Resumo: A cana-de-açúcar (Saccharum spp.) é uma das gramíneas cultivadas mais importantes do mundo e o Brasil é hoje o maior produtor e exportador mundial de cana, de açúcar de cana e etanol de cana, com o estado de São Paulo sendo o maior produtor. Entretanto, esta cultura é ameaçada por várias pragas e doenças. Dentre essas pragas, uma das mais importantes na região centro-sul é a cigarrinha-das-raízes (Mahanarva fimbriolata), que assumiu este posto devido ao aumento da colheita mecanizada. Por meio da técnica de cDNA-AFLP este trabalho teve como objetivo identificar genes possivelmente envolvidos na resistência a ninfas de Mahanarva fimbriolata em cana-de-açúcar. Para isso, foi utilizada uma variedade suscetível (SP80-1816) e uma resistente (SP83-5073). Ambas foram infestadas com ninfas da cigarrinha 77 dias após o plantio e amostras de raízes foram coletadas antes da infestação (controle) e 1, 2 e 7 dias após a infestação, posteriormente foi feita a extração de RNA, síntese de cDNA e o cDNA-AFLP. Após a análise dos fragmentos foram encontrados 16 fragmentos expressos somente na variedade resistente. Nove fragmentos foram encontrados no 2º dia de infestação e sete no 7º dia. Dentre os 16 fragmentos, sete foram recuperados e sequenciados. Seis fragmentos não apresentaram homologia com proteínas já descritas e um fragmento, de 129 pb, apresentou homologia com uma proteína putativa associada à senescência. Todos os fragmentos podem representar genes relacionados com a resistência da planta contra o inseto / Abstract: The sugarcane (Saccharum spp.) is the most important grass cultivated in the world and Brazil is now the world's largest producer and exporter of sugarcane, sugarcane sugar and sugarcane ethanol, with the São Paulo state being the largest producer. However, this culture is threatened by several pests and diseases. Among these pests, one of the most important in south-central region is the froghopper-roots (Mahanarva fimbriolata), that took this position because of increased mechanization. Through cDNA-AFLP technique this study aimed to identify genes possibly involved in resistance to nymphs of Mahanarva fimbriolata in sugarcane. Therefore we used a susceptible variety (SP80-1816) and a resistant (SP83-5073). Both were infested with spitlebugs 77 days after planting and root samples were collected before infestation (control) and 1, 2 and 7 days after infestation, was later made the extraction of RNA, cDNA synthesis and cDNA-AFLP. After fragments analyses were found 16 fragments expressed only in the resistant variety. Nine fragments were found on day 2 of infection and seven on day 7. Among the 16 fragments, seven were recovered and sequenced. Six fragments showed no homology to described proteins and a fragment of 129 bp showed homology to a putative protein associated with senescence. All fragments could represent genes related to the plant resistance against the insect / Orientadora: Maria Inês Tiraboschi Ferro / Coorientador: Miguel Angelo Mutton / Banca: Lucia Maria Carareto Alves / Banca: Marcelo de Almeida Silva / Mestre

Malgré la complexité du processus de transformation, plusieurs systèmes modèles ont été développés dans lesquels les cellules normales se transforment d'une manière progressive par l'introduction d’éléments génétique. Ici, seules les cellules transformées rentrent en apoptose induite par TRAIL. Comme le milieu des cellules sénescentes a déjà été impliqué dans certaines caractéristiques tumorales (la prolifération, l'invasion et d'autres), notre objectif était d'évaluer soneffet potentiel sur l'acquisition de la sensibilité à TRAIL. Lorsque toutes les cellules de ce système de transformation sont incubés avec le milieu conditionné de cellules sénescentes (CMS) la sensibilisation à TRAIL a été observée dans les cellules pré-transformées mais pas dans les cellules immortalisées ou normales. Ainsi, nous avons conclu que les différentes étapes de la transformation fournissent un contexte cellulaire et moléculaire particulier qui agit en synergie avec le CMS afin de promouvoir la sensibilisation à TRAIL. Ces observations mettent l'accent sur le rôle spécifique descellules sénescentes et leur phénotype sécrétoire. Nous avons ensuite étudié les mécanismes activés dans les cellules pré-transformées par le CMS et responsables de leur sensibilisation à TRAIL. Nos résultats suggèrent un rôle clé de l'axe Myc-FLIPL dans la signalisation activée par le CMS. / Despite the complexity of the transformation process, several model systems have been developed in which normal cells are transformed in a step-wise manner by introduction of genetic elements (telomerase, SV40ER viral genes and oncogenes). In this system, only the transformed cells go into TRAIL-induced apoptosis. As senescent-secreted factors have already been involved in promoting some of the tumor characteristics (proliferation, invasion and others), our objective was to assess its potential effect on the acquisition of sensitivity to TRAIL. When all the cells of the transformation system were incubated with conditioned medium of senescent cells (CMS) the sensitization to TRAIL was observed only in the pre-transformed, but never in normal or immortalized cells. Thus, we concluded that the different steps of transformation provide a cellular and molecular context that acts in synergy with the CMS to promote TRAIL sensitivity. These observations emphasize the specific role of senescent cells and their secretory phenotype. We then studied the mechanisms activated inthe pre-transformed cells by the CMS, responsible for their sensitization to TRAIL. Our results suggest a key role of the Myc-FLIPL axis in signaling activated by the CMS.

