Normal somatic cells have a limited proliferative capacity in vivo and in vitro, termed senescence and later, thought to contribute to molecular and cellular organismal aging. There are several studies that demonstrate the importance of the GH/IGF axis in longevity, aging and cellular senescence. One primary component of the IGF signaling involves IGFBP-3. It is well documented that IGFBP-3 levels are significantly increased in senescent human diploid fibroblasts however IGFBP-3 function is not known in this system. Interestingly, Werner syndrome fibroblasts, commonly used as a model of cellular aging, have upregulated IGFBP-3 levels in young and late passage cells compared to age matched normal fibroblasts. It is known that suppression of p38 MAPK activity in WS fibroblasts can reverse the senescence and promotes cell proliferation. As increased IGFBP-3 expression is associated with cellular senescence, and suppression of p38 MAPK can reverse senescence in WS fibroblasts, it is hypothesized that “IGFBP-3 can induce senescence, by activating the p38 MAPK signaling pathway.” Our studies demonstrate IGFBP-3 and novel IGFBP-3R can induce senescence in young fibroblasts, while suppression of IGFBP-3 in pre-senescent fibroblasts, can delay the onset of replicative senescence. We identified ROS accumulation in IGFBP-3/IGFBP-R-induced senescent cells which we speculated may be signaling p38 MAPK activation. Inhibition of ROS accumulation suppressed p38 signaling and prevented IGFBP-3/IGFBP-3R-induced senescence. To evaluate the sequence of activation we inhibited p38 activity prior to senescence induction. Interestingly, p38 inhibition prevented IGFBP-3/IGFBP-3R-induced senescence, suggesting IGFBP-3 signals ROS induction which activates p38 signaling. We next examined the significance of IGFBP-3R in IGFBP-3-induced senescence. Suppression of endogenous IGFBP-3R inhibits IGFBP-3-induced senescence. We aimed to identify a possible regulatory mechanism for IGFBP-3 upregulation. Using sequence analysis software we identified 3 possible highly conserved miRNA sequences aligned to IGFBP-3. miR-19a appeared to have the most significant downregulated expression in late passage fibroblasts compared to early passage. Furthermore, overexpression miR-19a in late passage cells, significantly decreased IGFBP-3 expression, suggesting miR-19a may silence IGFBP-3 expression in senescence. Making a direct mechanistic connection between senescence and aging is significant and unraveling how IGFBP-3/IGFBP-3R can induce senescence could prove beneficial in understanding the aging process.

Most organisms experience progressive declines in physiological function as they age. A number of studies in a variety of species support a strong link between oxidative damage, age-related functional declines and life span determination. Here, manipulating the expression levels of superoxide dismutase (SOD) isoenzymes SOD1 and SOD2, resulted in altered functional senescence and survival characteristics in Drosophila. Overexpression of cytosolic Sod1 using the yeast GAL4/UAS system conferred a 30-34% increase in mean life span and resulted in an attenuated senescence of odor avoidance behavior in aging flies. Tissue-specific Sod1 overexpression selectively in the nervous system or muscle failed to reproduce these delayed aging phenotypes suggesting that Sod1 overexpression in these tissues alone was not primarily responsible for the aging effects observed. Graded reduction of mitochondrially localized Sod2 expression in a series of Sod2 mutants led to progressive reductions in life span, accelerated age-related functional declines, mitochondrial oxidative damage and neuronal cell death. Tissue-specific Sod2 knock-down using RNA interference revealed that muscle is a key tissue underlying the accelerated age-related functional decline and mortality observed upon loss of SOD2. Sod2 knock-down in the musculature caused a degenerative phenotype consisting of a dramatic reduction in muscle mitochondrial content and ATP levels, elevated cell death and progressive locomotor dysfunction which culminated in early-onset mortality. Collectively, these studies highlight the important role of SOD enzymes in protecting against the impact of oxidative damage on senescence and survival. These findings also lend further support to the oxidative damage hypothesis of aging.

