Efficiency of rumen microbial protein synthesis in cattle grazing tropical pastures as estimated by a novel technique

The efficiency of microbial protein synthesis (EMPS) in cattle grazing a range of tropical pasture types was examined using a new method of intra-jugular infusion of CrEDTA to estimate urinary excretion of purine derivatives (PD). Seven pasture types were studied in south-east Queensland, Australia, over a 13-month period. These included native tropical grass (C4) pasture (major species Heteropogon contortus and Bothriochloa bladhii) studied in the early wet, the wet/dry transition and the dry season; introduced tropical grass (C4) pasture (Bothriochloa insculpta) in the mid wet season; two introduced tropical legume species (C3), (Lablab purpureus and Clitoria ternatea); and the temperate grass (C3) pasture, ryegrass (Lolium multiflorum). There was a large range in EMPS across pasture types: 26-209 g microbial crude protein (MCP)/kg digestible organic matter intake (DOMI). Estimated rumen degradable protein (RDP) supply (42-525 g/kg DOMI) was the major factor associated with EMPS across the range of pasture types studied. EMPS in steers grazing all tropical grass pastures was low (<130 g/kg DOMI) and limited by RDP supply. Negative linear relationships (P<0.05) between EMPS and both neutral detergent fibre (NDF) and acid detergent fibre (ADF) concentrations in extrusa were evident. However, non-fibre carbohydrate in extrusa, total non-structural carbohydrate concentration in plucked pasture leaf, rumen fluid and particle dilution rate, protozoal concentration in rumen fluid and rumen fluid pH were not correlated with EMPS. It was concluded that EMPS was well below 130 g MCP/kg DOMI when cattle grazed unfertilised, tropical grass pastures in south-east Queensland and that RDP was the primary limiting nutrient. High EMPS was associated with very high RDP, vastly in excess of RDP requirements by microbes

Rare earth elements and neodymium isotopes in world river sediments revisited

Over the past decades, rare earth elements (REE) and their radioactive isotopes have received tremendous attention in sedimentary geochemistry, as tracers for the geological history of the continental crust and provenance studies. In this study, we report on elemental concentrations and neodymium (Nd) isotopic compositions for a large number of sediments collected near the mouth of rivers worldwide, including some of the world’s major rivers. Sediments were leached for removal of non-detrital components, and both clay and silt fractions were retained for separate geochemical analyses. Our aim was to re-examine, at the scale of a large systematic survey, whether or not REE and Nd isotopes could be fractionated during Earth surface processes. Our results confirmed earlier assumptions that river sediments do not generally exhibit any significant grain-size dependent Nd isotopic variability. Most sediments from rivers draining old cratonic areas, sedimentary systems and volcanic provinces displayed similar Nd isotopic signatures in both clay and silt fractions, with ΔεNd (clay-silt) < |1.| A subtle decoupling of Nd isotopes between clays and silts was identified however in a few major river systems (e.g. Nile, Mississippi, Fraser), with clays being systematically shifted towards more radiogenic values. This observation suggests that preferential weathering of volcanic and/or sedimentary rocks relative to more resistant lithologies may occur in river basins, possibly leading locally to Nd isotopic decoupling between different size fractions. Except for volcanogenic sediments, silt fractions generally displayed homogeneous REE concentrations, exhibiting relatively flat shale-normalized patterns. However, clay fractions were almost systematically characterized by a progressive enrichment from the heavy to the light REE and a positive europium (Eu) anomaly. In agreement with results from previous soil investigations, the observed REE fractionation between clays and silts is probably best explained by preferential alteration of feldspars and/or accessory mineral phases. Importantly, this finding clearly indicates that silicate weathering can lead to decoupling of REE between different grain-size fractions, with implications for sediment provenance studies. Finally, we propose a set of values for a World River Average Clay (WRAC) and Average Silt (WRAS), which provide new estimates for the average composition of the weathered and eroded upper continental crust, respectively, and could be used for future comparison purposes.

