The development of cellular senescence is not consistent in cultured bovine fibroblasts. Numerous factors may be contributing to the variable onset of cellular senescence, including oxidative stress, telomere shortening and DNA damage. Recent results indicate that cellular senescence is also associated with markers of DNA replication. This suggests that cells may be more likely to enter a senescent state depending on their cell cycle progression. The purpose of this study was to determine the effect of cell cycle phase on the development of stress-induced premature cellular senescence (SIPS). Bovine fibroblasts were synchronized at various cell cycle phases, followed by treatment with increasing doses of H2O2. Senescent cells were detected using SA-β-galactosidase staining assay. As H2O2 dosage increased, the amount of cell death by necrosis increased in both unsynchronized and cell cycle synchronized groups, while the amount of senescence varied depending on cell cycle phase. Our results suggest that the S phase of the cell cycle is the most resistant to oxidative damage, the G2/M phase is the most susceptible, and the G1/S phase is the most likely to enter senescence as a protective measure following low doses of H2O2. Increased senescence also resulted from an increased recovery time post-H2O2 treatment, and gene expression studies suggest SIPS bovine fibroblasts senesce via the p53-independent pathway. An improved understanding of SIPS has important implications for improving cloning efficiency and understanding age-related diseases.

Magistrinio darbo problema – ne tik Lietuvai, bet ir visam pasauliui būdingas demografinis gyventojų senėjimas. Senstanti visuomenė susiduria su tokiomis problemomis, kaip pagyvenusių ir senų žmonių sveikatos apsauga, pagalbos teikimas, jų išstūmimo iš darbo rinkos didėjimas bei pačios visuomenės neigiamas požiūris. Magistriniame darbe siekiama įvertinti individualius žmonių poreikius senatvėje, išsiaiškinti gerovės senatvėje sąlygas bei nustatyti šių sąlygų tendencijas ir kryptis. Duomenys, mokslinė literatūra analizuojami, taikant analitinį – aprašomąjį bei lyginamąjį metodus, atliekant makroaplinkos veiksnių analizę ir anketinę apklausą. Tyrimo objektas - Utenos apskritis, periodas – 1995 – 2006 metai. Magistrinis darbas sudarytas iš įvado, penkių skyrių, išvadų, rekomendacijų, literatūros sąrašo, santraukos lietuvių ir anglų kalbomis bei priedų. Pirmojoje darbo dalyje analizuojami, lyginami ir apibendrinami įvairioje mokslinėje literatūroje pateikiami pagyvenusių ir senų žmonių sąvokos apibrėžimai, atskleidžiama senėjimo problema, pagyvenusių žmonių gerovės samprata bei požiūris į senatvę. Taip pat pateikta lyginamoji mokslinės literatūros, nagrinėjančios vyresnio amžiaus žmonių socialines ir ekonomines problemas, jų individualius poreikius senatvėje, analizė ir apibendrinimai. Antroji darbo dalis skirta išanalizuoti ir palyginti pagyvenusių ir senų žmonių gerovę reglamentuojančią Lietuvos Respublikos, Europos Sąjungos, Jungtinių Tautų teisinę bazę. Trečiojoje darbo... [to full text] / The problem analyzed in the Master’s Degree paper is the demographic senescence of the population, specific not only to Lithuania but throughout the world. The senescent community encounters the following problems: health care of the elderly, service provision, elimination of the elderly from the labor market, negative attitude of the society. The aim of the paper is to determine the individual needs of the elderly, define welfare conditions, tendencies and trends. Analytical - descriptional, as well as comparative methods were used, while analyzing the data and scientific literature. Besides, the analysis of macro environment factors and questionnaires were used. The object of the paper is Utena County. Time period is 1995-2006. The paper is comprised of the introduction, five parts, conclusions, recommendations, literature list, summaries, both, Lithuanian and English, and the annexes. In the first part of the paper the concepts of the elderly and senior people are analyzed, compared and generalized on the basis of the examples from the scientific literature. The problem of the senescence is revealed, as well as the attitude of the elderly towards welfare and the old age is presented. Furthermore, comparative analysis and generalizations of the social and economic problems and individual needs of the elderly are presented. In the second part the analysis and comparisons of the legislation of Lithuania, the European Union and the United Nations concerning the welfare of the... [to full text]