It is important to understand mechanisms that facilitate expansion of two common shrubs, Morella cerifera and Baccharis halimifolia in coastal environments. The purpose of my study was to investigate the physiological and structural changes that occur as leaves age. Photosynthesis, incident light, chlorophyll, and leaf C:N ratios were quantified for young, intermediate, and old leaves (distal, central and proximal leaves, respectively). Leaf structural differences were also compared. Leaves did not change morphologically with age. Light decreased with leaf age and during winter months. Photosynthesis showed no seasonal or age related patterns. Chlorophyll increased initially and then declined with age due to self shading. Nitrogen content was highest during spring. Seasonality and leaf age had unique effects on the two study species. Understanding senescence adaptations of these two shrubs can help explain their abundance in coastal ecosystems.

Cyclin Dependent Kinase-2 Associated Protein-1 (CDK2AP1) plays an important role in cell cycle regulation, by inhibiting CDK2 and by targeting it for proteolysis. It is also known to bind the DNA polymerase alpha-primase complex and regulate the initiation step of DNA synthesis. Its overexpression has been shown to inhibit growth, reduce invasion and increase apoptosis in a number of cancer cell lines. In studies in which mouse embryonic stem cells (mESCs) with targeted deletion of the Cdk2ap1 gene were used, Cdk2ap1 was shown to be required for epigenetic silencing of Oct4 during differentiation. The goal of this thesis was to examine the role of CDK2AP1 in somatic cells (primary human dermal fibroblasts (HDFs)) and human embryonic stem cells (hESCs) and specifically assess its impact on proliferation, self-renewal and differentiation. In the first part of this study, using a short-hairpin RNA (shRNA) approach, we investigated the effect of CDK2AP1 downregulation in HDFs. Outcomes indicated: (a) reduced proliferation, (b) premature senescence, (c) cell cycle alterations, (d) DNA damage, and (e) an increase in p53, p21, and the p53-responsive apoptotic genes BAX and PUMA. Simultaneous downregulation of p53 and CDK2AP1 in HDFs confirmed that observed phenotype was p53 dependent. In the second part of this study, using a shRNA approach, we investigated the role of CDK2AP1 on hESC fate associated with self-renewal and differentiation. We found that CDK2AP1 knockdown in hESCs resulted in: (a) reduced self-renewal (b) enhanced differentiation (c) cell cycle alterations and (d) increase in p53 expression. Results indicate that the knockdown of CDK2AP1 in hESCs enhances differentiation and favors it over a self-renewal fate. Thus, this study has successfully identified novel functions for CDK2AP1, as its knockdown has a significant impact on self-renewal, differentiation and senescence. Results obtained from this study could contribute to development of directed differentiation strategies for generating uniform populations of differentiated phenotypes from hESCs for clinical applications.

Title of Dissertation: FUNCTION OF INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN 7(IGFBP7) IN HEPATOCELLULAR CARCINOMA By Dong Chen. Purpose: Hepatocellular carcinoma (HCC) is a highly virulent malignancy with no effective treatment, thus requiring the development of innovative and effective targeted therapies. The oncogene Astrocyte Elevated Gene-1 (AEG-1) plays a seminal role in hepatocarcinogenesis and profoundly downregulates Insulin-like Growth Factor Binding Protein-7 (IGFBP7). The present study focuses on analyzing potential tumor suppressor functions of IGFBP7 in HCC and the relevance of IGFBP7 downregulation in mediating AEG-1 function. Experimental Design: IGFBP7 expression was detected by immunohistochemistry in HCC tissue microarrays by real-time PCR and ELISA in human HCC cell lines. Dual Fluorescence in situ hybridization was performed to detect loss of heterozygosity at the IGFBP7 locus. Stable IGFBP7- overexpressing clones were established in the background of AEG-1- overexpressing human HCC cells and were analyzed for in vitro proliferation, senescence, in vivo tumorigenesis and angiogenesis. HCC cell lines infected with an adenovirus expressing IGFBP7 (Ad.IGFBP7) were analyzed by using in vitro cell cycle, apoptosis, in vivo tumorigenesis assays. Results: IGFBP7 expression is significantly downregulated in both human HCC patients’ samples and cell lines compared to normal liver and hepatocytes. IGFBP7 expression was