Genealogy reconstruction is widely used in biology when relationships among entities are studied. Phylogenies, or evolutionary trees, show the differences between species. They are of profound importance because they help to obtain better understandings of evolutionary processes. Pedigrees, or family trees, on the other hand visualize the relatedness between individuals in a population. The reconstruction of pedigrees and the inference of parentage in general is now a cornerstone in molecular ecology. Applications include the direct infer- ence of gene flow, estimation of the effective population size and parameters describing the population’s mating behaviour such as rates of inbreeding. In the first part of this thesis, we construct genealogies of various types of cancer. Histopatho- logical classification of human tumors relies in part on the degree of differentiation of the tumor sample. To date, there is no objective systematic method to categorize tumor subtypes by maturation. We introduce a novel algorithm to rank tumor subtypes according to the dis- similarity of their gene expression from that of stem cells and fully differentiated tissue, and thereby construct a phylogenetic tree of cancer. We validate our methodology with expression data of leukemia and liposarcoma subtypes and then apply it to a broader group of sarcomas and of breast cancer subtypes. This ranking of tumor subtypes resulting from the application of our methodology allows the identification of genes correlated with differentiation and may help to identify novel therapeutic targets. Our algorithm represents the first phylogeny-based tool to analyze the differentiation status of human tumors. In contrast to asexually reproducing cancer cell populations, pedigrees of sexually reproduc- ing populations cannot be represented by phylogenetic trees. Pedigrees are directed acyclic graphs (DAGs) and therefore resemble more phylogenetic networks where reticulate events are indicated by vertices with two incoming arcs. We present a software package for pedigree reconstruction in natural populations using co-dominant genomic markers such as microsatel- lites and single nucleotide polymorphism (SNPs) in the second part of the thesis. If available, the algorithm makes use of prior information such as known relationships (sub-pedigrees) or the age and sex of individuals. Statistical confidence is estimated by Markov chain Monte Carlo (MCMC) sampling. The accuracy of the algorithm is demonstrated for simulated data as well as an empirical data set with known pedigree. The parentage inference is robust even in the presence of genotyping errors. We further demonstrate the accuracy of the algorithm on simulated clonal populations. We show that the joint estimation of parameters of inter- est such as the rate of self-fertilization or clonality is possible with high accuracy even with marker panels of moderate power. Classical methods can only assign a very limited number of statistically significant parentages in this case and would therefore fail. The method is implemented in a fast and easy to use open source software that scales to large datasets with many thousand individuals.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2002. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 112-132).

A side effect of generating an immune system for defense against invading pathogens is the potential to develop destructive cells that recognize self-tissues. Typically, through the "education" of developing immune cells, the organism inactivates potentially self-destructive cells, resulting in what is called self-tolerance. I proposed to explore the molecular mechanisms responsible for the induction and maintenance of tolerance. Our lab has developed a model of induced immune tolerance to skin and islet allografts utilizing a donor-specific transfusion of spleen cells and a brief course of anti-CD40L antibody. Because the difficulty in isolation of tolerant T cells from this system is prohibitive to performing large screens on these cells directly, I have chosen to study an in vitro CD4+Th1 cell line, A.E7, which can be made anergic via stimulation through the T cell receptor in the absence of costimulation. I hypothesized that anergized T cells upregulate genes that are responsible for the induction and maintenance of anergy and therefore exhibit a unique RNA expression profile. I have screened anergic cells using Affymetrix GeneChips and identified a small number of genes that are differentially expressed long-term in the anergic population compared to mock-stimulated and productively activated controls. The results have been confirmed by quantitative RT-PCR for each of the candidates. One of the most promising, the zinc-finger transcription factor Egr-2, was verified to be expressed long-term by western blotting, demonstrating perfect correlation between Egr-2 protein expression and the anergic phenotype. Silencing Egr-2 gene expression by siRNA in A.E7 T cells prior to anergy induction rescues the cells from the inability to phosphorylate ERK-1 and ERK-2 and also results in increased proliferation in response to antigen rechallenge. In this study I report that Egr-2 is specifically expressed long-term in anergic cells, protein expression correlates inversely with responsiveness to antigen rechallenge, and that Egr-2 is required for the full induction of anergy in T cell clones.