Nicotine, nornicotine, anabasine and anatabine are four major alkaloids in tobacco, of which nicotine is predominant. In many tobacco cultivars and also in other Nicotiana species, nicotine is converted to nornicotine, which in turn gives rise to potent carcinogen NNN. Nicotine to nornicotine conversion via nicotine-N-demethylation is mediated by the CYP82E family of P450 enzymes. Tobacco (Nicotiana tabacum) converts in senescing leaves, while its diploid progenitors N.tomentosiformis and N.sylvestris convert in both green and senescing and only in senescing leaves, respectively. Previously it has been shown that N.tomentosiformis has different active conversion loci in green and senescing leaves. The green leaf conversion enzyme CYP82E3 is inactivated in tobacco by a single amino acid substitution, while the senescing leaf converter enzyme CYP82E4 is active in tobacco, which gave tobacco a ‘senescing leaf converter’ phenotype. In nonconverter tobacco, CYP82E4 shows transcriptional silencing. The nicotine-N-demethylase gene NsylCYP82E2 involved in nicotine to nornicotine conversion in senesced leaves of N. sylvestris was isolated. NsylCYP82E2 is active in N. sylvestris, but it has become inactivated in tobacco through mutations causing two amino acid substitutions. The conversion factor from N.sylvestris was characterized and a model for the alkaloid profile evolution in the amphidiploid N.tabacum from its diploid progenitors was proposed. Regulation of conversion phenomenon was tested under different spatio-temporal conditions and various stresses. The promoter region for NtabCYP82E4 was isolated and promoter-reporter construct was used to determine that NtabCYP82E4 is specifically induced only during senescence. This pattern correlates with the nornicotine accumulation as measured by alkaloid profiling. Thus the regulatory regions of NtabCYP82E4 represent a senescence specific promoter. In another project functional characterization of tobacco EIN2 (NtabEIN2) was undertaken. EIN2 from tobacco and N.sylvestris were cloned, their genomic structure was deduced and NtabEIN2 was silenced using RNAi approach. Silenced plants showed significant delay in petal senescence and abscission; as well as anther dehiscence, pod maturation, pod size, seed yield and defense against tobacco hornworm. Mechanism of delayed petal senescence phenotype, including possible cross-talk with Auxin Response Factor 2 and potential involvement of tasiRNA3 were also investigated.

När ett blad gulnar genomgår det både morfologiska och metaboliska förändringar. Denna process benämns senescence och en förbättrad förståelse av dess mekanismer är viktiga både ur ett grundvetenskapligt perspektiv och för potentiella bioteknologiska applikationer. Denna avhandling rapporterar om flera viktiga aspekter relaterade till de cellulära och metaboliska mekanismer som sker under senescencen med tonvikt på mitokondriernas bidrag till denna process. I ett första steg utvecklade vi metoder för att isolera antingen mycket funktionella mitokondrier eller mycket rena mitokondrier från blad av Arabidopsis thaliana. Dessa metoder användes sedan till för att studera mitokondriella bidrag till cellens redox balans och att uppskatta mitokondriernas kapacitet under senescence-processen. Framför allt jämfördes induktionen av senescencen berodende på olika mörkerbehandlingar av Arabidopsis. Jämförelse mellan