Age Scandinavian society's contact with and adaptation of foreign material culture through the incorporation of comparative examples, in order to explore what this illustrates about the Scandinavian people in general. The conclusion reached is that 555783 was likely made in Frankia then transported to Scandinavia through unknown means. There it was modified from a mount on a sword belt with male, martial associations into a piece of fastening-jewellery associated with female costume and display. It was also determined that the adoption and adaption of foreign material culture was primarily a way of displaying foreign connections in a way better suited to their own aesthetic and material preferences. / En av de mest rikligt förekommande smyckestyperna från vikingatiden i Skandinavien är treflikiga spännen. Den här uppsatsen undersöker livshistorien av ett treflikigt spänne (555783) som upptäcktes i Birka. Genom ett arkeologiskt objektbiografiskt tillvägagångssätt undersöker denna uppsats spännet konstruktion, ursprung, förflyttning, förändring och deposition. Undersökningen illustrerar hur spänne 555783 och liknande exempel utvecklades i det skandinaviska samhället jämfört med i sin ursprungskultur. Vidare syftar denna uppsats till att belysa ett bredare mönster i vikingatida skandinavers kontakt med och anpassning av främmande materiell kultur. Uppsatsen besvarar detta genom jämförelse med andra liknande exempel. Slutsatsen ernådd är att spänne 555783 troligen har tillverkats i det Frankiska riket och sedan transporterats till Skandinavien på ett okänt sätt. Där modifierades den från ett beslag på ett svärdsbälte med manliga, krigiska associationer till ett smycke förknippat med kvinnlig uppvisning av status. Det fastställdes också att inlemmandet och modifieringen av främmande materiell kultur i första hand var ett sätt att visa utländska kopplingar som var anpassat till deras egna estetiska och materiella preferenser.

Human acidic fibroblast growth factor (FGF-1) is a member of the £]-trefoil superfamily and exhibits a characteristic three-fold tertiary structure symmetry. However, evidence of this symmetry is not readily apparent at the level of the primary structure. This suggests that while selective pressures may exist to retain (or converge upon) a symmetric tertiary structure, other selective pressures have resulted in divergence of the primary structure during evolution. Using intra-chain and homologue sequence comparisons for 19 members of this family of proteins, we have designed mutants of FGF-1 that constrain a subset of core-packing residues to three-fold symmetry at the level of the primary structure. The consequences of these mutations upon structure, stability, folding and unfolding kinetics have been evaluated using a combination of x-ray crystallography, differential scanning calorimetry, isothermal equilibrium denaturation and stopped flow protein refolding/unfolding kinetics. An alternative core packing group has been introduced into FGF-1. The alternative core is very similar from the wild type (WT) core with regard to structure, stability, folding and unfolding kinetics. The remaining asymmetry within the protein core is related to asymmetry in the tertiary structure. The removal of tertiary structure asymmetry greatly increases protein stability and results in a conversion from three-state to a two-state folding pathway. The tertiary structure asymmetry is intimately linked to functional regions of the protein. Surprisingly, upon deletion of the functional insertions, the mutant protein is approximately 80 times more potent than the wild type form as determined by functional bioassays. The results show that the ƒÒ-trefoil superfold is compatible with a three-fold symmetric constraint upon the core region, as might be the case if the superfold arose as a result of gene duplication/fusion events. Furthermore, this new protein arrangement can form the basis of a structural "building block" that can greatly simplify the de novo design of ƒÒ-trefoil proteins by utilizing symmetric structural complementarity. This study implies that a symmetric architecture of the £]-trefoil fold is kinetically and thermodynamically ¡§fit¡¨. / Dissertation / PhD

Submitted by Andrea Pereira (andrea.pereira@unipampa.edu.br) on 2017-03-10T16:36:46Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Análise da Secagem de Cornichão-Akel FKanaan.pdf: 1613401 bytes, checksum: 62cca34779fa01d06bb57914687636da (MD5) / Approved for entry into archive by Andrea Pereira (andrea.pereira@unipampa.edu.br) on 2017-03-10T16:37:11Z (GMT) No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Análise da Secagem de Cornichão-Akel FKanaan.pdf: 1613401 bytes, checksum: 62cca34779fa01d06bb57914687636da (MD5) / Made available in DSpace on 2017-03-10T16:37:11Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Análise da Secagem de Cornichão-Akel FKanaan.pdf: 1613401 bytes, checksum: 62cca34779fa01d06bb57914687636da (MD5) Previous issue date: 2014-09-03 / A crescente necessidade da utilização de espécies forrageiras de estação fria na região sul do país vem determinando uma grande demanda por sementes de qualidade. Entre as leguminosas se destaca o cornichão (Lotus corniculatus L.), que é uma leguminosa forrageira oriundo da Europa e da região mediterrânea. Esta leguminosa