For the first time in human history, more people worldwide suffer from obesity than are undernourished. Numerous health complications are associated with obesity including cardiovascular disease, Type 2 Diabetes, cancers of reproductive tissues, stroke, depression, anxiety disorders, and Alzheimer’s disease. A deeper understanding of the anti-adipogenic effects and mechanism of action of sesquiterpene lactones may have pharmacological import in the continuing search for therapeutic modalities to ameliorate the effects of this global obesity epidemic. Dehydroleucodine (DhL), 11,13-dihydro-dehydroleucodine (DH-DhL), and dehydroparashin-B (DhP), sesquiterpene lactones extracted from or derived from compounds extracted from Artemisia douglasiana, were investigated for their anti-adipogenic effects on 3T1-L1 preadipocytes. Dehydroleucodine inhibited the expression of C/EBPa and PPARg, and also strongly blocked the expression of C/EBPβ, an early stage biomarker of early adipogenesis, in a concentration-dependent manner. Dehydroleucodine arrested the cell cycle at the G0/G1 phase, increased p27 and decreased both cyclins A and D and their partners (e.g., CDK2 and CDK4). Furthermore, DhL downregulated expression of histone demethylase JMJD2 as well as repressed the expression of histone methyltransferase MLL4, which in turn diminished the expression of C/EBPb and PPARg, respectively. 11,13-dihydro-dehydroleucodine blocked the accumulation of lipid droplets and inhibited the expression of PPARγ and C/EBPβ. Collectively, the results indicate that the inhibition of early stage preadipocyte differentiation by DH-DhL may be associated with cell cycle arrest at the G0/G1 phase. Dehydroparashin-B significantly decreased the accumulation of lipid content and downregulated the expression of CEBPβ, PPARγ and CEBPα as well as FAS. Interestingly, the addition of DhP inhibited the number as well as the size of the lipid droplets during the differentiation of 3T3-L1 preadipocytes. Taken together, this data suggests that DhP has an important inhibitory effect on cellular pathways regulating adipocyte differentiation.

Orientador: Rinaldo Cesar de Paula / Banca: Dilermando Perecin / Banca: Miguel Luiz Menezes Freitas / Resumo: Com o objetivo de avaliar a eficiência da seleção precoce e tamanho ótimo de parcelas em Eucalyptus spp., foram usados dados de dois testes clonais avaliados quanto ao crescimento em altura (ALT), diâmetro à altura do peito (DAP) e volume individual de madeira (VOL) aos 25, 50 e 72 meses de idade. O delineamento experimental nos dois testes clonais foi o de blocos casualizados, com 30 tratamentos (clones), seis repetições, sendo um deles com seis plantas por parcelas (teste clonal 1) e o outro com 10 plantas (teste clonal 2). Foram obtidas as estimativas de coeficiente de determinação genotípico e de correlações genotípicas entre os caracteres nas idades juvenis e na idade de rotação. Para verificar a viabilidade da aplicação da seleção precoce foi simulada a seleção de 30% dos clones nas idades juvenis e na idade de rotação, para cada um dos caracteres e idades avaliadas, obtendo-se as estimativas de ganhos com a seleção direta e indireta. Para verificar o tamanho de parcelas, foi utilizado o coeficiente de repetibilidade calculado através dos métodos: Análise de Variância, dos Componentes Principais e o de Análise Estrutural, os dois últimos calculados através da matriz de correlação. Houve diferenças significativas entre os clones avaliados nos dois experimentos para todos os caracteres e idades. Com os resultados obtidos, recomenda-se a seleção precoce sobre DAP praticada em torno de dois anos de idade. O uso de seis repetições constituídas de pelo menos quatro plantas por parcela e a avaliação em apenas uma idade proporciona coeficiente de determinação superior a 80%. / Abstract: Aiming evaluate the Eucalyptus spp. early selection efficiency and optimum size of plots, were used data of height, diameter at breast height and individual wood volume evaluated in two clonal tests at 25, 50, and 72 months old. The experimental design in both clonal tests was randomized blocks, using 30 treatments (clones), 6 replications, at 3.0 x 3.0m spacing and linear plots, and one of them with 6 plants per plot (clonal test 1) and the other with 10 plants (clonal test 2). Variance analysis to each trait and age was done to each experiment. The estimates of genotypic determination coefficient and the correlation between the early and mature age traits were obtained. To check the viability of early selection, was simulated the selection in young and mature age, adopting 30% of selection to each one of the traits and ages evaluated, estimating the gains by the direct and indirect selection. To check the size of plots, we used the repeatability coefficient calculated by the methods: Analysis of Variance, and the Principal Components of Structural Analysis, the last two calculated through the matrix of correlation. There were significant differences among clones evaluated in the two experiments to all traits and ages. From the results obtained is suggested the practice of early selection from 2 years old age to eucalyptus clonal tests. The use of six replications consisting of at least four plants per plot and evaluation at one age provides a coefficient of determination more than 80%. / Mestre

