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

Neste estudo foram avaliados os efeitos farmacológicos do vemurafenibe (inibidor BRAFV600E) e da cloroquina (inibidor de autofagia) na viabilidade celular e crescimento tumoral das sublinhagens de melanoma:G361 pLKO que expressa dermicidina e G361 IBC I com silenciamento da expressão. As células G-361 responderam a vemurafenibe (2 μM) e cloroquina (100 μM), isoladamente ou combinadas, com aumento apoptose, e redução das taxas de senescência. Vemurafenibe (50 mg/kg / 21 dias) inibiu o crescimento tumoral em camundongos imunodeficientes independente da expressão da DCD. A combinação com Cloroquina (30 mg/kg) a cada 24 horas, acelerou, enquanto a cada 72 horas, reduziu o crescimento tumoral. Os tumores apresentaram alterações morfológicas e núcleos atípicos; e não expressaram os marcadores S100, HMB-45, Mela-A ou citoqueratinas. Este trabalho confirmar a eficácia do vemurafenibe e sugere o potencial adjuvante da cloroquina no tratamento de melanomas. O estudo também confirma o papel da dermcidina como oncogne e fator de crescimento de células de melanoma maligno. / In this study we evaluated the pharmacological effects of vemurafenib ( inhibitor BRAFV600E) and chloroquine (autophagy inhibitor) in cell viability and tumor growth of two melanoma cell lines identified as G-361 pLKO, which expresses dermcidin, and G361 IBC I which silenced DCD expression. G-361 melanoma cells responded to vemurafenib (1-2 μM) and chloroquine (50-100 μM) alone or combined, with increased apoptosis rates, while decreasing senescent cells. Vemurafenib (50 mg/kg / 21 days) inhibited melanoma growth in immunodeficient mice independent of dermicidin. Chloroquine (30 mg/kg) in combination with vemurafenib, accelerated (at 24 hour interval), and reduced (at 72 hours interval), melanoma growth. Tumor tissues showed atypical cell morphology and nuclear histological patterns and melanocytic differentiation biomarkers S100, HMB-45, Melan-A or pancytokeratins were not. This work confirms the efficacy of vemurafenib and suggests potential adjuvant effect of chloroquine. It also confirms the role of dermcidin as growth factor and oncogene for melanoma cells.

A ocorrência de estresses abióticos e bióticos em plantas imaturas de soja, o que inclui a presença de nematoides, podem resultar em morte prematura da planta ou maturação forçada e produzir sementes esverdeadas e de baixo potencial fisiológico. Assim, o objetivo deste trabalho foi avaliar a presença de Meloidogyne javanica e Pratylenchus brachyurus em plantas de soja e a relação com o desenvolvimento da planta, com a ocorrência de sementes esverdeadas e com o potencial fisiológico das sementes. Para isso, foram conduzidos três experimentos. O experimento I foi realizado utilizando plantas da cultivar Pintado, as quais foram submetidas à diferentes tratamentos de inóculo inicial e avaliadas quanto à fenologia e à produtividade e as sementes quanto ao potencial fisiológico. No experimento II foi analisada a resistência de dez cultivares de soja à M. javanica e P. brachyurus. No experimento III foram utilizadas plantas de duas cultivares de soja (TMG-115RR e M 7908 RR, previamente selecionados no experimento II), dois tratamentos de população de nematoides (0 e 7000 nematoides) e dois tratamentos de estresse hídrico (com e sem), cujas plantas foram avaliadas quanto a fenologia e produção de vagens e sementes, as raízes quanto à multiplicação de nematoides e as sementes quanto as determinações do grau de umidade, do potencial fisiológico (testes de germinação, tetrazólio, envelhecimento acelerado, condutividade elétrica, emergência de plântulas em areia e análise computadorizada de imagens de plântulas), ocorrência de sementes esverdeadas (método visual e fluorescência de clorofila) e citometria de fluxo. Com os resultados do experimento I, observou-se que a presença de M. javanica e de P. brachyurus em plantas de soja, independentemente da população inicial, provoca a redução do ciclo da planta devido exclusivamente à diminuição do estádio de senescência destas, porém a qualidade de sementes não foi afetada com população inicial de nematoides mais elevada (2400 espécimes). Todas as cultivares testadas no experimento II foram classificadas como suscetíveis, porém a cultivar TMG-115RR e M 7908 RR foram escolhidas para a condução do experimento III por permitirem boa reprodução dos nematoides em estudo e por serem consideradas por algumas empresas produtoras de sementes com sendo sensíveis a ocorrência de sementes esverdeadas. Finalmente, com os resultados do experimento III verificou-se que a população inicial de 7000 nematoides afeta, negativamente, a produção, a ocorrência de sementes esverdeadas e a qualidade de sementes. Entretanto, a intensidade do efeito de M. javanica e de P. brachyurus no desenvolvimento de plantas de soja, na