Tesi sul tema "Fragment size"

The assessment of fragmentation is an important aspect of the design and planning of any excavation. The distribution of fragment sizes in situ helps assess the requirement of explosive energy to excavate the rock material. In addition, the information can also be used to evaluate the ground water flow, leaching characteristics and the requirement of additional rock handling equipment in construction projects. In the block cave mining method, the assessment of in situ and secondary fragmentation is an integral part of the design of the excavations at the extraction level and the selection of material handling systems for transporting the ore to the processing stations. Secondary blasting requirements can also be estimated based on the fragment size distributions developed for the block cave. Methods of estimating fragment size distributions in block cave mines have been based on joint set parameters estimated from structural mapping in available excavations or outcrops. While this is acceptable in the absence of any other means of assessing the fragmentation, the results can often be misleading since the structural mapping is often carried out in limited areas and the results applied uniformly to the entire deposit. This new study proposes to use the core piece lengths information gathered from the boreholes to develop in situ and primary fragmentation estimates for block cave operations. Under this proposed study, drill core piece lengths from an exploration program in Indonesia will be used along with structural mapping data to develop estimates for in situ and primary fragment size distributions. Methods for estimating secondary fragmentation from primary fragmentation will be evaluated and the estimates of secondary fragmentation from the different methods will be compared with the actual fragmentation characteristics observed at the drawpoints. The primary assumption in the development of primary fragment size distributions from drill core data is that each drill hole piece represents one in situ rock block. The relationships between the joint spacings and lengths of the different joint sets, evaluated from the joint set characteristics gathered from available excavations, outcrops or oriented core drilling programs, can be utilized for estimating the shape of the rock blocks.

The success of rock fragmentation by blasting and subsurface fluid flow depends on many variables, including in-situ fragmentation. This study presents select data from a review of over 13000 feet of core samples drilled at seven different mine properties in the United States and Chile. Two-dimensional computer simulations of fractures can be used to quantify the volume of rock required to calculate a stable estimate of mean fragment size. Spatial periodicity of fragment size measurements is indicated through vertical variograms and is used as the basis for a statistical analysis for the zoning of rock masses. A more reliable quantitative method to characterize the size distribution of in-situ rock using digital image processing of drill core photographs is also included. Furthermore, a complete experimental analysis is provided for the comparison of currently used correction methods used to develop the full volumetric fragment size distribution curve.

Ce travail pose la question du sens et de la fonction des statuettes féminines gravettiennes. L'hypothèse d'une utilisation en contexte rituel menant à la fragmentation intentionnelle des sculptures, centrale dans la recherche européenne, est testée dans cette étude. La recherche est appliquée à la série de statuettes animales et féminines de Kostienki 1.1, site gravettien de la plaine russe daté des environs de 23 000 ans avant notre ère, une des plus riches du Paléolithique supérieur avec 297 pièces sculptées dont 289 en calcaire et 8 en ivoire. La classification des pièces selon leur place dans la chaîne de fabrication (support, ébauche, pièce achevée) et leur intégrité (entière, fragmentaire, fragment), étape préalable indispensable à l'inventaire des représentations, a nécessité de développer une approche technique. Trois outils principaux ont été utilisés à cette fin : l'observation de récurrences morphologiques et techniques, la reconstitution par défaut et l'expérimentation. L'étude a permis de caractériser un système technique peu connu auparavant (la sculpture en ronde-bosse gravettienne), d'identifier de nouveaux codes de représentation distants du réalisme visuel et de remettre en question la nature fragmentaire de la majorité des représentations. Ainsi, il ne s'agit pas de la fragmentation des représentations mais de la représentation du corps féminin fragmenté. Outre le thème de la fragmentation du corps, le thème de la gestation caractérise également les représentations féminines du site. L'interprétation iconographique de ces thèmes, jointe à l'analyse des indices témoins de l'usage matériel des statuettes, mène à proposer de nouvelles hypothèses fonctionnelles pour ces pièces.

Devido a atual situação de degradação da vegetação remanescente do estado de São Paulo, existe com relativa urgência uma grande necessidade de estudos que apoiem práticas de monitoramento e conservação dos fragmentos florestais para melhor gerenciá-los. Parte destas demandas é referente ao campo de amostragem, um conjunto de técnicas específicas para pesquisas ecológicas, onde a unidade amostral (parcela) é um dos fatores determinantes de sua eficiência. O objetivo deste estudo foi definir e propor o tamanho ideal de parcelas para otimizar o inventário florestal de áreas remanescentes de Floresta Estacional Semidecidual (FES) e de Cerrado (CER), assim como também de Floresta Estacional Semidecidual em processo de regeneração (FESreg). O trabalho foi realizado na Estação Experimental de Ciências Florestais de Itatinga - SP (EECFI/ESALQ/USP), visando à otimização da amostragem para as variáveis diâmetro à altura do peito (DAP), altura total, densidade populacional, área basal, volume total, volume de fuste, volume de galho, biomassa e índices de Shannon, de Simpson e de Riqueza. O delineamento experimental foi realizado com o auxílio de uma grade amostral, na qual foram distribuídas aleatoriamente 15 parcelas de 1.000 m2 cada, sendo cinco repetições em cada uma das três fisionomias florestais estudadas, onde todos os indivíduos com circunferência à altura do peito (CAP) acima de 16 cm foram mensurados e identificados. Cada unidade amostral foi divida em 10 subparcelas, permitindo assim avaliar os tamanhos de 100 m2 (10X10 m), 200 m2 (10x20 m), 300 m2 (10x30 m), 400 m2 (10x40 m e 20x20 m), 500 m2 (10x50 m), 600 m2 (20x30 m), 800 m2 (20x40 m) e 1.000 m2 (20x50 m). Os tratamentos (tamanhos) foram analisados através de análise de variância, métodos da curvatura máxima e esforço amostral. Com base nos resultados, recomenda-se respectivamente para Cerrado, Floresta Estacional Semidecidual e Floresta Estacional Semidecidual em regeneração o uso de parcelas de 200 m2, 200 m2 e 600 m2 para estimar qualquer uma das variáveis dendrométricas ou parcelas de 200 m2 para estimativas apenas de diversidade. Esses resultados são bastante úteis por embasarem uma série de pesquisas ambientais, deste modo, aperfeiçoando operações de inventário florestal para que o processo de amostragem não tenha esforço amostral desnecessário sem prejuízo da precisão.
Because of advanced status of degradation of the natural forest of the state of São Paulo, there is a relatively urgent need for the conduction of studies to support monitoring and conservation practices of forest fragments for their better management. Of these needs refers to sampling, with the use specific techniques for ecological research, in which characteristics of the sampling unit is of outmost importance. The objective of this study was to define and propose optimal plot sizes for inventories remnant areas of Semideciduous Forest (FES) and Cerrado (CER), as well as of Semideciduous forest in regeneration process (FESreg). The study was conducted at Estação Experimental de Ciências Florestais de Itatinga - SP (EECFI/ESALQ/USP), aiming to optimize the sampling for the variables diameter at breast height (DBH), total height, density, basal area, wood volume, volume of stem, branch volume, biomass as well as Shannon, Simpson and Richness indexes. The experiment was carried out with the aid of a sampling grid, in which 15 sampling units of 1,000 m2 each were randomly determined, with five replicates in each type of forest five. In each unit, all trees with circumference (girth) at breast height (GBH) above 16 cm were measured and identified. Each unit was divided into 10 subplots, so as to allow the evaluation in areas of 100 m2 (10x10 m), 200 m2 (10x20 m), 300 m2 (10x30 m), 400 m2 (10x40 and 20x20 m), 500 m2 (10x50 m), 600 m2 (20x30 m), 800 m2 (20x40 m) and 1,000 m2 (20x50 m). Treatments (sizes) were analyzed by analysis of variance, maximum curvature methods and sampling effort. The results indicated that for areas of Cerrado, Semideciduous Forest and Semideciduous Forest in regeneration plot sizes of 200 m2, 200 m2 and 600 m2 respectively should be used to estimate any of the dendrometric variables or plot size of 200 m2 should be used for estimates of diversity only. These results are very useful for a number of environmental researches, improving forest inventory operations by preventing unnecessary sampling effort without jeopardizing accuracy.

Barriers to fish movement can cause aquatic habitat fragmentation by reducing the amount of available habitat. The primary goal of my research was to investigate applications of population genetic analysis tools as indicators of barrier effects on brook trout populations in fragmented subwatersheds. In chapter1, I tested the hypothesis that brook trout population genetic differentiation (FST) above and below barriers will differ in relation to barrier height and gradient. I also tested the hypothesis that average gene diversity per locus (H) and the numbers of alleles (A) differed between samples below and above each barrier. There was no significant difference in average number of alleles (A) or average gene diversity per locus (H) between the above- and below-barrier samples, but linear regression identified a statistically significant relationship between barrier height and FST values. Unrooted neighbor-joining consensus trees of Cavalli-Sforza and Edwards (1967) chord distances provided evidence of genetic differentiation between samples of resident brook trout above and below natural barriers. Additionally, average total allelic diversity (A), average gene diversity per locus (H), average number of private alleles per locus per sample, and total alleles per sample differed between Level III Ecoregions. In chapter 2 I tested the hypothesis that the presence of a barrier, total habitat potentially isolated above a barrier (km), road density, and percent forest cover within a subwatershed (USGS 6th-level Hydrologic Units) were significant habitat fragmentation factors affecting the effective population size (Ne) of brook trout in the Blue Ridge Level III Ecoregion. Multivariable linear regression indicated that total habitat above the barrier (km) and road density were significant variables retained in the model to predict Ne. In chapter 3, the objective of the study was to infer relationships between barriers and family structure in brook trout populations. Maximum likelihood analysis of pairwise kinship relationships between above- and below-barrier individuals indicated the presence of parent-offspring relationships between above- and below-barrier individuals at six sites in the Blue Ridge Level III Ecoregion and five sites in the Northern Lakes and Forests Level III Ecoregion, which indicated movement of individuals between the above- and below-barrier locations.
Ph. D.

Es ADCs (Antibody-Drug Conjugates) correspondent à une nouvelle stratégie thérapeutique anti-tumorale particulièrement prometteuse. Néanmoins, les ADCs actuellement utilisés en clinique sont obtenus par conjugaisons chimiques, resultant en des mixtures hétérogènes impactant négativement leurs pharmacocinétiques et leurs performances in vivo.Récemment, différentes strategies de couplage site-spécifique ont été développées afin de réduire cette hétérogénéité. Dans cette thèse, nous rapportons le développement d’une nouvelle technologie CovIsoLink™ (Covalently Isopeptide Crosslinking) permettant la génération d’ADCs par utilisation de nouveaux peptides glutamine Q-Tag présentant des affinités optimisées par rapport à des peptides disponibles (ZQG, LLQG) pour une enzyme bactérienne la transglutaminase (mTG).La preuve de concept de cette technologie a été réalisée par insertion de ces peptides Q-Tag en C-ter de la région codant pour la chaine lourde des anticorps anti-HER2 (Trastuzumab). Nous avons ainsi pu démontrer la conjugaison homogène et reproductible de différentes drogues sans contamination par des chaines d’anticorps non conjuguées. Nous avons pu montrer que l’immunoréactivité et la capacité d’internalisation de ces ADCs n’étaient pas altérées par la conjugaison et qu’ils présentaient in vitro et in vivo, des propriétés de lyse de cellules tumorales similaires au Trastuzumab emtansine (Kadcyla®), actuellement en clinique. Par ailleurs, afin de généraliser notre technologie à différents formats d’anticorps nous avons générés des fragments Fab et scFv et évalué leur fonctionnalité. Ainsi, nous avons pu prouver que l’utilisation de nouveaux peptides optimisés Q-Tag substrat de la transglutaminase permettait une stratégie de couplage alternative plus homogène par couplage de différentes molécules sans espèce contaminante non couplée
Antibody-drug conjugates (ADCs) are a powerful class of therapeutic agents, demonstrating success in the treatment of several malignancies. The currently approved ADCs are produced by chemical conjugations and exist as heterogeneous mixtures that negatively influence the pharmacokinetics and in vivo performance. Recently many of site-specific conjugation technologies have been developed to reduce heterogeneity and batch-to batch variability. Microbial transglutaminase (mTG) has been demonstrated as efficient tool for site-specific conjugation. In this thesis we report the development CovIsoLink™ (Covalently Isopeptide Crosslinking) technology for the generation of homogenous immunoconjugates using a novel glutamine donor peptides (Q-tag) with improved affinity compared to the known peptides (ZQG, LLQG). As a proof of concept, the peptides sequences were engineered into the heavy chain C-terminal of Trastuzumab antibody. We demonstrated the reproducible and homogeneous conjugation of Q-tagged Trastuzumab with different payloads, without any unconjugated species. The ADCs were evaluated in series of in vitro and in vivo assays. We confirmed that the immunoreactivity and internalisation are not altered by the conjugation. Furthermore similar in vitro and in vivo tumor cell killing potency was demonstrated than Trastuzumab emtansine (Kadcyla®), which is already used in the clinic. Morover we extend our site-specific conjugation technology to antibody fragments (Fab and scFv), evaluating their functionality by conjugation with AlexaFluor488-cadaverine and in antigen binding assays. Thus, using novel glutamine donor peptides, our technology provides an alternative enzymatic conjugation strategy for the engrafment of different payloads resulting in homogeneous batches, without unconjugated species

