L’entose est une forme de mort cellulaire non apoptotique caractérisée par l’internalisation d’une cellule cible vivante dans une cellule hôte vivante. Ce processus de cannibalisme cellulaire qui est également connu sous le nom de « cellule dans une cellule » est retrouvé dans de nombreux cancers humains. Au cours de mes travaux de thèse, nous avons développé différents modèles d’entose in vitro et avons débuté l’identification des protéines qui répriment l’entose en combinant un criblage de petits ARN interférants à une approche de microscopie confocale. Nous avons découvert que la protéine suppressive de tumeur TP53 ainsi que son isoforme Δ133TP53 bloquent le processus d’internalisation cellulaire et l’entose. La perte de l’expression de la protéine TP53 ou de Δ133TP53 entraîne une libération extracellulaire d’adénosine triphosphate ainsi que l’activation du récepteur purinergique P2Y2, deux évènements cellulaires qui aboutissent à l’internalisation d’une cellule par une autre cellule. De plus, nous avons constaté que les cellules cannibales deviennent énescentes à la suite de l’induction de la protéine p21WAF1.Mes travaux de recherche révèlent l’existence d’une nouvelle modalité d’induction de la sénescence cellulaire. De façon surprenante, nous avons également observé que l’induction de la sénescence par l’oncogène RasV12ou à la suite de stress (réplicatifouoxydatif) déclenchait le cannibalisme cellulaire, suggérant que le cannibalisme cellulaire est une caractéristique des cellules sénescentes. L’ensemble de mes travaux de recherche souligne le lien étroit qui existe entre le cannibalisme cellulaire et la senescence. / Entosis is a non-apoptotic cell death process of live internalized cell inside the host/cannibal cell. In human cancers, commonly "cell-in-cell" cytological features have been observed over the period of time. In this study we have established in vitro models of entosis and initiated the identification of entotic repressors by developing fluorescent confocal microscopy screening of small interfering RNA. We identified that TP53 and one of its isoform 133TP53 specifically inhibits the cell internalization. Loss of TP53 or 133TP53 expression increases extracellular ATP release and the consequent activation of purinergic P2Y2 receptors, which signal for engulfment. Cannibal cells activate a senescence program through p21WAF1 induction, revealing a new modality of induction of cellular senescence that can occur in the absence of TP53 or 133TP53. Senescence induced by oncogenic RasV12 and by replicative or oxidative stresses also results in cellular cannibalism, suggesting that cannibalism is a common feature of senescent cells. Altogether, our results provide evidence that cellular cannibalism and senescence are tightly linked.

A técnica de cultura de tecidos permite a propagação rápida e maciça de propágulos geneticamente semelhantes, isentos de doenças, sendo amplamente empregada para a obtenção de gemas adventícias e embriões somáticos visando principalmente, a multiplicação clonal sob ação de reguladores de crescimento. Resultados satisfatórios foram obtidos com a espécie Bactris gasipaes Kunth. por meio da regeneração direta de gemas adventícias e de embriões somáticos, no entanto, as consequências decorrentes da prolongada manutenção in vitro de espécies perenes como a pupunheira, não estão elucidadas, sendo que as pesquisas mais expressivas ocorrem com espécies anuais e restritas a órgãos específicos. Considerando que o tempo de cultivo pode promover a senescência e a habituação de determinados tecidos aos reguladores de crescimento, afetando consideravelmente o potencial morfogênico, o objetivo principal do presente trabalho foi investigar a ocorrência destes processos em folhas, raízes e bases caulinares de plântulas e microplantas com um e oito anos de cultivo, respectivamente. Para tanto, o processo de senescência foi monitorado por meio de análises histológicas, ultraestruturais e histoquímicas à detecção de substâncias ergásticas e à fragmentação do DNA, ao passo que o processo de habituação, foi monitorado por meio de análises morfofisiológicas, histológicas e histoquímicas. Os resultados pertinentes ao processo de senescência evidenciaram a ocorrência de intenso processo de morte celular programada nas células de diversos tecidos nas estruturas analisadas das