ocorrência de sementes esverdeadas e no seu potencial fisiológico depende da população inicial e da espécie do nematoide, da cultivar e das condições ambientais as quais as plantas são submetidas. / Abiotic and biotic stresses in immature soybean plants, including nematodes presence, can result in premature plant death and can produce green seeds with low physiological potential. The aim of this study was to evaluate the Meloidogyne javanica and Pratylenchus brachyurus presence on soybean plants and the relationship with plant development, the green seeds occurrence and the seed physiological potential. For this, three experiments were done. The experiment I was carried out using the cultivar Pintado, which was inoculated with different nematode population and the phenology, productivity, and the seed physiological potential were evaluated. In experiment II it was analyzed the resistance of ten soybean cultivars to M. javanica and P. Brachyurus. In experiment III plants of two soybean cultivars (TMG-115RR and M 7908 RR, chosen by experiment II) were submitted to two nematode population treatments (0 nematodes and 7000) and two levels of water stress (with and without) and they were evaluated as phenology and production of pods and seeds, the multiplication of nematodes on roots and the determinations of moisture content, physiological potential (germination, tetrazolium, accelerated aging, electrical conductivity, sand seedling emergence and seedling computerized image analysis), the occurrence of green seeds (visual method and chlorophyll fluorescence) and flow cytometry on seeds. It was observed in the results of experiment I that the M. javanica and P. brachyurus on soybean plants, regardless of the initial population, decreases of cycle plant due the decrease on senescence phase, however, the seed quality was unaffected with higher initial nematode population (2400 nematodes). All the cultivars in experiment II were classified as susceptible, but the cultivar TMG-115RR and M 7908 RR were chosen for the experiment III because they allowed a good nematode reproduction and because some seed companies considered them as sensible to soybean green seed occurrence. Finally, the results of experiment III showed that the initial population of 7000 nematodes affected negatively the production, the soybean green seeds occurrence and the seed quality. However, the effect of M. javanica and P. brachyurus in the soybean plantas development, in the soybean green seeds occurrence and their physiological potential depends on the initial nematode population and species, cultivar and environmental conditions which the plants are exposed.

O objetivo desta pesquisa foi avaliar a influência do envelhecimento e da perda dos elementos dentais nos níveis totais de imunoglobulina secretória do tipo A (SIgA) na saliva. Foram selecionados 76 pacientes (entre 20 e 87 anos), os quais foram divididos em três grupos de acordo com sua faixa etária e condição bucal: adultos jovens com idades de 20 a 40 anos (Grupo I ou Grupo controle); idosos com idade entre 65 e 78 anos, desdentados parciais, portadores de prótese total unimaxilar (Grupo II) e idosos com idade entre 65 e 87 anos, desdentados totais, portadores de prótese total bimaxilar (Grupo III). Os níveis totais de imunoglobulina secretória do tipo A na saliva foram determinados por meio da técnica de ensaio imunoenzimático em fase sólida ( ELISA – Enzyme-linked Imunosorbent Assay). Após obtenção dos dados experimentais foi empregada a análise de variância de ANOVA com dois fatores (sexo e grupo) para verificar o efeito significante da interação destes fatores. Os níveis totais de imunoglobulina do tipo A secretória na saliva não apresentaram, em média, diferenças significantes entre os três grupos. Em relação ao fator gênero, ou sexo, em média, homens e mulheres apresentaram comportamentos de SIgA diferentes nos grupos. Para o grupo controle o nível total de SIgA dos homens foi maior que o das mulheres enquanto que para o grupo III o nível total de SIgA das mulheres foi maior que dos homens e para o grupo II não foi observada diferença significante dos níveis de SIgA entre homens e mulheres. Pela análise comparativa dos grupos I e III foi observada diferença significante no sexo feminino, o que não foi observado quando comparados os dois grupos experimentais (Grupos II e III). Os resultados desta pesquisa sugerem que não há influência direta dos fatores envelhecimento e perda dental sobre os níveis totais de imunoglobulina secretória do tipo A na saliva. Estes resultados mostraram a influência do gênero sobre os níveis de imunoglobulina secretória do tipo A. Entretanto, a influência do gênero não é bem conhecida e merece mais estudos. / The aim of this study was to evaluate the influence of senescence and teeth loss on secretory immunoglobulin A (SIgA) levels in saliva. Seventy-six