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A wide variety of adherent mammalian cells can be induced into a reversible state of metabolic arrest (quiescence) by conversion to non-adherent multicellular aggregates. These "spheroids" can be maintained at room temperature under oxygen- and nutrient-deprived conditions for extended periods of time (weeks) as well as converted back to viable proliferating monolayers. Herein it is shown that HEK293 spheroid arrest and recovery requires the co-activation of both NF-kB and JNK, and chemical inhibition of either NF-κB nuclear translocation or JNK phosphorylation leads to cell death. Cytokine profiling within the aggregates during the arrest and recovery process is suggestive that a cyclical cascade was in operation, leading to endogenous cytokine production of TNF-Alpha, IL-1Beta, and IL-8, thereby propagating the cellular stress signal within cells as well as throughout the aggregate. Cytokines exist <i>in vivo</i> as mixtures, yet tissue culture studies delineating how cells respond to these molecules are often performed using individual effectors added exogenously. Are the results obtained in these studies true representations of physiological responses? As HEK293 multicellular aggregates (spheroids) survive long term arrest by endogenous cytokine (TNF-α and IL-1β) and chemokine (IL-8) signaling, adherent monolayer cells were evaluated for their ability to provide a "spheroid signal response" when exposed to TNF-α, IL-1β and IL-8 individually, and in combination, at concentrations observed in the aggregates. The spheroid signal transduction response was only observed when all three cytokines were present, demonstrating that signal transduction cascade mechanisms are cytokine-profile dependent. To determine if similar processes were involved in the arrest and recovery of multicellular aggregates derived from other cell types, the responses of primary human foreskin fibroblasts (HFF-2) and a glioblastoma cell line (T98G) were characterized, utilizing the procedures developed in the HEK293 study. Both the T98G and HFF-2 cell lines entered and exited from the long term arrest utilizing an autocrine response. However, while the carcinoma cell line was dependent upon NF-κB for survival, its signaling partner was Gadd45α and signaling occurred through the p38 pathway. Primary fibroblast arrest and recovery proceeded through the p38 pathway as well, but was independent of NF-κB. Thus, three different cell types and transformation states (HEK293, HFF-2, and T98G) provided three different routes to survival, all with cyclical cytokine production and signaling. These routes cannot be measured or modulated effectively in adherent monolayers. Multicellular aggregates provide higher ordered systems that can be used to describe signaling pathways within a cell, highlighting the role of autocrine responses and the synergistic relationships between cytokines and neighboring cells. / Ph. D.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Les changements de forme et les mouvements des cellules constituant les tissus relèvent de la morphogenèse épithéliale. Dans les tissus segmentés les compartiments antérieurs et postérieurs représentent des domaines morphogénétiques indépendants constitués de lignées cellulaires distinctes et séparées par des barrières biophysiques. Le laboratoire a montré que lors de la fermeture dorsale de l’embryon de drosophile, certaines cellules des segments centraux de l’ectoderme, appelées « Cellules Mixer » (CMs), sont reprogrammées pour traverser la frontière segmentale dans un phénomène qui prend le nom de « mixing ». La reprogrammation des CMs est JNK dépendante induisant l’expression de novo du gène engrailed (en). La mise au point de nouveaux outils génétiques a permis de révéler le rôle de deux familles de gènes impliqués dans les mécanismes de reprogrammation et de mixing : le gène Polycomb (Pc) et les gènes Hox. La technique de DNA-FISH, qui analyse l’interaction entre Pc et le PRE d’en, a ainsi montré que la voie JNK induit l’expression de novo d’en par dé-répression de l’activité Pc dans les CMs. De manière intéressante l’analyse approfondie des mutants Pc a dévoilé que les gènes Hox abdominal-A (abdA) et Abdominal-B (AbdB) contrôlent le domaine du mixing. Des expériences de gain et perte de fonction ont par la suite confirmé le rôle positif d’abdA et le rôle négatif d’AbdB dans le mixing. En conclusion, l’ensemble des résultats obtenus ont permis de dévoiler la présence d’un réseau génétique composé de par JNK, en, Pc et les gènes Hox contrôlant les mécanismes de reprogrammation cellulaire et de remodelage des frontières segmentales au cours du développement normal. / Tissue morphogenesis relies on patterned cell shape changes and movements taking place in specific morphogenetic domains. In segmented tissues, anterior and posterior compartments represent independent morphogenetic domains which are made of distinct lineages separated by boundaries. We previously reported on a rare event leading to the exchange of specific ‘Mixer Cells’ (MCs) between compartments of the ectoderm. During dorsal closure, MCs, which are of anterior origin, cross the boundary to