Host cellular regulatory networks in dengue virus-human interactions

Dengue fever is one of the most important mosquito-borne diseases worldwide and is caused by infection with dengue virus (DENV). The disease is endemic in tropical and sub-tropical regions and has increased remarkably in the last few decades. At present, there is no antiviral or approved vaccine against the virus. Treatment of dengue patients is usually supportive, through oral or intravenous rehydration, or by blood transfusion for more severe dengue cases. Infection of DENV in humans and mosquitoes involves a complex interplay between the virus and host factors. This results in regulation of numerous intracellular processes, such as signal transduction and gene transcription which leads to progression of disease. To understand the mechanisms underlying the disease, the study of virus and host factors is therefore essential and could lead to the identification of human proteins modulating an essential step in the virus life cycle. Knowledge of these human proteins could lead to the discovery of potential new drug targets and disease control strategies in the future. Recent advances of high throughput screening technologies have provided researchers with molecular tools to carry out investigations on a large scale. Several studies have focused on determination of the host factors during DENV infection in human and mosquito cells. For instance, a genome-wide RNA interference (RNAi) screen has identified host factors that potentially play an important role in both DENV and West Nile virus replication (Krishnan et al. 2008). In the present study, a high-throughput yeast two-hybrid screen has been utilised in order to identify human factors interacting with DENV non-structural proteins. From the screen, 94 potential human interactors were identified. These include proteins involved in immune signalling regulation, potassium voltage-gated channels, transcriptional regulators, protein transporters and endoplasmic reticulum-associated proteins. Validation of fifteen of these human interactions revealed twelve of them strongly interacted with DENV proteins. Two proteins of particular interest were selected for further investigations of functional biological systems at the molecular level. These proteins, including a nuclear-associated protein BANP and a voltage-gated potassium channel Kv1.3, both have been identified through interaction with the DENV NS2A. BANP is known to be involved in NF-kB immune signalling pathway, whereas, Kv1.3 is known to play an important role in regulating passive flow of potassium ions upon changes in the cell transmembrane potential. This study also initiated a construction of an Aedes aegypti cDNA library for use with DENV proteins in Y2H screen. However, several issues were encountered during the study which made the library unsuitable for protein interaction analysis. In parallel, innate immune signalling was also optimised for downstream analysis. Overall, the work presented in this thesis, in particular the Y2H screen provides a number of human factors potentially targeted by DENV during infection. Nonetheless, more work is required to be done in order to validate these proteins and determine their functional properties, as well as testing them with infectious DENV to establish a biological significance. In the long term, data from this study will be useful for investigating potential human factors for development of antiviral strategies against dengue.

Effects of early nutrition on the wnt and bone morphogenic protein pathways in the developing intestine