forrageira tem na versatilidade a sua maior vantagem, visto que apresenta produção de forragem na primavera, no verão e no outono, possui resistência à seca e ao encharcamento temporário do solo e apresenta boa persistência da massa verde. Além disso, as sementes deste tipo de espécie podem ser misturadas com espécies de inverno, o que aumenta o rendimento da matéria seca, dá uma melhor distribuição da forragem ao longo do ano e agrega uma melhora na qualidade das pastagens. Apesar dessas vantagens, as sementes de cornichão comercializadas possuem ainda uma baixa qualidade. Esse fato é decorrente de um sistema de fiscalização ineficiente, associado à dificuldade de produção e beneficiamento do produto. Outro problema que o produtor de sementes desta espécie enfrenta é a dormência tegumentar, isto é, o revestimento da semente (tegumento) é impermeável e não permite que a água necessária para a germinação chegue ao interior da semente. Na literatura, diversos trabalhos têm relatado que cada vez mais há um aumento na demanda de sementes forrageiras em função do melhoramento do campo nativo e das pastagens cultivadas. Diante de um mercado tão promissor, a obtenção de sementes de melhor qualidade ainda apresenta dificuldades que vão desde a fase de produção no campo, onde as sementes são colhidas precocemente em relação ao ponto de maturidade fisiológica devido ao processo de perda natural, até seu beneficiamento, onde o processo de secagem apresenta-se como uma etapa limitante que necessita de uma atenção muito especial para evitar perdas fisiológicas no produto final. De um modo geral, a secagem é definida como a remoção, voluntária ou não, total ou parcial, de uma fase líquida ou gasosa de qualquer material através da transferência de calor e de massa. A secagem tem em sua essência a finalidade de assegurar a conservação de um produto por meio da redução do seu teor de água. No caso de materiais orgânicos, como é o caso de sementes, a redução no teor de água deve ser efetuada até o ponto em que a concentração de açúcares, ácidos, sais e outros componentes, sejam suficientemente elevados para reduzir a atividade de água e inibir, portanto, o desenvolvimento de micro-organismos. Na tecnologia de produção de sementes de alta qualidade a secagem é tida com uma das etapas de maior importância, pois permite a redução do teor de água em níveis adequados para o armazenamento e comercialização (12 a 15 % de umidade), preserva as sementes de alterações físicas e químicas induzidas pelo excesso de umidade, torna possível a manutenção da qualidade inicial durante o armazenamento e possibilita colheitas próximas da maturação fisiológica. Entretanto, o processo de secagem, seja ele qual for, deve acontecer de forma controlada e de maneira uniforme a fim de evitar elevados gradientes de umidade e temperatura no interior do material que podem provocar a perda da qualidade do produto final. De acordo com o que foi exposto, o presente trabalho de mestrado teve como objetivo realizar um estudo sobre o processo de secagem de sementes de cornichão (Lotus corniculatus L.) em um secador de leito fixo com fluxo de ar paralelo. Para alcançar tal objetivo, este trabalho foi dividido nas seguintes etapas: caracterização física e fisiológica das sementes, análise do processo de reumidificação das sementes, estudo das isotermas de dessorção das sementes reumidificadas e avaliação dos efeitos da variação da temperatura, da velocidade do ar e da altura da bandeja sobre a cinética de secagem e qualidade fisiológica do produto final. Os dados experimentais mostraram que os resultados obtidos para a umidade em base úmida das sementes secas ficaram na faixa de 5 % para as secagens realizadas a 60 °C e em aproximadamente 8 % para as secagens realizadas a 40 °C. No que se refere à germinação, os valores encontrados estão na ordem de 44 a 59 % para as secagens realizadas neste estudo. Dentre as secagens, a que obteve o maior valor de germinação, com valor de 59 %, foi a realizada na condição de T = 40 °C, v = 1,0 m/s e h = 1,0 cm. / The growing necessity for the use of cool-season forage species in the southern region of the country has indicated a high demand for quality seeds. Among the legumes, the birdsfoot trefoil (Lotus corniculatus L.) is a forage legume which is originated in Europe and in the Mediterranean region. This versatility in the forage legume has its biggest advantage, since it presents forage production in the spring, summer and autumn, is resistant to drought and temporary waterlogging and shows good persistence of green mass. Furthermore, seeds of such kind can be mixed with winter species, which increases the yield of dry matter, gives a better distribution of the material throughout the year and adds an improvement in the quality of pastures. Despite these advantages, the seeds of birdsfoot marketed still have a poor quality. This fact is a result of an inefficient system of monitoring and difficulties associated to production and processing of the product. Another problem is that the seed producer of this species is facing cutaneous numbness, that is, the seed coat (tegument) is impermeable and does not allow the water, necessary for germination to reache the interior of the seed. In the literature, several studies have reported that there is an increasing demand for an increase