patients (20 to 87 years old) were selected and classified in three groups according to their age and oral dental state: young adults were aged 20-40 years (Group I or Control group); elderly subjects were aged 65-78 years and wore maxillary or mandibular denture (Group II); and edentulous old subjects were aged 65-87 years and wore maxillary and mandibular denture (Group III). The secretory immunoglobulin A levels were determined by the Enzyme-linked imunosorbent assay (ELISA method). All results were correlated using ANOVA statistical analysis with two factors (sex and group) to verify the significant effect of these factors. The secretory immunoglobulin A levels were not significant differences among the average values of the three groups. In gender relation , men and women showed the mean rate of SIgA levels different in the groups. The men SIgA levels of control group showed greater when compared to women levels. In Group III the women levels were greater when compared to men levels. And to Group II statistical analysis demonstrated no significant difference between the SIgA levels of men and women. The analysis showed significant differences in the women levels when compared to Groups I and III. No differences of levels were demonstrated when compared to Groups II and III. These results suggests that the senescence and teeth loss do not have a direct relationship to the secretory immunoglobulin A levels in whole saliva. The se results showed that there is influence of gender in the secretory immunoglobulin levels. However, the influence of gender is not well known and further studies are still necessary.

La sénescence réplicative (SR) dans les fibroblastes humains primaires est causée par l’érosion des télomères et est contrôlée par p53. La régulation dynamique de l’activation de p53 est essentielle pour l’induction de la sénescence ; cependant, les mécanismes moléculaires sous-jacents ne sont pas clairement établis. Nous montrons, dans les cellules surexprimant les isoformes Δ133/Δ160p53, que ces isoformes s’oligomérisent avec p53, conduisant ainsi à la stabilisation d’une forme inactive de p53. A l’inverse, l’inactivation des isoformes endogènes Δ133/Δ160p53 induit l’accumulation de la protéine p53 et l’activation de son activité transcriptionnelle. La surexpression de Δ133/Δ160p53 inhibe les fonctions de p53, en particulier son activité transcriptionnelle et son rôle dans l’arrêt du cycle après un dommage à l’ADN. Nous avons remarqué que les protéines Δ133/Δ160p53 et p53 sauvage possédaient des conformations différentes. Les protéines Δ133/Δ160p53 sont reconnues pas l’anticorps Pab40 : elles adopteraient une conformation similaire à un mutant de conformation de p53. Enfin, nous observons qu’une faible expression de l’ARNm Δ133/Δ160TP53 coïnciderait avec la durée de l’activation transcriptionnelle de p53 lors de la SR, indiquée par l’accumulation de l’ARNm d’un effecteur majeur de p53, p21. L’augmentation de l’expression de Δ133/Δ160TP53 à un temps tardif au cours de la SR est corrélée à l’accumulation du marqueur de sénescence p16INK4a et à celle de la cytokine pro-inflammatoire IL-6. En conséquence, les isoformes Δ133/Δ160p53 contrôleraient l’activité de p53 dans l’arrêt du cycle et sur le phénotype sécrétoire des cellules sénescentes. / Telomere attrition in primary human fibroblasts induces replicative senescence by activation of the tumour suppressor p53. Fine-tuned activation of p53 is essential for senescence induction; however, the mechanisms underlying the regulation of p53 activity during senescence have not been clearly established yet. We report here that in cells that express the Δ133/Δ160p53 isoforms, these p53 isoforms oligomerize with p53, leading to the stabilization of the transcriptionally inactive form of p53. Conversely, endogenous Δ133/Δ160p53 silencing increases the level of p53 and p53-dependent transcriptional activity to promote cell cycle arrest. Overexpressed Δ133/Δ160p53 repress p53 functions, including gene transcription activation and growth inhibition, upon DNA damage. We also found that Δ133/Δ160p53 and wild-type p53 have different structural conformations. Δ133/Δ160p53 adopt a more unfolded conformation recognized by the Pab240 antibody, indicating that these p53 isoforms have a p53 mutant-like conformation. Finally, we observed that low level of Δ133/Δ160TP53 mRNA coincided with the duration of p53 transcriptional activation in replicatively senescent fibroblasts, as indicated by the upregulation of CDKN1A (p21) mRNA expression, a downstream effector of p53. Δ133/Δ160p53TP53 was upregulated at a later stage when the senescence marker p16INK4a and the pro-inflammatory interleukin-6 (IL-6) were also induced. Therefore, p53 activity on growth suppression and senescence-associated secretory phenotype may be differentially regulated by its Δ133/Δ160p53 isoforms.