Infant formula is consumed by the majority of infants in the United States for at least part of the first year of life. Infant formula lacks many of the bioactive compounds that are naturally occurring in breast milk. Because of this, there has been an increased interest by the companies that manufacture infant formula to include additives that would potentially allow formula to more closely mimic breast milk activity. One such ingredient currently being added to infant formula is prebiotics. Prebiotics are non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth of specific healthful bacteria in the colon. It is speculated that prebiotics replicate the activity of breast milk oligosaccharides, which through the production of butyrate by intestinal microbiota, may interact with the Wnt/BMP pathways. The Wnt/BMP pathways regulate intestinal stem cells, which determine the growth, development and maintenance of the intestine. Therefore, the objective of this study was to explore the effects that the addition of prebiotics to formula have on the regulation of the Wnt/BMP pathways when fed to neonatal piglets, a model commonly used in the study of infant nutrition. Piglets (n=5) were randomized into sow-reared (SR), fed control formula (F), or fed formula with added prebiotics (F+P). Fructooligosaccharides (FOS) (2 g/L) and polydextrose (PDX) (2 g/L) were chosen as the prebiotics for this study, because this combination had been less studied than other combinations. Ileum and ascending colon were collected at 7 and 14 days-of-age. Dry matter content, pH, and short chain fatty acid (SCFA) content was measured. The mRNA expression of β-catenin, sFRP3, sFRP4, frizzled 6, DKK1 (Wnt pathway), gremlin (BMP pathway), TNF-a, HNF-4α and osteopontin (OPN) was measured by RT-qPCR. Piglets fed the F+P diet had greater acetate concentration and lower pH in the ileum at day 14 and in the colon at day 7 and day 14 than F piglets. Butyrate concentrations were highest in SR with F+P not differing from F in ileum at day 14 and colon at day 7 and day 14. Effects of age were seen in all genes, with the exception of OPN, sFRP-3 and sFRP-4. On day 7, no effect of diet was observed in the ileum, however, mRNA expression of DKK1 and frizzled 6 were greater in F+P than SR (p≤0.05). On day 14, gremlin expression was lower and OPN was greater in the ileum of SR piglets compared to F and F+P. Also on day 14, HNF-4α mRNA expression was greater in both ileum and colon of F+P piglets and sFRP3 mRNA expression was greater in the colon than F or SR . In summary, differences were observed between gene expression of F+P and SR piglet intestines, but the supplementation of 2 g/L scFOS and 2 g/L PDX to formula did not shift expression of genes in the Wnt/BMP pathways to be more similar to SR than F. As the Wnt/BMP pathway is known to exist in a gradient along the crypt-villus axis, with Wnt expression dominating in the crypt region and BMP expression dominating in the villi, it was possible that pooling whole tissue reduced our ability to detect treatment effects that would be concentrated in either region. A method was therefore developed to remove intestinal epithelial cells along the villus-to-crypt axis. Twenty-five-day-old F and SR piglets were euthanized and ileal tissue was collected and placed in a dissociation buffer in a shaking water bath. Exfoliated cells were removed at increasing time points from 5 to 100 minutes in order to remove cells along the villus-to-crypt axis. After the final incubation, remaining mucosal tissue was removed using a sterile glass microscope slide and pooled with the final exfoliated cell isolation. After each cell collection, a section of tissue was fixed in formalin for histomorphological examination. Expression of genes in the Wnt/BMP pathways, along with crypt marker genes (CDK5 and v-myb), were measured in both whole ileal tissue, pooled epithelial cells, and separate epithelial cell isolations from the same piglet. The expression of β-catenin, HNF-4α, TNF-α, TGF-β and the crypt marker v-myb matched the expected villus-to-crypt pattern in cells collected after 10 (incubation 1), 30 (incubation 2) and 60 (incubation 3) minutes. However, expression of expression in cells collected after 100 minutes (incubation 4) was variable, which may be due to the fact that crypt cells were not efficiently removed and the presence of unwanted non-epithelial tissue. Gremlin, OPN, DKK1, sFRP3 and sFRP4 expression was not statistically different along the villus-to-crypt axis. Frizzled 6 and CDK5 did not express as we had predicted, with expression highest towards the villi. In summary, the epithelial cell collection method used was not entirely successful. While much of the gene data suggests that cells were removed along the villus-to-crypt axis through the first three incubations, the last incubation, which involved scraping the tissue, removed non-epithelial components of the mucosa, while leaving the crypts intact. In conclusion, the addition of 2 g/L PDX and 2 g/L scFOS did not cause gene expression of the Wnt/BMP pathways to mirror either F or SR expression. New isolation methods to extract cells along the crypt-villus axis should be considered, including the use of a laser capture microdissection. While this combination of prebiotics did not yield the intended effects, future research should be done on other combinations, such as the inclusion of galactooligosaccharides (GOS), which is commonly added to food products including infant formula.

Potencial antifúngico, antioxidante e inibidor da produção de aflatoxina por extratos fenólicos de Chlorella sp. e Spirulina platensis

A contaminação fúngica acarreta alterações na qualidade nutricional e no valor econômico de produtos alimentícios podendo causar danos patológicos em plantas, animais e humanos. A identificação da atividade antioxidante, antifúngica e antimicotoxinas, em extratos de microalgas com propriedade de inibir a multiplicação de fungos e subseqüente produção de micotoxinas abre a perspectiva de empregar substâncias mais eficientes e com maior ação específica contra estes microorganismos. Entre os compostos com propriedades inibidoras de radicais livres, de crescimento fúngico e produção de micotoxinas, destacam-se os compostos fenólicos, que podem inibir a atividade metabólica microbiana, dificultando a atividade de enzimas. Neste estudo foram avaliados o poder de inibição de multiplicação fúngica de Rhizopus oryzae e Aspergillus flavus pelos extratos fenólicos de Chlorella sp. e Spirulina platensis, bem como sua atividade antioxidante, e a atividade antimicotoxinas da última microalga contra Aspergillus flavus. O conteúdo de fenóis totais foi em média 1000 µgfenóis/g Spirulina platensis e 600 µgfenóis/g Chlorella sp., sendo que o acido gálico e o cafeíco foram identificados como compostos majoritários na Spirulina platensis. As determinações de glicosamina (parede celular) e ergosterol (membrana celular) mostraram-se bons indicativos do desenvolvimento microbiano permitindo uma boa estimativa da inibição dele. O extrato fenólico de Spirulina platensis apresentou capacidade de inibir cerca de 50% a formação da parede e da membrana celular para ambos os fungos estudados e de 100% a produção de aflatoxina B1 até o 10º dia de cultivo do Aspergillus flavus. Além disso, o extrato metanólico de Spirulina platensis inativou 53,5% o DPPH reativo, limitou o escurecimento enzimático ocasionado pela peroxidase em 55% e inibiu a peroxidação lipídica em 46% após 14 dias de armazenamento sob luz. Estes resultados mostram que a ação antifúngica, antimicotoxinas e antioxidante está naturalmente presente em alguns tecidos microbianos e que encontrar a forma de extraí-los e aplicá-los como conservantes alimentícios é muito promissor para substituição aos antifúngicos e outros conservantes químicos.