individuellt mörklagda blad med hela mörklagda växter visade en betydande skillnad i metabolisk strategi mellan de två mörkerbehandlingarna. Genom att integrera data från mätningar av fotosyntes, respiration och konfokal mikroskopi med transcriptomics- och metabolomics-profiler föreslår vi att metabolismen hos blad från helt mörklagda växter antar ett ”stand-by läge” för att kunna bibehålla fotosynteskapaciteten så länge som möjligt. I kontrast till detta visar mitokondrier från individuellt mörklagda blad en hög aktivitet och kan därmed producera energi och kolskelett för degraderingen av cellkomponenter, vilket möjliggör återvinning av näringsämnen. Vi har även studerat dynamiken av det mikrotubulibaserade cytoskelettet under mörkerindicerad senescence. Mitokondriernas rörlighet påverkades av en tidig nedbrytning av mikrotubuli hos individuellt mörklagda blad men inte hos blad där hela växten mörkerbehandlats. Dessutom verkade ett flertal mikrotubuliassocierade proteiner (MAP’s) att vara involverade i buntningen av mikrotubuli runt kloroplasterna. Sammanfattningsvis belyser det arbete som presenteras i denna avhandling ett flertal viktiga steg med avseende på metabolisk anpassning och andra cellulär mekanismer i Arabidopsisblad som utsätts för långvarig mörkerbehendling. Specifikt föreslår vi att mitokondrierna bidrar med speciella och viktiga funktioner under bladens senescence eftersom mitokondriernas roll under långvarig mörkerbehandling av blad verkar bero på den totala statusen av metabolismen hos växten. / When switching from green to yellow, a leaf undergoes both morphological and metabolic changes. This process is known as senescence and improved understanding of its mechanisms is important both from a fundamental scientific perspective but also for biotechnological applications. The present thesis reports on several important aspects regarding the cellular and metabolic mechanisms occurring during leaf senescence with an emphasis on the mitochondrial contribution to this process. As a first step, we developed methods to isolate either highly functional crude mitochondria or highly purified mitochondria from leaves of Arabidopsis thaliana. These methods were further used to study mitochondrial contributions to cellular redox homeostasis and to estimate the mitochondrial capacities in leaves undergoing senescence. In particular, we compared the induction of senescence by different dark treatments in Arabidopsis. The comparison between individually darkened leaves and leaves from whole darkened plants revealed different metabolic strategies in response to darkness. Integrating data from measurements of photosynthesis, respiration and confocal laser microscopy with transcriptomics and metabolomics profiling, we suggested that metabolism in leaves of the whole darkened plants enter a “stand-by mode” with low mitochondrial activity in order to maintain the photosynthetic machinery for as long as possible. In contrast, mitochondria from individually darkened leaves are more active and may provide energy and carbon skeletons for the degradation of cell constituents, facilitating the retrieval of nutrients. We also investigated the dynamic of the microtubular cytoskeleton during dark-induced senescence. Mitochondrial mobility was affected by an early disruption of the microtubules in individually darkened leaves but not in whole darkened plants. In addition, several microtubules associated proteins (MAPs) seemed to be involved in the bundling of the microtubules around the chloroplasts. Altogether, the work presented in this thesis highlights several important steps regarding the metabolic adjustments and the cellular mechanisms in Arabidopsis leaves submitted to prolonged darkness. In particular, we suggest the mitochondria to fulfill specific and important functions during leaf senescence since the role of mitochondria in leaves experiencing prolonged darkness appears very dependant on the whole metabolic status of the plant.