in forage seed due to the improvement of native grassland and cultivated pastures. Faced with such a promising market, obtaining better quality seed still presents difficulties ranging from the production phase of the field, where the seeds are harvested early in relation to physiological maturation due to the natural loss process until its processing where the drying process is presented as a limiting step that requires particular attention to avoid losses in physiological final product. In general, drying is defined as the removal voluntary or not, full or partial, of a liquid or gaseous phase of any material by the transfer of heat and mass. Drying has its essence in order to ensure the preservation of a product through reduction of water content. In the case of organic materials, such as seeds, reduction in water content must be performed to the extent that the concentration of sugars, acids, salts and other components are sufficiently high to reduce the water activity and inhibit, therefore, the growth of microorganisms. In the production of high quality seeds the drying technology is seen with one of the most important steps, since it allows reducing the water content suitable for storage and marketing levels (12 to 15% moisture), preserves the seeds from physical changing and chemical induced excess moisture, makes it possible to maintain the initial quality during storage and enables the next harvest physiological maturity. However, the drying process, whatever it may be, should occurs in a controlled and uniform manner in order to avoid high temperature and humidity gradients within the material that could result in loss of quality of the final product. Towards to it, this paper aimed to conduct a study on the drying of seeds of birdsfoot trefoil (Lotus corniculatus L.) in a fixed bed dryer with parallel air flow. To accomplish this goal, this study was divided into the following steps: physical and physiological seed characterization, analysis of rewetting seed process, study of desorption isotherms of the rewetted seeds and evaluation the effects of temperature, air velocity and the height of the tray on the drying kinetics and physiological quality of the final product. The experimental data showed that the results for the dry seeds of UBU were in the range of 5 % to drying carried out at 60 ° C and about 8% to drying carried out at 40 ° C. With regard to seed germination, the values are found in a range of 44 to 59% for the drying carried out in this study. The drying condition whom presented the highest germination value about 59 % was performed on T = 40 ° C, v = 1,0 m/s and h = 1,0 cm.

Conventional production of meat products from ruminant animals in the United States requires inputs including the cultivation and nitrogen fertilization of annual grains such as corn and barley, and transportation of cattle and grain to feedlots. Consumers have concerns about the impact of feedlot conditions on animal health, and about the implications of pharmaceutical inputs such as growth hormones and antibiotics on the environment and human health. These concerns have led to a growing interest in pasturefinished meat production by consumers. Such smaller-scale livestock production systems can be healthier and lower-stress for animals, are integrated into local food systems and are more transparent to consumers, and have higher potential profitability for producers than traditional ruminant production methods. There is a strong market for pasture-finished beef products, and prices for naturally or organically raised beef have remained well above feedlot-produced product prices. There is also concern about the impact of ruminant production on the environment, including air and water pollution from feedlot production and greenhouse gasses that are emitted from ruminant animals during feed digestion. This thesis project explored the potential of a beef production system based on perennial legumes, including the non-bloating legume birdsfoot trefoil (BFT; Lotus corniculatus L.) for producing meat products from cattle while reducing concentrate feeding and methane production. The condensed tannins that are produced by BFT bind proteins in the rumen but allow them to be digested in the abomasum and intestines, which in turn leads to better utilization of forage nutrients during the finishing period and higher gains or milk production. The higher digestibility of legumes compared with grasses reduces methane emissions in cattle both through higher digestibility of the forage and through direct impacts on methanogens operating in the rumen. As reported in this thesis, steers finished on BFT gained significantly more weight per day than steers fed another perennial forage legume, cicer milkvetch, but did not gain as rapidly as feedlot-fed steers. At the end of summer grazing, the blood plasma of pasture-fed steers was lower in saturated and omega-6 fatty acids and higher in transvaccenic and omega-3 fatty acids than the blood plasma of feedlot-fed steers. When beef cows grazed grass and legume pastures, enteric methane emissions were lower on the legume pastures than the grass pasture. These results demonstrate that, compared with other feed sources, perennial legume pastures used for cattle production can improve cattle gains and reduce environmental impacts.