Inibição de linhagens do complexo Fusarium Graminearum por compostos naturais e sintéticos

Quando produtos alimentícios e especiarias são contaminados por micotoxinas é quase impossível detoxificar utilizando processos usuais da indústria de alimentos ou durante o preparo doméstico. Por isso, controlar o crescimento do fungo e a produção de toxinas é uma demanda para garantir a segurança alimentar. Os agrotóxicos são rotineiramente utilizados como estratégia para proteger as plantas de doenças provocadas pela contaminação fúngica. No entanto, eles estão associados a efeitos adversos ao sistema nervoso central e periférico, têm ação imunodepressora e são cancerígenos. Em virtude disso, o objetivo deste trabalho foi estudar a inibição do desenvolvimento, do potencial toxigênico e da expressão gênica de linhagens do Complexo Fusarium graminearum por compostos naturais comparativamente aos fungicidas azoxistrobina e trifloxistrobina. Do farelo de arroz, foram extraídos o γ-orizanol e os ácidos fenólicos (EFF). Das sementes de nim foram extraídos os ácidos fenólicos (EFN), totalizando três extratos naturais. A capacidade antioxidante dos extratos foi verificada pelo consumo do radical livre DPPH• , capacidade de captura do radical ABTS●+, redução do ferro e inibição da oxidação enzimática. Os mecanismos de inibição de três linhagens de F. graminearum foram avaliados através da determinação de compostos estruturais (glicosamina e ergosterol) e da atividade de enzimas do metabolismo primário (α- amilase e proteases). Foram determinadas as micotoxinas de Fusarium: deoxinivalenol (DON), 15 acetildeoxinivalenol (15AcDON), 3 acetildeoxinivalenol (3AcDON), nivalenol (NIV) e zearalenona (ZEA). A expressão dos genes Tri1 e Tri5 foi determinada a fim de verificar se ocorria modificação da expressão gênica nas linhagens do Complexo F. graminearum ocasionada pela presença dos antifúngicos. O EFF apresenta atividade antioxidante destacada em relação aos demais extratos naturais para inibir a iniciação do processo, a propagação do radical livre e a catálise enzimática. A presença dos compostos naturais mostrou efeito promissor como antifúngico para as linhagens, sendo que a concentração necessária para inibir 50% do crescimento radial das colônias (MIC50) foi 0,9 g/kg para γ-orizanol; 0,032 g/kg para EFF e 0,037 g/kg para EFN, portanto, os extratos fenólicos são mais eficazes para inibição de F. graminearum do que o γ-orizanol. Os extratos naturais afetaram as atividades das enzimas α-amilase e proteases. Também ocorreu a redução da formação de componentes estruturais (glicosamina e ergosterol). Os extratos naturais se destacaram pela capacidade de inibição de micotoxinas produzidas pela biomassa fúngica, com destaque para o EFN sobre a produção de DON, 15AcDON, 3AcDON e ZEA. Sendo assim, é possível dizer que há uma relação direta entre a atividade antioxidante na inibição do fungo e na manifestação do seu potencial toxigênico. Além disso, esse estudo contribuiu com a elucidação do mecanismo de ação dos antifúngicos naturais estudados. Ocorre modificação na expressão gênica quando a linhagem é submetida ao tratamento com antifúngico, havendo uma relação direta entre a expressão do gene Tri5 e a produção de DON.

A altura de dossel e a adubação nitrogenada da pastagem podem afetar a nutrição nitrogenada do milho, num sistema de integração lavoura pecuária?