microplantas, sendo que estes eventos foram escassos e limitados às bases caulinares nas plântulas. Além disso, foi observada a presença elevada de plastoglóbulos no interior dos cloroplastos e de compostos fenólicos nas estruturas foliares e radiculares das microplantas. Já, em relação aos resultados obtidos à detecção do processo de habituação nestas microplantas, quando comparados às plântulas, foram detectados problemas relacionados ao alongamento da parte aérea e do sistema radicular, bem como alterações morfológicas nas raízes e uma pronunciada redução no potencial morfogênico das células pré-procambiais em relação às plântulas. Estes resultados evidenciam que a manutenção in vitro de pupunheiras por longos períodos promoveu o envelhecimento dos propágulos em decorrência à senescência generalizada, bem como provavelmente os conduziu ao processo de habituação aos reguladores de crescimento ANA e/ou BAP, inviabilizando a propagação em grande escala desta espécie. / The tissue culture technique allows rapid and massive spread of propagules genetically similar, free from diseases, being the technique widely employed to obtain adventitious buds and somatic embryos mainly targeting the clonal multiplication under the action of growth regulators. Satisfactory results have been obtained with the specie Bactris gasipaes Kunth. through direct regeneration of adventitious buds and somatic embryos, however, the consequences from prolonged in vitro maintenance of perennial species such as peach palm are not clear, and the most significant research occur with annual species and restricted to specific organs. Whereas the cultivation time can promote senescence and habituation of certain tissues to the growth regulators, affecting considerably the morphogenic potential, the main objective of this study was to investigate the occurrence of these processes in leaves, roots and stem bases of seedlings and microplants with one and eight years of cultivation, respectively. Thus, the senescence process was monitored by histological, ultrastructural and histochemical detection of ergastic substances and DNA fragmentation, whereas the habituation process was monitored by analyses histologic, histochemic and morpho-physiological. The relevant results from the senescence process showed the occurrence of an intensive process of programmed cell death in cells of various tissues of the analised microplants structures, and these events were rare and limited to the stem bases in the seedlings. Furthermore, it was observed the high presence of plastoglobules inside chloroplast and phenolic compounds in the leaf and root structure of microplants. Already, the results obtained in relation to the detection of the habituation process in these microplants, when compared to the seedlings, were detected problems related to the elongation of shoots and roots, as well as morphological changes in roots and a pronounced reduction in the morphogenic potential of pre procambial cells compared to seedlings. These results demonstrate that the in vitro maintenance of peach palm for long periods promoted the aging of seedlings due to senescence widespread and probably led to the habituation process, to the growth regulators NAA and/or BAP, difficulting the propagation on a large scale of this species.

Die schlechte Prognose der meisten disseminierten Tumorerkrankungen ist häufig in einer vorbestehenden oder erworbenen Resistenz gegenüber Zytostatika begründet. Da die meisten Zytostatika mit zellulären Strukturen interagieren, war lange angenommen worden, dass der antineoplastische Effekt unmittelbar durch massive Zellschädigung bewirkt wird. Hieraus folgte, dass Chemoresistenz auf Mechanismen beruhen müsse, welche das Zytostatikum an der Wechselwirkung mit seiner intrazellulären Zielstruktur hindern. Arbeiten der letzten Jahre haben jedoch gezeigt, dass die meisten Zytostatika indirekt über DNA-Schädigung ein relativ uniformes, genetisch kodiertes Zelltod-Programm auslösen, demzufolge postuliert wurde, dass auch Apoptosedefekte für "Multi-Drug-Resistenz" verantwortlich sein könnten. Allerdings ist der tatsächlich Beitrag zytostatika-induzierter Apoptose am Therapieerfolg nicht geklärt, wobei der Wahl geeigneter Testsysteme eine wesentliche Bedeutung für diese Kontroverse zuzukommen scheint. Gegenstand der vorliegenden Arbeit ist daher die