hCLCA2 and hCLCA4 are chloride channel regulators that are expressed in normal breast epithelial cells and frequently downregulated in breast cancers. Recent investigations revealed that these two proteins may have a role in suppressing breast cancer progression. In this thesis, I will address their role in maintaining epithelial differentiating and inhibiting cell proliferation of breast epithelial cells. The epithelial to mesenchymal transition (EMT) is a developmental program in which epithelial cells downregulate their cell-cell junctions, acquire spindle cell morphology and exhibit cellular motility. In breast cancer, EMT facilitates invasion of surrounding tissues and correlates closely with cancer metastasis and relapse. We found previously that the candidate tumor suppressor hCLCA2 is a p53-inducible proliferation-inhibitor that is frequently lost in breast cancer. We show here that another member of the CLCA gene family, hCLCA4, is expressed in mammary epithelial cells and is similarly downregulated in breast tumors and in breast cancer cell lines. Like CLCA2, the gene is stress-inducible, and ectopic expression inhibits colony formation. Transcriptional profiling studies revealed that hCLCA4 and hCLCA2 together are markers for mammary epithelial differentiation, and both are downregulated by TGF beta. Moreover, knockdown of either on in immortalized cells by shRNAs caused downregulation of epithelial marker E-cadherin, while mesenchymal markers N-cadherin, vimentin, and fibronectin were upregulated, indicating an EMT program. Double knockdown of hCLCA2 and hCLCA4 enhanced the mesenchymal profile. These findings suggest that hCLCA4 and hCLCA2 play complementary but distinct roles in epithelial differentiation. Clinically, low expression of hCLCA2 and hCLCA4 signaled lower relapse-free survival in breast cancers. Cellular senescence is a program of irreversible cell cycle arrest in response to stressors such as DNA damage, ROS, telomere erosion, or oncogene activation. It is one of the primary tumor suppression mechanisms mediated by p53 and is often disabled in cancer cells. However, the downstream signaling pathway whereby p53 induces cellular senescence remains incomplete. We reported previously that hCLCA2 was a p53 inducible gene that is downregulated with breast cancer progression. We and other group noticed that hCLCA2 was induced in parallel with several types of senescence. Lentiviral transduction of CLCA2 into MCF7 cells inhibited cell proliferation and cells showed senescence phenotype. To investigate the mechanism biochemically, we used pAd-Easy to express hCLCA2 in the model breast cancer cell line CA1d. A protein expression profile of these cells over a 6 day period revealed induction of p21, p53, and the DNA damage-response pathway. To test whether hCLCA2 is required for the cellular senescence process, hCLCA2 was knocked down in HMLE. The knockdown cells (KD) and negative control were treated with a low concentration of doxorubicin, and cell proliferation was measured. The KD cells were more resistant to growth inhibition by doxorubicin. Moreover, a time course experiment showed that induction of SA beta-galactosidase, DNA damage response, and lysosomal markers IFI30 and CTSS was delayed in the knockdown cells. These results suggest that hCLCA2 plays an important role in DNA damage response and the senescence program.

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.