We conducted a field experiment near Abelardo Luz, Santa Catarina, from October 2012 to April 2014, to evaluate the effect interaction of nitrogen fertilization and height of canopy over the N nutrition of corn subsequently grown to pasture. The data belonging to this thesis are related to the first two production cycles obtained in sorghum pasture (2012/2013), oat (2013) and corn crop (2013/2014). In the evaluation of forage sorghum and oat it was used the same experimental design, consisting of randomized complete block in a factorial arrangement (2 x 2) with three replications. The first factor was considered canopy height (Low and High) and the second factor was the fertilization of cover crop pasture (0 and 200 kg N.ha-1). In phase I and II, the combination of factors evaluators were prepared in the same experimental unit. For corn crop the design was a randomized complete block in a factorial design (2X2X4X6) with three replications. Factors considered in corn were: canopy height of pasture (Low and High), nitrogen application times (NG - nitrogen in the grains and NP - nitrogen in pasture), nitrogen fertilization in corn (0, 100, 200 and 300 kg N.ha-1) and time (46, 53, 60, 67, 76 and 103 days after sowing the maize). In phases I and II, in general the use of N in the pasture increased the productive potential of the pasture and animal management and canopy height has changed the dynamics of structural components and botanical pasture. In cold conditions for long periods and not acclimatized plants the adoption of high nitrogen fertilization and height high grazing pasture leave vulnerable to damage caused by the freezing of plants. The anticipation of nitrogen fertilization on pasture positively affects the corn crop by increasing the accumulated dry matter and N content in the plant. Nitrogen nutritional content of corn with the anticipation of fertilization in pasture is suitable for obtaining high crop production in integrated crop-livestock system. When used nitrogen only coverage in corn sufficiency level in the nitrogen nutrition is achieved with the use of 100 kg N.ha-1. With the use of 200 kg N ha -1 NG and NP no difference in nitrogen content and nitrogen nutrition index.

A microestrutura e o desenvolvimento dos órgãos genitais masculinos de preás (Galea spixii) criados em cativeiro

O preá do semiárido nordestino (Galea spixii) é um roedor pertencente à família Caviidae. São encontrados nas regiões da Caatinga e do Cerrado Brasileiro e se reproduz ao longo do ano, apresentando um período de gestação de 48 dias e uma ninhada de 2 a 4 crias. O objetivo deste estudo foi caracterizar histologicamente os componentes estruturais dos órgãos genitais de preás machos relacionando com a evolução cronológica destes órgãos na espécie. Foram utilizados para análise animais ao nascimento e aos 15, 30, 45, 60, 75, 90, 105, 120 e aos 150 dias de idade. Fragmentos do epidídimo, ducto deferente, pênis e glândulas sexuais acessórias foram coletados, fixados e processados para descrição em microscopia de luz. O epidídimo apresentou epitélio colunar simples e em cada fase sexual notou-se diferença quanto ao tamanho do lúmen tubular e à presença de espermatozóides no lúmen aos 45 dias de idade. O epitélio do ducto deferente no preá mostrou-se pseudo-estratificado colunar com crescente presença de estereocilios com o avanço da idade. A glândula vesicular no preá apresentou uma mucosa com pregueamento variado, de acordo com a fase do desenvolvimento sexual. A próstata mostrou-se pouco desenvolvida, com lúmen pequeno nos preás ao nascimento e aos 15 dias de idade; aos 45 dias mostrou-se com um pregueamento do epitélio variável. Os órgãos genitais masculinos do preá passaram por transformações morfológicas no decorrer da idade e com o desenvolvimento sexual, isso colaborou para a determinação do início da fase da puberdade, que na espécie em estudo foi aos 45 dias de idade.

Deconjugated Bile Salts Produced by Extracellular Bile-Salt Hydrolase-Like Activities from the Probiotic Lactobacillus johnsonii La1 Inhibit Giardia duodenalis In vitro Growth

Giardiasis, currently considered a neglected disease, is caused by the intestinal protozoan parasite Giardia duodenalis and is widely spread in human as well as domestic and wild animals. The lack of appropriate medications and the spread of resistant parasite strains urgently call for the development of novel therapeutic strategies. Host microbiota or certain probiotic strains have the capacity to provide some protection against giardiasis. By combining biological and biochemical approaches, we have been able to decipher a molecular mechanism used by the probiotic strain Lactobacillus johnsonii La1 to prevent Giardia growth in vitro. We provide evidence that the supernatant of this strain contains active principle(s) not directly toxic to Giardia but able to convert non-toxic components of bile into components highly toxic to Giardia. By using bile acid profiling, these components were identified as deconjugated bile-salts. A bacterial bile-salt-hydrolase of commercial origin was able to mimic the properties of the supernatant. Mass spectrometric analysis of the bacterial supernatant identified two of the three bile-salt-hydrolases encoded in the genome of this probiotic strain. These observations document a possible mechanism by which L. johnsonii La1, by secreting, or releasing BSH-like activity(ies) in the vicinity of replicating Giardia in an environment where bile is present and abundant, can fight this parasite. This discovery has both fundamental and applied outcomes to fight giardiasis, based on local delivery of deconjugated bile salts, enzyme deconjugation of bile components, or natural or recombinant probiotic strains that secrete or release such deconjugating activities in a compartment where both bile salts and Giardia are present.