also found to inversely correlate with the stages and grade of HCC. Genomic deletion of IGFBP7 was identified in 26% of HCC patients. Forced overexpression of IGFBP7 in AEG-1 overexpressing HCC cells inhibited in vitro growth and induced senescence. When injected into nude mice, in vivo growth was profoundly suppressed, potentially as a result of inhibition of both angiogenesis and IGF1R activation by IGFBP7. Ad.IGFBP7 profoundly inhibited viability and induced apoptosis in multiple human HCC cell lines by inducing Reactive Oxygen Species (ROS) and activating a DNA damage response. N-acetylcysteine could neutralize ROS and rescue the cells from apoptosis. In early phase after Ad.IGFBP7 infection, activation of cell cycle control proteins like Rb, p53, ATM, ATR, CHK1 and CHK2 were identified and G2/M cell cycle arrest was recorded by FACS. Ad.IGFBP7 infection resulted in the activation of p38 MAPK, and a p38 MAPK inhibitor SB 203580 could block the apoptotic process. In orthotopic xenograft models of human HCC in athymic nude mice, intravenous administration of Ad.IGFBP7 profoundly inhibited primary tumor growth and intra-hepatic metastasis. In a nude mouse subcutaneous model, xenografts from human HCC cells were established in both flanks and only left- side tumors received intratumoral injection of Ad.IGFBP7. Ad.IGFBP7 markedly inhibit growth of both left-sided injected tumors and right-sided un- injected tumors by profound suppression of angiogenesis. Conclusion: The present findings provide evidence that IGFBP7 functions as a novel putative tumor suppressor for HCC and establish the corollary that IGFBP7 downregulation can effectively modify AEG-1 function. Targeted overexpression of IGFBP7 may be a potential novel and effective therapy for HCC.

Les télomères sont des structures nucléo-protéiques présentes à l'extrémité des chromosomes. Ils sont un des facteurs garant de la stabilité génomique. Ils assurent la protection des extrémités des chromosomes et leur entière réplication. Les dysfonctionnements du télomère sont impliqués dans la tumorigénèse et le vieillissement.Un des rôles majeurs des télomères est d'éviter que les extrémités des chromosomes ne soient reconnues comme des cassures double brin de l'ADN et traitées comme telles par la machinerie de réparation. Cependant, de nombreuses protéines impliquées dans la reconnaissance et le métabolisme des cassures double brin, comme la protéine Tel1 et le complexe MRX par exemple, sont présentes au niveau des télomères et participent au maintien de leur taille par la télomérase. En s'appuyant sur cette analogie, j'ai étudié le rôle télomérique de l'endonucléase Sae2 et de l'hélicase Sgs1, impliquées dans l'étape de dégradation du brin 5' qui précède la réparation des cassures double brin de l'ADN par recombinaison.Les rôles des protéines Sae2 et Sgs1 ont été étudiés sur les télomères natifs et sur les télomères érodés lors de la sénescence réplicative. L'ensemble de mes résultats suggèrent que, bien que les télomères érodés en absence de télomérase soient reconnus comme une cassure double brin de l'ADN et traités comme tels par les nucléases et hélicases, le rôle majeur de Sae2 et Sgs1 au niveau des télomères natifs serait de les protéger contre des recombinaisons illégitimes au cours de leur réplication. / Telomeres are nucleoprotein complexes that protect the extremities of linear chromosomes, avoiding end-to-end fusions and nucleolytic degradation of chromosome ends. The failure of cells to properly maintain telomeres can be an important source of chromosome instability involved in cancer progression and aging.A major role of telomeres is to prevent chromosome ends from being recognized as damage-induced double-strand DNA breaks (DSBs). However, many proteins involved in recognition and processing of DSBs are also involved in telomeres maintenance, like Tel1 and MRX. Based on this analogy, I have studied the role at telomeres of the role of the endonuclease Sae2 and the helicase Sgs1, two proteins that have a key function in the processing of DSBs through nucleolytic degradation of their 5' end.The role of protein Sae2 and Sgs1 has been studied at native and eroded telomeres. My results showed that eroded telomeres, in telomerase deficient cells, are recognized and resected as a double-strand break DNA by a set of nucleases and helicases including Sae2 and sgs1. In contrast, the main role of Sae2 and Sgs1 at native telomeres would be to protect telomeres against illegitimate recombination during replication.

