Orientador: Louis Bernard Klaczko / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T03:22:48Z (GMT). No. of bitstreams: 1 Rocha_FelipeBastos_D.pdf: 4246569 bytes, checksum: 8297db94661890abb9ddc71ef0280318 (MD5) Previous issue date: 2013 / Resumo: Apesar de descrever a dinâmica populacional da variação genética, a teoria da genética de populações não descreve como a interação entre genótipos e os ambientes onde estes se desenvolvem gera distribuições fenotípicas. As normas de reação representam uma possível estratégia para tal descrição; elas representam a resposta fenotípica de cada genótipo para uma variável ambiental e, com isso, expõem a variação causada pela plasticidade fenotípica e capturam o fenômeno da interação genótipo-ambiente. Neste trabalho, Drosophila mediopunctata foi utilizada como organismo modelo. Ela é uma espécie do grupo tripunctata, do gênero Drosophila, polimórfica para inversões do segundo cromossomo e para um padrão de pigmentação conspícuo nos tergitos abdominais, formado por fenótipos que podem apresentar de zero a três pintas escuras. Na primeira parte deste trabalho, é apresentado um teste da independência genética entre a plasticidade fenotípica e o valor médio do polimorfismo de pigmentação. Foram utilizadas oito estirpes homocariotípicas para inversões do cromossomo II com diferentes valores fenotípicos médios para analisar a variação de normas de reação do número de pintas à temperatura. As normas de reação desse caráter foram parábolas cujas curvaturas estão correlacionadas ao valor médio, indicando que a variação dessas duas características pode ser determinada por pleiotropia. O mesmo padrão foi observado em heterozigotos resultantes de cruzamentos entre estirpes com normas de reação de curvaturas diferentes, evidenciando que a variação das normas de reação do número de pintas de D. mediopunctata é previsível por uma regra simples de associação entre forma (curvatura) e valor médio. Na segunda parte deste trabalho é apresentada uma análise de 40 normas de reação de cinco caracteres diferentes na qual se buscou estabelecer um padrão geral de forma das normas de reação e verificar as consequências desse padrão para estudos que investigam a variação da plasticidade fenotípica com modelos e métodos baseados na linearidade. Os resultados apresentados mostram que a forma típica das normas de reação é não-linear. Quando analisadas com um desenho experimental apropriado apenas para curvas lineares, i.e. com somente três ambientes, tais curvas apresentam um padrão imprevisível de variação, fundamentando um conceito de interação genótipo-ambiente associado à imprevisibilidade. Além disso, o uso de um modelo linear para descrever a variação da plasticidade fenotípica em normas de reação não lineares leva à perda de informação e, em alguns casos, a artefatos que embasam conclusões falsas. Considerando os resultados e conclusões obtidos, é proposta uma nova visão sobre normas de reação e plasticidade fenotípica, baseada no uso de um modelo parabólico e em desenhos experimentais mais abrangentes e detalhados, que permitiriam descrever a forma e variação das normas de reação sem perder fenômenos e padrões importantes / Abstract: Despite describing the population dynamics of genetic variation, the theory of population genetics lacks a description of how genotype and developmental environment interact to generate a phenotypic distribution. Such a description could be achieved through reaction norms, which give the phenotypic response of individual genotypes to a given environmental variation and describe the variation due to phenotypic plasticity and genotype-environment interaction. Here, we used Drosophila mediopunctata as a model organism. This species belongs to the tripunctata group of the genus Drosophila, and is polymorphic for second chromosome inversions. D. mediopunctata specimens display a conspicuous pigmentation polymorphism in the abdomen, with phenotypes ranging from zero to three dark spots in the last three tergites. In the first part of this study, we describe a test of the genetic independency between phenotypic plasticity and mean phenotypic value for the pigmentation polymorphism of D. mediopunctata. We analyzed the reaction norms of the number of abdominal spots in response to temperature of eight strains homozygous for second chromosome inversions and with different mean phenotypic values. The reaction norms were parabolic, and their curvature was correlated with the mean phenotypic value, suggesting that the variation of these two traits may be determined by pleiotropy. The same pattern was observed in heterozygous genotypes resulting from crosses between strains with different reaction norm curvatures. These results show that the variation of reaction norms of the number of dark abdominal spots of D. mediopunctata is predictable by a simple association rule between shape (curvature) and mean value. In the second part of this study we show the results from the analysis of 40 reaction norms of five different traits, which had two aims: establishing a general pattern of reaction norms shape; and verifying the impact of this pattern for studies that investigate the variation of phenotypic plasticity using models and methods based on a linearity assumption. The results show that the typical reaction norm shape is nonlinear. A pattern of unpredictable variation emerges when these curves are analyzed with only three environments, suggesting that the association of genotype-environment interaction with unpredictability may be due to the use of an experimental design only suitable for linear curves. Furthermore, the use of a linear model to describe the variation of nonlinear reaction norms leads to loss of information and, in some cases, artifacts which support false conclusions. Considering these results, we propose a new vision of reaction norms and phenotypic plasticity which is based on the use of a parabolic model and on more comprehensive and detailed experimental designs which would describe the shape and variation of reaction norms without losing important patterns and phenomena / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

In response to telomere shortening, oxidative stress, DNA damage or aberrant activation of oncogenes, normal somatic cells exit the cell cycle and enter an irreversible growth arrest termed senescence. The limited proliferative capacity imposed by senescence on cells impedes the accumulation of mutations necessary for tumorigenesis and prevents proliferation of cells at risk of neoplastic transformation. Opposite to the tumor suppressor function, accumulation of senescent cells in adult organisms is thought to contribute to aging by depleting the renewal capacity of tissues and stem/progenitor cells, and by interfering with tissue homeostasis and functions. The Antagonistic Pleiotropy Theory of senescence proposes that senescence is beneficial early in life by acting as a tumor suppressor, but harmful late in life by contributing to aging. Recent studies have provided evidence strongly supporting the tumor suppressor function of senescence, however, direct evidence supporting the role of senescence in aging remains largely elusive. In this thesis, I describe studies to test the Antagonistic Pleiotropy Theory of senescence in tumorigenesis and aging. The approach that I have taken is to alter the senescence response in vivo by changing the expression of a senescence regulator in mice. The consequence of altered senescence response on tumorigenesis and stem cell self-renewal was investigated. The senescence regulator I studied is Smurf2, which has been shown previously to activate senescence in culture. I hypothesized that the senescence response will be impaired by Smurf2 deficiency in vivo. Consequently, Smurf2-deficient mice will develop tumors at an increased frequency, but also gain enhanced self-renewal capacity of stem/progenitor cells with age. I generated a Smurf2-deficient mouse model, and found that Smurf2 deficiency attenuated p16 expression and impaired the senescence response in primary cells and tissues. Smurf2-deficient mice exhibited an increased susceptibility to spontaneous tumorigenesis, indicating that Smurf2 is a tumor suppressor. At the premalignant stage of tumorigenesis, a defective senescence response was documented in the Smurf2-deficient mice, providing a mechanistic link between impaired senescence response and increased tumorigenesis. The majority of tumors developed in Smurf2-deficent mice were B-cell lymphomas with an origin in germinal centers of the spleen and a phenotype resembling human diffuse large B-cell lymphoma (DLBCL). I discovered that Smurf2 mediated ubiquitination of YY1, a master regulator of germinal centers. Stabilization of YY1 in the absence of Smurf2 was responsible for increased cell proliferation and drove lymphomagenesis in Smurf2-deficient mice. Consistently, a significant decrease of Smurf2 expression was observed in human primary DLBCL samples, and more importantly, a low level of Smurf2 expression in DLBCL correlated with poor survival prognosis. Moreover, I found that hematopoietic stem cells (HSCs) in Smurf2-deficient mice had enhanced function compared to wild-type controls. This enhanced stem cell function was associated with increased cell proliferation and decreased p16 expression, suggesting that defective senescence response in Smurf2-deficient mice leads to increased self-renewal capacity of HSCs. My study, for the first time, offers direct genetic evidence of an important tumor suppressor function for Smurf2 as well as its function in contributing to stem cell aging. Collectively, these findings provide strong evidence supporting the Antagonistic Pleiotropy Theory of senescence in tumorigenesis and aging.