Guided Migration and Collective Behavior: Cell Dynamics during Cancer Progression

This dissertation focuses on gaining understanding of cell migration and collective behavior through a combination of experiment, analysis, and modeling techniques. Cell migration is a ubiquitous process that plays an important role during embryonic development and wound healing as well as in diseases like cancer, which is a particular focus of this work. As cancer cells become increasingly malignant, they acquire the ability to migrate away from the primary tumor and spread throughout the body to form metastatic tumors. During this process, changes in gene expression and the surrounding tumor environment can lead to changes in cell migration characteristics. In this thesis, I analyze how cells are guided by the texture of their environment and how cells cooperate with their neighbors to move collectively. The emergent properties of collectively moving groups are a particular focus of this work as collective cell dynamics are known to change in diseases such as cancer. The internal machinery for cell migration involves polymerization of the actin cytoskeleton to create protrusions that---in coordination with retraction of the rear of the cell---lead to cell motion. This actin machinery has been previously shown to respond to the topography of the surrounding surface, leading to guided migration of amoeboid cells. Here we show that epithelial cells on nanoscale ridge structures also show changes in the morphology of their cytoskeletons; actin is found to align with the ridge structures. The migration of the cells is also guided preferentially along the ridge length. These ridge structures are on length scales similar to those found in tumor microenvironments and as such provide a system for studying the response of the cells' internal migration machinery to physiologically relevant topographical cues. In addition to sensing surface topography, individual cells can also be influenced by the pushing and pulling of neighboring cells. The emergent properties of collectively migrating cells show interesting dynamics and are relevant for cancer progression, but have been less studied than the motion of individual cells. We use Particle Image Velocimetry (PIV) to extract the motion of a collectively migrating cell sheet from time lapse images. The resulting flow fields allow us to analyze collective behavior over multiple length and time scales. To analyze the connection between individual cell properties and collective migration behavior, we compare experimental flow fields with the migration of simulated cell groups. Our collective migration metrics allow for a quantitative comparison between experimental and simulated results. This comparison shows that tissue-scale decreases in collective behavior can result from changes in individual cell activity without the need to postulate the existence of subpopulations of leader cells or global gradients. In addition to tissue-scale trends in collective behavior, the migration of cell groups includes localized dynamic features such as cell rearrangements. An individual cell may smoothly follow the motion of its neighbors (affine motion) or move in a more individualistic manner (non-affine motion). By decomposing individual motion into both affine and non-affine components, we measure cell rearrangements within a collective sheet. Finally, finite-time Lyapunov exponent (FTLE) values capture the stretching of the flow field and reflect its chaotic character. Applying collective migration analysis techniques to experimental data on both malignant and non-malignant human breast epithelial cells reveals differences in collective behavior that are not found from analyzing migration speeds alone. Non-malignant cells show increased cooperative motion on long time scales whereas malignant cells remain uncooperative as time progresses. Combining multiple analysis techniques also shows that these two cell types differ in their response to a perturbation of cell-cell adhesion through the molecule E-cadherin. Non-malignant MCF10A cells use E-cadherin for short time coordination of collective motion, yet even with decreased E-cadherin expression, the cells remain coordinated over long time scales. In contrast, the migration behavior of malignant and invasive MCF10CA1a cells, which already shows decreased collective dynamics on both time scales, is insensitive to the change in E-cadherin expression.

Guided Migration and Collective Behavior: Cell Dynamics during Cancer Progression