O fragmento de anticorpo scFv foi expresso pela levedura Pichia pastoris recombinante utilizando o promotor AOX1, em presença de metanol, como fonte de carbono e como modelo de expressão. Este anticorpo é considerado uma possível alternativa para o diagnóstico e tratamento da aterosclerose. Foi realizado um estudo inicial da produção do fragmento de anticorpo e do crescimento da levedura com dois bancos de trabalho diferentes, variando a fonte de carbono (glicose e glicerol). Os resultados obtidos demonstraram que as máximas concentrações celulares e a maior produção de anticorpo foram atingidas com o estoque de trabalho mantido com glicerol como fonte de carbono e de preservação. Posteriormente, foi realizado um estudo em shaker e biorreator a partir de um planejamento fatorial completo 23 em que se estudaram as variáveis tempo de indução (24, 28 e 32 horas), temperatura de indução (10, 20 e 30°C), e concentração de indutor (metanol 0,5, 1,0 e 1,5%) sobre a variável resposta produção do fragmento de anticorpo. A única variável que apresentou efeito estatisticamente significativo, com nível de confiança de 95%, foi a concentração de indutor para os dois sistemas de produção. As melhores produções de fragmento de anticorpo foram de 27,8 mg/L em shaker e de 41,3 mg/L em biorreactor. O terceiro estudo consistiu em avaliar a produção do fragmento de anticorpo em shaker pela levedura em distintas fontes de carbono (sacarose, glicerol, glicose e sacarose/glicose 50%). Neste estudo a analise de ANOVA não apresentou diferenças estatísticas significativas. Foi realizado um estudo para melhorar a quantificação do fragmento de anticorpo visando diminuir o número de etapas para evitar perdas no processo. Após obter um novo protocolo para a quantificação, realizou-se um novo estudo para avaliar a produção com as diferentes fontes de carbono. Com a analise dos resultados observou-se que todas as fontes de carbono apresentaram efeitos estatisticamente significativos, sendo que com a sacarose se obteve a maior produção com 93,7 mg/L do fragmento de anticorpo. Estes resultados foram comparados e analisados com observações realizadas ao microscópio da levedura, que demonstraram que a melhor produção de anticorpo foi obtida no momento que a levedura apresentava diâmetros menores que 0,895 µm. Seguidamente foi realizado um estudo para determinar fontes alternativas de carbono e nitrogênio (farelo de arroz e farelo de trigo) para produzir o fragmento de anticorpo, em substituição de subtratos como peptona de caseína e casaminoacidos, a maior produção obtida neste caso foi de 27,9 mg/L quando foi trocado o casaminoacidos pelo farelo de trigo. Finalmente foi avaliado o efeito do kLa no crescimento da levedura e a produção do fragmento de anticorpo scFv, os resultados demonstraram que a melhor condição para o crescimento da levedura foi com kLa 96,12 h-1, por outro lado a melhor condição para a produção do fragmento de anticorpo foi de 12,24 h-1 com relação a produção final de 8,12 mg/L de anticorpo/ g/L de célula.
The scFv antibody fragment was expressed in the recombinant yeast Pichia pastoris by using AOX1 promoter, in the presence of methanol, as carbon source and as a model for expression. This antibody is considered as a possible alternative for the diagnosis and treatment of atherosclerosis. An initial study was conducted in the production of the antibody fragment and the growth of yeast with two different work benches, varying the carbon source (glucose and glycerol). The results showed that the maximum cell concentrations, and the best production of antibody, were achieved with maintained working stock with glycerol as carbon source and preservation. Subsequently, a study was conducted in shaker and bioreactor, from a 23 complete factorial design, in which the variables studied were time induction (24, 28 and 32 hours), temperature induction (10, 20 and 30°C), and concentration of the inducer (methanol 0.5, 1.0 and 1.5%) on the response variable production of the antibody fragment. The only variable that had a statistically significant effect, with a confidence level of 95%, was the inducer concentration for the two production systems. The best yields of the antibody fragment were of 27.8 mg/L for the shaker and 41.3 mg/L for the bioreactor. The third study was to assess the production of antibody fragment into the shaker with the yeast at different carbon sources (sucrose, glycerol, glucose and sucrose/glucose 50%). In this study, the ANOVA analysis did not show statistically significant differences. We conducted planning to improve the quantification of antibody fragment in order to decrease the number of steps to avoid losses in the process. After getting a new protocol for the quantification, we carried out a new study to evaluate the production with different carbon sources. The analysis of the results showed that all carbon sources showed statistically significant effects, among which sucrose obtained the highest yield 93.7 mg/L of antibody fragment. These results were compared and analyzed through a microscope, in which with the observations of the yeast showed that the best antibody production was obtained when the yeast had sizes smaller than 0.895 microns. Next, a study was conducted to determine alternative sources of carbon and nitrogen (rice bran and wheat bran) to produce the antibody fragment in place of casein peptone and casaminoacids as substrates. The highest yield obtained in this case was 27.9 mg/L, when casamino was replaced by the wheat bran. Finally, we evaluated the effect of kLa in yeast growth and production of the antibody fragment scFv, the results showed that the best condition for the growth of the yeast was 96.12 h-1, on the other hand, the best condition for the production antibody fragment was 12.24 h-1 with a final relation yield of 8.12 mg/L antibody/g/L cell.

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Este estudo foi realizado em dez fragmentos de floresta ripária localizados no sul do Brasil (30°09’S, 51°36’W; 46 m de altitude). Entre as dez florestas ripárias selecionadas, cinco estavam circundadas por pastejo de gado e as outras cinco estavam circundadas por plantações de eucalipto. Foi avaliada a sustentabilidade de Campomanesia rhombea, Diospyros inconstans, Myrciaria cuspidata e Sebastiania commersoniana através de análises da estrutura de tamanho, as quais foram realizadas em duas escalas. Em escala local, foram consideradas as populações presentes nos fragmentos circundados por pastejo de gado ou por plantações de eucalipto e, em escala regional considerou-se também áreas florestais maiores, tomadas como referência (Inventário Florestal Contínuo do Rio Grande do Sul). Em escala local, nas populações presentes em fragmentos circundados por plantações de eucalipto, prevaleceram indivíduos de pequenas classes de tamanho, e em áreas expostas ao pastejo de gado, as mesmas espécies apresentaram uma falha
This studied was carried in ten riparian forest fragments located southern Brazil. Of the ten riparian forests selected for study, five were embedded in pasturelands and five in eucalyptus plantations. We assessed the population sustentability of Campomanesia rhombea, Diospyros incontans, Myrciaria cuspidata and Sebastiania commersoniana through analyses of size structure, which were carried out at two scales. At a local scale, we consider populations in fragments surrounded by pastures or eucalyptus forest plantations, and at a regional scale we also consider larger forest tracts taken as reference areas (Rio Grande do Sul Forest Inventory databank). At local scale, the populations in fragments embedded by eucalyptus plantations prevailed small size classes individuals, and in areas exposed to cattle ranching, the same species seem to have a recurring failure of small individuals. Regional analyses included only established individuals with DBH > 1.6 cm. In this scale, populations in fragments surrounded by

Using the overlapping sub-fragments, i.e. P-, S- and SP-domains of CRT in different binding assays, we determined that the S-domain has the strongest affinity to bind to C1q and MBL. This makes the structure of the S-domain most relevant for understanding the binding interactions to C1q and MBL and the sterical inhibition of the complement activation by competing with the respective C1q or MBL associated serine proteases for binding. The CRT S-domain structure determined in this work has an extended hairpin fold stabilised by a short 3 residues beta-sheet holding the two opposite strands together. Two hydrophobic cores, each containing two conserved tryptophan residues, are located on either sides of this beta-sheet and further stabilise this fold. Comparison of 2D [1H, 15N]-HSQC spectra indicated higher internal mobility of the S-domain than of the other active fragments, suggesting that the dynamical properties are important for the protein activity. Detailed multi-field relaxation analysis showed high dynamics in the hairpin with more restrictions where secondary structure elements are present. The interaction with MBL and C1q was determined to be optimal under low ionic strength conditions, indicative of electrostatic interactions.;The interaction probably involves the tip of the hairpin in the CRT S-domain structure that has a high concentration of negatively charged residues, and a complimentary positively charged region of the MBL and C1q collagen-like domains. The flexibility of the hairpin is likely to be a key element for CRT, allowing it to adopt its conformation for binding to multiple ligands and is expected to be the key factor for the enhanced activity of the S-domain.

The Cameroon Highlands that run along the Cameroon-Nigeria border are an important source of biodiversity. Not only are they rich in species and high in endemics, but biota from West Africa have not been studied as extensively relative to other parts of the Afrotropics, or the tropics in general. Threatening these rare and diverse habitats is anthropogenic pressure, which fragments forests and changes local animal communities. This thesis wished to address the impact of humans on seed dispersal and recruitment processes on selected tree species in forests on the Mambilla Plateau - a montane region in Nigeria's north-east. Research was conducted at Ngel Nyaki Forest Reserve, a conservation area established by the Nigerian Montane Forest Project. The reserve comprises a moderately-large forest patch (Ngel Nyaki Forest) and many small riparian fragments embedded in a grassland matrix. Cattle grazing and burning of this grassland are major threats to the survival of forest in this area.Hunting of local wildlife for bushmeat is also of concern, considering many of the region’s large-mammalian fauna are now locally extirpated (e.g. elephants) or at low abundances (many primate species). Loss of large-bodied frugivorous species has the potential to negatively impact the recruitment of large-seeded tree species that solely rely on them as seed dispersers. In this study, the ability for scatterhoarding rodents to act as surrogate dispersers for large-seeded species is tested. While much research has been carried out on secondary rodent dispersal in the Neotropics, work in the Afrotropics is still in its infancy. Because the outcome of plant-rodent interactions (i.e. predated or dispersed) may vary with season, habitat, or traits of the seed species in question, a number of experiments were established to quantify how local rodents at Ngel Nyaki may or may not be acting as effective dispersers. Additionally, the benefits of rodent dispersal were examined by creating an experiment that simulated secondary dispersal on seedling recruitment. The results of this study demonstrated that rodents can act as effective dispersers in Afromontane forests, but this is influenced by habitat, seasonal abundance of resources, and palatability of seed species. Furthermore, it was demonstrated that burial of seeds by rodents can increase the establishment probability of a seed by protecting it from removal by other rodents. However, while rodents play a strong driver of seed survival, it was also demonstrated that seedling mortality factors (such as herbivory) can also be heavy filters to seedling success. It is hoped that the results of this study will help to inform better management decisions and understand how the composition of the forest might change in the future.