A descondensação da cromatina exerce um papel central nas diversas etapas do processo de carcinogênese abrindo o genoma para a ação de fatores de transcrição, exercendo papel na progressão e resistência tumoral. Bromodomínios e proteínas com terminal extra, como o BRD4, são leitores epigenéticos que regulam a expressão gênica e, portanto, também estão envolvidos na patogênese do câncer. O objetivo do presente estudo foi estudar o efeito da inibição do BRD4 no carcinoma espinocelular de cabeça e pescoço (CECP). Para esse propósito, foi utilizado JQ1, inibidor de BRD4, em concentração de 1uM, nas linhagens de carcinoma de cabeça e pescoço HN6, HN12 e HN13. Foi analisado os níveis de BRD4, H4 acetilada e SIRT1 fosforilado através de reações de imunofluorecência e p16ink4 por imunohistoquímica. Foi realizado western blot para checar os níveis de p53 e p53 acetilado. Ensaio de formação de colônias e câmera de invasão foram realizados para testar o efeito do inibidor na proliferação e invasão celular. Através da citometria de fluxo foi analisado o efeito da apoptose com a marcação de caspase-3 clivada, do ciclo celular através da reação por iodeto de propídio e ainda da população de células tronco tumorais pela análise de ALDH e CD44. Por fim, foi realizado modelo xenográfico subcutâneo para analisar o efeito do JQ1. Os resultados mostraram diminuição significativa da expressão de BRD4 e H4ac após tratamento com JQ1. As linhagens celulares mostraram redução na capacidade de invasão e de formação de colônias quando submetidas ao JQ1. Não foram encontradas diferenças em relação ao número de células caspase-3 clivada positivas. Por outro lado, foi encontrado um maior número de células na fase G1 do ciclo celular após o uso do inibidor estudado. As células tratadas com JQ1 mostraram menor expressão de p-SIRT1 o que levou a uma diminuição da acetilação do p53 e um aumento na expressão de p16ink4. Paralelamente, foi encontrado uma diminuição na população de células positivas para ALDH e CD44. Houve diminuição do crescimento do tumor no modelo xenográfico tratado com JQ1 quando comparado ao veículo. Nos tecidos derivados do ensaio in vivo, houve uma diminuição nos marcadores p16ink4, pSIRT1 além de acúmulo de H2AX. Conclui-se que o uso de JQ1 resulta na disrupção do crescimento do CECP associado a ativação de senescência, indução de dano de DNA além de reduzir a população de células tronco tumorais. Esses novos achados indicam que o BRD4 é um importante modificador epigenético nos CECP sendo um viável alvo terapêutico. / Chromatin descondensation plays a central step in the various stages of the carcinogenesis process opening the genome for transcription factors playing a role in tumor progress and resistance. Bromodomains and extra terminal family, as BRD4, are epigenetics readers that regulate gene expression thus they are also involved in cancer pathogenesis. The objective of this project was studied the effect of BRD4 in head and neck squamous cell carcinoma (HNSCC). For this purpose, JQ1, a BRD4 inhibitor, was used in 1uM concentration, in HN6, HN12 and HN13 head and neck carcinoma cell lines. The levels of BRD4, acetylates h4 and phosphorylated SIRT1 were analyzed by immunofluorescence and p16ink4 labeling by immunohistochemistry. Western blot was performed to check the levels of p53 and acetylated p53. Colony assay and invasion chamber were performed to test the inhibitory effect on cell proliferation and invasion. The effect of apoptosis with the cleaved caspase-3 labeling, the cell cycle by propidium iodide and of the population of tumor stem cells by the analysis of ALDH and CD44 was analyzed through flow cytometry. Finally, a subcutaneous xerographic model was performed to analyze the effect of JQ1. A significant decrease in the expression of BRD4 and H4ac was found after application of JQ1. The cell lines results showed a reduction in the capacity of invasion and also formation of colonies when submitted to JQ1. No differences were found in relation to the number of cells caspase-3 cleaved positives. On the other hand, a large number of cells were found in G1 arrest of cell cycle after use of the BRD4 inhibitor studied. Cells treated with JQ1 showed lower expression of p-SIRT1 which led to a decrease in p53 acetylation and an increase in p16ink4 expression. In parallel, a decrease of ALDH and CD44 positive cells population was found. A decrease in tumor growth was discovered when treated by JQ1 if compared to the vehicle. In tissues samples derived from the in vivo assay, there was a decrease in p16ink4, pSIRT1 markers in addition to -H2Ax accumulation. In conclusion JQ1 causes HNSSC tumor growth disruption associated a senescence activation, DNA damage and a reduce number of cancer stem cells. These new findings indicate that BRD4 is an important genetic modifier in HNSSC and is a viable therapeutic target.