integrate the adjacent posterior compartment through de novo expression of the posterior determinant Engrailed (En). This reprograming process is dependent on JNK signalling and is restricted to the central abdominal region. Here, we show that JNK signalling represses Polycomb (Pc) expression and that loss of Pc leads to an absence of MCs reprogramming. FISH-DNA coupled to immunostaining further shows that MCs fate transition is accompanied by a release of the en promoter from the repressing Pc bodies. Interestingly, our genetic data reveal that spatial control of MCs reprograming depends on the activity of the Hox genes abdominal-A (abdA) and Abdominal-B (AbdB). In their respective domains, abd-A promotes mixing while abd-B behaves as a strong repressor, thus restricting cell mixing to the central abdominal region. Together, these results provide new insights into the mechanisms of developmental reprogramming, showing that segment boundary plasticity relies on regional control of cell remodelling involving a gene regulatory network composed of JNK, en, Pc, and Hox activities.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Made available in DSpace on 2017-07-10T14:17:06Z (GMT). No. of bitstreams: 1 Luiz Caetano.pdf: 1961056 bytes, checksum: 05aefff53c5671351bad5763039bd60b (MD5) Previous issue date: 2013-11-10 / Among the several organic alterations arising from obesity, chronic inflammation is associated with the balance of cytokines TNF-&#945;, IL-1&#946;, IL-6, IL-2, IFNg, IL-4 and IL-10, and there is evidence the amino acid taurine (Tau) has anti-inflammatory effect. Therefore, this study investigated the inflammatory profile in plasma and retroperitoneal adipose tissue of MSG-obese rats, supplemented or not, with the TAU. Male Wistar rats received subcutaneous injections of MSG (4mg/kg body weight/day) or hyperosmotic saline during the first 5 days of life, composing the control (CON) and MSG groups. After 21 days, half of each group received TAU 2.5% in drinking water, and separated into 04 groups: CON, CON with TAU (CTAU), MSG and MSG with TAU (MTAU). At 120 days of age, the animals were euthanized. The MSG rats showed an increase in Lee Index, retroperitoneal and perigonadal fat pads deposition, insulin and triglycerides plasmatic concentrations and HOMA-IR, when compared to CON animals, showing that the treatment with MSG led to obesity. The TAU supplementation attenuated retroperitoneal fat deposition, as well as TG concentration. The MSG treatment did not alter the expression of JNK and I&#954;B&#945;. However, the supplementation with TAU increased 61% the expression of IkB&#945; in CTAU group compared to the CON and 107% in the MTAU animals compared to the MSG. The expression of TNF-&#945;, IL-1&#946; and IL-6 in the retroperitoneal adipose tissue were similar in the four groups of animals, as well as plasma concentrations of TNF-&#945;, IL-1&#946;, IL-6, IL-2, IFN&#947;, IL-4 and IL-10. It is possible to conclude that neonatal treatment with MSG does not influence the inflammatory profile of the animals. We also conclude that the TAU increased 61% of IkB&#945; protein expression in the control group and 107% in the MSG-obese animals, without affecting the inflammatory cytokines. Thus we suggest that TAU can exert their anti-inflammatory effects in adipose tissue, via NF-kB / Dentre as várias alterações orgânicas decorrentes da obesidade, está o processo inflamatório crônico associado ao balanço das citocinas TNF-&#945;, IL-1&#946;, IL-6, IL-2, IFN&#947;, IL-4 e IL-10, e, há evidências de que o aminoácido taurina (TAU) possui efeito anti-inflamatório. Assim, neste trabalho investigamos o perfil inflamatório plasmático e do tecido adiposo retroperitoneal de ratos obesos-MSG, suplementados ou não, com o aminoácido TAU. Ratos Wistar receberam injeções subcutâneas de MSG (4mg/kg de peso corporal/dia) ou salina hiperosmótica, durante os primeiros 5 dias de vida e foram distribuídos nos grupos MSG e CON, respectivamente. Após os 21 dias de vida, metade de cada grupo recebeu 2,5% de TAU na água de beber, sendo separados nos grupos CON, CON + TAU (CTAU), MSG e MSG + TAU (MTAU). Aos 120 dias de vida os animais foram eutanasiados. Ratos MSG apresentaram obesidade acompanhada de hipertrigliceridemia e resistência à insulina (RI). Todavia, não afetou a expressão de I&#954;B&#945; e JNK. A suplementação com TAU aumentou 61% a expressão do IkB&#945; no grupo CTAU em relação ao grupo CON e 107% nos animais MTAU em comparação com os obesos-MSG. As expressões de TNF-&#945;, IL-1&#946; e IL-6 no tecido adiposo retroperitoneal foram semelhantes nos 4 grupos de animais estudados, assim como as concentrações plasmáticas do TNF-&#945;, IL-1&#946;, IL-6, IL-2, IFN&#947;, IL-4 e IL-10. É possível concluir que o tratamento neonatal com MSG não influencia o perfil inflamatório dos animais. Concluímos também que a TAU aumentou a expressão proteica do IkB&#945; nos animais controle e MSG, sem afetar as citocinas inflamatórias. Desta forma sugerimos que a TAU possa exercer seus efeitos anti-inflamatórios no tecido adiposo, via NF-&#954;B

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Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal