El síndrome de Down constituye la cromosomopatía más frecuente que ocurre en uno de cada 700 a 1000 nacimientos y está causado por la trisomía completa o por una parte del cromosoma 21 humano (HSA21). Aún se desconoce cómo la presencia del cromosoma 21 extra da lugar al fenotipo característico de este síndrome. En este sentido la participación de las especies reactivas de oxígeno (ROS) ha sido propuesta como uno de los mecanismos que intervienen en la patogénesis del mismo. Dicho mecanismo se fundamenta en la sobreexpresión de al menos 16 genes del HSA21 relacionados con el metabolismo de las especies reactivas de oxígeno (ROS) y con la generación de energía mitocondrial. Uno de estos genes es el que codifica una importante enzima del sistema antioxidante celular, el gen SOD1, propuesto como potencial culpable del estrés oxidativo inusual en los individuos con SD. En condiciones normales, los ROS, producidos in vivo principalmente por la respiración aeróbica, se eliminan de la célula por la acción de las enzimas antioxidantes, superóxido dismutasa (SOD), catalasa (CAT) y glutatión peroxidasa (GPx). La Cu/Zn superóxido dismutasa (SOD1) convierte el radical superóxido a peróxido de hidrógeno, el cual es eliminado por la glutatión peroxidasa y/o catalasa a agua y oxígeno. La sobreexpresión de la SOD1 puede producir un desequilibrio en la relación de las enzimas antioxidantes (SOD1, GPx y CAT) generando estrés oxidativo y podría resultar en el daño oxidativo a biomoléculas tales como ácidos grasos poliinsaturados en los lípidos de las membranas, proteínas esenciales y el DNA, ya que existe una variabilidad en los niveles de enzimas antioxidantes dentro de la población con SD, lo que puede estar relacionado con una desregulación compleja que afecte no sólo a los genes del HSA21 sino también en otros cromosomas. Así, el daño celular puede ser inducido por los ROS y asociarse a algunas de las alteraciones celulares en el SD, causando diversas patologías y conducir a un envejecimiento prematuro. Se obtuvieron 18 muestras de fibroblastos primarios fetales humanos, 9 de ellos con síndrome de Down (FT21) y 9 controles (FC), en los cuales se evaluó la disminución de la capacidad endógena antioxidante debido a la sobreexpresión de la SOD1, causando un exceso en la producción intracelular de ROS y el origen prematuro de estrés oxidativo asociado al daño oxidativo a lípidos y proteínas, así como a una disfunción mitocondrial. Se analizaron varios marcadores de senescencia celular con la finalidad de contribuir al conocimiento de un nuevo aspecto de la patología de este síndrome, el envejecimiento prematuro. Estos mecanismos fisiopatológicos podrían estar relacionados con la aparición y establecimiento de la senescencia celular prematura en los fibroblastos con trisomía del cromosoma 21 (FT21). / Down syndrome is the most common chromosomal disorder that occurs in 1 in 700 to 1,000 births and is caused by trisomy full or part of human chromosome 21 (HSA21). It is still unknown how the presence of the extra chromosome 21 results in the phenotype of this syndrome. In this sense the involvement of reactive oxygen species (ROS) has been proposed as one of the mechanisms involved in the pathogenesis of same. This mechanism is based on the overexpression of at least 16 genes related HSA21 metabolism of reactive oxygen species (ROS) and mitochondrial energy generation. One of these genes encoding is an important cellular antioxidant enzyme system, the SOD1 gene, proposed as a potential culprit unusual oxidative stress in individuals with DS. Under normal conditions, the ROS produced in vivo mainly by aerobic respiration of the cells are removed by the action of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). The Cu/Zn superoxide dismutase (SOD1) converts the superoxide radical to hydrogen peroxide, which is eliminated by glutathione peroxidase and/or catalase into water and oxygen. SOD1 overexpression can produce an imbalance in the ratio of antioxidant enzymes (SOD1, GPx and CAT) generating oxidative stress and may result in oxidative damage to biomolecules such as polyunsaturated fatty acids in the membrane lipids, proteins and essential DNA, since there is a variability in the levels of antioxidant enzymes in the DS population, which can be related to a complex deregulation affects not only Hsa21 genes but also on other chromosomes. Thus, cell damage can be induced by ROS and associated with some of the cellular changes in the DS, causing various diseases and lead to premature aging. Eighteen samples were obtained from primary human fetal fibroblasts, 9 with Down syndrome (TF21) and 9 normal (NF), which was evaluated in decreasing endogenous antioxidant capacity due to overexpression of the SOD1, causing excess in intracellular production of ROS and oxidative stress origin associated premature oxidative damage to lipids and proteins, as well as mitochondrial dysfunction. We analyzed several markers of cellular senescence in order to contribute to the knowledge of a new aspect of the pathology of this syndrome, premature aging. These pathophysiological mechanisms may be related to the emergence and development of premature cellular senescence in fibroblasts with trisomy 21 (FT21).