Orientador: Akebo Yamakami / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Eletrica e de Computação / Made available in DSpace on 2018-08-11T03:11:24Z (GMT). No. of bitstreams: 1 Rampazzo_PriscilaCristinaBerbert_M.pdf: 1295026 bytes, checksum: ad0738bc161445ec5b9f0db0db565f09 (MD5) Previous issue date: 2008 / Resumo: O objetivo desta dissertação é explorar a utilização de um Sistema Imunológico Artificial, baseado no princípio de Seleção Clonal, na resolução de problemas de Otimização Multiobjetivo. Os Sistemas Imunológicos Artificiais apresentam, em sua estrutura elementar, as principais características requeridas para a resolução de problemas de Otimização Multiobjetivo: exploração, explotação, paralelismo, elitismo, memória, diversidade, mutação e clonagem proporcionais à afinidade e população dinâmica. A abordagem proposta utiliza o conceito de Pareto dominância e factibilidade para identificar os anticorpos (soluções) que devem ser clonados. Nos experimentos, foram consideradas algumas situações importantes que podem aparecer nos problemas reais: presença de restrições (lineares e não-lineares) e formato da Fronteira de Pareto (convexa, côncava, contínua, descontínua, discreta, não-uniforme). Na maioria dos problemas, o algoritmo obteve resultados bons e competitivos quando comparados com as propostas da literatura. Palavras-chave: Otimização Multiobjetivo, Algoritmos Bio-inspirados, Sistemas Imunológicos Artificiais, Seleção Clonal / Abstract: The aim of this work is to explore an Artificial Immune System, based on the Clonal Selection principle, in the solution of Multiobjective Optimization problems. Artificial Immune Systems have, in their elementary structure, the main characteristics required to solve Multiobjective Optimization problems: exploration, exploitation, paralelism, elitism, memory, diversity, mutation and proliferation proportional to the affinity, and dynamic repertorie. The proposed algorithm uses the Pareto dominance concept and feasibility to identify the antibodies (solutions) that must to be cloned. In the experiments, some important situations that occurs in real problems were considered: the presence of constraints (linear and non-linear) and Pareto Front format (convex, concave, continuous, discontinuous, discrete, non-uniforme). In the major part of the problems, the algorithm obtains good and competitive results when compared with approaches from the literature. Keywords: Multiobjective Optimization, Bio-inspired Algorithms, Artificial Immune Systems, Clonal Selection / Mestrado / Telecomunicações e Telemática / Mestre em Engenharia Elétrica

A produtividade florestal é definida pelo ambiente de produção em que o povoamento está inserido, pela implantação de materiais genéticos superiores e pelo seu correto manejo silvicultural. A correta prescrição de recomendações silviculturais e a execução das operações de maneira padronizada tem por objetivo eliminar ou minimizar as restrições ao crescimento para cada árvore. Apesar das melhorias silviculturais que ocorreram nas últimas décadas, sabe-se que ainda é possível ganhos em produtividade através do monitoramento, detecção e correção precoce de desvios na qualidade silvicultural. Assim, o objetivo deste trabalho foi investigar um índice para o monitoramento da qualidade silvicultural utilizando uma rede de inventário florestal. Para isto, foram realizadas três etapas de trabalho: i) Definição de índices de uniformidade das variáveis dendrométricas; ii) Padronização dos chamados intervalos ótimos de uniformidade (IOU) e iii) Validação da metodologia via sua aplicação numa rede de inventário florestal para um único clone comercial. Na primeira etapa utilizaram-se três ensaios da rede BEPP (Brasil Eucalyptus Produtividade Potencial) com diferentes níveis de produtividade para estabelecer os índices adequados em caracterizar a uniformidade silvicultural. Na segunda etapa utilizaram-se cinco testes clonais de Eucalyptus no Estado de São Paulo para validar o conceito de Intervalo Ótimo de Uniformidade. Na última etapa, os índices e conceito do IOU foram aplicados em escala comercial em uma rede de parcelas de inventário florestal instalada em 12.000 hectares de plantios clonais de Eucalyptus no Nordeste do Estado de São Paulo, com cerca