L'hypertension artérielle pulmonaire (HTAP) idiopathique ou associée à une maladie sous-jacente est un trouble inexpliqué dont les formes graves, chez l'adulte comme le nouveau-né, restent mortels et pour lequel à ce jour, aucun traitement satisfaisant n'est disponible. Elle est caractérisée par une augmentation anormale de la pression artérielle pulmonaire (PAP) moyenne supérieur à 25 mmHg au repos et 30 mmHg à l'effort alors que chez un sujet sain elle est comprise entre 10 et 15 mmHg. L'hyperplasie des cellules musculaires lisses artérielle pulmonaire (CML-AP) est le principal déterminant du processus de remodelage vasculaire pulmonaire qui sous-tend l'hypertension artérielle pulmonaire.Il existe cependant des similitudes entre le cancer et l'hypertension artérielle pulmonaire. En effet, certains dysfonctionnements cellulaires et métaboliques sont communs aux deux maladies. En effet, des anomalies sont souvent retrouvées à la fois dans le cancer et dans les pathologies prolifératives, comme une mutation du gène codant pour le facteur de transcription p53, l'inactivation post-transcriptionnelle de p53 via l'inhibition de son interaction avec son régulateur négatif MDM2 (murine à double minute 2), l'augmentation de l'activité de la télomérase ainsi que la translocation de la sous-unité TERT vers la mitochondrie.Un moyen thérapeutique de choix dans le cancer ou autre pathologie proliférative est l'induction d'une sénescence cellulaire.Dans notre première étude, nous avons induit la sénescence des CML-AP en inhibant l'interaction p53-MDM2 en utilisant la Nutlin-3a, antagoniste spécifique et puissant de la liaison p53-MDM2, afin de limiter sa dégradation par le protéasome et ainsi augmenter son activité. Dans notre deuxième étude nous avons induit une sénescence des CML-AP en inhibant génétiquement ou pharmacologiquement la télomérase. Ces deux études ont montré que l'induction d'une sénescence cellulaire prévient et réverse partiellement l'HTAP dans différents modèles expérimentaux, en diminuant le nombre de CML-AP prolifératives et en augmentant le nombre de cellules sénescentes (p21-positives) sans induire davantage d'apoptose. Nous avons également confirmé l'implication de l'activité de la télomérase dans le développement de l'hypertension artérielle pulmonaire expérimentale ainsi que la translocation de la sous unité TERT dans la mitochondrie au cour de l'HTAP.La sénescence cellulaire semble donc être une cible thérapeutique de choix dans le traitement de l'hypertension artérielle pulmonaire. / Pulmonary artery hypertension (PH) occurring as an idiopathic condition or associated with an underlying disease is an unexplained disorder whose sever forms in adults and neonates are fatal and for which no satisfactory treatment is available. PH is characterized by an abnormal increase in pulmonary artery pressure (PAP) greater than 25 mmHg at rest and 30 mmHg during exercise while in a healthy person is between 10 and 15 mmHg. Hyperplasia of pulmonary artery smooth muscle cells (PA-SMCs) is the primary determinant of the pulmonary vessel remodeling process that underlies PH.However, similarities exist between cancer and PH. Indeed, some cellular and metabolic dysfunctions are common to both diseases. Abnormalities are often found in both cancer and proliferative disorders, such as an inactivating mutation in the gene encoding the transcription factor p53, a post-transcriptional p53 inactivation via interaction of the p53 protein with its negative regulator MDM2 (mouse double minute 2), increasing the telomerase activity and translocation of the TERT subunit to mitochondria.A selecting means in therapeutic cancer or other proliferative diseases is the induction of cellular senescence.In our first study, we induced CML-AP senescence by inhibiting the p53-MDM2 interaction using Nutlin-3a, a specific and potent antagonist of the p53-MDM2 binding, to limit its degradation by the proteasome and thus increase its activity. In our second study we induced CML-AP senescence by genetical or pharmacological inhibition of telomerase. Both studies showed that the induction of cellular senescence in PA-SMCs prevents and partially reverses PH in different experimental models, by reducing the number of proliferative CML-AP and increasing the number of (p21-positive) senescent cells without inducing more apoptosis. We also confirmed the involvement of telomerase activity and the translocation of the TERT subunit in the mitochondria during PH development.