Stress can be a driving force for new evolutionary changes leading to local adaptation, or may be responded to with pre-existing, ancestral tolerance mechanisms. Using brachionid rotifers (microzooplankton) as a study system, I demonstrate roles of both conserved physiological mechanisms (heat shock protein induction) and rapid evolution of traits in response to ecologically relevant stressors such as temperature and hydroperiod. Rapid evolution of higher levels of sex and dormancy in cultures mimicking temporary waters represents an eco-evolutionary dynamic, with trait evolution feeding back into effects on ecology (i.e., reduced population growth). I also reveal that prolonged culture in a benign laboratory environment leads to evolution of increased lifespan and fecundity, perhaps due to reduction of extrinsic mortality factors. Potential mechanisms (e.g., hormonal signals) are suggested that may control evolvability of facets of the stress response. Due to prior studies suggesting a role of progesterone signaling in rotifer sex and dormancy, the membrane associated progesterone receptor is assayed as a candidate gene that could show positive selection indicating rapid divergence. Despite some sequence variation that may contribute to functional differences among species, results indicate this hormone receptor is under purifying selection. Detailed analyses of multiple stress responses and their evolution as performed here will be imperative to understanding current patterns of local adaptation and trait-environment correlations. Such research also is key to predicting persistence of species upon introduction to novel habitats and exposure to new stressors (e.g., warming due to climate change). Perhaps one of the most intriguing results of this dissertation is the rapid, adaptive change in levels of sex and dormancy in a metazoan through new mutations or re-arrangements of the genetic material. This suggests species may be able to rapidly evolve tolerance of new stressors, even if standing genetic variation does not currently encompass the suite of alleles necessary for survival.

Diss. (sammanfattning) Umeå : Sveriges lantbruksuniv. / Härtill 6 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet. / Härtill 4 uppsatser.

L'interaction symbiotique entre la bactérie du sol Sinorhizobium meliloti et la plante de la famille des légumineuses Medicago sp. conduit au développement d’un nouvel organe racinaire: la nodosité. Au sein de cet organe, les bactéries différenciées en bactéroïdes, réduisant l’azote atmosphérique en ammoniac directement assimilable par la plante, favorisant ainsi sa nutrition azotée. En échange, la plante, grâce à son activité photosynthétique, fournit aux bactéroïdes des composés carbonés. Cette association à bénéfice mutuel n’est toutefois pas permanente. En effet, quelques semaines seulement après l'établissement de la symbiose, une sénescence définie par une dégradation des bactéroïdes puis des cellules végétales, est observée. Cette étape du développement nodositaire est aujourd’hui encore peu étudiée et mal comprise.L’objectif premier de ce travail était donc d’analyser le rôle du bactéroïde dans cette rupture symbiotique. Pour cela, nous nous sommes plus particulièrement intéressés au rôle des systèmes Toxine Antitoxine (TA) de type VapBC de S. meliloti. En effet, ces opérons sont, dans la littérature, connus pour être impliqués dans la réponse aux stress, la persistance et/ou la mort bactérienne ainsi que la survie de la bactérie au sein de la cellule hôte. Dans un premier temps, nous avons développé une analyse globale du rôle des 11 systèmes VapBC chromosomiques de S. meliloti dans l’interaction symbiotique par des analyses in silico et de phénotypes de mutants d'invalidation du gène de la toxine en interactions avec Medicago sp. Deux études ont été réalisés de façon plus détaillées sur deux modules vapBC (VapBC5 et VapBC7). / The symbiotic interaction between the soil bacterium Sinorhizobium meliloti and the legumes plant Medicago sp. led to the development of a new root organ: the nodule. In this nodule differenciated bacteria into bacteroids, reducing atmospheric nitrogen into ammonia directly assimilated by the plant, thus promoting its nitrogen nutrition. In exchange, the plant, thanks to its photosynthetic activity, provides carbon compounds to the bacteroids. This mutual benefit association is however not permanent. Indeed, just weeks after the establishment of the symbiosis, senescence defined by a degradation of Bacteroides and plant cells, is observed. This stage of development is poorly understood in particularly about bacterial signal.The primary objective of this study was therefore to analyze the role of bacteroids in this symbiotic rupture. For this, we are particularly interested in the role of VapBC toxin antitoxin systems (TA) of S. meliloti. Indeed, in the literature, they are known to be involved in the stress response, persistence and / or bacterial death and the survival of the bacteria within the host cell. At first, we developed a global analysis of the role of 11 VapBC chromosomal systems in S. meliloti symbiotic interaction. After an in silico study, we studied the symbiotic phenotype with Medicago sp., Of each of the bacterial toxin mutants invalidation. Given the results, we, as a second step, developed a detail analysis of phenotypes obtained with two of these mutants: vapC5- and vapC7-.