The purpose of this thesis is to investigate associations between parasite infection and host life-history variation in the wild Soay sheep population of the islands of St Kilda, NW Scotland. Studying host-parasite interactions in wild animal populations is of interest because of the importance of heterogeneity in resource availability, genetics, and environmental conditions in determining resistance to parasites, with implications for human populations and wildlife conservation and management. However, very few studies are able to investigate these associations in a longitudinal manner, which is essential in order to understand how infection is associated with life-history variation across ages and environmental conditions. In this thesis, I investigate associations between parasite resistance and ageing and the importance of maternal effects on offspring parasite resistance. I also establish the shape of natural selection on parasite resistance, and associations between measures of parasite burden and antibody responses. The principle findings of the analyses presented in this thesis are: i) Adult sheep of both sexes show a decline in parasite resistance in old age which is consistent with senescence. Furthermore, the rate of decline in parasite resistance with age is accelerated in individuals that have experienced more stressful environmental conditions over their lifespan. ii) Aspects of maternal phenotype and lamb early life performance are significantly associated with parasite resistance in lambs. Some of these effects persist into adult life and may even affect late-life changes in parasite resistance with age. iii) Analysis of ageing in five female reproductive traits shows that the contributions of individual senescence, terminal effects, and selective disappearance vary across traits, and that therefore multiple traits should be studied in order to understand ageing more fully. Most strikingly, there was no evidence for significant senescence in the probability of producing twins. iv) The first estimate of the strength of natural selection on parasite resistance in a longitudinally-monitored population provided evidence for positive selection on parasite resistance in lambs but not adults. Selection in lambs also varied across environmental conditions, being stronger in years of more favourable conditions. v) Analysis of associations between estimates of parasite burden and antibody responses showed that an estimate of parasite burden was not correlated with either a general or parasite-specific antibody response. However, antibody responses were positively correlated, and there was some evidence for a genetic correlation between the two in lambs but not adults.

Submitted by DHARA CARLESSO ZAMPIVA (dhara.zampiva@univates.br) on 2018-08-20T17:17:13Z No. of bitstreams: 3 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2018EdinaAparecidadosReisBlasi.pdf: 4920714 bytes, checksum: 936b932d7a6e49e5bca50678cab5e1d9 (MD5) 2018Edina_artigo.pdf: 1100636 bytes, checksum: a4919dc64d214daf7c76333e0b77f3a0 (MD5) / Rejected by Ana Paula Lisboa Monteiro (monteiro@univates.br), reason: Inserir o Lattes do autor. on 2018-09-11T18:28:07Z (GMT) / Submitted by DHARA CARLESSO ZAMPIVA (dhara.zampiva@univates.br) on 2018-09-17T17:18:08Z No. of bitstreams: 3 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2018EdinaAparecidadosReisBlasi.pdf: 4920714 bytes, checksum: 936b932d7a6e49e5bca50678cab5e1d9 (MD5) 2018Edina_artigo.pdf: 1100636 bytes, checksum: a4919dc64d214daf7c76333e0b77f3a0 (MD5) / Approved for entry into archive by Ana Paula Lisboa Monteiro (monteiro@univates.br) on 2018-10-03T16:31:01Z (GMT) No. of bitstreams: 3 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2018EdinaAparecidadosReisBlasi.pdf: 4920714 bytes, checksum: 936b932d7a6e49e5bca50678cab5e1d9 (MD5) 2018Edina_artigo.pdf: 1100636 bytes, checksum: a4919dc64d214daf7c76333e0b77f3a0 (MD5) / Made available in DSpace on 2018-10-03T16:31:01Z (GMT). No. of bitstreams: 3 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2018EdinaAparecidadosReisBlasi.pdf: 4920714 bytes, checksum: 936b932d7a6e49e5bca50678cab5e1d9 (MD5) 2018Edina_artigo.pdf: 1100636 bytes, checksum: a4919dc64d214daf7c76333e0b77f3a0 (MD5) Previous issue date: 2018-08-20 / High levels of Schizotetranychus oryzae phytophagous mite infestation on rice leaves can severely affect pro- ductivity. Physiological characterization showed that S. oryzae promotes a decrease in chlorophyll concentration and the establishment of a senescence process in rice leaves. Late-infested leaves also present high levels of superoxide radical and hydrogen peroxide accumulation, along with high levels of membrane integrity loss, which is indicative of cell death. To better understand the rice molecular responses to high levels of mite in-festation, we employed the Multidimensional Protein Identification Technology (MudPIT) approach to identify differentially expressed proteins. We identified 83 and 88 proteins uniquely present in control and late-infested leaves, respectively, along with 11 and one proteins more abundant in control and late-infested leaves, re-spectively. S. oryzae infestation induces a decreased abundance of proteins related to translation, protease in-hibition, and photosynthesis. On the other hand, infestation caused increased abundance of proteins involved in protein modification and degradation. Our results also suggest that S. oryzae infestation interferes with in-tracellular transport, DNA structure maintenance, and amino acid and lipid metabolism in rice leaves. Proteomic data were positively correlated with enzymatic assays and RT-qPCR analysis. Our findings describe the protein expression patterns of late-infested rice leaves and suggest several targets which could be tested in future bio-technological approaches aiming to avoid the population increase of phytophagous mite in rice plants.

Introdução: As síndromes de Li-Fraumeni (LFS) e Li-Fraumeni Like (LFL) são síndromes hereditárias de predisposição a câncer frequentemente associadas à mutações germinativas no gene TP53. Devido à importância de p53 e diversidade de processos celulares que ela regula, várias vias de sinalização podem ser afetadas pela doença. Nesse estudo discutimos o impacto da mutação p.R337H na proliferação, senescência, autofagia, população e funcionalidade mitocondrial. Métodos: As taxas de proliferação foram avaliadas pelo ensaio de Population Doubling. Os experimentos de senescência, autofagia, massa total e funcionalidade mitocondrial foram realizados por citometria de fluxo. Resultados: As células contendo a mutação proliferaram mais do que as células controle. Além disso, as células mutadas não ativaram autofagia sob tratamento de Rapamicina nem senescência sob tratamento de Doxorubicina ou Cisplatina e exibiram maior população mitocondrial, mas com funcionalidade inalterada após os tratamentos. Conclusão: os dados sugerem que a mutação p.R337H em TP53 afeta a indução de senescência realizada por p53 e suas funções pró-autofágicas, bem como seu controle. As células mutadas proliferam mais do que células sem mutação em TP53 e exibiram maior massa mitocondrial sem perda de funcionalidade após o tratamento com Doxorubicina. / Background: Li-Fraumeni (LFS) and Li-Fraumeni Like (LFL) syndromes are hereditary cancer predisposition syndromes frequently associated with germline mutation in TP53. Due to the importance of the protein p53 and its regulation of several important cellular processes, impairment in some pathways can be implicated. Here we discuss the impact of p.R337H TP53 mutation on proliferation, senescence, autophagy, mitochondrial population and functionality. Methods: Growth rates were assayed with Population Doubling assay. Senescence and autophagy were assessed through flow cytometry and functionality and total population of mitochondria were also analyzed through flow cytometry. Results: mutated cells proliferated more than control cells. TP53 mutated cells didn’t build up autophagy under Rapamycin treatmend nor senescence under Doxorubicin or Cisplatin treatments and showed more mitochondrial mass, but no alterations in mitochondrial functionality after Doxorubicin treatment. Conclusion:data suggests that p.R337H TP53 mutation affect senescence induction by p53 and pro-autophagic actions of p53. Mutated cells proliferate more than control cells and exhibited larger mitochondrial mass without effects in their functionality in response to Doxorubicin treatment.