Les microparticules (MP) sont des vésicules de la membrane plasmique émises après stress cellulaire. Nous avons étudié le rôle des MPs leucocytaires extraites de la rate de rats comme marqueur du vieillissement et effecteurs de la senescence et de la dysfonction endothéliales induites par les fortes concentrations de glucose (HG). L’émission basale de MP augmente avec l’âge qui favorise leur génération en réponse au LPS ou au PMA/ionophore A23187 (MPLPS, MPPMA/I). Les MP de rats âgés mais pas de jeunes induisent la sénescence de cellules endothéliales primaires d’artères coronaires (AC) de porc. MPLPS ou MPPMA/I de rats jeunes, mais pas MPCTL (cellules non traitées) réduisent la relaxation dépendante de l’endothélium d’anneaux d’AC en réponse à la bradykinine avec sous-expression de eNOS, surexpression de COX2, ICAM-1, VCAM-1. HG favorise l’émission des MP de rate. Dans les AC en HG, la vasoconstriction en réponse au U46619l est diminuée de manière dépendante du SGLT1/2 et de l’EDHF. / Microparticles (MP) are plasma membrane vesicles shed from stimulated cells. We investigated whether leukocyte MP extracted from rat spleen are reliable markers of aging and effectors of high glucose (HG)-induced endothelial senescence and dysfunction. Data indicate that ageing enhances MP shedding from spleen cells of middle-age and aged rats and raises MP release in response to LPS, or to PMA and ionophore A23187. Of note, MP from aged but not young rats induced senescence of porcine coronary artery primary endothelial cells. In young rats, MPLPS, MPPMA/I but not from resting cells (MPCTL) reduced the endothelial-dependent relaxation of coronary artery rings (CAR) in response to bradykinin with down-regulation of eNOS, up-regulation of COX-2, ICAM-1, VCAM-1. HG enhanced early and late MP release from spleen cells. Prolonged exposure to HG potentiated endothelial dysfunction in CAR and altered vasoconstriction in response to U46619l in a SGLT1/2 and EDHF dependent manner.

A base evolucionaria da senescência é um problema biológico de longa data. Senescência, no sentido da deterioração progressiva de um organismo, distingue-se de envelhecimento, ou a mera passagem do tempo. Como, pelo menos em mamíferos, senescência e envelhecimento ocorrem simultaneamente, os termos são tomados erroneamente como sinônimos. A senescência parece explicada pelo desgaste do organismo, levando a um paradoxo frente aos mecanismos de seleção natural, pois, sendo a maquinaria biológica portadora de mecanismos de auto-reparo, esperáramos o aprimoramento destes, com gradual eliminação da deterioração associada ao envelhecimento. Historicamente, procurou-se resolver este paradoxo imaginando-se que a senescência conferisse uma vantagem adaptativa, mas este argumento, que requereria distinguir os indivíduos mais velhos, é circular. A partir de meados do século XX, três hipóteses prevaleceram. O acúmulo de mutações, proposto por Medawar (1951), considera que o decaimento gradual da força de seleção com a idade favorece o acúmulo de genes deletérios expressos em idades avançadas. O antagonismo pleiotrópico, proposto por Williams (1957), defende que genes ligados a características benéficas e deletérias poderiam ser, em certas condições, favoravelmente selecionados. Finalmente, a teoria da soma descartável, proposta por Kirkwood (1975), sugere que organismos são favoravelmente selecionados quando investem em Reprodução mesmo em detrimento da manutenção somática. A descrição dinâmica dos fenômenos envolvidos em cada hipótese e a contribuição relativa de cada uma é, ainda, objeto de debate. O presente trabalho visa ao desenvolvimento de modelos computacionais estocásticos que mimetizem as condição para cada uma delas. Iniciamos o desenvolvimento pela ordem histórica, testando o mecanismo proposto por Medawar, que é o objeto desta tese. A teoria do acúmulo de mutações encontrou críticos que sugerem que este mecanismo levaria os efeitos deletérios à sincronia em idades muito avançadas, tornando a senescência um fenômeno repentino e limitado a tais idades. Isto contrariaria a observação experimental, pois a senescência é um processo gradual e mesmo animais silvestres exibem fenômenos senescentes detectáveis precocemente. Para manter a modelagem compatível com o conhecimento atual sobre genética de populações, incluímos neste modelo os efeitos da seleção, da deriva genética e de diferentes taxas de mutação. Como previsto, em nossas simulações a moda das idades de manifestação dos genes deletérios se estabilizou apenas em idades muito avançadas, próximas ao término das distribuições etárias. Também como esperado, estas distribuições terminaram em idades mais precoces nos cenários de maior mortalidade extrínseca. No entanto, em todas as nossas simulações, houve distribuição mais ou menos larga das idades de manifestação em torno da moda. A