Bien que faiblement représenté dans les tissus, le collagène V joue un rôle essentiel dans l’élaboration des matrices extracellulaires et dans la morphogenèse des tissus conjonctifs. Des mutations sur ses gènes sont à l’origine du syndrome d’Ehlers-Danlos classique, et une dérégulation de l’expression de ce collagène est observée dans certaines pathologies dont le cancer. Les mécanismes moléculaires conduisant aux défauts observés dans des situations pathologiques sont encore largement méconnus. Les collagènes sont des protéines modulaires pouvant exercer leur fonction par l’intermédiaire de leurs domaines collagéniques et non-collagéniques. L’objectif de mon projet de thèse a été de réaliser une caractérisation biochimique et fonctionnelle du collagène V par production recombinante de fragments dérivés de ce collagène et production d’anticorps spécifiques. Deux fragments de la chaîne 1(V) ont été plus particulièrement ciblés. (1) Le domaine N-propeptidique, impliqué dans la régulation de la fibrillogénèse est partiellement clivé au cours de la maturation du collagène V. Ce clivage conduit à la libération du domaine TSPN dont la fonction dans les tissus n’est pas connue. (2) Le fragment HepV, issu d’un domaine collagénique, contient un site de liaison à l’héparine et représente un site privilégié pour nouer des interactions avec les protéoglycannes à héparanes sulfates matriciels et cellulaires. Nos résultats montrent que ces fragments confèrent au collagène V des propriétés biologiques importantes, notamment dans le cancer du sein et dans la régulation de l'angiogenèse, processus pendant lesquels de nombreux fragments dérivés des collagènes sont libérés, par protéolyse, dans le micro-environnement cellulaire
Collagen V plays a key role in extracellular matrix organisation and in connective tissue formation even though it is poorly expressed in tissues. Mutations in its genes are responsible for the classical Ehlers-Danlos syndrome and deregulation of its expression is observed in different diseases including cancer. The molecular mechanisms underlying the defects observed in pathological situations are poorly understood. Collagens are modular proteins composed of a succession of collagenous and non-collagenous domains, which are both involved in collagen functions. The aim of my PhD project was to perform a biochemical and functional characterization of different collagen V regions by generating recombinant fragments and specific antibodies. Two regions of the 1(V) chain have been selected for this study : (1) the N-propeptide domain, which has been involved in the regulation of fibril formation. The N-propeptide is partially cleaved during maturation releasing the TSPN domain which function is unknown. (2) The HepV fragment is located in the collagenous domain and encompasses a heparin binding site that can bind matrix and/or membranar heparan sulfate proteoglycans. Overall, our results show that these fragments are responsible for important biological functions, notably in breast cancer and in the regulation of angiogenesis, events during which intense matrix remodelling occurs

Master of Science
Department of Animal Sciences and Industry
Terry A. Houser
Nursery age pigs (n=198) were used to evaluate the difference in the occurrence of injection site abscesses between needle-free jet injection and conventional needle-and-syringe injection systems. Pigs were fed for 21 d prior to treatment administration to acclimate the pigs to the environment of the Kansas State University Segregated Early Weaning (SEW) unit. On d 21 each pig was injected with aluminum hydroxide adjuvant in the neck and ham with needle-free jet injection (Pulse Needle-Free Systems, Lenexa, KS) and conventional needle-and-syringe injection. Needle-free and conventional needle-and-syringe injections were randomly assigned to pig side yielding a total of 396 injections per treatment with a total of 792 injections sites. Immediately prior to injection, the external surface of the injection sites were contaminated with an inoculum of Arcanobacterium pyogenes, a bacterium commonly associated with livestock abscesses. The pigs were then fed for a period of 27 or 28 d. On d 27 or d 28 the pigs were humanely euthanized and sent to the Kansas State Veterinary Diagnostics Laboratory where necropsies were performed and the injection sites harvested for histopathological evaluation. The needle-free jet injection system was associated with more injection site abscesses than the conventional needle-and-syringe injection method for both neck (P=0.0625) and ham (P=0.0313) injection sites. Twelve abscesses were found at injection sites administered via needle-free jet injection method while only 1 abscess was found with the conventional needle-and-syringe injection method. 5 abscesses were found at the neck injection sites and 8 abscesses were found at ham injection sites. There were no significant differences seen in tissue granulation resulting from reaction to the adjuvant. In summary, the implementation of needle-free jet injection systems in market hog production will be beneficial to eliminate needles and needle fragments in meat products but, when in the presence of Arcanobacterium pyogenes, it may increase the occurrence of injection site abscesses in pork carcasses that will need to be trimmed in pork processing plants. Although more abscesses were associated with needle-free jet injection, their occurrence was observed at a very low rate given that all injection sites were intentionally contaminated prior to injection.

This thesis represents one of the first systematic, detailed spatial analyses of artifacts at the mid-19th century Hudson's Bay Company's Fort Vancouver Village site, and of clay tobacco pipe fragments in general. Historical documents emphasize the multi-cultural nature of the Village, but archaeologically there appears to be little evidence of ethnicity (Kardas 1971; Chance and Chance 1976; Thomas and Hibbs 1984:723). Following recent approaches to cultural interaction in which researchers examined the nuanced uses of material culture (Lightfoot et al 1998; Martindale 2009; Voss 2008); this study analyzed the spatial distribution of tobacco pipe fragments for behavioral information through a practice theory approach (Bourdieu 1977; Ortner 2006). The analysis aimed to determine the role of tobacco smoking in the Village. It evaluated tobacco smoking as a significant and social behavior, the visibility of maintenance behaviors in the clay pipe distributions, and evidence of ethnic variation in tobacco consumption. Spatial patterning characteristics were compiled from the few behavioral studies of clay pipe fragments (Davies 2011; Fox 1998: Hamilton 1990; Hartnett 2004; Hoffman and Ross 1973, 1974; King and Miller 1987), and indications of ethnic specific behaviors from archaeological and historical evidence (Burley et al 1992; Jacobs 1958; Jameson 2007). Distributional maps examined three pipe assemblage characteristics: fragment frequency, use wear fragment frequency, and the bowl to stem fragment ratio, to define smoking locations on the Village landscape. Visibility of maintenance and refuse disposal behaviors in the size distribution of fragments was measured through the Artifact Size Index (ASI) (Bon Harper and McReynolds 2011). This analysis also tested two possible indications of ethnic variation: differential use of stone vs. clay pipes, and consumption rates as reflected through clay pipe assemblages. The commonality of tobacco smoking locations across the landscape suggests a significant, social, and shared practice between households. Analysis of maintenance behaviors and ethnic variation proved inconclusive. This study demonstrates the value of spatially analyzing clay pipe fragment distributions for behavioral information. The insight gained from examining multiple spatial patterns suggests future studies can benefit from analyzing the spatial distribution of diagnostic characteristics of pipes and other artifact types.

Antibodies are proteins capable of specific interactions to a wide range of molecules. These interactions are facilitated by the complementary determining regions (CDR).

Carcinomas are the most common of human cancers and they release significant amount of cytokeratins (CK) in the necrotic areas of the tumors. The CKs stay in the tumor, since they have low solubility. The antibody studied in this thesis, the anti-CK 8 antibody TS1, has shown to be effective in tumor targeting and is proposed to be useful in therapy.

Single-chain antibodies (scFv) are recombinant antibodies which are much smaller than the intact IgG. This is an advantage when used in tumor therapy, since they can penetrate the tumors more easily than the larger IgG. Moreover, they are expressed by one single gene which make them easy to modify, for example by site-directed mutagenesis.

The anti-idiotypic antibody αTS1 can be used to clear the TS1 form the circulation and thereby clear the body from non-tumor bound TS1 in therapy. To be able to modify the binding of an antibody to its antigen and or anti-idiotype, these interactions must be studied. In this study this is accomplished by chemical modifications of the IgGs TS1 and αTS1 and the antigen CK 8. Guided by these results, amino acid residues were mutated by using site-directed mutagenesis in the TS1-218 scFv and the effects were studied. From mutational study results, the functional epitope could be mapped and it was found that there are mainly tyrosines, but also charged residues, serine and a tryptophan that are important for both interactions. The binding of TS1-218 to both αTS1 and CK 8 could be improved by changing the negatively charged side-chains by mutations to their corresponding amide or alanine.

Both the IgG and scFv versions of TS1 were administered in vivo. The IgG αTS1 was used to clear the TS1 from the circulation by forming immune complexes. The immune complexes, consisting of four or more antibodies, were mainly metabolized by the liver. The scFv TS1-218 could localize to the tumor in a tumor xenograft mouse model, although a higher uptake would be desired in a therapeutic strategy. The scFv was cleared rapidly by the kidneys, but the clearance could be slowed by pre-formed immune complexes with anti-TS1 scFv in vitro, prior to administration in vivo.

Orientador: Alexandre Magno Sebbenn
Banca: Mario Luiz Teixeira de Moraes
Banca: Ananda Virgínia de Aguiar
Resumo: O fluxo e padrões de dispersão de pólen foram investigados em um pequeno fragmento florestal isolado da espécie arbórea neotropical, polinizada por insetos da Copaifera langsdorffii, por meio da análise de paternidade e oito locos microssatélites, também foi investigado a coancestria e o tamanho efetivo populacional dentro de progênies para a conservação e recuperação ambiental. Sementes de polinização aberta (20 a 25 sementes) foram coletadas de 15 árvores matrizes de um fragmento, onde todos os indivíduos adultos foram previamente mapeados, medidos e genotipados para oito locos microssatélites. Vinte sementes foram coletadas da árvore vizinha mais próxima (1,2 km) do fragmento. Os níveis de diversidade genética foram significativamente maiores nos adultos do que nas progênies. Níveis significativos de endogamia foram detectados em progênies (F = 0,226), o que foi atribuído principalmente ao cruzamento entre parentes. A partir da análise de paternidade, baixos níveis de autofecundação (s = 8%) e imigração de pólen (m = 8%) foram observados no fragmento florestal, mas níveis muito altos foram detectados na árvore isolada (s = 20%; m = 75%), indicando que o fragmento e a árvore não estão reprodutivamente isolados e são conectados por dispersão de pólen a longas distancias (máximo detectado 1,420 m). Dentro do fragmento, o padrão de dispersão de pólen foi o vizinho próximo, com cerca de 49% do pólen se dispersando até 50 m. O tamanho efetivo populacional da árvore-matriz foi baixa, indicando a necessidade de se coletar muitas sementes de árvores (mínimo de 76 árvores) para fins de conservação. Em termos gerais, os resultados mostraram que o fragmento e a árvore isolada pela fragmentação florestal não estão reprodutivamente isoladas, embora o isolamento espacial parecesse aumentar a taxa de autofecundação e cruzamentos correlacionados
Abstract: Pollen flow, dispersal and patterns were investigated in a small and isolated forest fragment of the neotropical, insect pollinated tree Copaifera langsdorffii, using paternity analysis and eight microsatellite loci, we also investigated the coancestry and effective population size of progeny array for conservation and environmental restoration purpose. Open-pollinated seeds (20 to 25 seeds) were collected from 15 seed trees of forest fragment, where all adults trees were previously mapped, measured and genotyped by eight microsatellite loci. Twenty seeds were also collected from the neighbour tree (1.2 km) of the forest fragment. Levels of genetic diversity were significantly higher in adults than offspring. Significant levels of inbreeding were detected in offspring (F=0.226), which was attributed mainly to the mating among relatives. From paternity analysis, low levels of selfing (s=8%) and pollen immigration (m=8%) were observed in the forest fragment, but very high levels were detected in the isolated tree (s=20%; m=75%), indicating that the forest fragment and the tree are not reproductive isolated and are connected by long pollen dispersal (maximum detected 1,420 m). Within the forest fragment, the pattern of pollen dispersal was the near neighbor with about 49% of the pollen being dispersed until 50 m. The effective population size of the progeny array was low, indicating the necessity to collect seeds from many seed trees (minimum of 76 trees) for conservation purposes. In general terms, the results showed that the fragment and the tree isolated by forest fragment are not brooked the genetic connectivity, although the spatial isolation seems increase selfing rate and correlated mating
Mestre

Les aminoglycosides, des dérivés aminés de saccharides, interfèrent avec le mécanisme de synthèse des protéines chez les bactéries en se fixant au site de décodage de l'ARN de transfert aminoacylé (site A) situé en 3' de l'ARN ribosomique 16S. Au cours de ce travail de thèse, les structures de trois complexes entre des fragments d'ARN incorporant le site A et les aminoglycosides paromomycine, tobramycine et généticine, ont été résolues par cristallographie aux rayons X à 2,40-2,54 Å. L'analyse des structures montre que la reconnaissance et la fixation spécifiques des aminoglycosides au site A font intervenir de nombreuses liaisons hydrogène directes et pontées par des molécules d'eau. Dans ces structures, la partie néamine commune aux aminoglycosides (cycles I et II) s'intercale dans l'hélice de manière similaire : le cycle I (non plan) forme une pseudo paire de bases avec l'adénine 1408 ; la néamine oblige les adénines 1492 et 1493 à pointer hors de l'hélice. La comparaison des structures 3D de ces trois complexes offre des explications moléculaires aux différents résultats de biochimie et de microbiologie, ainsi qu'à certains phénomènes de résistances et de toxicités. Les conformations du site A et des aminoglycosides au sein de ces complexes sont similaires à celles du site A et de la paromomycine au sein de la sous-unité ribosomique 30S. Ainsi, la stratégie développée permet une description des interactions et des modes de fonctionnement des aminoglycosides proche du contexte naturel mais plus précise, essentielle à notre connaissance du système ARN/aminoglycoside. De ces résultats découlent des lignes directrices laissant envisager sous un jour nouveau la conception d'antibiotiques moins sujets aux résistances et moins toxiques.