Forages containing condensed tannins (CT) have potential to reduce the environmental impact of dairy farming. In 3 studies, I hypothesized that feeding CTcontaining birdsfoot trefoil (Lotus corniculatus, BFT) would result in improved nutrient utilization and lactational performance of dairy cows compared with control forages of the respective experiments. Improved milk components, reduction in waste N, and overall improved N efficiency were hypothesized for BFT-fed cows compared to those cows fed alfalfa hay (Study 1) or grass-based diets (Study 2). In addition, a decrease in in vitro methane production and improved rumen fermentation due to diets based on BFT pasture and concentrate supplementation compared with grass pasture-based diets was the hypothesis of the third study. Study 1 showed BFT-hay diets improved lactational performance through increased energy-corrected milk yield and increased milk protein yield, resulting in improved N utilization efficiency compared with the alfalfa hay diet. Total volatile fatty acids concentration tended to increase, and greater microbial protein yield was exhibited by cows fed BFT compared to other diets tested. Therefore, BFT can replace alfalfa hay in dairy diets and showed improved feed and N utilization efficiencies and lactational performance. Study 2 determined that pasture nutrient content increased for BFT pasture compared to the mixed grass control, contributing to increases in milk yield most weeks during the 2-year study. Energy-corrected milk yield increased most weeks by BFT-grazed cows due to increased milk yield, although milk protein concentration was similar between treatments. Cows grazing BFT pasture increased N efficiency coupled with decreased milk urea N secretion in the first, but not the second year, suggesting an environmental advantage over traditional grass-based pastures depending on the effect of growing conditions on pasture quality at time of grazing. Study 3 showed that offering BFT pasture to continuous cultures without or with barley grain or total mixed ration supplements reduced methane production and altered rumen microbial populations. The reduced methane production on the continuous cultures was likely due to direct and/or indirect effects of CT on rumen microbiota. Overall, diets including BFT showed improved nutritive, lactational, and environmental benefits by decreasing N waste and methane production over typical alfalfa hay-based dairy diets and grass pastures.

Understanding the origin of protein folds, and the mechanism by which evolution has generated them, is a critically important step on a path towards rational protein design. Modifying existing proteins and designing our own novel folds and functions is a lofty but achievable goal, for which there are many foreseeable rewards. It is believed that modern proteins may have arisen from a primordial set of peptide precursors, which were initially only pseudo-stable or stable only as complexes with RNA, and later were able to self-assemble into multimeric complexes that resembled modern folds. In order to experimentally examine the feasibility of this theory, an attempt was made at reconstructing the evolutionary path of a beta-trefoil. The beta-trefoil is a naturally abundant fold or superfold, possessing pseudo-threefold symmetry, and usually having a sugar-binding function. It has been proposed that such a fold could arise from the triplication of just one small peptide on the order of 40-50 amino acids in length. The evolutionary path of a ricin, a family within the beta-trefoils known to possess a carbohydrate binding function was the chosen template for evolutionary modelling. It was desirable to have a known function associated with this design, such that it would be possible to determine if not only the fold, but also the function, could be reconstructed. A small peptide of 47 amino acids was designed and expressed. This peptide not only trimerized as expected, but possessed the carbohydrate binding function it was predicted to have. In an evolutionary model of the early protein world, the gene for this peptide would undergo duplication and later, triplication, eventually resulting in a completely symmetrical beta-trefoil, which would represent the first modern beta-trefoil fold. Such a completely symmetrical protein was also designed and expressed by triplicating the gene for the aforementioned small peptide. This hypothetical first modern beta-trefoil is: well folded, stable, soluble, and appears to adopt a beta-trefoil fold. Together these results demonstrate that an evolutionary model of early life: that proteins first existed as self-assembling modular peptides, and subsequent to gene duplications or fusions, as what we now recognize as modern folds, is experimentally consistent and not only generates stable structures, but those with function, which of course is a prime requisite of evolution. Moreover the results show that it may be possible to use this modular nature of protein folding to design our own proteins and predict the structure of others.