distribuição alargou-se com o aumento da probabilidade de mutação, sugerindo que as idades de manifestação podem espalhar-se ao longo da vida, chegando, em alguns cenários, à manutenção de alelos com manifestação ao nascer. Isto é compatível com o modelo de alelos ineptos utilizado em genética populacional, em que diferentes variantes concorrem até atingirem um equilíbrio entre seleção, mutação e deriva genética. Considerando-se o critério demográfico para senescência, em que a mortalidade aumenta em função da idade, podemos dizer que as nossas simulações evoluíram populações senescentes. Embora nossos dados não contrariem as outras teorias da senescência, claramente mostram que a perda da força de seleção não é um mecanismo suficiente para a senescência. Especialmente com alelos de expressão tardia, as forças de deriva podem ser preponderantes e devem ser levadas em conta para explicar a evolução da senescência / The evolutionary basis of senescence is a long standing biological problem. Senescence, in the sense of progressive deterioration of an organism, is distinguished from aging, or the mere passage of time. Because, at least in mammals, aging and senescence occur simultaneously, the terms are wrongly taken as synonyms. Senescence seems explained by the wear of the organism, leading to a paradox facing the mechanisms of natural selection, because, being the biological machinery bearer of self-repair mechanisms, we would expect their improvement, with gradual elimination of the deterioration associated with aging. Historically, there were attempts to resolve this paradox by supposing that senescence confers an adaptive advantage, but this argument would require that older individuals were previously distinct from younger ones and is, therefore, circular. From mid-twentieth century, three hypotheses have prevailed. The mutation accumulation, proposed by Medawar (1951), proposes that the gradual decay of the force of selection with age favors the accumulation of deleterious genes expressed in advanced ages. The antagonistic pleiotropy, proposed by Williams (1957), argues that genes linked to benecial and deleterious traits could, under certain conditions, be favorably selected. Finally, the disposable soma theory, proposed by Kirkwood (1975), suggests that organisms are positively selected when they invest in reproduction even at the expense of somatic maintenance. The dynamic description of the phenomena involved in each mechanism and the relative contribution of each one is still debated. The present work aims to develop stochastic computer models that mimic the conditions for each. We started by historical order, testing the mechanism proposed by Medawar, which is the subject of this thesis. The theory of mutation accumulation found critics who suggest that this mechanism would cause deleterious eects to synchronize in very advanced ages, causing senescence to be a sudden phenomenon limited to these ages. This is a contradiction with the experimental observation, because senescence is a gradual process and even wild animals exhibit senescent phenomena detectable in young ages. To keep the model consistent with the current knowledge on population genetics, this model included the eects of selection, genetic drift and dierent mutation rates. As predicted, in our simulations the mode of the age of onset of deleterious genes stabilized only in very advanced ages, near the end of the age distribution. Also as expected, these distributions ended in earlier ages in scenarios of higher extrinsic mortality. However, in all our simulations, there was more or less wide distribution of ages of onset around the mode. The distribution is enlarged with increased probabilities of mutation, suggesting that the ages of onset may spread throughout life, including, in some scenarios, the maintenance of alleles with manifestation at birth. This is consistent with the innite alleles\' model used in population genetics, where dierent variants compete until achieving an equilibrium between selection, mutation and genetic drift. Considering the demographic criterion for senescence, in which mortality increases with age, we can say that our simulations evolved senescent populations. Although our data do not con ict with the other theories of senescence, they clearly show that the falling force of selection with age is not a sucient mechanism for senescence. Especially with alleles of late expression, the forces of genetic drift may be prominent and should be taken into account to explain the evolution of senescence