Thesis (Ph.D.) -- University of Maryland, College Park, 2009.
Thesis research directed by: Marine, Estuarine, Environmental Sciences Graduate Program. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
O fluxo e padrões de dispersão de pólen foram investigados em um pequeno fragmento florestal isolado da espécie arbórea neotropical, polinizada por insetos da Copaifera langsdorffii, por meio da análise de paternidade e oito locos microssatélites, também foi investigado a coancestria e o tamanho efetivo populacional dentro de progênies para a conservação e recuperação ambiental. Sementes de polinização aberta (20 a 25 sementes) foram coletadas de 15 árvores matrizes de um fragmento, onde todos os indivíduos adultos foram previamente mapeados, medidos e genotipados para oito locos microssatélites. Vinte sementes foram coletadas da árvore vizinha mais próxima (1,2 km) do fragmento. Os níveis de diversidade genética foram significativamente maiores nos adultos do que nas progênies. Níveis significativos de endogamia foram detectados em progênies (F = 0,226), o que foi atribuído principalmente ao cruzamento entre parentes. A partir da análise de paternidade, baixos níveis de autofecundação (s = 8%) e imigração de pólen (m = 8%) foram observados no fragmento florestal, mas níveis muito altos foram detectados na árvore isolada (s = 20%; m = 75%), indicando que o fragmento e a árvore não estão reprodutivamente isolados e são conectados por dispersão de pólen a longas distancias (máximo detectado 1,420 m). Dentro do fragmento, o padrão de dispersão de pólen foi o vizinho próximo, com cerca de 49% do pólen se dispersando até 50 m. O tamanho efetivo populacional da árvore-matriz foi baixa, indicando a necessidade de se coletar muitas sementes de árvores (mínimo de 76 árvores) para fins de conservação. Em termos gerais, os resultados mostraram que o fragmento e a árvore isolada pela fragmentação florestal não estão reprodutivamente isoladas, embora o isolamento espacial parecesse aumentar a taxa de autofecundação e cruzamentos correlacionados
Pollen flow, dispersal and patterns were investigated in a small and isolated forest fragment of the neotropical, insect pollinated tree Copaifera langsdorffii, using paternity analysis and eight microsatellite loci, we also investigated the coancestry and effective population size of progeny array for conservation and environmental restoration purpose. Open-pollinated seeds (20 to 25 seeds) were collected from 15 seed trees of forest fragment, where all adults trees were previously mapped, measured and genotyped by eight microsatellite loci. Twenty seeds were also collected from the neighbour tree (1.2 km) of the forest fragment. Levels of genetic diversity were significantly higher in adults than offspring. Significant levels of inbreeding were detected in offspring (F=0.226), which was attributed mainly to the mating among relatives. From paternity analysis, low levels of selfing (s=8%) and pollen immigration (m=8%) were observed in the forest fragment, but very high levels were detected in the isolated tree (s=20%; m=75%), indicating that the forest fragment and the tree are not reproductive isolated and are connected by long pollen dispersal (maximum detected 1,420 m). Within the forest fragment, the pattern of pollen dispersal was the near neighbor with about 49% of the pollen being dispersed until 50 m. The effective population size of the progeny array was low, indicating the necessity to collect seeds from many seed trees (minimum of 76 trees) for conservation purposes. In general terms, the results showed that the fragment and the tree isolated by forest fragment are not brooked the genetic connectivity, although the spatial isolation seems increase selfing rate and correlated mating

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O fragmento florestal e os testes de progênies, com a função de auxiliar a conservação, reabilitação dos processos ecológicos e promover a conservação da biodiversidade, tem um papel importante nos processos de restauração de áreas degradadas e diminuir os efeitos negativos da destruição dos ambientes naturais. Este trabalho objetivou conhecer a estrutura populacional, distribuição espacial e tamanho efetivo de espécies arbóreas nativas presente em um fragmento florestal de cerrado, além de estimar a variabilidade genética num teste de progênies misto de Myracrodruon urundeuva; Terminalia argentea e Astronium fraxinifolium; a partir dos carácteres silviculturais em Selvíria – MS, com vistas de transformá-las em área para subsidiar coleta de sementes. No fragmento florestal foram mensurados e identificados 1187 indivíduos com DAP ≥ 10 cm, que compõem o dossel arbóreo da floresta. Em uma área de 0,89 ha foram amostrados, pertencentes a 60 espécies distribuídas em 31 famílias, com destaque para a família Fabaceae com 11 espécies. O índice de ShannonWeaver apresentou um H’ = 2,98 nats ind-1 , o que pode ser considerado como uma diversidade média. As espécies de maior Índice de Valor de Importância (IVI) foram Copaifera landesdorffii, Diptychandra aurantiaca, Parapiptadenia rigida, Qualea grandiflora, Qualea parviflora, Vochysia tucanorum, Tapirira guianensis, Astronium fraxinifolium, Xylopia aromatica, e Mabea fistulifera. Assim, dentre as espécies encontradas com o maior IVI podem ser classificadas como potenciais para a locação de árvores matrizes para subsidiar a coleta de sementes. Portanto, dispõem de tamanho efetivo que permite a formação de novas populações, a partir da coleta de sementes nessa área de Reserva legal. No teste de progênies misto aos 22 anos, os valores altos observados para o caráter SOB (M. urundeuva - 88,0%, A. fraxinifolium – 85,9% e T. argentea - 59,6%), indicam boa adaptação das espécies em estudo e potencial para uso em reflorestamentos. As estimativas do coeficiente de variação genética oscilaram de 3,03% a 49,50%, estimando o menor valor obtidos para a variável DAP nas três espécies. As herdabilidades individuais variaram de baixa (0,14) para T. argentea, e alta (>0,40) para M. urundeuva e A. fraxinifolium no caráter DAP. A proporção sexual para as duas espécies foi tendenciosa para sexo masculino, sendo que a proporção geral foi de 1,5 machos para 1 fêmea. O tamanho efetivo populacional variou conforme a proporção de machos e fêmeas, sendo que o que mais se aproximou do valor real de indivíduos reprodutivos foi Myracrodruon urundeuva. Portanto, a seleção dos indivíduos dentro dos testes de progênies não deve considerar apenas o caráter de crescimento (DAP), mas também com base na proporção sexual e no tamanho efetivo populacional para a sua futura transformação em um Pomar de Sementes por Mudas. As informações obtidas com este estudo são úteis para fins de conservação e melhoramento genético das espécies, como também para seleção de matrizes potenciais para recuperação ambiental, o que será fundamental para restauração ecológica em áreas do Bolsão Sul-Mato-Grossense.
The forest fragment and progeny tests, with the function of assisting conservation, rehabilitation of ecological processes and promoting biodiversity conservation, play an important role in the restoration of degraded areas and reduce the negative effects of the destruction of natural environments. This work aimed to know the population structure, spatial distribution and effective size of native tree species present in a cerrado forest fragment, besides estimating the genetic variability in a test of mixed progenies of Myracrodruon urundeuva; Terminalia argentea and Astronium fraxinifolium; from silvicultural characters in Selvíria - MS, with a view to transforming them into an area to subsidize seed collection. In the forest fragment were measured and identified 1187 individuals with DBH ≥ 10 cm, which make up the arboreal forest canopy. In an area of 0,89 ha were sampled, belonging to 60 species distributed in 31 families, with emphasis on the Fabaceae family with 11 species. The Shannon-Weaver index showed a H '= 2,98 nats ind-1, which can be considered as a mean diversity. The species with the highest Import Value Index (IVI) were Copaifera landesdorffii, Diptychandra aurantiaca, Parapiptadenia rigida, Qualea grandiflora, Qualea parviflora, Vochysia tucanorum, Tapirira guianensis, Astronium fraxinifolium, Xylopia aromatica, and Mabea fistulifera. Thus, among the species found with the highest IVI can be classified as potential for the lease of matrix trees to subsidize the collection of seeds. Therefore, they have an effective size that allows the formation of new populations, from the collection of seeds in this area of legal reserve. In the progenies test mixed at 22 years, the high values observed for SOB (M. urundeuva – 88,0%, A. fraxinifolium – 85,9% and T. argentea – 59,6%) indicated a good adaptation of the Species and potential for use in reforestation. Estimates of the coefficient of genetic variation ranged from 3,03% to 49,50%, estimating the lowest value obtained for the DAP variable in the three species. The individual heritabilities ranged from low (0,14) for T. argentea, and high (> 0,40) for M. urundeuva and A. fraxinifolium on the DAP character. The sex ratio for both species was biased for males, with the general proportion being 1,5 males for 1 female. The effective population size varied according to the proportion of males and females, and the one that most approached the real value of reproductive individuals was Myracrodruon urundeuva. Therefore, the selection of individuals within progeny tests should not only consider the growth character (DBH), but also on the basis of the sex ratio and the effective population size for its future transformation into a Seedling Seed Orchard. The information obtained from this study is useful for the conservation and genetic improvement of the species, as well as for the selection of potential matrices for environmental restoration, which will be fundamental for ecological restoration in areas of the Bolsão Sul-mato-grossense.

Orientador: Mario Luiz Teixeira de Moraes
Abstract: The forest fragment and progeny tests, with the function of assisting conservation, rehabilitation of ecological processes and promoting biodiversity conservation, play an important role in the restoration of degraded areas and reduce the negative effects of the destruction of natural environments. This work aimed to know the population structure, spatial distribution and effective size of native tree species present in a cerrado forest fragment, besides estimating the genetic variability in a test of mixed progenies of Myracrodruon urundeuva; Terminalia argentea and Astronium fraxinifolium; from silvicultural characters in Selvíria - MS, with a view to transforming them into an area to subsidize seed collection. In the forest fragment were measured and identified 1187 individuals with DBH ≥ 10 cm, which make up the arboreal forest canopy. In an area of 0,89 ha were sampled, belonging to 60 species distributed in 31 families, with emphasis on the Fabaceae family with 11 species. The Shannon-Weaver index showed a H '= 2,98 nats ind-1, which can be considered as a mean diversity. The species with the highest Import Value Index (IVI) were Copaifera landesdorffii, Diptychandra aurantiaca, Parapiptadenia rigida, Qualea grandiflora, Qualea parviflora, Vochysia tucanorum, Tapirira guianensis, Astronium fraxinifolium, Xylopia aromatica, and Mabea fistulifera. Thus, among the species found with the highest IVI can be classified as potential for the lease of matrix trees to subsidize t... (Complete abstract click electronic access below)
Resumo: O fragmento florestal e os testes de progênies, com a função de auxiliar a conservação, reabilitação dos processos ecológicos e promover a conservação da biodiversidade, tem um papel importante nos processos de restauração de áreas degradadas e diminuir os efeitos negativos da destruição dos ambientes naturais. Este trabalho objetivou conhecer a estrutura populacional, distribuição espacial e tamanho efetivo de espécies arbóreas nativas presente em um fragmento florestal de cerrado, além de estimar a variabilidade genética num teste de progênies misto de Myracrodruon urundeuva; Terminalia argentea e Astronium fraxinifolium; a partir dos carácteres silviculturais em Selvíria – MS, com vistas de transformá-las em área para subsidiar coleta de sementes. No fragmento florestal foram mensurados e identificados 1187 indivíduos com DAP ≥ 10 cm, que compõem o dossel arbóreo da floresta. Em uma área de 0,89 ha foram amostrados, pertencentes a 60 espécies distribuídas em 31 famílias, com destaque para a família Fabaceae com 11 espécies. O índice de ShannonWeaver apresentou um H’ = 2,98 nats ind-1 , o que pode ser considerado como uma diversidade média. As espécies de maior Índice de Valor de Importância (IVI) foram Copaifera landesdorffii, Diptychandra aurantiaca, Parapiptadenia rigida, Qualea grandiflora, Qualea parviflora, Vochysia tucanorum, Tapirira guianensis, Astronium fraxinifolium, Xylopia aromatica, e Mabea fistulifera. Assim, dentre as espécies encontradas com o maior IVI pod... (Resumo completo, clicar acesso eletrônico abaixo)
Doutor