Le tabagisme aggrave de nombreuses maladies inflammatoires chroniques (BPCO, maladie de Crohn, arthrite rhumatoïde, psoriasis) associées à la sous-population de lymphocytes T CD4+ (LT CD4+) inflammatoires sécrétant l’IL-17 (Th17). Le tabagisme est associé à une augmentation en nombre et en proportion, du nombre de Th17 systémiques et pulmonaires, contrastant avec la rareté habituelle de ces cellules dans les tissus périphériques-sites d’inflammation. Le tabagisme est associé à l’augmentation de la sénescence des cellules pulmonaires récemment impliquée dans la pathogénèse de la BPCO. Les mécanismes responsables de l’augmentation des Th17 chez les fumeurs sont encore inconnus, et le rôle potentiel de la sénescence dans cette augmentation et la modification des fonctions des Th17 n’a jamais été exploré.Dans ce travail, nous avons fait l’hypothèse que les Th17 présentent une susceptibilité augmentée à la sénescence induite par l’exposition à la fumée de cigarette, en comparaison des autres sous-populations de LT CD4+, qui serait responsable de l’augmentation des Th17, et contribuerait à l’aggravation de leur potentiel inflammatoire. Nous avons analysé la susceptibilité des Th17 à la sénescence induite par l’exposition au condensat de FC (CFC), et le rôle de la voie de signalisation ERK1/2 dans ce phénomène. Nous avons également analysé le rôle des espèces réactives de l’oxygène (ERO), impliquées, d’une part dans la régulation de l’activation de ERK1/2, et d’autre part dans la sénescence cellulaire.Des lymphocytes T CD4+ CCR6+ Th17 et T CD4+ CCR6- quiescents de donneurs sains ont été exposés in vitro au CFC. La production d’ERO est mesurée en analysant l’oxydation de la sonde H2DCF-DA (cytométrie de flux). La sénescence est évaluée en analysant l’expression de p16INK4a (ImmunoFluorescence). L’analyse de l’expression des cytokines (Luminex/CBA) évalue le potentiel inflammatoire des cellules.Nos résultats montrent que les LT CD4+ Th17 quiescents exposés au CFC présentent une augmentation de différents marqueurs de sénescence : activité -galactosidase, expression des inhibiteurs du cycle cellulaire p16INK4a et ATF3. L’exposition au CFC modifie le profil sécrétoire des Th17 quiescents et induit leur sécrétion d’IL-8. Nous montrons que la voie des MAPKs ERK1/2 est impliquée dans l’induction du phénotype sénescent des Th17 en réponse à une exposition au CFC. La sur-expression de p16INK4a est associée à une activation et une translocation nucléaire de ERK1/2 plus importante dans les Th17. Le traitement antioxydant par la NAC, diminue l’expression des ERO et de p16INK4a induite par l’exposition au CFC dans toutes les sous-populations de LT CD4+, mais maintient la production d’ERO et l’expression de p16INK4a plus importantes dans les Th17 en comparaison des autres sous-populations de LT CD4+. Le traitement par l’inhibiteur du complexe III de la chaîne respiratoire mitochondriale, l’antimycine, maintient la production d’ERO plus importante dans les Th17, mais l’expression de p16INK4a est diminuée à des niveaux comparables dans tous les LT CD4+. En revanche, le traitement par l’agent découplant, le FCCP, diminue l’expression de p16INK4a et la production d’ERO à des niveaux comparables dans tous les LT CD4+, et abroge les différences de production d’ERO entre les Th17 et les T CD4+ CCR6-.Nous montrons que les lymphocytes Th17 humains présentent une susceptibilité plus importante à la sénescence induite par l’exposition au CFC en comparaison des autres sous-populations de LT CD4+. Nos résultats suggèrent que l’activité mitochondriale basale plus importante dans les Th17, est responsable de la plus grande susceptibilité des Th17 à la sénescence après exposition au CFC. Enfin, nous montrons pour la première fois qu’un découplage modéré de la chaîne respiratoire mitochondriale est une solution efficace pour prévenir la sénescence des Th17 et pourrait être une stratégie anti-inflammatoire dans les maladies chroniques associées aux Th17 / Smoking worsens chronic inflammatory diseases (COPD, Crohn disease, rheumatoid arthritis, psoriasis) associated with inflammatory CD4+ IL-17 secreting lymphocytes (Th17). Smoking is associated with an absolute number and proportion increase, at both systemic and pulmonary levels, of Th17 cells. This increase of Th17 cells in smokers contrasts with their usual rarity in peripheral tissues and inflammatory sites. Most of the knowledge about the effects of cigarette smoke exposure comes from COPD studies, and, in COPD increased senescence of pulmonary cells has been associated to the pathogenesis of the disease. The mechanisms responsible for the increase of Th17 cells are still unknown, and the potential role of senescence in this increase and functional modifications of Th17 in smokers has never been explored.In