Leaves are responsible for the majority of the fixed carbon in most plant species. Along leaf development, the photosynthetic capacity increases until the organ reaches maturity. Consequently, at the onset of senescence the leaves have the highest photosynthetic activity, then, as the chloroplasts are dismantled and the photosynthetic machinery is degraded, leaves gradually lose the rate of carbon assimilation. Although the capacity to fix carbon declines as senescence progresses, nutrient remobilization from macromolecule degradation nourishes the developing sink organs. In this regard, delaying leaf senescence stands out as a promising strategy to increase plant yield as extends the window of time with maximum carbon fixation rate. Another approach that is receiving much attention is the manipulation of chlorophyll degradation once it potentially regulates photosynthetic capacity and affects the nutritional quality of harvestable organs. As chlorophyll is degraded, the released phytol is recycled and can be either stored (i.e. as fatty acid phytyl esters), used for chlorophyll synthesis or be incorporated in tocopherol biosynthesis. Tocopherols have high nutraceutical value due to their antioxidant properties. However, the majority of the studies regarding senescence and chlorophyll degradation were carried out in the model plant Arabidopsis thaliana or grasses, creating a knowledge gap about these processes in fleshy fruit-bearing plants of human diet interest. In this regard, the tomato, Solanum lycopersicum, is an excellent model not only for the genetic and genomic resources, but also for its agronomic and nutritional importance. Thus, this project aims to extend what is known about the effects of chlorophyll degradation and senescence manipulation over the metabolism and yield of tomato plants, as well as fruit nutritional quality. In order to evaluate the consequences of alteration in chlorophyll degradation, first the enzymes chlorophyllase and pheophytinase, both capable of dephytylating the chlorophyll molecule, were identified and characterised. An extensive phylogenetic, evolutive and transcriptional analysis allowed the identification of two groups of chlorophyllases, one putatively involved in the response to different stimuli, while the other may act in chlorophyll homeostasis. As for pheophytinase, only one group was identified, being related to physiologically programmed processes that trigger chlorophyll degradation (i.e. leaf senescence and fruit ripening). Given this scenario, pheophytinase was chosen to be constitutively knocked-down in order to evaluate the effects over the metabolism of leaves and fruits. As consequence of this manipulation, transgenic plants were impaired in the leaf senescence-associated chlorophyll breakdown, but, although with an initial delay, fruit ripening-associated degreening was not compromised. Several photosynthetic and biochemical parameters were signs of photoinhibition, possibly due to a deficiency in chlorophyll recycling in leaves. This led to an increase in sugar exportation towards fruits, ultimately increasing soluble sugar content of ripe fruits. However, as a consequence, carotenoid levels were reduced, what, at least partially, it was compensated by an increase in tocopherol content. The results indicated that pheophytinase plays a role beyond senescence-associated chlorophyll degradation and its manipulation led to the development of fruit with increased soluble sugars and tocopherols at the cost of lowering carotenoid levels. Thus, these evidences support the manipulation of chlorophyll breakdown as a strategy for improvement of fleshy fruit plants. In order to address the effects of senescence over yield and fruit quality, the transcription factor ORESARA1, which has been widely characterised in A. thaliana and is considered a key regulator of senescence initiation, was targeted. After a comprehensive phylogenetic analysis and the characterization of the regulatory mechanisms, one putative ortholog was selected to be silenced. As consequence of this manipulation, leaves displayed increased chlorophyll content. Moreover, as senescence was delayed, the extent of photosynthetic activity of the leaves was also expanded. As the number of fruits was increased in the knockdown lines, this reflected in an increment in the harvest index. Ripe fruits accumulated more soluble sugars and tocopherols. Collectively, the results support the manipulation of leaf senescence as a strategy for tomato yield improvement. Altogether, data obtained enhance the knowledge about the impacts of chlorophyll degradation and leaf senescence over the metabolism of fleshy-fruit plants, providing strategies to increase yield and nutritional quality of fruits / As folhas, para a maioria das espécies vegetais, são o principal órgão responsável pela fixação de carbono. Durante o desenvolvimento foliar, o potencial fotossintético aumenta até a folha atingir a sua maturidade. Consequentemente, no momento que o programa de senescência se inicia, a folha apresenta a maior taxa de fotossíntese, a qual passa então a declinar conforme o cloroplasto se desorganiza