Etablierung eines transgenen Lymphom-Modells, in welchem chemotherapeutische Effekte an spontan entstandenen Tumoren mit definierten genetischen Läsionen in ihrer natürlichen Umgebung untersucht werden können. Hierbei konnte gezeigt werden, dass Mutationen in apoptose-relevanten Genloci wie p53, INK4a/ARF oder bcl2 sowohl die Manifestation myc-transgener Lymphome dramatisch beschleunigen, als auch den Therapieerfolg kompromittieren. Neben Apoptose wurde darüberhinaus prämature Seneszenz, ein terminaler Zellzyklus-Arrest, als prognose-relevantes Chemotherapie-Effektorprogramm identifiziert. Damit dokumentiert die vorliegende Arbeit einen wichtigen Zusammenhang von Gendefekten, die während der Tumorigenese erworben wurden, und später evidenter Chemoresistenz, wobei manche Mutationen bereits vor Zytostatika-Exposition Resistenz begründen können. Die Identifikation und pharmakogenomische Charakterisierung potentiell resistenz-vermittelnder Gene und Mutationen in relevanten Testsystemen wird für die Entwicklung spezifischerer, aber weniger toxischer "targeted Therapeutics" von großer Bedeutung sein. / Intrinsic or acquired chemoresistance is the major cause for the adverse outcome of disseminated malignancies. The fact that most anticancer agents bind to subcellular targets prompted the assumption that drug-induced cytotoxicity must be a direct consequence of severe cellular damage. Hence, chemoresistance was thought to arise from mechanisms that prevent or disrupt the drug-target interaction. By contrast, more recent data suggested that DNA damage caused by most, if not all, anticancer agents may trigger a relatively uniform, genetically encoded cell death program. In turn, defects in the apoptotic machinery should account for multi-drug resistance as well. However, due to technical limitations of current test systems, it has been difficult to assess the overall contribution of apoptotic cell death to treatment outcome. In the studies presented here, a transgenic mouse lymphoma model was established in order to exploit drug responses of spontaneously developed malignancies growing at their natural sites but harboring defined genetic defects. Using this model, alterations in apoptosis-related gene loci such as p53, INK4a/ARF or bcl2 result in both dramatic acceleration of myc-driven lymphomagenesis and compromised treatment responses. Importantly, not only apoptosis, but premature senescence, a terminal cell-cycle arrest, was found to impact on treatment outcome. In essence, this work describes and important connection between cancer genes and cancer therapy, i.e. genetic defects acquired during tumorigenesis may already co-select for chemoresistance prior to any drug encounter. The identification and pharmacogenomic evaluation of resistance conferring candidate genes and mutations using adequate test systems is likely to play a key role in the development of novel, more specific but less toxic so called "targeted Therapeutics".

A goiaba é uma fruta saudável para consumo, oferece níveis elevados de licopeno, vitamina C e fibras e constitui-se numa boa opção ao processamento mínimo. Entretanto, o ponto de colheita e o controle da senescência são questões importantes para o sucesso do processamento. O objetivo do trabalho foi definir o melhor estádio de maturação para o processamento mínimo, em rodelas, de goiabas Kumagai e Pedro Sato, bem como utilizar meios de controlar a senescência desses frutos, como o 1- Metilciclopropeno (1-MCP) e atmosfera modificada. Primeiramente, foi conduzida uma avaliação dos melhores estádios de maturação das goiabas para o processamento mínimo. Os frutos de ambas as variedades foram colhidos em 3 estádios de maturação definidos pela cor da casca em verde, verde-claro e verde-amarelado. Análises físicoquímicas e sensoriais ocorreram no início do experimento e a cada 3 dias durante 9 dias. As goiabas do estádio verde obtiveram notas abaixo do limite de aceitabilidade quanto à aparência durante as avaliações. Porém, nas goiabas dos estádios de maturação mais avançados, foram observados intensa perda de firmeza e escurecimento da polpa na região placentária, características de senescência. No segundo experimento, goiabas dos estádios verde-claro e verde-amarelado foram submetidas, antes do processamento, ao tratamento com 1-MCP por 0, 3, 6 e 12 horas, para escolha do tempo