this study, we hypothesized that Th17 present a higher susceptibility to cigarette smoke-induced senescence, as compared to other CD4+ T lymphocytes subsets, which could be responsible for the increased number and proportion of Th17 in smokers, and the higher inflammatory potential of these cells. We analyzed senescence susceptibility of Th17 exposed to cigarette smoke condensate (CSC), and the potential role of ERK1/2 signaling pathway in this phenomenon. We also analyzed the potential role of reactive oxygen species (ROS) known to be implicated, on one hand in ERK1/2 activation, and on another hand in cellular senescence.Quiescent CCR6+ Th17 and CCR6- CD4+ T lymphocytes of healthy donors are exposed in vitro to cigarette smoke extract (CSE). ROS production is measured by H2DCF-DA oxidation (flow cytometry). Senescence is evaluated by p16INK4a expression (ImmunoFluorescence). Expression of relevant cytokines (Luminex/CBA) evaluated inflammatory potential.Our results show that quiescent CD4+ Th17 exposed to CSE present an increase of senescence markers: -galactosidase activity and expression of cell cycle inhibitors p16INK4a and ATF3. Moreover, CSE exposure modifies Th17 secretion pattern and increases IL-8 secretion. Our results also show that ERK1/2 MAPK pathway is implicated in Th17 senescent phenotype induction upon CSE exposure. The overexpression of p16INK4a is associated with a higher activation and a nuclear translocation of ERK1/2 in Th17 cells. Treatment with the anti-oxidant NAC reduces CSE-induced ROS and p16INK4a expression in all CD4+ T cell subsets, but the higher production of ROS and higher p16INK4a expression in Th17 as compared to other CD4+ T cells are maintained. Treatment with mitochondrial complex III inhibitor, antimycine, maintains the higher production of ROS in Th17 as compared to other CD4+ T lymphocytes, whereas p16INK4a expression is reduced to the same level in all subsets. Conversely, treatment with the mitochondrial decoupling agent, FCCP, reduces p16INK4a expression to the same level in Th17 as in other CD4+ T cell subsets, and abrogates the difference of ROS production between Th17 compared to CCR6- CD4+ T lymphocytes.We show that human Th17 lymphocytes present a higher senescence susceptibility to CSE exposure as compared to other CD4+ T lymphocytes sub-populations. Moreover, our results suggest that a higher mitochondrial activity in Th17 in steady state is responsible for the Th17 higher senescence susceptibility upon CSC exposure. Finally, we show for the first time, that mild mitochondrial respiratory chain uncoupling is an effective solution to prevent Th17 senescent phenotype and could represent an anti-inflammatory strategy in Th17-associated chronic inflammatory diseases

Neste trabalho analisamos a influência da variabilidade climática sobre um ecossistema representado principalmente por cerrado sensu stricto, na Gleba Pé de Gigante, em Santa Rita do Passa Quatro, SP, durante o período de 2001 a 2007. Os dados coletados para esta análise são provenientes da torre micrometeorológica localizada no Parque Estadual de Vassunuga, cuja instalação está associada ao desenvolvimento do projeto temático Interação Biosfera- Atmosfera Fase 2: Cerrados e Mudanças de Uso da Terra. As propriedades físicas do clima utilizadas para análise foram a precipitação, a temperatura do ar e a radiação solar. Um levantamento teórico da biomassa acima e abaixo do solo foi realizado para caracterizar a vegetação quanto ao potencial de estoque de carbono existente. A biomassa da área coberta por campo cerrado foi de 67,1 Mg.ha-1, da área coberta por cerrado sensu stricto, 185,6 Mg.ha-1 e da área coberta por cerrado denso, 242,7 Mg.ha-1. Uma relação entre estoques de carbono e fluxos de CO2 foi estabelecida, onde uma tonelada de carbono em cerrado sensu stricto é capaz de assimilar, em média, 0,27 KgC.ha-1.dia-1 da atmosfera. A combinação de diferentes intensidades das propriedades climáticas formam condições ambientais variadas que contribuem para o estado da vegetação e sua produtividade. O principal parâmetro usado para avaliar o estado da vegetação foi o albedo, tanto para a faixa espectral da radiação visível (albedo solar) como para a faixa da radiação fotossintéticamente ativa (albedo RFA). O comportamento sazonal do albedo permitiu verificar que a vegetação apresentou-se fortemente condicionada pela variabilidade climática, que ditou o ritmo da funcionalidade ecossistêmica. De maneira geral, a precipitação, a temperatura do ar e a oferta de energia solar oscilam de forma proporcional ao longo das estações, caracterizando dois períodos distintos: um período com condições favoráveis ao desenvolvimento vegetal, de outubro a março, e um período de estresse, de abril a setembro. Os valores mínimos e máximos de