Idiopathic pulmonary fibrosis (IPF) is a disease of unknown aetiology, characterised by the progressive and irreversible scarring of parenchymal lung tissue that leads to respiratory failure and death. The disease is understood to be driven by an impaired and aberrant wound healing response, with an inappropriate reactivation of developmental signalling. The greatest risk factor for the disease is age, which is a process intimately associated with an increase in the burden of senescent cells. Such cells acquire a unique secretory phenotype and are known to have a significant impact on their local micro-environment. It was hypothesised that an alteration in epithelial-mesenchymal secretory communication, due to senescent-like changes in the fibroblast phenotype, may detrimentally contribute to lung homeostasis. An in vitro model of the lung airway was established in which primary human lung fibroblasts (HLFs) were co-cultured with human bronchial epithelial cells (HBECs). HBECs were cultured on a semi-permeable, transwell insert and co-cultured with either normal (NHLF), fibrotic (FHLF) or senescent fibroblasts. Over 72 hrs of co-culture, wound healing was assessed, via an epithelial scratch assay, and epithelial regeneration was measured, via trans-epithelial electrical resistance. Co-culture with NHLFs improves epithelial regeneration, however, FHLFs and senescent cells in co-culture show a diminished ability to promote epithelial regeneration and wound repair. The secretory repertoire of these cells contains elevated levels of IL-6, CXCL8, CXCL1 and GCSF (when assessed at both an RNA and protein level), factors strongly associated with the senescent phenotype. Targeting this secretome via treatment with the JAK 1/2 inhibitor Ruxolitinib attenuates these impairments and may point towards a new therapeutic strategy for the treatment of IPF.

Interleukin-1 alpha (IL-1a) is a powerful inflammatory cytokine that modulates both innate and adaptive immunity. As such, IL-1a is implicated in the development of multiple inflammatory and autoimmune diseases including atherosclerosis, arthritis and cancer. Therefore, understanding the mechanisms that regulate IL-1a activity is extremely important. For many years, pro-IL-1a was considered to be a fully active alarmin. However, we have previously shown that the removal of the pro-domain by calpain, a protease that is activated upon necrosis, significantly increases IL-1a bioactivity. The work presented in this thesis demonstrates that multiple proteases from diverse biological systems cleave and activate IL-1a. We therefore suggest that IL-1a is an important signalling hub that integrates diverse proteolytic danger signals to alert the immune system. In particular we have identified the inflammatory caspase, caspase-5, as a novel and potent activator of IL-1a. We show that caspase-5 directly cleaves pro-IL-1a during the activation of the non-canonical inflammasome by cytosolic LPS, which mimics intracellular bacterial infection. We also demonstrate that caspase-5-cleaved IL-1a mediates the senescence-associated secretory phenotype (SASP), which drives the deleterious effects of senescent cells in multiple age-related diseases. Therefore, therapeutically targeting caspase-5 may be of interest for pathologies mediated by the non-canonical inflammasome and/or senescent cells. Finally we find that rs17561, a common IL1A polymorphism, reduces active IL-1a release. We find that blood from minor allele homozygotes releases significantly less IL-1a than major allele homozygotes upon LPS stimulation. Therefore, genotyping patients under consideration for anti-IL-1a therapy could predict who would be likely to respond well to the treatment. In conclusion, the work presented in this thesis enhances our understanding of how IL-1a activity is regulated. The identification of both the caspase-5-mediated pathway of IL-1a activation and the defect conferred by the rs17561 SNP could have important clinical implications for the treatment of multiple inflammatory diseases.