Μηχανισμοί οι οποίοι εξασφαλίζουν τη σωστή διαδοχή των φάσεων του κυτταρικού κύκλου συμβάλλουν στη διασφάλιση της γονιδιωματικής σταθερότητας των κυττάρων. Η αδειοδότηση της αντιγραφής του DNA, η συγκρότηση επί των αφετηριών της αντιγραφής του DNA του προ-ανιγραφικού συμπλόκου, καθορίζει τη σωστή χρονικά και τοπικά έναρξη της αντιγραφής. Βασικό συστατικό αυτού του συμπλόκου είναι ο παράγοντας Cdt1. Η Geminin προσδένεται στο Cdt1, αναστέλοντας τη δράση του από την S μέχρι και την Μ φάση, παρεμποδίζοντας, έτσι, την αδειοδότηση της αντιγραφής. Παρά το οτι φυσική αλληλεπίδραση των δύο πρωτεϊνών έχει δειχθεί τόσο in vitro όσο και in vivo, προηγούμενες μελέτες δείχνουν οτι έκφραση των Cdt1 και Geminin εντοπίζεται σε διαφορετικές φάσεις του κυτταρικού κύκλου. Τα φυσιολογικά κύτταρα, ανάλογα με τα μηνύματα που δέχονται, είτε παραμένουν σε μιτωτικό κύκλο, είτε εξέρχονται από αυτόν προς φάση ηρεμίας (ή G0), διαφοροποίηση ή γήρανση. Αυστηρός συντονισμός των παραπάνω διαδικασιών είναι απαραίτητος προκειμένου να διασφαλιστεί η ομοιόσταση των πολύπλοκων δομών των ιστών των μεταζώων. Προηγούμενες μελέτες προτείνουν το σύστημα της αδειοδότησης της αντιγραφής του DNA ως έναν βασικό ρυθμιστή της εξόδου από τον κυτταρικό κύκλο και της επανεισόδου στη G1. Οι παράγοντες Cdt1 και Geminin ρυθμίζονται αρνητικά κατά την έξοδο των κυττάρων σε G0, ενώ έκφρασή τους χαρακτηρίζει διαιρούμενα κύτταρα. Σε αντίθεση με τις άλλες καταστάσεις εκτός κυτταρικού κύκλου, λίγα είναι γνωστά αναφορικά με τη ρύθμιση των Cdt1 και Geminin κατά την κυτταρική γήρανση. Στο πρώτο μέρος της διατριβής εστιαστήκαμε στη μελέτη του προτύπου έκφρασης των Cdt1 και Geminin κατά τη διάρκεια του κυτταρικού κύκλου πρωτογενών ανθρώπινων ινοβλαστών, και στη σύγκρισή του με εκείνο των καρκινικών κυττάρων. Διαπιστώσαμε οτι τόσο η ενδοκυτταρική εντόπιση όσο και η ικανότητα των Cdt1 και Geminin να εκφράζονται σε συγκεκριμένες φάσεις του κυτταρικού κύκλου, δεν διαφοροποιούνται στους πρωτογενείς φυσιολογικούς ινοβλάστες σε σχέση με κύτταρα που προέρχονται από καρκινικό ιστό. Επιπλέον, δείξαμε οτι ο παράγοντας Cdt1 εκφράζεται αποκλειστικά σε BrdU-αρνητικά κύτταρα, σε αντίθεση με την Geminin, η οποία δείχνει να συσσωρεύεται σταδιακά μετά την έναρξη της S φάσης, ενώ δεν εντοπίστηκε συνέκφραση των δύο πρωτεϊνών στο χρονικό παράθυρο της G1/S μετάβασης. Στο δεύτερο μέρος της εργασίας εστιαστήκαμε στη μελέτη της έκφρασης του παράγοντα αδειοδότησης Cdt1 και του αρνητικού ρυθμιστή αυτού, Geminin, κατά την είσοδο των κυττάρων σε κυτταρική γήρανση και εξετάσαμε την πιθανή λειτουργική εμπλοκή τους στην εξέλιξη του φαινομένου. Δείξαμε οτι, ενώ οι παράγοντες Cdt1 και Geminin διατηρούν τη σωστή ενδοκυτταρική εντόπιση και το σωστό πρότυπο έκφρασης κατά τη διάρκεια του κυτταρικού κύκλου, υφίστανται αρνητική ρύθμιση σε κύτταρα που εισέρχονται σε κυτταρική γήρανση, τόσο αναπαραγωγική όσο και πρόωρη, επαγόμενη από οξειδωτικό στρες. Το γεγονός οτι η μείωση της έκφρασης της Geminin προηγήθηκε της εμφάνισης του γηρασμένου φαινοτύπου, μας ώθησε στην περαιτέρω διερεύνιση του λειτουργικού ρόλου