La sénescence cellulaire, arrêt stable de la prolifération cellulaire, est accompagnée de la sécrétion de nombreux facteurs pro-inflammatoires (programme sécrétoire associé à la sénescence appelé SASP). La sénescence est induite par divers stimuli, et joue un rôle clé dans de multiples contextes physiopathologiques. Cependant, la régulation de la sénescence est encore mal comprise. Notre laboratoire a récemment identifié le récepteur inositol 1,4,5-trisphosphate de type 2 (ITPR2, canal calcique du ER) comme nouveau régulateur de la sénescence. L'expression du gène ITPR2 est réprimée dans la plupart des cancers, mais sa régulation transcriptionnelle est peu connue. Dans ce contexte, le premier objectif de ma thèse était de caractériser de nouveaux régulateurs de l’expression d’ITPR2. Par un criblage (siRNA) et une analyse Nanostring, nous avons identifié le récepteur nucléaire RXRA comme répresseur transcriptionnel d’ITPR2. Nous avons montré que dans les fibroblastes primaires humains, le knockdown de RXRA induit l’expression d’ITPR2 et de ce fait la signalisation calcique, la production d’espèces réactives de l’oxygène (ROS), le dommage de l’ADN et finalement la sénescence via l’activation de la voie p53-p21. Inversement, la surexpression constitutive de RXRA retarde la sénescence réplicative. Les molécules du SASP, induisant ou renforçant la sénescence, peuvent réguler la signalisation calcique. Le deuxième objectif de ma thèse était d’étudier le rôle du SASP et la participation de la signalisation calcique dans celui-ci. Nous avons observé que le SASP induit la sénescence cellulaire accompagnée d’une différenciation neuroendocrine (NED) dans des cellules de cancer du sein. Le SASP induit une accumulation de calcium dans le cytoplasme qui paraît être impliquée dans la régulation de la NED. Une analyse de données d’échantillons de tumeurs du sein humaines et observé que les échantillons positifs pour la NED présentent des marques de sénescence / Cellular senescence is a stable proliferation arrest accompanied with senescence-associated secretory phenotype (SASP). Senescence is induced by diverse stimuli such as telomere shortening and oncogene activation and plays key roles in many physiopathological contexts like embryonic development, cancer and aging. However the molecular mechanisms regulating senescence remain partially understood. Our laboratory recently identified a new senescence regulator: the inositol 1,4,5-trisphosphate receptor type 2 (ITPR2), an ER calcium release channel. ITPR2 is repressed in many cancers, but its transcriptional regulation is barely known. Therefore, the first aim of my thesis was to characterize new ITPR2 regulators. Through siRNA screen and Nanostring analysis, we identified the nuclear receptor RXRA as a transcriptional repressor of ITPR2. We found that in primary human fibroblasts, RXRA knockdown induces ITPR2 expression and thereby calcium signaling, reactive oxygen species (ROS) production, DNA damage and ultimately senescence through p53-p21 axis. Conversely, RXRA overexpression delays replicative senescence. SASP has been described to induce/reinforce senescence, and most of the SASP factors are able to regulate calcium signaling through their receptors. The second aim of my thesis was to investigate the role of the SASP and the participation of calcium signaling in it. We observed that the SASP induces senescence accompanied with a neuroendocrine differentiation (NED) in some breast cancer cells. Interestingly, SASP triggers calcium accumulation in the cytoplasm which seems to be involved in the regulation of NED. We then analyzed human breast tumor datasets and observed that NED-positive samples display some senescence marks: functional p53, low proliferation level and Sprouty 2 expression. Altogether, my work identified RXRA as a new senescence regulator and showed calcium signaling is involved in SASP-induced NED in breast cancer cells

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