ideal de exposição dos frutos ao produto. As análises físicoquímicas seguiram de acordo com o experimento anterior. A determinação da atividade respiratória e da produção de etileno foram realizadas diariamente durante 9 dias. O pico respiratório e a elevação da concentração de etileno foram evidenciados no dia do processamento em ambas as variedades e em todos os tratamentos. Isto ocorre, provavelmente, devido às injúrias ocasionadas pelo processamento. Contudo os tratamentos em que as goiabas ficaram expostas ao 1-MCP por 12 horas reduziram a atividade respiratória e a produção de etileno das goiabas, mantendo a qualidade físicoquímica durante o armazenamento. No terceiro experimento foram estudados seis materiais de embalagem, os quais foram selecionados em função da manutenção de qualidade dos produtos minimamente processados. As análises físico-químicas ocorreram no início e ao final dos 9 dias de armazenamento. Nesse período, o monitoramento da composição gasosa foi realizado diariamente. As embalagens de polipropileno e polietileno de baixa densidade, sob atmosfera modificada passiva, foram eficientes, permitindo a conservação e manutenção da qualidade das goiabas. No último experimento foi avaliada a combinação dos melhores resultados obtidos anteriormente. O processamento mínimo das duas variedades ocorreu simultaneamente, com objetivo de se obter um mix de goiabas brancas e vermelhas, proporcionando, assim, um visual mais atrativo. Goiabas do estádio verde-amarelado, minimamente processadas, tratadas por 12 horas com 1-MCP e embaladas com filme de polipropileno de 52µm conseguiram manter sua qualidade, viabilizando, assim, a combinação das técnicas de controle da senescência. / The guava is a healthy fruit, offer high levels of lycopene, vitamin C and fibers, being a good option to the minimal processing. However, the harvest point and senescence control are important for the processing success. The objective of this work was to define the best ripening stage for the minimal processing, in round slices, of Kumagai and Pedro Sato guavas, as well as means of controlling the senescence of theses fruits, as the 1-methylcyclopropene (1-MCP) and modified atmosphere. Firstly, the evaluation of the best ripening stages was conducted. The fruits of both varieties were harvested at 3 ripening stages defined by the skin color in green, light-green and yellowish-green. In the beginning of the experiment, physical-chemical and sensorial analyses took place every 3 days during 9 days. The green ones obtained low acceptability grades as to their appearance during evaluations. However, the yellowishgreen ones lost their firmness and gained a browning flesh. In the second experiment, the light-green and yellowish-green guavas were submitted, before the processing, to treatment with 1-MCP per 0, 3, 6 and 12 hours, in order to choose the best time of exposure of the fruit to the product. The physical-chemical analyses were equal to the previous experiment. The respiratory activity and ethylene production were achieved daily during 9 days. The respiratory peak and the increase of ethylene concentration were observed in the day of the processing in both varieties as well as in all treatments, probably due to injuries caused by the cuts. However, the treatment in which the guavas were exposed to 1-MCP for 12 hours had their respiratory activity and ethylene production reduced, thus keeping the physical-chemical qualities storage. In the third experiment, six packaging materials were studied, being selected according to the quality of the guavas. The physical-chemical analyses took place in the beginning and the end of the 9 days of storage. In this period the monitoring of gas composition was accomplished daily. The packagings of polypropylene and low density polyethylene films, under passive modified atmosphere, were efficient in keeping the quality of guavas. In the last experiment, the combination of the best results of the previous experiments was evaluated. The minimal processing of the two varieties was performed simultaneously, obtaining a mix of white and red guavas, thus achieving a more attractive look. Yellowish-green guavas treated with 1-MCP per 12 hours and packed with polypropylene film (52µm) the keeping the quality, thus enabling the combination of two techniques of senescence control.