This dissertation focuses on gaining understanding of cell migration and collective behavior through a combination of experiment, analysis, and modeling techniques. Cell migration is a ubiquitous process that plays an important role during embryonic development and wound healing as well as in diseases like cancer, which is a particular focus of this work. As cancer cells become increasingly malignant, they acquire the ability to migrate away from the primary tumor and spread throughout the body to form metastatic tumors. During this process, changes in gene expression and the surrounding tumor environment can lead to changes in cell migration characteristics. In this thesis, I analyze how cells are guided by the texture of their environment and how cells cooperate with their neighbors to move collectively. The emergent properties of collectively moving groups are a particular focus of this work as collective cell dynamics are known to change in diseases such as cancer. The internal machinery for cell migration involves polymerization of the actin cytoskeleton to create protrusions that---in coordination with retraction of the rear of the cell---lead to cell motion. This actin machinery has been previously shown to respond to the topography of the surrounding surface, leading to guided migration of amoeboid cells. Here we show that epithelial cells on nanoscale ridge structures also show changes in the morphology of their cytoskeletons; actin is found to align with the ridge structures. The migration of the cells is also guided preferentially along the ridge length. These ridge structures are on length scales similar to those found in tumor microenvironments and as such provide a system for studying the response of the cells' internal migration machinery to physiologically relevant topographical cues. In addition to sensing surface topography, individual cells can also be influenced by the pushing and pulling of neighboring cells. The emergent properties of collectively migrating cells show interesting dynamics and are relevant for cancer progression, but have been less studied than the motion of individual cells. We use Particle Image Velocimetry (PIV) to extract the motion of a collectively migrating cell sheet from time lapse images. The resulting flow fields allow us to analyze collective behavior over multiple length and time scales. To analyze the connection between individual cell properties and collective migration behavior, we compare experimental flow fields with the migration of simulated cell groups. Our collective migration metrics allow for a quantitative comparison between experimental and simulated results. This comparison shows that tissue-scale decreases in collective behavior can result from changes in individual cell activity without the need to postulate the existence of subpopulations of leader cells or global gradients. In addition to tissue-scale trends in collective behavior, the migration of cell groups includes localized dynamic features such as cell rearrangements. An individual cell may smoothly follow the motion of its neighbors (affine motion) or move in a more individualistic manner (non-affine motion). By decomposing individual motion into both affine and non-affine components, we measure cell rearrangements within a collective sheet. Finally, finite-time Lyapunov exponent (FTLE) values capture the stretching of the flow field and reflect its chaotic character. Applying collective migration analysis techniques to experimental data on both malignant and non-malignant human breast epithelial cells reveals differences in collective behavior that are not found from analyzing migration speeds alone. Non-malignant cells show increased cooperative motion on long time scales whereas malignant cells remain uncooperative as time progresses. Combining multiple analysis techniques also shows that these two cell types differ in their response to a perturbation of cell-cell adhesion through the molecule E-cadherin. Non-malignant MCF10A cells use E-cadherin for short time coordination of collective motion, yet even with decreased E-cadherin expression, the cells remain coordinated over long time scales. In contrast, the migration behavior of malignant and invasive MCF10CA1a cells, which already shows decreased collective dynamics on both time scales, is insensitive to the change in E-cadherin expression.

Contribuição para a valorização, geoconservação e gestão da jazida fossilífera de Cacela (Parque Natural da Ria Formosa, Algarve, Portugal)

Dissertação de mest. em Gestão e Conservação da Natureza, Faculdade de Ciências do Mar e do Ambiente, Univ. do Algarve, 2004

O tempo de permanência como caracterizador do espaço habitacional

para obtenção do grau de Mestre em Arquitectura, apresentada na Universidade de Lisboa - Faculdade de Arquitectura.

Initiation and early crack growth in VHCF of stainless steels : Experimental and theoretical analysis

Mechanical fatigue is a failure phenomenon that occurs due to repeated application of mechanical loads. Very High Cycle Fatigue (VHCF) is considered as the domain of fatigue life greater than 10 million load cycles. Increasing numbers of structural components have service life in the VHCF regime, for instance in automotive and high speed train transportation, gas turbine disks, and components of paper production machinery. Safe and reliable operation of these components depends on the knowledge of their VHCF properties. In this thesis both experimental tools and theoretical modelling were utilized to develop better understanding of the VHCF phenomena. In the experimental part, ultrasonic fatigue testing at 20 kHz of cold rolled and hot rolled stainless steel grades was conducted and fatigue strengths in the VHCF regime were obtained. The mechanisms for fatigue crack initiation and short crack growth were investigated using electron microscopes. For the cold rolled stainless steels crack initiation and early growth occurred through the formation of the Fine Granular Area (FGA) observed on the fracture surface and in TEM observations of cross-sections. The crack growth in the FGA seems to control more than 90% of the total fatigue life. For the hot rolled duplex stainless steels fatigue crack initiation occurred due to accumulation of plastic fatigue damage at the external surface, and early crack growth proceeded through a crystallographic growth mechanism. Theoretical modelling of complex cracks involving kinks and branches in an elastic half-plane under static loading was carried out by using the Distributed Dislocation Dipole Technique (DDDT). The technique was implemented for 2D crack problems. Both fully open and partially closed crack cases were analyzed. The main aim of the development of the DDDT was to compute the stress intensity factors. Accuracy of 2% in the computations was attainable compared to the solutions obtained by the Finite Element Method.