albedo solar sobre o cerrado sensu stricto, durante o período analisado, oscilou entre 15% (novembro/dezembro) e 9% (setembro/outubro) e, o albedo RFA oscilou entre 2% (fevereiro/março) e 6% (setembro/outubro). Na escala interanual, observou-se o aumento do albedo RFA em 2006 após um período de três anos de queda contínua da precipitação, entre 2003 e 2006, sendo 2006 o ano menos chuvoso de toda a série considerada. Em 2007, os valores de albedo RFA foram bem mais baixos do que os calculados para os demais anos, respondendo rapidamente ao alto índice de precipitação ocorrido na estação chuvosa entre 2006 e 2007. Embora tenha sido observado uma resposta relativamente rápida do albedo RFA à recuperação do estresse hídrico na escala sazonal, o padrão do albedo na escala interanual é distinto: entre 2003 e 2006, período em que se observou taxas negativas de precipitação consecutivas, o albedo RFA diminuiu ou ficou com valores aparentemente constantes, apresentando valores mais altos somente em 2006. Desta forma, conclui-se que o estado da vegetação é condicionado principalmente pelo índice de precipitação, uma vez que a temperatura do ar e a quantidade de radiação solar não apresentam variações bruscas na região considerada. Considerando-se a importância da estimativa de albedo RFA como um parâmetro para estimar a variação sazonal do estado da vegetação, sugeriu-se um ajuste linear simples para a estimativa de albedo RFA em cerrado sensu stricto com base nos valores de IVDN, cuja variância explicada foi igual a 0,68. / In this work we analyze the climatic variability influence over a woodland savannah ecosystem at Gleba Pe de Gigante, Santa Rita do Passa Quatro, SP, during the 2001-2007 period. The data collected for this analysis are from a micrometeorological tower located at Vassununga State Park, which was installed under the thematic project called Biosphere- Atmosphere Interaction Phase 2: Savannah and Land Use Change. The physical climate properties used for this analysis were precipitation, air temperature and solar radiation. A theoretical survey for above and below ground biomass was made to characterize the existing carbon stock potential related to the vegetation. The total biomass estimated at grassland savannah was 67.1 Mg.ha-1, at woodland savannah was 185.6 Mg.ha-1 and at dense savannah was 242.7 Mg.ha-1. A relationship between carbon stocks and CO2 fluxes was established where one tone of carbon in woodland savannah absorbs an average of 0.27 KgC.ha-1.day-1 from the atmosphere. The combination of different climate properties and intensities generates different environmental conditions that lead to the vegetation state and its productivity. The main physical parameter considered to evaluate vegetation state was the albedo, which was shared in two spectral bands: visible spectrum (solar albedo) and photosynthetic active radiation (PAR albedo). The seasonal pattern of albedo allows checking that vegetation was strongly conditioned by climatic variability, which dictates the ecosystem functionality rhythm. Generally, precipitation, air temperature and solar radiation vary in a proportional way along the year, providing two different periods related to vegetation status: one period characterized by favorable conditions to vegetal development (October-March) and another by stressing conditions (April-September). Maximum and minimum values for solar albedo at woodland savannah varied, respectively, between 15% (November/December) and 9% (September/October); for PAR albedo, maximum and minimum values varied between 6% (September/October) and 2% (February/March). At annual scale, PAR albedo rose in 2006, after a four years period of falling precipitation rate, between 2003 and 2006. 2006 was the drier year among the others. In 2007, the PAR albedo values were much lower than those calculated for the remaining years, promptly responding to the high precipitation rate observed in the previous rainy season, 2006-2007. Even though a quick response in PAR albedo was noticed due to the recovered water stress in seasonal scale, the albedo pattern in annual scale held a different way: between 2003 and 2006, period characterized by consecutive and negative precipitation rates, vegetation was apparently associated to stable values of PAR albedo, presenting higher values only in 2006. Considering these results, we conclude that the vegetation state is mainly conditioned by precipitation rate, once the air temperature and solar radiation had not presented high variation in the study region. Based on the importance of PAR albedo as a parameter to estimate seasonal vegetation status, a simple linear adjustment according for woodland savannah PAR albedo based on NDVI values was suggested, which explained variance by NDVI was equal to 0.68.