The purpose of this study was to determine if the histomorphometric analysis of age could be used to determine the age at death of subadults from the Commissary Archaeological Site. The histomorphometric analysis was used because of the fragmented condition of the bones. Sections from the left femur were obtained and processed for light and electron microscopy. Using the regression formula of Ahlqvist and Damsten and Ubelaker, age was estimated from the percentage of Haversian canals per 1 mm unit of bone. Results show the subadult population ranged in age from 0.9- 16.7 years. The correlation between the thickness of compact bone, the midshaft width, and the marrow cavity width to that of the assigned age supports the validity of the regression formula. The study demonstrates that the histomorphometric analysis can be applied to estimate age in subadult skeletons. This study further provides the first scientific analysis of age for the subadult skeletons of the Commissary site, an Early Late Woodland Native American population.
Department of Anthropology

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Os objetivos deste trabalho foram verificar o sistema de reprodução entre e dentro de frutos, a distância e padrões de fluxo de pólen, os níveis de endogamia e diversidade genética, a depressão por endogamia e a distribuição espacial de genótipos em duas populações de Hymenaea stigonocarpa: a primeira está presente em um fragmento e a segunda em árvores isoladas na pastagem. Para tanto, foram mapeadas e medidas todas as árvores adultas reprodutivas existentes nos dois locais. Foram coletadas sementes de 15 árvores matrizes no fragmento florestal e em 20 árvores matrizes isoladas na pastagem, sendo 30 sementes por árvore. Esta coleta propiciou a instalação de um teste de progênies na Fazenda de Ensino, Pesquisa e Extensão da FEIS/UNESP. Foram feitas a genotipagem de todas as árvores adultas de ambas as populações e de todas as progênies. Adicionalmente, dentro do fragmento foram também amostrados, mapeados e medida a altura de juvenis. As análises genéticas permitiram avaliar os efeitos da depressão por endogamia para altura e sobrevivência, e as análises dos genótipos foram feitas para seis locos microssatélites, já transferidos para a espécie. O estudo do sistema de reprodução foi baseado no modelo misto de reprodução e modelo de cruzamentos correlacionados. A análise de paternidade das sementes permitiu determinar a distância e o padrão de fluxo efetivo de pólen dentro das populações, bem como o fluxo gênico externo das áreas amostradas. A análise da distribuição espacial dos genótipos foi realizada para árvores adultas localizadas dentro dos fragmentos, utilizando-se estimativas do coeficiente de coancestria entre pares de indivíduos dentro de diferentes classes de distância, tornando-se possível estimar o tamanho efetivo de variância e, assim, estabelecer estratégias para a coleta de sementes. Os resultados permitiram entender o processo de reprodução que indica a presença marcante de progênies de irmãos-completos, formação da estrutura genética espacial, vizinhança genética reprodutiva e depressão por endogamia na geração descendente proveniente de ambas as populações de H. stigonocarpa. A coleta de sementes deve ser feita em árvores espaçadas de pelo menos 350 m de distância em 78 árvores com 30 sementes cada árvore para reter o tamanho efetivo de referência de 150 nas amostras para garantir o estabelecimento destas gerações futuras em longo prazo na conservação “ex situ” e/ou reflorestamento em áreas degradadas.
The aim of this work were to study the mating system within and among fruits, distance and pollen flow patterns, inbreeding levels and genetic diversity, inbreeding depression and the spatial distribution of genotypes in two populations of Hymenaea stigonocarpa: first is present in a fragment and the second in isolated trees in the pasture. Therefore, were mapped and measures all existing reproductive adult trees in both sites. It was collected seed of 15 seed trees in the forest fragment and in 20 isolated trees in the pasture, with 30 seeds per tree. This collection allowed the installation of a progeny test in Farm of Teaching, Research and Extension from FEIS/UNESP. It was made genotyping of all adult trees of both populations and all progenies. Additionally, within the fragment were also sampled, mapped and measured the height of juveniles. Genetic analysis allowed to evaluate the effects of inbreeding depression for height and survival, and analysis of genotypes were made for six microsatellite loci, already transferred for the species. The mating system study was based on the mixed mating model and correlated mating model. The paternity analysis of the seeds allowed to determine the distance and the pattern of effective flow of pollen within the fragments, as well as the outside gene flow of the sampled areas. The spatial distribution analysis of genotypes was done for adult trees located within of the fragments, using estimates coancestry coefficient between pairs of individuals within different distances classes, making it possible to estimate the variance effective size and thereby establish strategies for seed collection. The results allowed understanding the process of mating that indicates the strong presence of progeny full-sibs, formation of spatial genetic structure, reproductive genetic neighborhood and inbreeding depression in the descending generation from both populations of H. stigonocarpa. Seed collection should be done in trees spaced of at least 350 m away in 78 trees with 30 seeds each tree to retain the effective size of the reference 150 in the samples to ensure the establishment of these future generations in the long-term conservation "ex situ "and / or reforestation in degraded areas.
CNPq: 141028/2012-2

Orientador: Alexandre Magno Sebbenn
Resumo: Os objetivos deste trabalho foram verificar o sistema de reprodução entre e dentro de frutos, a distância e padrões de fluxo de pólen, os níveis de endogamia e diversidade genética, a depressão por endogamia e a distribuição espacial de genótipos em duas populações de Hymenaea stigonocarpa: a primeira está presente em um fragmento e a segunda em árvores isoladas na pastagem. Para tanto, foram mapeadas e medidas todas as árvores adultas reprodutivas existentes nos dois locais. Foram coletadas sementes de 15 árvores matrizes no fragmento florestal e em 20 árvores matrizes isoladas na pastagem, sendo 30 sementes por árvore. Esta coleta propiciou a instalação de um teste de progênies na Fazenda de Ensino, Pesquisa e Extensão da FEIS/UNESP. Foram feitas a genotipagem de todas as árvores adultas de ambas as populações e de todas as progênies. Adicionalmente, dentro do fragmento foram também amostrados, mapeados e medida a altura de juvenis. As análises genéticas permitiram avaliar os efeitos da depressão por endogamia para altura e sobrevivência, e as análises dos genótipos foram feitas para seis locos microssatélites, já transferidos para a espécie. O estudo do sistema de reprodução foi baseado no modelo misto de reprodução e modelo de cruzamentos correlacionados. A análise de paternidade das sementes permitiu determinar a distância e o padrão de fluxo efetivo de pólen dentro das populações, bem como o fluxo gênico externo das áreas amostradas. A análise da distribuição espacial d... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The aim of this work were to study the mating system within and among fruits, distance and pollen flow patterns, inbreeding levels and genetic diversity, inbreeding depression and the spatial distribution of genotypes in two populations of Hymenaea stigonocarpa: first is present in a fragment and the second in isolated trees in the pasture. Therefore, were mapped and measures all existing reproductive adult trees in both sites. It was collected seed of 15 seed trees in the forest fragment and in 20 isolated trees in the pasture, with 30 seeds per tree. This collection allowed the installation of a progeny test in Farm of Teaching, Research and Extension from FEIS/UNESP. It was made genotyping of all adult trees of both populations and all progenies. Additionally, within the fragment were also sampled, mapped and measured the height of juveniles. Genetic analysis allowed to evaluate the effects of inbreeding depression for height and survival, and analysis of genotypes were made for six microsatellite loci, already transferred for the species. The mating system study was based on the mixed mating model and correlated mating model. The paternity analysis of the seeds allowed to determine the distance and the pattern of effective flow of pollen within the fragments, as well as the outside gene flow of the sampled areas. The spatial distribution analysis of genotypes was done for adult trees located within of the fragments, using estimates coancestry coefficient between pairs of ... (Complete abstract click electronic access below)
Doutor

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Cariniana estrellensis e Cariniana legalis são uma das maiores árvores dos biomas florestais da Amazônia e Mata Atlântica, sendo atualmente vulneráveis à extinção devido ao intenso desmatamento desses biomas. Estratégias para conservação in situ e ex situ são urgentes e estudos de diversidade genética e fluxo de genes são chaves e para esses propósitos. Assim, investigamos a diversidade genética, a estrutura genética espacial (SGS) e o fluxo histórico de genes em populações fragmentadas de ambas as espécies, utilizando marcadores de microssatélites. Todas as árvores encontradas nas populações foram mapeadas, medidas para o diâmetro na altura do peito (DAP) e amostrado o cambio de casca. O índice de fixação (F), em alguns casos, foi significativamente maior em árvores com menor DAP, indicando que as árvores menores apresentam um maior parentesco do que as maiores. Foi detectada SGS significativa para populações de ambas as espécies (60-350 m), indicando um padrão de dispersão de genes de isolamento pela distância (IBD). Para ambas as espécies, foi observada alta imigração de semente (38,5-61,5%) e pólen (80,1-100%), mostrando que as populações não são isoladas geneticamente. Não foi detectada autofecundação, mas o cruzamento entre árvores relacionadas foi detectado nas espécies (8,9-12,5%), sugerindo uma seleção mais forte contra árvores de autofecundação do que se originou do cruzamento entre árvores relacionadas. A distância de dispersão de pólen e sementes em C. estrellensis atingiu longa distância (> 3 km) do que em C. legalis (máximo de 385 m). No entanto, o pólen e as sementes em C. estrellensis e o pólen em C. legalis foram dispersos em um padrão de IBD. Os resultados sugerem que as populações estudadas são adequadas para conservação in situ e ex situ.
Cariniana estrellensis and Cariniana legalis, two of the largest trees in the Amazon and Atlantic Forest biomes, are currently vulnerable to extinction due to the intense deforestation of these biomes. Strategies for in and ex situ conservation are urgent, and studies of genetic diversity and gene flow are key aspects needed to develop these strategeis. Thus, we investigate the genetic diversity, spatial genetic structure (SGS), and historical gene flow in fragmented populations of both species, using microsatellite markers. All trees found in the study populations were mapped, measured for diameter at breast height (DBH), and sampled for bark cambium. Our results show that in some cases, fixation index (F) was significantly higher in trees with lower DBH, indicating that smaller trees have higher levels of inbreeding than larger ones. Significant SGS was detected in populations of both species (60-350 m), indicating a gene dispersal pattern of isolation by distance (IBD). For both species, we found high seed (38.5-61.5%) and pollen (80.1-100%) immigration demonstrating that populations are not genetically isolated. No self-fertilization was detected, but we did find evidence of mating among related trees (8.9-12.5%), suggesting stronger selection against selfed individuals than those originated from mating among relatives. Pollen and seed dispersal distance for C. estrellensis reached longer distances (> 3 km) than for C. legalis (maximum of 385 m). However, pollen and seeds of C. estrellensis and pollen of C. legalis were dispersed in an IBD pattern. The results suggest that the studied populations are suitable for in and ex situ conservation.