Made available in DSpace on 2014-06-11T19:26:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-11-09Bitstream added on 2014-06-13T20:54:10Z : No. of bitstreams: 1 moraes_fe_me_jabo.pdf: 1680634 bytes, checksum: 35809b1bab51b8d182c7d496667f751f (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A cana-de-açúcar (Saccharum spp.) é uma das gramíneas cultivadas mais importantes do mundo e o Brasil é hoje o maior produtor e exportador mundial de cana, de açúcar de cana e etanol de cana, com o estado de São Paulo sendo o maior produtor. Entretanto, esta cultura é ameaçada por várias pragas e doenças. Dentre essas pragas, uma das mais importantes na região centro-sul é a cigarrinha-das-raízes (Mahanarva fimbriolata), que assumiu este posto devido ao aumento da colheita mecanizada. Por meio da técnica de cDNA-AFLP este trabalho teve como objetivo identificar genes possivelmente envolvidos na resistência a ninfas de Mahanarva fimbriolata em cana-de-açúcar. Para isso, foi utilizada uma variedade suscetível (SP80-1816) e uma resistente (SP83-5073). Ambas foram infestadas com ninfas da cigarrinha 77 dias após o plantio e amostras de raízes foram coletadas antes da infestação (controle) e 1, 2 e 7 dias após a infestação, posteriormente foi feita a extração de RNA, síntese de cDNA e o cDNA-AFLP. Após a análise dos fragmentos foram encontrados 16 fragmentos expressos somente na variedade resistente. Nove fragmentos foram encontrados no 2º dia de infestação e sete no 7º dia. Dentre os 16 fragmentos, sete foram recuperados e sequenciados. Seis fragmentos não apresentaram homologia com proteínas já descritas e um fragmento, de 129 pb, apresentou homologia com uma proteína putativa associada à senescência. Todos os fragmentos podem representar genes relacionados com a resistência da planta contra o inseto / The sugarcane (Saccharum spp.) is the most important grass cultivated in the world and Brazil is now the world’s largest producer and exporter of sugarcane, sugarcane sugar and sugarcane ethanol, with the São Paulo state being the largest producer. However, this culture is threatened by several pests and diseases. Among these pests, one of the most important in south-central region is the froghopper-roots (Mahanarva fimbriolata), that took this position because of increased mechanization. Through cDNA-AFLP technique this study aimed to identify genes possibly involved in resistance to nymphs of Mahanarva fimbriolata in sugarcane. Therefore we used a susceptible variety (SP80-1816) and a resistant (SP83-5073). Both were infested with spitlebugs 77 days after planting and root samples were collected before infestation (control) and 1, 2 and 7 days after infestation, was later made the extraction of RNA, cDNA synthesis and cDNA-AFLP. After fragments analyses were found 16 fragments expressed only in the resistant variety. Nine fragments were found on day 2 of infection and seven on day 7. Among the 16 fragments, seven were recovered and sequenced. Six fragments showed no homology to described proteins and a fragment of 129 bp showed homology to a putative protein associated with senescence. All fragments could represent genes related to the plant resistance against the insect

Made available in DSpace on 2015-03-26T13:36:37Z (GMT). No. of bitstreams: 1 texto completo.pdf: 407532 bytes, checksum: b633523fe7dd877278a161e6da72a14a (MD5) Previous issue date: 2008-10-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Physiological changes and oxidative stress were studied in soybean leaves, Glycine max, variety MG / BR 46 (Conquista) during their development. In a group of plants the reproductive organs were removed, in an attempt to extend their life cycle, retarding their leaf senescence. In the control plants the floweres were not removed and the plants followed their normal cycle until natural senescence. Control plants developed normally, exhibiting progressive degradation of chlorophylls and carotenoids (in low proportion), with the characteristic yellow color of senescent leaves. On the other hand, plants with no flower kept their leaves green until the end of the experiment. The degradation of the pigments led to a sharp decline in carbon assimilation rates (A) in control plants, in which also occurred a decrease in stomatal conductance (gs) and increase in the ratio between the internal and ambient CO2 concentration (Ci/Ca). In contrast, plants without flowers showed a minor drop in A, gs and Ci/Ca. The proportion of electrolyte leakage increased in the control plants throughout their development. Leaves of control plants showed lower levels of hydrogen peroxide (H2O2), in contrast with a great increase observed in plants with no flowers. The low activity of superoxide dismutase (SOD) in control plants in addition to the high activity of peroxidases (POX) and ascorbate peroxidase (APX) are likely responsible for the low levels of H2O2 in those plants. Conversely, the intense activity of SOD and the low performance of POX and APX in plants without flowers might have contributed to the high levels of hydrogen peroxide. Catalase (CAT) activity dropped continuously in the leaves of the two kind of plants, indicating that this enzyme does not play a fundamental role in the removal of reactive oxygen species during this senescence. In both kind of plants, the activity of glutathione reductase had initially increased, followed by a sharp decrease until the end of the experiment. The results of this experiment suggest that oxidative stress is not the determinant factor associated with leaf senescence process in soybean plants. / Foram avaliadas diversas alterações fisiológicas e o estresse oxidativo ao longo do desenvolvimento de folhas de soja, Glycine max, variedade MG/BR 46 (Conquista), em dois diferentes grupos de plantas: com órgãos reprodutores intactos, cujas plantas seguiram o ciclo normal até a senescência e com órgão reprodutores removidos, a fim de prolongar o ciclo de vida, retardando a senescência foliar. Foram avaliados: teores de clorofilas e carotenóides, parâmetros de trocas gasosas, atividades de algumas enzimas do sistema antioxidativo, teores de peróxido de hidrogênio, além de danos celulares. As folhas analisadas das plantas desenvolvendo normalmente apresentaram progressiva degradação de clorofilas e carotenóides (em menor proporção), fato que resultou na coloração amarela característica de folhas senescentes, enquanto as plantas desfloradas mantiveram suas folhas verdes. A degradação dos pigmentos resultou em queda acentuada das taxas de assimilação líquida de carbono (A) nas plantas com órgãos reprodutores intactos, além de ter ocorrido queda na condutância estomática (gs) e aumento na razão entre a concentração interna e ambiente de CO2 (Ci/Ca). Ao contrário, as plantas com órgãos reprodutores removidos apresentaram menor queda em A, gs e Ci/Ca, que se mantiveram constantes após a ligeira queda inicial. O percentual de extravasamento de eletrólitos em plantas em senescência natural aumentou ao longo do desenvolvimento, mas não foi acompanhado de aumento de aldeído malônico (MDA). Em plantas desfloradas o extravasamento foi inicialmente constante, seguido de queda com o início da senescência, mas, por outro lado, os níveis finais de MDA foram duas vezes maiores que os iniciais. As folhas das plantas intactas apresentaram baixos teores de peróxido de hidrogênio (H2O2), ao contrário das plantas desfloradas. A baixa atividade da dismutase do superóxido (SOD) em plantas senescendo normalmente, além das atividades elevadas de peroxidase (POX) e peroxidase do ascorbato (APX), devem ter sido as responsáveis pelos baixos níveis de H2O2 nessas plantas. Inversamente, a atividade intensa de SOD e a baixa atuação de POX e APX em plantas desfloradas contribuíram para os altos teores de peróxido de hidrogênio. A catalase (CAT) teve sua atividade em queda ao longo do experimento, nas folhas de ambos os tratamentos, indicando que a enzima não teve participação importante na remoção de H2O2. Nos trifólios das plantas dos dois tratamentos, a redutase da glutationa (GR) teve sua atividade inicialmente elevada, seguida de queda drástica até o final do experimento. Tais resultados indicam que o estresse oxidativo não foi o fator determinante da senescência foliar natural das plantas de soja utilizadas no presente experimento.

Made available in DSpace on 2016-12-06T17:42:05Z (GMT). No. of bitstreams: 1 PGPV14DA006.pdf: 2258194 bytes, checksum: 11c3c8a7148c0f9fa9b1f7bd89ddfa6f (MD5) Previous issue date: 2014-04-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Defoliation processo d apples trees in the Brazilian conditions occurs naturally throughout autumm. The period is critical to controlo f leaf disease in postharvest. This disease cause premature defoliation and other diseases can to start their infection through injuries of fall leaf. Apart from the physical, the phenomenon on falling leaves involves anatomical and biochemical changes in the base the petiole, called the abscission zone. The objective of this research was to evaluate the effects of defoliation with 1 g L¯¹ ethephon, AVG 0,06 g L¯¹, calcium chloride 23 g L¯¹ and abscisic acid (ABA) 1,5 g L¯¹ in mature apple trees Fuji Standart and Fuji Raku Raku . A method to observe the formation of na abscission layer, and characterize the process was definwed. To follow ecophysiological effects of ethephon and ABA, the activity of peroxidase enzyme and total protein, temporal falling leaves and functionality characterization based on the presence of chlorophyll and nitrogen translocation were