Senescent cells interact with the surrounding microenvironment achieving both pro- oncogenic and tumour-suppressive outcomes. In addition to autocrine and paracrine signalling mediated by factors of the senescence-associated secretory phenotype (SASP), we have recently identified that NOTCH1 can drive a unique form of senescence in adjacent cells via juxtacrine signalling. Here, we show that NOTCH1 signalling confers a dramatic impact on chromatin structure during senescence. RAS-induced senescent (RIS) fibroblasts often develop chromatin structures called senescence-associated heterochromatic foci (SAHF). We find that NOTCH1 inhibits SAHF formation at least partially through transcriptional repression of a critical structural component, high-mobility group A (HMGA). Using ATAC-sequencing (assay for transposase accessible chromatin) we demonstrate that nucleosome positioning is substantially altered in RIS and that this re-distribution is also antagonised by NOTCH1, resulting in a distinct chromatin landscape. Importantly, normal or cancer cells that express the NOTCH ligand jagged-1 can drive similar chromatin structural changes in adjacent cells in a cell-cell contact dependent manner. In addition, using a highly optimised chromatin immunoprecipitation (ChIP-seq) protocol and the proximity ligation assay ‘Hi-C’, we demonstrate that HMGA proteins are directly involved in the formation of long-range interactions in RIS cells that may underpin SAHF formation. These ChIP-seq data have also allowed us to identify a unique HMGA1 binding profile, potentially suggesting a novel role for HMGA1 in gene regulation. Together, our data indicate that NOTCH signalling, both cell-autonomously and non-cell-autonomously, can repress HMGA1, a multi-faceted protein that regulates nucleosome positioning (1D structure), SAHF formation (3D structure) and potentially mRNA abundance.

Ce travail scientifique a abordé la problématique du vieillissement des îlots pancréatiques et l’effet de la senescence endothéliale et des microparticules (MPs) sur la fonction des îlots. Nous avons exploré l’impact du vieillissement du pancréas sur la morphologie, le devenir et la fonction de l’îlot pancréatique par analyse comparative entre pancréas de rats jeunes et d’âge moyen et le rôle des MPs endothéliales pro-sénescentes sur la fonction des îlots et leur sénescence prématurée. Nos résultats in vivo montrent que le pancréas est un organe précocement sensible au stress oxydant s’accumulant avec l’âge. Il conduit à la surexpression des marqueurs procoagulants et de senescence sans apparition d’apoptose. In vitro, les MPs de cellules endothéliales sénescentes ont un effet pro-sénescent sur les îlots pancréatiques isolés de rats jeunes avec une activité SA-β-galactosidase caractéristique, la surexpression des marqueurs p53, p21 et p16 et la réduction de la capacité de la sécrétion d’insuline en réponse au glucose. L’ensemble de nos résultats in vivo et in vitro désigne la contribution de la sénescence endothéliale comme une cause probable à la dysfonction de greffon. / This scientific work has tackled the question of the pancreatic islets aging and the effect of endothelial senescence and microparticles (MPs) on islet function. We investigated the impact of aging on pancreas morphology, fate and on the function of the pancreatic islet by comparative analysis between pancreas in young and middle-aged rats, as well as the role of pro-senescent endothelial MPs on islet function and their premature senescence. Our in vivo data show that the pancreas is an early sensitive organ to oxidative stress accumulating with age and leading to overexpression of the procoagulant and senescence markers without appearance of apoptosis. In vitro, MPs of senescent endothelial cells have a pro-senescent effect on pancreatic islets isolated from young rats with characteristic SA-β-galactosidase activity, overexpression of p53, p21 and p16 markers and reducing the ability of insulin secretion in response to glucose. Altogether, our in vivo and in vitro data indicate the contribution of endothelial senescence as a possible contributor to graft dysfunction.

La sénescence est un état d’arrêt prolifératif mis en place par les cellules en réponse à des dommages à l’ADN. Elle est considérée comme un mécanisme de protection qui s’oppose à l’initiation et au développement d’un cancer. Or, les mécanismes de sénescence et la capacité des cellules à s’échapper de cet état et à générer des cellules transformées semblent varier selon les types cellulaires. Chez les kératinocytes humains normaux de peau (NHEKs), la sénescence est transitoire et débouche pour la plupart des cellules sur une mort par autophagie et, pour environ une sur dix mille, sur une émergence néoplasique post-sénescence. Les cellules émergentes présentent des caractères de transformation et accumulent des mutations et des délétions. Cet échappement néoplasique de la sénescence n’est jamais observé dans les fibroblastes normaux de peau (NHDFs) qui, au contraire, une fois en sénescence sont bloqués irréversiblement dans le cycle cellulaire.J’ai participé dans un premier temps à l’étude du rôle de l’autophagie dans la balance échappement néoplasique et mort des NHEKs sénescents. Nous avons pu démontrer que les progéniteurs de cellules néoplasiques ont une activité autophagique modérée plus faible que ceux qui subissent la mort. Ainsi, ils échappent à la mort par autophagie tout en gardant un niveau d’activité autophagique de ménage suffisant pour éliminer leurs composés altérés par le stress oxydant et être capable de ré-entrer en mitose.J’ai ensuite cherché à caractériser les dommages oxydants mutagènes impliqués dans l’échappement néoplasique. Ma stratégie a été d’analyser de façon comparative les NHEKs par rapport aux NHDFs, puisque les uns mais non les autres développent une émergence néoplasique. J’ai ainsi pu constater que le taux de cassures augmente à la sénescence dans les deux types cellulaires, mais que ces cassures sont de nature différente, uniquement des SSBs (Single Strand Breaks) pour les NHEKs et principalement des DSBs (Double Strand Breaks) pour les NHDFs. L’accumulation de DSBs à la sénescence des NHDFs s’accompagne d’une induction robuste de la voie DDR (DNA Damage Response), d’une activation la voie p53-p21 et d’un arrêt stable dans le cycle cellulaire. Dans le cas des NHEKs, l’augmentation du taux de SSBs est la conséquence de l’augmentation du niveau de stress oxydant et de la perte de l’expression et de l’activité de la PARP1. Ceci contribue à une agglomération aberrante de XRCC1 au niveau des cassures engendrant une induction de la voie p38MAPK - p16INK4a et un arrêt dans le cycle cellulaire caractéristique de la sénescence. D’une manière paradoxale, l’échappement néoplasique de la sénescence dépend également de cette accumulation de SSBs non réparés. Ainsi, la nature des dommages à l’ADN influence le devenir des cellules sénescentes. Les DSBs renforcent la stabilité de l’arrêt du cycle cellulaire alors que les SSBs promeuvent l’acquisition de mutations et l’échappement néoplasique. / Senescence is a permanent cell-cycle arrest activated in response to DNA damage. If a cell escapes from this state, it should inherit mutations and could potentially initiate a tumor. NHDFs (Normal Human Dermal Fibroblasts) display a classical irreversible and stable senescence plateau. In contrast, senescent NHEKs (Normal Human Epidermal Keratinocytes) experience two different outcomes. Most of them undergo autophagic cell death and about one on 10000 spontaneously resumes mitosis and generates clones of transformed, mutated and tumorigenic cells.I contributed in a first time to studying the role of macroautophagy in the cell death / post-senescence neoplastic emergence balance of senescent NHEKs. We have shown that macroautophagy plays antagonistic roles during senescence, inducing cell death or promoting neoplastic transformation, depending on its level of activation. Indeed, the progenitors of post-senescent emergent cells display oxidative stress and autophagic activity levels slightly lower than the average, what allows them to avoid autophagic cell death and to ensure the quality control indispensable for mitosis re-entry.Since oxidative stress is the motor of the post-senescence neoplastic emergence in NHEKs, I wondered next whether oxidative stress could operate through the generation of some mutagenic DNA damage. I took advantage of the comparison of senescent NHEKs to NHDFs. I have shown that unlike NHDFs, NHEKs do not suffer from significantly shortened telomeres, nor accumulate DSBs, do not activate a DDR (DNA Damage Response) pathway and in consequence do not significantly activate the p53/p21 pathway. Instead, they suffer from a decrease in PARP1 expression, which compromises the repair of SSBs generated by oxidative stress. In consequence, SSBR foci, precisely XRCC1 foci, become persistent. These persistent foci initiate a signalization, through p38MAPK, which leads to up-regulation of p16INK4A and to cell cycle arrest. Notably, the accumulation of unrepaired SSBs is sufficient for the post-senescence neoplastic emergence phenomenon, in addition, paradoxically to its involvement in the onset of senescence.In conclusion, senescence results from the persistence of a DNA damage signalization, but the exact nature of the damages could vary in different cell types depending on their repair capacities and could dictate completely different outcomes. Namely, persistent DSBs, including telomeric ones, dictate a permanent tumor-suppressor cell cycle arrest, whereas persistent SSBs are permissive to mutation and senescence evasion.

A goiaba é uma fruta saudável para consumo, oferece níveis elevados de licopeno, vitamina C e fibras e constitui-se numa boa opção ao processamento mínimo. Entretanto, o ponto de colheita e o controle da senescência são questões importantes para o sucesso do processamento. O objetivo do trabalho foi definir o melhor estádio de maturação para o processamento mínimo, em rodelas, de goiabas Kumagai e Pedro Sato, bem como utilizar meios de controlar a senescência desses frutos, como o 1- Metilciclopropeno (1-MCP) e atmosfera modificada. Primeiramente, foi conduzida uma avaliação dos melhores estádios de maturação das goiabas para o processamento mínimo. Os frutos de ambas as variedades foram colhidos em 3 estádios de maturação definidos pela cor da casca em verde, verde-claro e verde-amarelado. Análises físicoquímicas e sensoriais ocorreram no início do experimento e a cada 3 dias durante 9 dias. As goiabas do estádio verde obtiveram notas abaixo do limite de aceitabilidade quanto à aparência durante as avaliações. Porém, nas goiabas dos estádios de maturação mais avançados, foram observados intensa perda de firmeza e escurecimento da polpa na região placentária, características de senescência. No segundo experimento, goiabas dos estádios verde-claro e verde-amarelado foram submetidas, antes do processamento, ao tratamento com 1-MCP por 0, 3, 6 e 12 horas, para escolha do tempo ideal de exposição dos frutos ao produto. As análises físicoquímicas seguiram de acordo com o experimento anterior. A determinação da atividade respiratória e da produção de etileno foram realizadas diariamente durante 9 dias. O pico respiratório e a elevação da concentração de etileno foram evidenciados no dia do processamento em ambas as variedades e em todos os tratamentos. Isto ocorre, provavelmente, devido às injúrias ocasionadas pelo processamento. Contudo os tratamentos em que as goiabas ficaram expostas ao 1-MCP por 12 horas reduziram a atividade respiratória e a produção de etileno das goiabas, mantendo a qualidade físicoquímica durante o armazenamento. No terceiro experimento foram estudados seis materiais de embalagem, os quais foram selecionados em função da manutenção de qualidade dos produtos minimamente processados. As análises físico-químicas ocorreram no início e ao final dos 9 dias de armazenamento. Nesse período, o monitoramento da composição gasosa foi realizado diariamente. As embalagens de polipropileno e polietileno de baixa densidade, sob atmosfera modificada passiva, foram eficientes, permitindo a conservação e manutenção da qualidade das goiabas. No último experimento foi avaliada a combinação dos melhores resultados obtidos anteriormente. O processamento mínimo das duas variedades ocorreu simultaneamente, com objetivo de se obter um mix de goiabas brancas e vermelhas, proporcionando, assim, um visual mais atrativo. Goiabas do estádio verde-amarelado, minimamente processadas, tratadas por 12 horas com 1-MCP e embaladas com filme de polipropileno de 52µm conseguiram manter sua qualidade, viabilizando, assim, a combinação das técnicas de controle da senescência. / The guava is a healthy fruit, offer high levels of lycopene, vitamin C and fibers, being a good option to the minimal processing. However, the harvest point and senescence control are important for the processing success. The objective of this work was to define the best ripening stage for the minimal processing, in round slices, of Kumagai and Pedro Sato guavas, as well as means of controlling the senescence of theses fruits, as the 1-methylcyclopropene (1-MCP) and modified atmosphere. Firstly, the evaluation of the best ripening stages was conducted. The fruits of both varieties were harvested at 3 ripening stages defined by the skin color in green, light-green and yellowish-green. In the beginning of the experiment, physical-chemical and sensorial analyses took place every 3 days during 9 days. The green ones obtained low acceptability grades as to their appearance during evaluations. However, the yellowishgreen ones lost their firmness and gained a browning flesh. In the second