de 2 anos de idade, e plantados no período de 1995 a 2009. Na fase de definição, o índice que representa a porcentagem do volume total existente em 50% das menores árvores plantadas (o que inclui as falhas de plantio) (PV50) se mostrou como o que melhor se adequou à proposta do trabalho, pois possui limites finitos (50% a 0%), contempla as falhas de plantio como parte do índice de uniformidade do povoamento e, indiretamente, representa a distribuição de classes de crescimento. As uniformidades aos 5, 9, 12 e 24 meses foram fortemente correlacionadas à uniformidade aos 6 anos (r² > 0,74) mostrando a possibilidade de monitoramento precoce para detecção de desvios de qualidade na silvicultura. Além disso, o índice PV50 inicial foi altamente correlacionado com a produtividade final (p < 0,001). Na fase de padronização, não se detectou diferença estatística (Tukey, 5%) do PV50 entre os testes clonais a despeito de suas distintas produtividades, evidenciando que o índice de uniformidade pode ser generalizado, independentemente da produtividade do sítio. O IOU do PV50 foi de 34 a 50 %, ou seja, parcelas amostrais que possuírem o PV50 dentro deste intervalo podem ser consideradas satisfatoriamente uniformes. Na etapa de validação, quando o conceito foi aplicado em escala comercial observou-se uma forte evolução temporal do PV50. Nos plantios realizados em 1995 a média do índice foi de 29% e elevou-se para 42% em 2009. O percentual de parcelas dentro do intervalo ótimo de uniformidade tendeu claramente a se elevar ao longo deste período, devendo estar relacionada com as melhorias nas principais operações silviculturais e seu monitoramento através do controle de qualidade. / Forest productivity is defined by the environment the population is inserted, by the implementation of genetically superior material and by the correct silvicultural management applied at this material. The correct prescription of technical recommendations and the execution of those operations according to acceptable quality standard are intended to eliminate or minimized the growth constrains. Despite the silvicultural improvements that have occurred in recent decades, it is known that it is still possible to obtain gains in productivity through monitoring, early detection and correction of deviations in the silviculture. The objective of this study was to investigate an index for monitoring the silvicultural quality using forest inventory networks. For this, there were three stages of work: i) Definition of uniformity indexes of dendrometric variables; ii) Standardization of so-called optimal range of uniformity (ORU) and validation of the methodology through its application in a network of forest inventory for a single commercial clone. In the first stage we used three tests of the network BEPP (Brazil Eucalyptus Potential Productivity) with different levels of productivity to establish the appropriate indexes to characterize the silvicultural uniformity. In the second stage we used five clonal test of Eucalyptus in Sao Paulo state to validate the concept of Optimum Range of Uniformity. In the last step, the indexes and the concept of IOU were applied on a commercial scale in a network of forest inventory plots installed in 12.000 hectares of Eucalyptus clonal plantations in northeastern of São Paulo state, with about 2 years old, and planted within 1995 to 2009. In the definition phase, the index that represents the percentage of the total volume of 50% of smaller trees planted (which includes the planting holes) (PV50) was shown as the best adapted to the purpose of this study because it has finite limits (50% to 0%), includes the holes of planting and indirectly represents the distribution of classes of growth. The uniformity at 5, 9, 12 and 24 months were strongly correlated to uniformity to 6 years (r² > 0.74) showing the possibility of early monitoring for detection of quality deviations in forestry. Furthermore, the initial rate PV50 was highly correlated with the final yield (p <0.001). At the stage of standardization, there was no statistical difference (Tukey, 5%) of PV50 among clonal tests despite their different yields, showing that the uniformity index can be generalized, irrespective of the productivity of the site. The ORU of PV50 was from 37 to 50%, i.e., sample plots which have the PV50 within this range can be considered satisfactory \"uniform\". In the validation phase, when the concept was applied on a commercial scale there was a strong temporal evolution of PV50. In the plantations made in 1995 the average of PV50 was 29% and increased to 42% in 2009. The percentage of plots within the optimal range of uniformity clearly tended to rise over this period and could be related to major improvements in forestry operations and their monitoring through quality control.