The epidemiology of foot-and-mouth disease at the wildlife-livestock interface in northern Tanzania

Foot-and-mouth disease (FMD), a disease of cloven hooved animals caused by FMD virus (FMDV), is one of the most economically devastating diseases of livestock worldwide. The global burden of disease is borne largely by livestock-keepers in areas of Africa and Asia where the disease is endemic and where many people rely on livestock for their livelihoods and food-security. Yet, there are many gaps in our knowledge of the drivers of FMDV circulation in these settings. In East Africa, FMD epidemiology is complicated by the circulation of multiple FMDV serotypes (distinct antigenic variants) and by the presence of large populations of susceptible wildlife and domestic livestock. The African buffalo (Syncerus caffer) is the only wildlife species with consistent evidence of high levels of FMDV infection, and East Africa contains the largest population of this species globally. To inform FMD control in this region, key questions relate to heterogeneities in FMD prevalence and impacts in different livestock management systems and to the role of wildlife as a potential source of FMDV for livestock. To develop FMD control strategies and make best use of vaccine control options, serotype-specific patterns of circulation need to be characterised. In this study, the impacts and epidemiology of FMD were investigated across a range of traditional livestock-keeping systems in northern Tanzania, including pastoralist, agro-pastoralist and rural smallholder systems. Data were generated through field studies and laboratory analyses between 2010 and 2015. The study involved analysis of existing household survey data and generated serological data from cross-sectional livestock and buffalo samples and longitudinal cattle samples. Serological analyses included non-structural protein ELISAs, serotype-specific solid-phase competitive ELISAs, with optimisation to detect East African FMDV variants, and virus neutralisation testing. Risk factors for FMDV infection and outbreaks were investigated through analysis of cross-sectional serological data in conjunction with a case-control outbreak analysis. A novel Bayesian modeling approach was developed to infer serotype-specific infection history from serological data, and combined with virus isolation data from FMD outbreaks to characterise temporal and spatial patterns of serotype-specific infection. A high seroprevalence of FMD was detected in both northern Tanzanian livestock (69%, [66.5 - 71.4%] in cattle and 48.5%, [45.7-51.3%] in small ruminants) and in buffalo (80.9%, [74.7-86.1%]). Four different serotypes of FMDV (A, O, SAT1 and SAT2) were isolated from livestock. Up to three outbreaks per year were reported by households and active surveillance highlighted up to four serial outbreaks in the same herds within three years. Agro-pastoral and pastoral livestock keepers reported more frequent FMD outbreaks compared to smallholders. Households in all three management systems reported that FMD outbreaks caused significant impacts on milk production and sales, and on animals’ draught power, hence on crop production, with implications for food security and livelihoods. Risk factor analyses showed that older livestock were more likely to be seropositive for FMD (Odds Ratio [OR] 1.4 [1.4-1.5] per extra year) and that cattle (OR 3.3 [2.7-4.0]) were more likely than sheep and goats to be seropositive. Livestock managed by agro-pastoralists (OR 8.1 [2.8-23.6]) or pastoralists (OR 7.1 [2.9-17.6]) were more likely to be seropositive compared to those managed by smallholders. Larger herds (OR: 1.02 [1.01-1.03] per extra bovine) and those that recently acquired new livestock (OR: 5.57 [1.01 – 30.91]) had increased odds of suffering an FMD outbreak. Measures of potential contact with buffalo or with other FMD susceptible wildlife did not increase the likelihood of FMD in livestock in either the cross-sectional serological analysis or case-control outbreak analysis. The Bayesian model was validated to correctly infer from ELISA data the most recent serotype to infect cattle. Consistent with the lack of risk factors related to wildlife contact, temporal and spatial patterns of exposure to specific FMDV serotypes were not tightly linked in cattle and buffalo. In cattle, four serial waves of different FMDV serotypes that swept through southern Kenyan and northern Tanzanian livestock populations over a four-year period dominated infection patterns. In contrast, only two serotypes (SAT1 and SAT2) dominated in buffalo populations. Key conclusions are that FMD has a substantial impact in traditional livestock systems in East Africa. Wildlife does not currently appear to act as an important source of FMDV for East African livestock, and control efforts in the region should initially focus on livestock management and vaccination strategies. A novel modeling approach greatly facilitated the interpretation of serological data and may be a potent epidemiological tool in the African setting. There was a clear temporal pattern of FMDV antigenic dominance across northern Tanzania and southern Kenya. Longer-term research to investigate whether serotype-specific FMDV sweeps are truly predictable, and to shed light on FMD post-infection immunity in animals exposed to serial FMD infections is warranted.