experiment, the light-green and yellowish-green guavas were submitted, before the processing, to treatment with 1-MCP per 0, 3, 6 and 12 hours, in order to choose the best time of exposure of the fruit to the product. The physical-chemical analyses were equal to the previous experiment. The respiratory activity and ethylene production were achieved daily during 9 days. The respiratory peak and the increase of ethylene concentration were observed in the day of the processing in both varieties as well as in all treatments, probably due to injuries caused by the cuts. However, the treatment in which the guavas were exposed to 1-MCP for 12 hours had their respiratory activity and ethylene production reduced, thus keeping the physical-chemical qualities storage. In the third experiment, six packaging materials were studied, being selected according to the quality of the guavas. The physical-chemical analyses took place in the beginning and the end of the 9 days of storage. In this period the monitoring of gas composition was accomplished daily. The packagings of polypropylene and low density polyethylene films, under passive modified atmosphere, were efficient in keeping the quality of guavas. In the last experiment, the combination of the best results of the previous experiments was evaluated. The minimal processing of the two varieties was performed simultaneously, obtaining a mix of white and red guavas, thus achieving a more attractive look. Yellowish-green guavas treated with 1-MCP per 12 hours and packed with polypropylene film (52µm) the keeping the quality, thus enabling the combination of two techniques of senescence control.

Cellular senescence is a distinct cell state, characteristic by cessation of cell proliferation and it is accompanied by specific morphological and biochemical alterations. Increasing and persisting incidence of senescence cells has been shown to have detrimental effect on an organism largely contributing to its ageing. Senescent cells also positively support tumour growth and can even stimulate carcinogenic transformation of surrounding cells. Moreover, senescence can be induced even in tumour cells spontaneously or by chemotherapy. Regardless of an initial stimuli and type of cells, there are two main senescence inducing pathways p16/pRb and p53/p21. Both senescent cells as well as senescent cancer cells seems to have modified apoptotic signalling at the level of mitochondria and Bcl-2 family proteins. In this study, we aimed to analyse effect of senescent state as well as pre-senescent (growth arrested state) induced by p16/pRb and p53/p21 signalling pathways on the response of H28 mesothelioma cancer cells-derived clonal cultures to various cell death-inducing stimuli. By inducible expression of p16 and p21 proteins in doxycycline-dependent manner, we forced cells to acquire senescent-like phenotype, which we detailly characterised. Our results showed that senescent-like phenotype, manifests...

In autologous stem cell therapy, cell characteristics determine the potency of stem cells for regeneration. Aging and ischemia are two factors that are often neglected in pre-clinical tests for stem cell therapy. Here, we characterized cardiac explant-derived cells (EDCs) with a focus on distinguishing the effect of age and ischemia and then we looked for the effects of the combination of the two factors. We observed that ischemia worsens the age effect on EDCs. EDCs that were derived from aged mice with a history of myocardial infarction showed the highest number of senescent cells with dysregulation of the DNA repair system resulting in activation of cell cycle checkpoints. We over-expressed the anti-senescence Mybl2 transcription factor in EDCs from ischemic aged mice. The senescent state, paracrine profile and superoxide dismutase antioxidant enzyme activity improved in these cells. In vivo, we observed a boost in the potency of the Mybl2-modified EDCs, with an increase in short-term engraftment leading to improved heart function in infarcted mice. In general, Mybl2 over-expression rejuvenates senescent EDCs.