Thesis (PhD(For))--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Widdringtonia whytei is a tropical endemic, fire-adapted pioneer coniferous tree species within natural fire-fragmented Afromontane forest patches in a confined area on Mulanje Mountain in Malawi. Natural and anthropogenic fires within the surrounding fire-prone landscape, insect attacks, and uncontrolled harvesting of mature trees for timber threaten the survival of W. whytei. This study investigated the effects of population fragmentation on the reproductive biology of W. whytei, through four specific studies: effects of population size, tree size and crown position on viable seed output; seed rain variation among population sizes; effects of temperature and light on viability and germination of W. whytei seeds; and the influence of population size on natural regeneration patterns. The study was conducted during 2008 and 2009 on three sites using three W. whytei population sizes: small (≤10 reproductively mature cone-bearing W. whytei trees), medium (11-20 cone-bearing trees) and large (>20 cone-bearing trees). Data were collected using field and laboratory experiments and a review of secondary information. The effect of fragment size and crown position on viable seed output and seed rain pattern was tested with a nested linear mixed model. The influence of stem diameter (dbh) on viable seed output was tested using a separate data set. The variation in seed rain was explored with a generalised linear model (GLM) with a negative binomial link function. Variations in seed germination was analysed with a Weibull regression model. The influence of fragment size on seedling regeneration was tested with a GLM (binary logistic regression). Viable seed output from W. whytei cones was very low (23%) and was not affected by population size, tree diameter or crown position. W. whytei population size influenced seed rain with large fragments having higher seed rain densities. Number of dispersed seeds was generally very low and poorly dispersed. W. whytei cones are semiserotinous and depends on moderate to severe fires for heavy seedfall and wider dispersal. Such fires did not occur during the study period. Temperature is a critical factor for seed germination. Seeds germinate between 15 and 25°C with the optimum at ~ 20°C either under light or dark conditions. Regeneration density followed the seed rain pattern indicating that seed availability is a critical factor for regeneration. Regular cool fires outside the forest patches and along the edges during the hot dry season, and the thick litter layer and shady conditions inside forest patches, caused most of the seedling mortality. This study highlighted the difficulties W. whytei experiences with low output of viable seed, limited seed dispersal and low seedling regeneration in and around forest patches, i.e. factors important for conservation management of this species. It is recommended that gaps with diameter equal to canopy height and occasional spot fires would promote seed dispersal onto exposed mineral soil for rapid seedling establishment and subsequent population growth. Such natural fire disturbance events may show the true seed rain and seedling establishment patterns in this species.
AFRIKAANSE OPSOMMING: Widdringtonia whytei is ‘n tropiese endemiese, vuuraangepaste pionier naaldhoutagtige boomsoort binne die natuurlike vuurgefragmenteerde Afromontane woudkolle binne ‘n beperkte area op Mulanje berg in Malawi. Natuurlike en menslike vure binne die omringende vuurgeneigde landskap, insekaanvalle, en onbeheerde kap van volwasse bome vir hout bedreig die voortbestaan van W. whytei. Hierdie studie het die effek van populasiefragmentasie op die voortplantingsbiologie van W. whytei ondersoek, deur vier spesifieke studies: die effek van populasiegrootte, boomgrootte en kroonposisie op produksie van lewenskragtige saad; saadreënvariasie tussen populasiegroottes; die effek van temperatuur en lig op lewenskragtigheid en ontkieming van W. whytei saad; en die invloed van populasiegrootte op natuurlike verjongingspatrone. Die studie is gedurende 2008 en 2009 uitgevoer op drie groeiplekke met drie W. whytei populasiegroottes: klein (≤10 keëldraende (volwasse) W. whytei bome), medium (11-20 keëldraende bome) en groot (>20 keëldraende bome). Data is versamel in veld- en laboratorium eksperimente asook 'n oorsig van sekondêre inligting. Die invloed van populasie fragmentgrootte en kroonposisie van bome op saadproduksie en die saadreënpatroon is getoets met ‘n geneste liniêre gemengde model. Die invloed van boomstamdeursnee op die produksie van kiemkragtige saad is met ‘n aparte datastel getoets. Die variasie in saadreënpatroon is met 'n algemene liniêre model (GLM) met 'n negatiewe binomiese skakelfunksie getoets. Variasies in ontkiemingspersentasie van sade is met 'n Weibull regressiemodel ontleed. Die invloed van fragmentgrootte op saailingverjonging is met 'n algemene linêre model (binêre logistiese regressie) getoets. Produksie van lewenskragtige saad in W. whytei keëls was baie lag (23%) en was nie deur populasiegrootte, boomstamdeursnee of kroonposisie beïnvloed nie. W. whytei populasiegrootte het saadreën beïnvloed en groot fragmente het groter saadreëndigthede gehad. Die aantal verspreide sade was in die algemeen baie laag en swak versprei. W. whytei keëls is semi-saadhoudend (serotinous) en is afhanklik van matige tot intense vure vir massiewe saadvrystelling en wyer saadverspreiding. Sulke vure het nie gedurende die studieperiode voorgekom nie. Temperatuur is ‘n kritiese factor vir saadontkieming. Sade ontkiem goed tussen 15 en 25°C met die optimum rondom ~ 20°C onder beide lig en donker toestande. Fragmentgrootte het nie die saailingverjonging in W. whytei beïnvloed nie. Verjongingsdigtheid het die saadreënpatroon gevolg, wat daarop dui dat saadbeskikbaarheid ‘n kritiese factor is vir saailingverjonging. Gereelde koel vure buite die woudkolle en langs die woudrand gedurende die droë warm seisoen, en die dik lae blaarval (litter) en skadutoestande binne ‘n woud, het meeste van die saailingmortaliteit veroorsaak. Hierdie studie het die probleme beklemtoon wat W. whytei ondervind met die lae produksie van kiemkragtige saad, saadverspreiding en saailingverjonging binne en rondom die woudkolle, m.a.w. faktore wat belangrik is vir bewaringsbestuur van hierdie boomsoort. Die aanbeveling word gemaak dat kroonopeninge met ‘n deursnee gelyk aan minstens die kroonhoogte, en sporadiese kolbrande binne die woudkolle kan saadverspreiding op blootgestelde minerale grond vir vinnige saailingvestiging en gevolglike populasiegroei bevorder. Sodanige vuurversteuringsgeleenthede mag die werklike patrone in saadreën en saailingvestiging in hierdie species toon.

La maladie d’Alzheimer touche plus de 40 millions de personnes dans le monde et résulte de l’agrégation du peptide beta-amyloïde de 40/42 résidus. En dépit de nombreuses études expérimentales et théoriques, le mécanisme de formation des fibres et des plaques n’est pas élucidé, et les structures des espèces les plus toxiques restent à déterminer. Dans cette thèse, je me suis intéressée à deux aspects. (1) La détermination du noyau de nucléation (N*) de deux fragments (Aβ)16-22 et (Aβ)37-42. Mon approche consiste à déterminer les paramètres OPEP du dimère (Aβ)16-22 en comparant des simulations Monte Carlo sur réseau et des dynamiques moléculaires atomiques par échange de répliques. Les paramètres fonctionnant aussi sur le trimère (Aβ)16-22 et les dimères et trimères (Aβ)37-42, j’ai étudié la surface d’énergie libre des décamères et mes simulations montrent que N* est de 10 chaines pour (Aβ)16-22 et est supérieure à 20 chaines pour (Aβ)37-42. (2) J’ai ensuite étudié les structures du dimère (Aβ)1-40 par simulations de dynamique moléculaire atomistique par échanges de répliques. Cette étude, qui fournit les conformations d’équilibre du dimère Aβ1-40 en solution aqueuse, ouvre des perspectives pour une compréhension de l’impact des mutations pathogènes et protectrices au niveau moléculaire
The neurodegenerative Alzheimer's disease (AD) is affecting more than 40 million people worldwide and is linked to the aggregation of the amyloid-β proteins of 40/42 amino acids. Despite many experimental and theoretical studies, the mechanism by which amyloid fibrils form and the 3D structures of the early toxic species in aqueous solution remain to be determined. In this thesis, I studied the structures of the eraly formed oligomers of the amyloid-β peptide and the critical nucleus size of two amyloid-β peptide fragments using either coarse-grained or all-atom simulations. First, at the coarse-grained level, I developed a lattice model for amyloid protein, which allows us to study the nucleus sizes of two experimentally well-characterized peptide fragments (Aβ)16-22 and (Aβ)37-42 of the Alzheimer's peptide (Aβ)1-42. After presenting a comprehensive OPEP force-field parameterization using an on-lattice protein model with Monte Carlo simulations and atomistic simulations, I determined the nucleus sizes of the two fragments. My results show that the nucleation number is 10 chains for (Aβ)16-22 and larger than 20 chains for (Aβ)37-42. This knowledge is important to help design more effective drugs against AD. Second, I investigated the structures of the dimer (Aβ)1-40 using extensive atomistic REMD simulations. This study provides insights into the equilibrium structure of the (Aβ)1-40 dimer in aqueous solution, opening a new avenue for a comprehensive understanding of the impact of pathogenic and protective mutations in early-stage Alzheimer’s disease on a molecular level

Les déchets plastiques s'accumulent depuis plusieurs décennies dans les océans où ils se fragmentent en particules appelés microplastiques lorsque leur taille est inférieure à 5 mm. Ces microplastiques sont retrouvés dans toutes les eaux du globe, dans les sédiments ainsi que dans de nombreux organismes marins. Le devenir physicochimique à long terme de ces particules et leur possible fragmentation en nanoplastiques sont complexes, encore peu documentés et nécessitent des études en laboratoire.Afin de comprendre les processus liés à la photodégradation et à la fragmentation des polymères dans l’environnement, mais également dans le but d’'appréhender l’évolution des fragments générés au cours de l’irradiation, un protocole de vieillissement accéléré en milieu abiotique a été mis en place sur des polymères modèles. Le suivi de l’oxydation et de la fragmentation des deux polymères étudiés,polyéthylène basse densité et polypropylène, a été mené à l’aide de techniques spectroscopiques (infrarouge, Raman), DSC, angles de contact, et microscopiques (lumière polarisée, MEB, AFM…).Ce travail a permis de mettre en évidence l’influence significative de l’environnement et de la morphologie initiale des polymères sur leurs cinétiques de vieillissement et leurs mécanismes de fissuration. Ainsi des distributions en nombres, tailles et formes de fragments très différentes ont été obtenues pour les deux polymères selon la présence d’eau. Enfin, après un long temps d’irradiation, des produits de dégradation ont pu être détectés mais la production significative de nanoplastiques n’a pas été démontrée. L'hypothèse d'une taille limite de fragmentation devrait être envisagée
Plastic wastes have been accumulating for several decades in the oceans where they break up into particles called microplastics when their size is less than 5 mm. These microplastics are found in all earth’s waters, in sediments and in many marine organisms. Their long-term physico-chemical fate and their possible fragmentation into nanoplastics are complex, still poorly documented and require laboratory studies.In order to understand the processes related to photodegradation and fragmentation of polymers, but also in order to understand the evolution of these fragments during irradiation, an accelerated aging protocol in abiotic conditions has been set up. The oxidation and fragmentation of two model polymers, low density polyethylene and polypropylene, were monitored using spectroscopic techniques (InfraRed, Raman), DSC, contact angles and microscopic technics (light microscopy, polarized light, SEM, AFM ...).This work has demonstrated a significant influence of the environment and the initial morphology of the polymers on their kinetics of aging and cracking mechanisms. This lead to significantly different distributions in numbers, sizes and shapes of the generated fragments. Moreover, after a long time of irradiaiton, other degradation products could be detected but the significant production of nanoplastics has not been demonstrated. The possibility of a size limit below which the fragmentation rate of plastics would strongly decrease should be considered

Les coordonnees atomiques du complexe entre le fab de l'anticorps monoclonal igg1() d1. 3 et son antigene specifique, le lysozyme de blanc d'uf de poule (hel), obtenues par cristallographie des rayons x, ont ete affinees a 2,5 a de resolution. Ce travail a permis d'ameliorer la precision de ce modele 'environ 0,4 a). Les principales caracteristiques de la liaison antigene-anticorps observees sont: forte complementarite des surfaces en contact (aire de contact de l'ordre de 650 a#2); l'epitope est forme de deux segments polypeptidiques distincts et eloignes dans la sequence du lysozyme; tous les cdr, mais pas le fr, font des contacts directs avec hel, du type van der waals ou liaison hydrogene; l'antigene ne change pas de conformation lors de la formation du complexe fab d1. 3-hel. Ce travail a mis en evidence la complexite des interactions antigene-anticorps, donnant les bases moleculaires de la specificite des fonctions immunitaires, et il a fourni une interpretation pour la facilite avec laquelle de nombreux antigenes peuvent echapper a la reponse humorale par mutation d'un petit nombre de residus a leur surface. Il a aussi permis pour la premiere fois une etude preliminaire de l'influence des molecules d'eau dans la liaison antigene-anticorps. L'existence d'espaces libres occupes par ces molecules d'eau, mais ou pourrait venir se placer la chaine laterale d'un residu d'un antigene heterologue, fournit une interpretation de l'existence de reactions croisees entre des anticorps monoclonaux et des antigenes heterologues. L'affinement du fab d1. 3-+hel a ete egalement une etape importante dans la determination des structures tridimensionnelles du fv d1. 3 bacterien et du fv d1. 3-hel. La comparaison des modeles des complexes fab d1. 3-hel et fv d1. 3-hel, et du fv d1. 3 montre: 1) les structures secondaires, tertiaires, quaternaires, et les conformations individuelles des residus dans le fv d1. 3 d'une part, et dans l'ensemble des domaines variables du fab d1. 3, d'autre part, sont tres proches; 2) fv d1. 3 et fab d1. 3 se lient au lysozyme de facon comparable, par un reseau d'interactions semblables, confirmant l'observation que ces deux fragments ont des specificites identiques, et justifiant l'emploi du fragment fv pour des reactions avec des antigenes specifiques, comme par exemple dans certaines applications therapeutiques; 3) le seul changement de conformation observe dans le fv d1. 3 lors de la liaison de celui-ci a l'antigene, est une faible difference dans la juxtaposition des domaines, de l'ordre des differences observees entre les structure de divers fab