της Geminin στην επαγωγή της κυτταρικής γήρανσης. Για το σκοπό αυτό, απορρυθμίσαμε τα επίπεδα έκφρασης της Geminin σε πρωτογενή φυσιολογικά κύτταρα ανθρώπου και ποντικού, αξιοποιώντας την τεχνολογία του RNAi και ρετροϊικά συστήματα υπερέκφρασης γονιδίων αντίστοιχα. Δείξαμε οτι η μείωση της έκφρασης της Geminin σε ανθρώπινους ινοβλάστες (χρησιμοποιώντας siRNAs αλλά και pSUPER πλασμιδιακούς φορείς αποσιώπησης γονιδίων που κατασκευάστηκαν ειδικά για την Geminin) επάγει αύξηση της κυτταρικής γήρανσης της καλλιέργειας. Επιπλέον, κύτταρα που στερούνταν της έκφρασης της Geminin ήταν πιο επιρρεπή σε γήρανση επαγόμενη από οξειδωτικό στρες, σε σχέση με τα κύτταρα-μάρτυρες. Ετεροζυγώτες για το γονίδιο της Geminin εμβρυικοί ινοβλάστες ποντικού εμφάνιζαν μεγαλύτερα ποσοστά κυτταρικής γήρανσης σε σχέση με τους αντίστοιχους ινοβλάστες αγρίου τύπου. Αντίθετα, αύξηση των επιπέδων της Geminin σε αγρίου τύπου εμβρυικούς ινοβλάστες ποντικού προκάλεσε μείωση της εμφανιζόμενης γήρανσης. Τέλος, η μείωση των επιπέδων έκφρασης του παράγοντα αδειοδότησης Cdt1 σε ανθρώπινα κύτταρα ήταν, επίσης, σε θέση να επάγει ισχυρό φαινότυπο κυτταρικής γήρανσης. Συνοψίζοντας, τα αποτελέσματα μας αναδεικνύουν την κρισιμότητα του ισοζυγίου Cdt1:Geminin στα κύτταρα, και προτείνουμε οτι η διατάραξη της ισορροπίας αυτής είναι ικανή να επάγει κυτταρική γήρανση, μέσω διαδικασιών όπως η υπεραδειοδότηση ή η υποαδειοδότηση της αντιγραφής του DNA. / Genome integrity relies on the strict alternation of S and M phases of the cell cycle, so that one and only round of DNA replication takes place per cell cycle. This is achieved through replication licensing, which involves the formation of a multi-protein complex, the pre-replicative complex, onto origins of replication. Cdt1 is a crucial component of this complex and Geminin, a small protein shown to tightly bind Cdt1, inhibits its licensing function from S to M phase, when licensing is illegitimate. Although previous experimental evidence shows that Cdt1 and Geminin are expressed in different phases of the cell cycle, physical interaction between these two proteins has been demonstrated in vitro as well as in vivo. The fate of a normal cell is not perpetual division. Cells may exit the mitotic cell cycle to enter quiescence, to terminally differentiate or to senesce. These “out-of-cycle-states” must be strictly regulated in order to establish and maintain the hierarchical organization of complex tissues in metazoa. Replication licensing has been proposed to coordinate cell-cycle exit and re-entry in vitro and in metazoan tissues. Cdt1 and Geminin down-regulation during exit to quiescence supports the idea that their expression correlates with cell proliferation. In contrast to other out-of-cycle states, little is known about the regulation of Cdt1 and Geminin expression during cellular senescence. Senescence refers to the irreversible resting state of cells grown for succeeding passages in culture, as a response to DNA damage caused by telomeres erosion. Other stimuli, such as oxidative or oncogenic stress, may