determined. The experiments of defoliation were consucted during autumm in comercial apple orchards in Vacaria, RS, in 2011 and 2012 and in Region of Maule, San Clemente, in Chile in 2013. Defoliants as ethephon, AVG, calcium chloride and ABA caused the diferente defoliation progresso n Fuji Standart . The spraying of defoliants in mid-april caused no effect on the buds differentiation and phenology of apple trees. In the situation of drought during summer and fall in the second year, defoliation with ethephon in Vacaria was equal to treatment with ABA in Chile. However, ethephon and calcium chloride as defoliant may affect fruit set and yield of Fuji Standart apple trees. The most effective defloiants were ethephon and ABA. Early spraying of ABA and ethephon on Fuji Raku Raku accelerated senescence process by promoting early defoliation compared to the control. Peroxidase enzyme was found in leaf abscission processo f Fuji Raku Raku . The peak of peroxidase enzyme ocurred in less than 24 hours after ABA spray, whereas to ethephon treatment peak was about 24 hours. Chlorophyll contente, obtained by a chlorophyll meter (SPAD 502) served to assess the functionality of the apple leaves on postharvest. SPAD index below 40 indicate senescente leaves, in the processo f formation of abscission layer. Ethephon and ABA, evenanticipating defoliation, allowed translocation of nitrogen and delas on phenological stages was not observed on apple trees. The processo f leaf abscission zone in apple trtees was observed and characterized by method in this research. The abscission zone occurs in proximal parto f petiole, with flattened and lignificated cells. The formation of the abscission layer occrus toward adaxial to abaxial surface, epidermis toward vascular tissues / O processo de desfolha de macieiras nas condições brasileiras ocorre naturalmente durante todo outono. O período é crítico para o manejo fitossanitário de pós-colheita para o controle de manchas foliares que causam desfolha precoce e de outras doenças que iniciam sua infecção através da lesão da queda da folha. Além de físico, o fenômeno de queda das folhas envolve transformações anatômicas e bioquímicas na base do pecíolo, chamada zona de abscisão. O objetivo desta pesquisa foi avaliar os efeitos da desfolha com etefon 1g L-1, AVG 0,06 g L-1, cloreto de cálcio 23 g L-1 e ácido abscísico (ABA) 1,5 g L-1, em macieiras Fuji Standard e Fuji Raku Raku em idade produtiva. Juntamente foi definido um método para observar a formação da camada de abscisão, e caracterizar este processo. Para acompanhar os efeitos ecofisiológicos do etefon e ABA, foi determinada a atividade das proteínas totais e enzima peroxidase, a queda temporal das folha, e a caracterização da sua funcionalidade com base na presença de clorofila e translocação de nitrogênio. Os experimentos de estudo da desfolha foram conduzidos, durante o outono, em pomares comerciais de macieira em Vacaria, RS, nos anos 2011 e 2012 e no Chile, na região de Maule, em San Clemente, em 2013. Como desfolhantes o etefon, o AVG e o cloreto de cálcio e o ABA causaram diferente progresso da desfolha em Fuji Standard . A pulverização de desfolhante em meados de abril, não causou efeito na diferenciação das gemas e na fenologia das plantas. Na situação de estiagem durante o verão e o outono no segundo ano, a desfolha com etefon em Vacaria foi igual ao tratamento com ABA no Chile. Porém, os defolhantes etefon e cloreto de cálcio podem afetar o pegamento das frutas e a produtividade da macieira cv, Fuji Standard. Os desfolhantes mais eficazes foram etefon e ABA. Quando se avaliou o efeito do ABA e etefon em Fuji Raku Raku , a senescência das folhas foi acelerada, promovendo a desfolha antecipada em comparacão com a testemunha. A enzima peroxidase foi constatada no processo de abscisão foliar de Fuji Raku Raku e teve seu pico de atividade em menos de 24 horas depois da aplicacão no tratamento de ABA, enquanto que etefon foi cerca de 24 horas. Os valores do teor de clorofila, obtidos por um medidor de clorofila (SPAD 502), serviram para estimar a funcionalidade das folhas de macieira após a colheita das frutas. Índices SPAD abaixo de 40 indicam folhas senescentes, em processo de formação da camada de abscisão. Etefon e ABA mesmo antecipando a desfolha, permitiram a translocação de nitrogênio e não causaram atrasos nos estágios fenológicos da macieira. O método utilizado para o estudo da abscisão permitiu observar e caracterizar a zona de abscisão foliar em macieiras durante todo o processo. Observou-se que a zona de abscisão ocorre na parte proximal do pecíolo, apresentando células achatadas e em processo de lignificação. A formação da camada de abscisão dá-se no sentido da face adaxial para a face abaxial, da epiderme em direção aos tecidos vasculares