In medicinal chemistry, two chemical fragments are considered bioisosteric if they bind to the same protein environment. Accordingly, looking at the same players from an opposite perspective, two protein environments can be considered chemoisosteric if they interact with the same chemical fragment. In this respect, this Thesis introduces the term chemoisosterism, which represents a new concept in drug discovery. Currently available crystal structures for protein-ligand complexes constitute a basis for the identification of chemoisosteric protein environments, of great utility for the construction of focused fragment chemical libraries. Under the premise that similar protein environments will probably bind to similar fragments, a novel approach to assess protein environment similarities is introduced and used to predict new chemoisosteric relationships. Examples of the potential applicability of chemoisosterism in fragment-based drug discovery are provided. The implications of chemoisosterism for drug polypharmacology are explored, leading to the speculation that the levels of polypharmacology observed in current drugs may just be a latent signature of the exploitation of chemoisosterism during evolution.
En química mèdica, dos fragments moleculars són considerats bioisostèrics si s’uneixen al mateix entorn de proteïna. Canviant la perspectiva sobre el mateix esdeveniment, dos entorns de proteïna poden ésser considerats quimioisostèrics si interaccionen amb el mateix fragment molecular. Aquesta Tesi introdueix el terme quimioisosterisme, un nou concepte en química farmacèutica. Les estructures actualment disponibles de complexos de proteïna i lligand constitueixen una font d’entorns de proteïna quimioisostèrics potencialment útils per a la construcció de llibreries de fragments moleculars dirigides a una proteïna diana en particular. Partint de la premissa que entorns de proteïna similars molt probablement interaccionaran amb fragments moleculars similars, aquesta Tesi presenta un nou mètode per a identificar entorns de proteïna similars, utilitzat per predir noves relacions quimioisostèriques. S’aporten també alguns exemples de potencials aplicacions del quimioisosterisme en la disciplina del descobriment de fàrmacs. Un anàlisis de les implicacions que té el quimioisosterisme en la polifarmacologia ens duu a la hipòtesis de que els nivells de polifarmacologia observats en la majoria de fàrmacs no són res més que una signatura de l’explotació del quimioisosterisme al llarg de l’evolució.

Concern regarding the health effects of indoor air quality has grown in recent years, due to the increased prevalence of many diseases, as well as the fact that many people now spend most of their time indoors. While numerous studies have reported on the dynamics of aerosols indoors, the dynamics of bioaerosols in indoor environments are still poorly understood and very few studies have focused on fungal spore dynamics in indoor environments. Consequently, this work investigated the dynamics of fungal spores in indoor air, including fungal spore release and deposition, as well as investigating the mechanisms involved in the fungal spore fragmentation process. In relation to the investigation of fungal spore dynamics, it was found that the deposition rates of the bioaerosols (fungal propagules) were in the same range as the deposition rates of nonbiological particles and that they were a function of their aerodynamic diameters. It was also found that fungal particle deposition rates increased with increasing ventilation rates. These results (which are reported for the first time) are important for developing an understanding of the dynamics of fungal spores in the air. In relation to the process of fungal spore fragmentation, important information was generated concerning the airborne dynamics of the spores, as well as the part/s of the fungi which undergo fragmentation. The results obtained from these investigations into the dynamics of fungal propagules in indoor air significantly advance knowledge about the fate of fungal propagules in indoor air, as well as their deposition in the respiratory tract. The need to develop an advanced, real-time method for monitoring bioaerosols has become increasingly important in recent years, particularly as a result of the increased threat from biological weapons and bioterrorism. However, to date, the Ultraviolet Aerodynamic Particle Sizer (UVAPS, Model 3312, TSI, St Paul, MN) is the only commercially available instrument capable of monitoring and measuring viable airborne micro-organisms in real-time. Therefore (for the first time), this work also investigated the ability of the UVAPS to measure and characterise fungal spores in indoor air. The UVAPS was found to be sufficiently sensitive for detecting and measuring fungal propagules. Based on fungal spore size distributions, together with fluorescent percentages and intensities, it was also found to be capable of discriminating between two fungal spore species, under controlled laboratory conditions. In the field, however, it would not be possible to use the UVAPS to differentiate between different fungal spore species because the different micro-organisms present in the air may not only vary in age, but may have also been subjected to different environmental conditions. In addition, while the real-time UVAPS was found to be a good tool for the investigation of fungal particles under controlled conditions, it was not found to be selective for bioaerosols only (as per design specifications). In conclusion, the UVAPS is not recommended for use in the direct measurement of airborne viable bioaerosols in the field, including fungal particles, and further investigations into the nature of the micro-organisms, the UVAPS itself and/or its use in conjunction with other conventional biosamplers, are necessary in order to obtain more realistic results. Overall, the results obtained from this work on airborne fungal particle dynamics will contribute towards improving the detection capabilities of the UVAPS, so that it is capable of selectively monitoring and measuring bioaerosols, for which it was originally designed. This work will assist in finding and/or improving other technologies capable of the real-time monitoring of bioaerosols. The knowledge obtained from this work will also be of benefit in various other bioaerosol applications, such as understanding the transport of bioaerosols indoors.

En Gaule méridionale la pratique des têtes coupées, décrite par la littérature antique, est attestée par les vestiges lapidaires et les restes humains mis au jour depuis plus d’un siècle. Des données inédites sur cette coutume ont été acquises suite à la découverte au Cailar (Gard, France), à partir de 2003, d’un ensemble de crânes humains fragmentés, d’armement manipulé et de plusieurs dizaines de monnaies, dispersés dans les phases de remblaiement d’une place publique entre la fin du IVe et la fin du IIIe s av. n. è. Cette dernière appartient à une agglomération fortifiée fondée dès le VIe s. av. n. è. Les fragments de crânes trouvés sur ce site constituent le sujet de cette étude. Ces os forment un corpus assez différent des restes généralement associés aux têtes coupées : ils sont nombreux, très fragmentés et largement mélangés et dispersés dans les couches. Pour comprendre les évènements qui ont conduit à la création d’un tel assemblage, il a été nécessaire de mettre en place des outils adaptés à leur étude. Après une remise en contexte archéologique régionale et locale, ce travail se propose de présenter les méthodes de l’archéothanatologie adaptées à l’étude de ce type d’ossements (quantification, modification osseuses). Dans un deuxième temps, sont détaillés l’outil d’enregistrement créé pour optimiser l’étude (base de données/géodatabase, SIG), et les méthodes d’analyses spatiales retenues pour étudier non seulement les traces et les fractures des pièces osseuses, mais aussi leur répartition sur le terrain. Les résultats obtenus sont multiples : données quantitatives et taphonomiques précises sur l’assemblage, proposition d’une méthode inédite pour l’analyse de la découpe et de la fragmentation sur des crânes humains fragmentés, analyse poussée de la répartition spatiale dans les trois dimensions. Tous ces éléments permettent de proposer des hypothèses solides quant à la chaîne opératoire qui concerne les têtes coupées : mode de récupération, traitement, rejet, mise en place du dépôt. Ce travail permet également de suggérer des pistes à approfondir ou à abandonner dans les méthodes usuelles d’analyses des grands ensembles fragmentés et dispersés
Once only known from Classical accounts, the practice of collecting and curating human heads by certain Iron Age groups in southern France has, for more than a century now, been evinced by materials including stone carvings and human remains. In particular, new evidence has been brought to light at the site of Le Cailar (Gard), a fortified site occupied from the end of the 6th century BCE. Specially, excavations carried out since 2003 have revealed an extensive deposit accumulated from the end of the 4th until the end of the 3rd century BCE, comprising fragmented human crania, purposefully deformed armaments, and many dozens of coins scattered across a public plaza, beneath a thick layer of rubble. The human cranial fragments discovered on this plaza are the subject of this study. These bones form a corpus quite different from the remains generally associated with severed heads: they are numerous, very fragmented, and largely mixed and dispersed in the levels. In order to understand the events that led to the creation of the assemblage, it has been necessary to adapt tools to this study. After putting the site into its local and regional context, this work proposes to present bioarchaeological methods adapted to the study of these types of bone (especially, quantification and modification of the bone). In a second time, the recording tool created to optimize the study is presented (database / geodatabase, GIS), then the spatial analysis methods used to study, not only the traces and the fractures of the parts bones, but also their distribution on the field. There are a lot of results: precise quantitative and taphonomic data about the assemblage, proposition of new method to study cut marks and fracturing, a hitherto unparalleled understanding of the process of how crania were distributed across the site (this last measured in three dimensions). All these elements permit to propose solid hypotheses regarding the process by which these heads were produced: the means of recuperation, treatment, disposal, and deposition. This work also suggests which avenues of research will or won't be useful to pursue in future projects of a similar nature

This thesis is focused on habitat preferences of carnivores in fragmented landscape. By using the scent station method, fine-scale utilization of forest-farm edges by mammalian predators in dependence on patch size was examined. Preferences for edge structures was detected and these structures were equally utilized by carnivores in large as well as in small forest fragments. This shows that edge effect was present in all fragments regardless the patch size.

The biological diversity of the planet is at great risk as a direct result of an ever-expanding human population and its associated activities. Landscape transformation to accommodate such activities leads to habitat loss and habitat fragmentation, often creating patches of relatively undisturbed habitat within a matrix of transformed areas that are often too small too support most species previously occupying the area and as a result loses its ecological integrity. A century ago the escarpment region of Mpumalanga consisted of large open plains covered with montane grassland dissected by montane forests and riparian vegetation alongside mountain streams. Today the grasslands and forests have almost disappeared from the area, the remaining patches mostly small fragments within a matrix of exotic tree plantations which have also dried up many of the rivers and streams in the area. The natural grassland areas persisting in the region are unique in habitat characteristics and floral species composition. It is also high in plant species richness, diversity and endemicity. The high degree of isolation experienced by these floral communities poses serious threats to both the floral and faunal species that currently exits within these isolated ‘islands’, many of which are endemic to the area and at great risk of extinction. These risks call for serious collaboration between the land owners (mostly forestry companies) and conservationists to assure the practical and necessary preservation and management of this unique and crucially valuable natural resource. This study aims to provide the first step into understanding the ecological principles associated with habitat fragmentation related specifically to the mountain grassland fragments within the afforestation matrix of the escarpment region of Mpumalanga, and to create a platform for the process of collaboration between land owners and conservation agencies to assess and manage these grassland patches. The aims of Chapter 2 included: 1 To determine if any marked human-induced disturbance to the plant communities in the grassland fragments has occurred. I do this in three ways: a) To compare the plant community composition of eighteen fragments with those of six control plots outside of the plantations where no marked disturbance to the grassland can be observed. b) To determine whether the plant assemblages in the fragments can be assigned to any of the natural and intact plant communities that Matthews (1993) described from a large-scale survey of undisturbed mountain areas. c) To determine the presence of any known intruder plant species within the fragments. 2 To assign conservation priorities to the remaining grassland fragments. The results obtained from the TWINSPAN analysis revealed six alliances of plant communities grouped hierarchically into four orders and two major classes of montane grassland. The DECORANA supported these results, indicating clear differences between communities 1.1 (Eriosema salignum – Loudetia simplex grassland of the wetter North region), 1.2 (Lobelia erinus – Panicum natalense grassland of the Transitional region), 2.1 (Parinari capensis – Eragrostis racemosa grassland) and 2.2 (Helichrysum rugulosum – Eragrostis racemosa grassland). Different plant communities revealed different combinations of geological characteristics, slope, aspect and elevation. The sample plots of isolated grassland fragments and those of large unfragmented areas compare well with each other - the species richness of experimental and control plots do not differ significantly. Also, the species composition of plots from the Wetter North, Transitional and Drier South regions show more variation than is evident between experimental and control plots. There was also good qualitative comparisons (quantitative comparisons were not possible as a results of sampling and analytic discrepancies) between the sample plots used in this study and the plant communities described by Graham Deall and Wayne Matthews. Some evidence of exotic invader plants was found within the sample plots, notably Pteridium aquilinum. Fortunately such species were localized in their distribution and restricted to community 1.1.2.2. The aims of Chapter 3 included the following: 1. To compare the faunal biodiversity in grassland fragments within afforested areas to that of control plots in large, relatively undisturbed grassland areas. 2. To determine to which degree the Coleoptera, Orthoptera, Lepidoptera and bird communities reflect recognized plant communities, and are restricted to specific plant communities. 3. To compare the habitat specificity (degree of stenotopy) of the different taxonomic groups and trophic levels of animals. 4. To make recommendations for the conservation of the Afromontane grassland fauna in the remaining grassland fragments in afforested areas. The sampling of 15602 beetles, grasshoppers and crickets were collected, with an average of 3900 per sampling period, revealed unique combinations of animal species linked to the different plant communities mentioned above. Significant differences were evident from the one-way analyses of similarity (ANOSIM) used to compare the faunal community structures of sample plots of the Wetter North, Transitional and Dryer South regions. The faunal community structures of the experimental and control plots of the Wetter North and Transitional regions did not differ significantly. The indexes of habitat specificity (fractions of species constricted to certain plant communities) indicated that the plants and butterflies were more habitat-specific than the Coleoptera, Orthoptera and Birds. The carnivorous insects showed a surprisingly high level of habitat specificity compared to the relatively low level of the phytophagous insects. This surprising trend was also evident in various insect families – Acrididae, Scarabaeidae and Nymphalidae had relatively high levels of habitat specificity compared to that of the Curculionidae and the Chrysomelidae. Chapter 4 has the following aims: 1. To quantify the effect of several environmental characteristics (slope, rainfall, geology, etc) on the faunal community structure of the grassland fragments. 2. To quantify the effects of degree of isolation on species richness, species diversity and assemblage structure of plants, insects and birds in grassland remnants. 3. To test for the effects of edges on the extant insect biodiversity in the grassland fragments inside plantations. 4. To quantify the effects of fragment size on species richness, species diversity and assemblage structure of plants, insects and birds. 5. To rank the grassland fragments in an order of conservation importance using factors such as biodiversity and uniqueness of the floral community. No clear relationship between fragment size and area sampled and species richness and/or – diversity was evident from the results. Indeed, the smallest area sampled had the fourth highest species richness and the largest area sampled had the fourth lowest species richness and species diversity. Regressions results did not show any significant effects of the geographical area sampled on the biodiversity estimates of the fragments. Therefore I assume that the estimates arrived at for the area sampled within each fragment is representative of that of the complete fragment. The Detrended correspondence analysis (DCA - using square root transformed abundance data) used, indicated the need to perform a gradient analysis using a redundancy analysis (RDA). The permutation test resulting from this analysis revealed a non-significant value for the first canonical axis, but a significant value for the first four canonical axes together. The ten species contributing the most to above-mentioned result include two Scarabs (Scarabaeidae – Aphodius sp 1 and Melolonthinae sp 2), two weevils (Curculionidae – Eudraces sp 1 and Curculionidae sp 42), one leaf beetle (Chrysomelidae – Asbecesta near capensis), one darkling beetle (Tenebrionidae – Lagria sp 1), one longhorn beetle (Cerambycidae – Anubis scalaris), one jewel beetle (Buprestidae – Buprestidae sp 1), one ladybird (Coccinellidae – Coccinellidae sp 4) and one Dor beetle (Bolboceratidae – Mimobolbus maculicollis). Of these ten beetles only three are not restricted to the Drier South Region (Anubis scalaris – Wetter North and Drier South, Lagria sp 1 – throughout and Eudraces sp 1 – throughout). The associated stepwise multivariate regression showed distance to the nearest grassland to be the only environmental characteristic to significantly influence the faunal community structure of the fragments. Slope was the environmental characteristic with the smallest effect. In contrast with the results from redundancy analysis, the analysis of similarity (ANOSIM) and t-tests did not reveal significant differences in the faunal community structure of fragments closer to – and further than one kilometre from the nearest grassland neighbour. This trend was the most evident for fragments of the Transitional region and the least obvious for the fragments of the study area as a whole. The SIMPER analysis showed that of the ten species contributing most to the dissimilarity between insect communities of fragments closer/further than one kilometre from the nearest grassland neighbour, eight were also in the group of ten species characterizing the faunal communities of either/both groups (contributing towards similarity). The insect communities found at 10, 20 and 50 metres from the edge of the grassland fragments did not differ significantly, nor did an ANOSIM performed separately for each of the three major plant communities reveal any significant edge-related differences. The ANOVA results for the individual species revealed only one (of 57 - in the Transitional region) having a distribution that differs significantly with respect to distance from the habitat edge:Eremnus sp. 2 was only found at 10 metres from the edge of the fragment, close to the plantations. Of all the groups, only bird diversity, bird richness and general faunal diversity showed significant relationships with fragment size. There was a non-significant trend for insects to biodiversity to be reduced in very small fragments. Most of the botanical data exhibited no significant relationship with fragment size. The species composition of control sites were not found to be significantly different from that of experimental fragments for all the faunal groups pooled together or for the fragments of the Transitional Region and the Wetter North Region. Using the four separate scores for birds, butterflies, beetles and grasshoppers, each fragment was assigned a total conservation score. The twenty-four fragments were then ranked in order of conservation importance. Fragments of the Wetter North had an average score of 65.3, fragments of The Transitional Region 66.6 and fragments of the Drier South 52.3. The results relating to this study has lead to the following conclusions: o It is concluded that afforestation and habitat fragmentation have not significantly impacted on the flora of the montane grassland of the study area since many of the grassland fragments surrounded by plantations are still easily identifiable as natural communities, described by Matthews and Deall in broader-scale surveys in the past. Also, no obvious invader – or disturbed plant communities are discernible even though some of them have been isolated for as long as 40 years. o The plant communities of conservation importance described by Matthews coincide with the important communities recognized in this study. Rare and endangered plant species, as well as species endemic to the region, are more often than not found on the scarce Black Reef quartzite of the region, which is more evident to the Northern part of the study area. Communities 1.1 and 1.2.2 are therefore of particular conservation importance, not only as a result of their scare geological base, but also because of the high risk associated with the few grassland examples left of these communities. o The high levels of habitat specificity of many of the taxonomic and trophic faunal groups indicate that many of the invertebrate taxa are probably endemic to the region, and that the plant endemicity encountered in the Afromontane grasslands is reflected by a similar degree of animal endemicity. o The similarity in faunal assemblages and diversity between isolated fragments and large areas of grassland emphasizes the conservation importance of the fragments, even when smaller than 5 Ha in extent. o Appropriate management of the grassland fragments within the plantations is therefore important for the conservation of the plant and animal taxa encountered there. Experimental management involving grazing, mechanical cutting, grazing and burning is needed to decide on an efficient management regime, so that the grassland biodiversity can be conserved in a planned way. Such work will also allow empirical testing the efficiency of the indicator species suggested above. o Isolated grassland fragments in this study represent largely unaffected natural plant and insect communities, differing little from large unfragmented grasslands in the study area. o Fragments found within afforested areas therefore have a high conservation importance, since they represent ‘natural’ grassland areas and are often the only representative of a particular plant community left in the area. o No significant edge effects on the faunal communities 10, 20 and 50 metres from the fragments’ edges exist as a result of afforestation in the area. o Birds (and probably other vertebrates in these grasslands) are affected by fragment size, while invertebrates are much less affected and plants do not show any measurable effect of fragment size. o Fragments in the wetter northern part of the study area, characterized by high levels of plant endemicity, have a higher conservation importance as judged by faunal biodiversity.
Dissertation (MSc (Zoology and Entomology))--University of Pretoria, 2005.
Zoology and Entomology
unrestricted

Thesis (M.S.)--University of Wisconsin--Madison, 1988.
Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 65-74).

Several studies have revealed that calcium-binding EF-hands associate in various arrangements resulting in unique calcium affinities and target interactions, yet similar structures. This study examines the effect of EF-hand association on the structure, calcium-binding, and target-enzyme activation of a novel fragment of calmodulin formed by the association of EF-hands 2 and 3 (CaM2/3). The results from CaM2/3 are compared to those for the N- and C-domain fragments of calmodulin (CaM1/2 and CaM3/4, respectively). Based on NMR spectroscopic analyses, CaM2/3 is unstructured in the absence of calcium, but upon binding calcium adopts a structure that is similar to both the N- and C-domains of calmodulin. CaM2/3 is composed of four α-helices and a short anti-parallel β-sheet between EF-hands 2 and 3. These EF-hands associate in a manner similar to those in the N- and C-domains of calmodulin. This association is facilitated by the conformationally-flexible hinge region between EF-hands. CaM2/3 exhibits stepwise calcium-binding with a Kd₁ = 30 ± 5 μM to EF-hand 3, and a Kd₂ = 1800 ± 400 μM to EF-hand 2. In native calmodulin, aromatic residues opposite the loop between EF-hands are an important part of its hydrophobic core. It is suggested here that these residues act as an "aromatic zipper" to hold the first and fourth helices of each domain together. In CaM2/3 this "aromatic zipper" is in a structurally redundant position adjacent to the loop between EF-hands, and this may result in weak calcium-binding affinity at EF-hand 2. Our studies reveal that CaM2/3 binds a peptide corresponding to the calmodulin-binding sequence on skeletal muscle myosin light chain kinase (M13). This affinity is lower than that measured for both CaM1/2 and CaM3/4. MI3 appears to bind to a hydrophobic binding pocket on CaM2/3 in two equally populated conformations. CaM1/2 and CaM3/4 have little or no ability to stimulate the activity of calcineurin. However, CaM2/3 has a limited ability to stimulate calcineurin, but also appears to inhibit the stimulatory effect of calmodulin on calcineurin. This investigation leads to a greater understanding of the effect of EF-hand pairing, on structure, calcium-binding affinity, and target interactions in calmodulin.
Pharmaceutical Sciences, Faculty of
Graduate

Recent improvements in structural genomics efforts have greatly increased the number of hypothetical proteins in the Protein Data Bank. Several computational methodologies have been developed to determine the function of these proteins but none of these methods have been able to account successfully for the diversity in the sequence and structural conformations observed in proteins that have the same function. An additional complication is the flexibility in both the protein active site and the ligand. In this dissertation, novel approaches to deal with both the ligand flexibility and the diversity in stereochemistry have been proposed. The active site analysis problem is formalized as a classification problem in which, for a given test protein, the goal is to predict the class of ligand most likely to bind the active site based on its stereochemical nature and thereby define its function. Traditional methods that have adapted a similar methodology have struggled to account for the flexibility observed in large ligands. Therefore, I propose a novel fragment-based approach to dealing with larger ligands. The advantage of the fragment-based methodology is that considering the protein-ligand interactions in a piecewise manner does not affect the active site patterns, and it also provides for a way to account for the problems associated with flexible ligands. I also propose two feature-based methodologies to account for the diversity observed in sequences and structural conformations among proteins with the same function. The feature-based methodologies provide detailed descriptions of the active site stereochemistry and are capable of identifying stereochemical patterns within the active site despite the diversity. Finally, I propose a Markov Random Field approach to combine the individual ligand fragment classifications (based on the stereochemical descriptors) into a single multi-fragment ligand class. This probabilistic framework combines the information provided by stereochemical features with the information regarding geometric constraints between ligand fragments to make a final ligand class prediction. The feature-based fragment identification methodology had an accuracy of 84% across a diverse set of ligand fragments and the mrf analysis was able to succesfully combine the various ligand fragments (identified by feature-based analysis) into one final ligand based on statistical models of ligand fragment distances. This novel approach to protein active site analysis was additionally tested on 3 proteins with very low sequence and structural similarity to other proteins in the PDB (a challenge for traditional methods) and in each of these cases, this approach successfully identified the cognate ligand. This approach addresses the two main issues that affect the accuracy of current automated methodologies in protein function assignment.