force mitotically competent cells to respond similarly, a phenomenon termed as Stress Induced Premature Senescence (SIPS). The first part of this work focused on the study of the expression patterns of Cdt1 and Geminin during the unperturbed cell cycle of primary human fibroblasts and compared to that of tumor-derived cell lines. The cell cycle specific expression and the intracellular localization of both proteins, as assessed at a single-cell level using indirect immunofluorescence and a new monoclonal antibody against Geminin, appear similar in primary fibroblasts compared to the cancer cells examined. Cdt1 is strictly expressed in BrdU-negative cells, whereas Geminin starts accumulating after S phase onset. The two proteins are, therefore, not co-expressed at the ”time-window” of G1/S transition of the cell cycle. We showed that Cdt1 levels, but not those of Geminin, are mainly regulated in a proteasome-dependent way during normal cell cycle of human primary and cancer cells. The second part of this work focused on the investigation of Cdt1 and Geminin during cellular senescence and their possible role in the establishment of the senescence phenotype. To this end, primary human fibroblasts were maintained in culture for succeeding passages in order to induce them to undergo replicative senescence. Alternatively, an H202-induced senescence protocol was applied to force cells to undergo premature senescence (Stress-Induced Premature Senescence/SIPS). We show that, although Cdt1 and Geminin retain their nuclear localization and are correctly expressed during specific phases of the cell cycle during both replicative and Η202-induced premature senescence, their expression levels are down-regulated. In SIPS-experiments, Geminin down-regulation is an early event during the establishment of the senescent-phenotype, as assessed by senescence-associated β-Galactosidase and BrdU incorporation assays. This prompted us to further examine Geminin’s functional significance in the establishment of cellular senescence. To achieve this, we interfered with Geminin expression levels in human and mouse cells. Using RNA interference techniques, we were able to show that Geminin depletion from human cells is able to induce a senescent-phenotype in a fraction of the treated culture. Similarly, Geminin-depleted human cells were more susceptible to Η202-induced premature senescence, compared to control cells. Heterozygotes for Geminin mouse embryonic fibroblasts were more prone to senescence compared to their control counterparts. In contrast, when Geminin was over-expressed in control mouse embryonic fibroblasts cultures, senescent phenotype was reduced. Finally, a strong senescent-phenotype was induced when the licensing regulator, Cdt1, was silenced within human cells. Taken together, we conclude that Cdt1:Geminin balance within cells is crucial, and when disturbed, is able to promote a senescent phenotype, possibly through a mechanism that involves over- or under-licensing of DNA replication.

No description available.

No description available.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

The factors influencing senescence in white clover (Trifolium repens L.) are of considerable importance to the pastoral sector of New Zealand’s economy. The plant hormones, ethylene and the cytokinins, have been implicated as having opposing influences on senescence. This project focused on the cytokinins. The rate limiting step in cytokinin biosynthesis is catalysed by isopentenyl transferase (IPT) and the primary enzyme in the degradation of cytokinins is cytokinin oxidase/dehydrogenase (CKX). Both IPT and CKX genes are present as multi-gene families. A reduction in the level of active cytokinins either via a decrease in IPT expression, or an increase in CKX expression, or both, would implicate the cytokinins in developmental leaf senescence in white clover. White clover grows in a sequential pattern with leaves at all stages of development making it a good model for studying leaf development and senescence. A decrease in leaf chlorophyll is used as a marker for the onset of senescence. A micro-scale chlorophyll analysis was developed using the NanoDrop™ thus allowing tissue from the same leaflet to be used for gene expression and chlorophyll measurements. The pattern of chlorophyll changes was similar to that shown by Hunter et al.(1999) and Yoo et al.(2003) in white clover stolons used for ethylene research. Reverse transcriptase PCR (RT-PCR) and BLAST analysis was used to identify five putative IPT genes and seven putative CKX genes from white clover. RT-PCR demonstrated the expression of seven of these genes (TrIPT1. TrIPT13, TrIPT15 TrCKX1, TrCKX2, TrCKX6). Analysis with quantitative real-time PCR showed expression of TrCKX2 increased markedly during leaf expansion and was consistently high during senescence, suggesting a potential role for CKX in facilitating the progression of senescence.

No description available.

This work presents a methodology to describe the dynamics and topology of biological regulatory networks through the application of graph theory. We model these networks using Boolean rules and simulations performed with algorithms implemented in MATHEMATICA 7.0. Through this methodology, we characterize the regulatory network of opening and closing of the stomata of a plant by abscisic acid (ABA), including the relationships between the network s elements during the dynamics and the description of the state-space by comparing each one of its elements. For the state of cellular replicative senescence in humans, the goal is to describe a regulatory network of proteins involving the mechanisms of Shelterin complex, DNA double-strand break signaling, and the cell cycle arrest in G1 phase. The network topology must involve a combination of pathways and modules to ensure the stability of the signal and the permanent cell-cycle arrest. The state senescent is also characterized by establishing a secretory phenotype (SASP) with both beneficial and harmful effects for the organism. Different pathways are identified along with the activation of NF-kB in the regulation of these phenotypes and in this sense, a regulatory network to regulate the SASP in replicative senescence is proposed and the main consequences of the SASP for human colon are identified via analysis of microarray data related to normal colon, inflamed colon, colonic adenoma and carcinoma of the colon. / Este trabalho apresenta uma metodologia para descrever a dinâmica e a topologia de redes biológicas regulatórias através da aplicação da teoria de grafos, da modelagem de redes a partir de regras booleanas e através do uso de simulações baseadas em algoritmos implementados no software MATHEMATICA 7.0. Por meio desta metodologia procurou-se caracterizar a rede de regulação de abertura e fechamento dos estômatos de uma planta pelo ácido abscísico (ABA), incluindo a relação entre os elementos da rede durante a dinâmica e a descrição do espaço de estados. No estudo do estado celular de senescência replicativa em humanos, o objetivo é descrever uma rede regulatória de proteínas envolvendo os mecanismos do complexo de Shelterin com a sinalização de duplas quebras no DNA e a sinalização da parada do ciclo celular na fase G1. Por meio desta rede, a topologia deve envolver a combinação de vias e módulos para garantir a estabilidade da sinalização e a parada permanente da célula no estado senescente. O estado senescente também é caracterizado pelo estabelecimento de um fenótipo secretor (SASP) com efeitos tanto benéficos quanto prejudiciais para o organismo. Diferentes vias estão identificadas juntamente com a ativação de NF-kB na regulação deste fenótipo e nesse sentido, uma rede regulatória do SASP em senescência replicativa é proposta e as principais consequências do SASP para o cólon humano são identificadas através da análise de dados de microarranjos que relacionam cólon normal, cólon inflamado, adenoma de cólon e carcinoma de cólon.