Les leucémies aigues myéloïdes (LAM) dérivent de progéniteurs hématopoïétiques dans lesquels se sont accumulés des événements génétiques conduisant à leur transformation. En établissant la hiérarchie clonale de multiples lésions récurrentes, nous démontrons que les événements impliquant les régulateurs de l'épigénétique sont les premiers événements dans le clone. A l'inverse, les mutations régulant la prolifération se produisent tardivement. Les événements précoces sont quasi constamment détectables en rémission complète. Les clones qui persistent servent de réservoir pour les rechutes, avec une variation qui augmente avec la durée de la rémission. Après xénogreffe, les échantillons de patients porteurs de lésions épigénétiques sont capables de repopulation hématopoïétique, ce qui est la signature fonctionnelle des événements pré-leucémiques. Cette hiérarchie est observée chez la majorité des patients. Cependant dans 1/3 des cas, il n'est pas retrouvé d'événement épigénétique, et on observe notamment des prédispositions germinales aux LAM, ou des mutations acquises de TP53. Chez 3 patients avec des LAM de novo, les mutations de TP53 sont associées à des mutations de DNMT3A dans un même clone. Cela suggère que les mutations de DNMT3A et de TP53 sont complémentaires pour obtenir une dominance clonale pré leucémique. En conclusion, dans la majorité des LAM, des hiérarchie clonales récurrentes, initiées par des lésions pré-leucémiques variées vont promouvoir l'expansion ou la variation clonale, jusqu'à l'émergence de la maladie. Ces données sont essentielles pour le développement de nouvelles stratégies personnalisées de suivi de la maladie résiduelle. / Acute myeloid leukaemia (AML) emerge from haematopoietic stem/progenitor cells that acquiregenomic or chromosomal aberrations, some being considered as pre-leukemic lesions. Here, byestablishing the chronological hierarchy of multiple driver lesions in AML, we show that most eventsaffecting epigenetic regulators - DNMT3A, TET2, ASXL1 mutations, as well as MLL and chromosome20q rearrangements - are the first lesions in the clone. In contrast to late mutations involving signallingpathways such as FLT3 or RAS, these early lesions are frequently detectable in complete remissionsample. By studying late relapses, we show that persistent clones behave as long-term relapsereservoirs, and variegate increasingly with delay to relapse. Cells from patients with early epigeneticdefects can repopulate bone marrow of xenotransplanted NOD/SCID/IL-2Rgc-null (NSG) mice withleukemic or non leukemic engraftment, a functional signature of pre-leukemic events. This genetichierarchy is observed in most patients but in one third of them lesions in epigenetic regulators are notthe first events. Some of these patients have genetic predisposition, or somatic mutations in TP53. Inthree de novo AMLs, but not in three secondary or therapy-related AMLs, these TP53 mutations wereaccompanied by DNMT3A mutations in a unique clone. This suggests that DNMT3A mutations maycomplement TP53 mutations to generate a dominant pre-leukemic clone. We conclude that in mostAMLs recurrent genetic hierarchies, initiated by distinct pre-leukemic lesions, promote progressiveclonal expansion or variegation, leading to the development of a full blown disease. These data areessential for the development of new personalised strategies for MRD evaluation.

Throughout society’s continual war against cancer, we have attempted pharmacological intervention only to find that tumors develop modes of resistance. It is well known that genetics play an integral role in cancer. Technological advances have greatly improved our ability to study cancer biochemistry beyond the genome by measuring changes in the expression and activity of RNA, proteins, and lipids in experimental models and human patients. As our techniques and technology to perform cancer research progresses, it is becoming more evident that cancer cells develop stress tolerance mechanisms at multiple levels within the central dogma, including altering mRNA expression, enzyme concentrations, and functional activity of cellular proteins and lipids. In the first chapter, I review previous discoveries demonstrating the importance of metabolic reprogramming in cancer cells and how shifts in metabolic pathways contribute to cancer progression and therapeutic challenges. I discuss how mass spectrometry is a multifunctional research tool that can be used to identify global shifts in gene expression, identify oncogenic roles of specific metabolites and corresponding metabolic pathways, conduct enzyme activity assays, and understand the effects of drugs on cell signaling and metabolic flux through specific pathways. While metabolic reprogramming is a complex and multifaceted concept, the following chapters focus on two specific stress tolerance pathways of lipid and protein metabolism we have shown to significantly promote cancer cell evolution, proliferation, and drug resistance in models of human pancreatic and colon cancer. I describe novel mass spectrometry-based lipidomics and proteomics methods we developed to measure and determine the biological impact of these pathways in each model. I discuss the contributions we have made toward increasing general knowledge of metabolic reprogramming networks in cancer and how they may be targeted in more specific and effective manners to sensitize cancers to therapeutic drugs. Specifically, the second chapter entails our study of a pro-survival lipid metabolic pathway driven by the sphingolipid modifying enzyme sphingosine kinase in a panel of differentially reprogrammed pancreatic cancer subclones. The third chapter describes our novel kinetic proteomics approach to identify how the cellular degradation system autophagy is used to selectively remodel the proteome of colon tumor cells in a xenograft mouse model of colon cancer. Lastly, I discuss how these and other projects completed during my graduate work lay a foundation for ongoing research to further our fundamental understanding of cancer metabolism and treatment development.