e a maquinaria fotossintética é degradada. Apesar da redução na fixação de carbono, o catabolismo de macromoléculas possibilita a remobilização de nutrientes para os órgãos dreno em desenvolvimento. Neste contexto, atrasar a senescência destaca-se como uma promissora estratégia para aumento da produtividade, uma vez que estende o período de máxima fixação de carbono das folhas. Outra estratégia que tem recebido atenção por, potencialmente, regular a capacidade fotossintética e afetar a qualidade nutricional dos órgãos coletáveis é a manipulação da degradação da clorofila. Durante o catabolismo deste pigmento, o fitol liberado é reciclado podendo ser armazenado (i.e. na forma de ésteres de fitil com ácidos graxos), ser utilizado na síntese de novas moléculas de clorofila ou ser incorporado na rota biossintética de tocoferóis. Estes últimos compostos, por seu potencial antioxidante, possuem alto valor nutracêutico. No entanto, a maior parte dos estudos sobre senescência e degradação de clorofila foi realizada na planta modelo Arabidopsis thaliana ou em gramíneas, tornando escassas as informações relativas a plantas com frutos carnosos de interesse para a dieta humana. Nesse âmbito, o tomateiro, Solanum lycopersicum, é um excelente modelo de estudo não apenas pela disponibilidade de recursos genético e genômicos, mas também pela importância agronômica e nutricional desta espécie. Assim, este trabalho pretende expandir o conhecimento acerca dos efeitos da manipulação da degradação de clorofila e da senescência sobre o metabolismo e produtividade do tomateiro, bem como sobre a qualidade nutricional dos frutos. De modo a se avaliar as consequências de alterações na degradação de clorofila, iniciou-se por identificar e caracterizar em tomateiro as enzimas clorofilase e feofitinase, as quais catalisam a defitilação da molécula de clorofila. Uma vasta análise filogenética, evolutiva e transcricional permitiu a identificação de dois grupos de clorofilases, um dos quais estaria envolvido na plasticidade de respostas a estímulos e o outro na homeostase dos níveis de clorofila. Já para feofitinase, somente um grupo foi identificado, o qual está relacionado a processos fisiologicamente programados que levam à degradação de clorofila (i.e. senescência foliar e amadurecimento de frutos). Dado o panorama obtido, a feofitinase foi escolhida para ser constitutivamente silenciada de modo a se avaliar as consequências para o metabolismo de folhas e frutos. Como consequência do silenciamento, as linhagens transgênicas mostraram-se incapazes de degradar clorofila durante a senescência, mas, embora com um atraso nas etapas iniciais, a degradação ao longo do amadurecimento de frutos não foi comprometida. Diversos parâmetros fotossintéticos e bioquímicos foram indicativos de fotoinibição, possivelmente em virtude de uma deficiência na reciclagem da clorofila em folhas. Isto acarretou em um aumento na exportação de açúcares para frutos, incrementando a concentração de açúcares solúveis nos frutos maduros, que, em contrapartida, resultou na queda nos teores de carotenoides. A queda nestes compostos antioxidantes foi, ao menos parcialmente, compensada por um aumento nos níveis de tocoferóis. Os resultados indicaram que a feofitinase possui um papel além da degradação de clorofila associada à senescência, e que sua manipulação leva ao desenvolvimento de frutos com maior teor de açúcares solúveis e de tocoferóis ao custo da redução no de carotenoides. Desta forma, estas evidências suportam a manipulação da clorofila como estratégia para o melhoramento de frutos carnosos. Para investigar o efeito da senescência sobre a produtividade e qualidade de frutos foi escolhido o fator de transcrição ORESARA1, o qual está amplamente caracterizado em A. thaliana e é considerado um regulador chave no desencadeamento deste processo. A partir de uma extensa análise filogenética e da caracterização de sua regulação, um putativo ortólogo foi selecionado como alvo para silenciamento. Como consequência desta manipulação, folhas apresentaram os níveis de clorofila incrementados. Além disto, taxas fotossintéticas maiores que as do genótipo controle foram mantidas por maior tempo indicando que a iniciação da senescência foi retardada. Assim, estas plantas produziram um maior número de frutos, consequentemente, aumentando o índice de colheita dessas linhagens. Os frutos maduros apresentaram maiores teores de açúcares solúveis e de tocoferóis. Os resultados demostraram que o retardo do início da senescência é uma estratégia efetiva para aumento da produtividade de tomateiro. Coletivamente, os resultados obtidos aprofundam o conhecimento acerta dos impactos da degradação de clorofila e senescência sobre o metabolismo de plantas com frutos carnoso, além de prover estratégias para se incrementar a produtividade e a qualidade nutricional de frutos