Abstract Coronary heart disease (CHD), a manifestation of atherosclerosis, is the leading single cause of death in Finland. CHD is affected by numerous genetic and environmental factors, their combined effects and interactions between them. Low HDL-cholesterol (HDL-C) is an independent risk factor for atherosclerosis and the most common dyslipidemia associated with early onset CHD, but the mechanisms regulating HDL-C levels and protecting from atherosclerosis are still not completely understood. Adiponectin is a hormone that is secreted by adipose tissue and has several anti-atherosclerotic effects. There is multiple evidence suggesting that adiponectin could protect against CHD via positive effects on HDL metabolism. Vascular endothelial growth factor (VEGF) is a potent angiogenic growth factor that has a potentially conflicting role in atherosclerosis; it may have protecting or predisposing effects. The objective of this thesis was to study the genetic background of HDL-C regulation and atherosclerosis. Three studies were executed using extended families with CHD or case-control setting, with samples collected from Northern Finland. In the first study, seven chromosomal regions showing suggestive evidence of linkage were identified for HDL-C regulation, using genome-wide linkage approach. In the second study, we found a strong correlation between HDL-C and adiponectin, but failed to show evidence of a shared genetic background. However, a genetic correlation between adiponectin and low-density lipoprotein-cholesterol was revealed. We also studied the genetic regulation of adiponectin, and for the first time its most active form, high-molecular weight adiponectin, and found suggestive evidence of linkage to three chromosomal regions. In the third study, it was discovered that the studied VEGF gene polymorphisms did not have a major effect on atherosclerosis quantified as carotid intima-media thickness or the risk of acute myocardial infarction (AMI). This thesis presents potential regions for the genetic regulation of HDL-C and adiponectin and gives new information about their relationship and the effect of VEGF polymorphisms in atherosclerosis. The strong correlation between adiponectin and HDL-C was further strengthened, but we failed to show a shared genetic background between them. / Tiivistelmä Sepelvaltimotauti, eräs valtimonkovettumataudin ilmentymä, on yleisin yksittäinen kuolinsyy maassamme. Taudin syntyyn vaikuttavat lukuisat geneettiset ja ympäristötekijät sekä niiden väliset yhteis- ja vuorovaikutukset. Pieni HDL-kolesterolipitoisuus on valtimonkovettumataudin itsenäinen riskitekijä ja yleisin kolesterolipoikkeavuus, joka liittyy varhain ilmenevään sepelvaltimotautiin. HDL-kolesterolin vaihtelun syitä ja tämän "hyvän kolesterolin" sepelvaltimotaudilta suojaavia vaikutusmekanismeja ei kuitenkaan pystytä täysin selittämään. Adiponektiini on rasvakudoksen tuottama hormoni, jonka sepelvaltimotaudilta suojaavan ominaisuuden on ehdotettu johtuvan siitä, että se vaikuttaisi HDL-kolesterolin aineenvaihduntaan. VEGF (vascular endothelial growth factor) on verisuonten sisäseinämissä vaikuttava kasvutekijä, jolla saattaa olla joko sepelvaltimotaudilta suojaavia tai sille altistavia vaikutuksia. Väitöskirjatyön tavoitteena oli tutkia HDL-kolesterolin ja valtimonkovettumataudin geneettistä taustaa. Kolmessa osatyössä tutkittiin suuria pohjoissuomalaisia sepelvaltimotautisukuja; lisäksi käytettiin väestö- ja potilasaineistoja. Ensimmäisessä tutkimuksessa löydettiin koko genomin kytkentäkartoitusmenetelmällä seitsemän kromosomialuetta, jotka saattavat vaikuttaa HDL-kolesterolin säätelyyn. Toisessa tutkimuksessa selvitettiin adiponektiinin, ja ensimmäistä kertaa myös sen aktiivisimman muodon, HMW-adiponektiinin geneettistä taustaa. Kytkentäanalyysissä saatiin viitteitä kolmesta adiponektiineja mahdollisesti säätelevästä kromosomialueesta. Havaittiin myös, että HDL-kolesterolin ja adiponektiinin pitoisuudet korreloivat vahvasti keskenään, mutta yhteistä geneettistä säätelytekijää ei pystytty osoittamaan. LDL-kolesterolin ja adiponektiinin välillä kuitenkin havaittiin geneettinen korrelaatio. Kolmannessa tutkimuksessa todettiin, ettei tutkituilla VEGF-geenin nukleotidimuutoksilla todennäköisesti ole merkittävää syy-yhteyttä valtimonkovettumatautiin kaulavaltimoiden sisäseinämän paksuudella tai sydäninfarktiriskillä mitattuna. Tämä tutkimus tuo uutta tietoa HDL-kolesterolin ja adiponektiinin geneettisestä säätelystä ja niiden suhteesta sekä VEGF-geenin nukleotidimuutosten osuudesta valtimonkovettumataudissa. Tutkimus vahvistaa edelleen HDL-kolesterolin ja adiponektiinin yhteyden, muttei pysty osoittamaan niille yhteistä geneettistä tekijää.