Au cours de cette étude, nous avons isolé 4 ADNc codant pour des protéines activatrices putatives de l'APC/C de tomate : SlCCS52A, SlCCS52B, SlCDC20-1, et SlCDC20-2. Des données obtenues par RT-qPCR et par hybridation in situ révèlent des profils d'expression différents au niveau tissulaire mais également au cours du développement du fruit de tomate, suggérant que différentes protéines activatrices pourraient assurer la modulation spatio-temporelle de l'activité de l'APC/C chez la tomate. De plus, les transcrits du gène SlCCS52A s'accumulent plus particulièrement dans le fruit durant la phase d'expansion cellulaire, tandis que les transcrits du gène SlCCS52B sont plutôt présents durant les premiers stades de développement, caractérisés par une forte activité de division cellulaire. Afin de préciser le rôle de SlCCS52A et SlCCS52B dans le contrôle du cycle cellulaire et de l'endocycle chez la tomate, ces gènes ont fait l'objet d'une étude fonctionnelle. La réduction de l'expression de SlCCS52A entraîne une réduction de la taille des fruits et de la taille cellulaire, qui s'accompagne d'une diminution du niveau de ploïdie. La surexpression de ce gène modifie la cinétique de développement des fruits. La mise en place de l'endocycle est retardée, mais l'augmentation de la ploïdie est plus rapide et la croissance relative du fruit est alors plus importante. Enfin, la réduction de l'expression de SlCCS52B entraîne une augmentation de l'expression de SlCCS52A au niveau des fruits, suggérant l'existence de mécanismes compensatoires. L'ensemble de ces résultats montrent que SlCCS52A est impliqué dans la mise en place de l'endoréduplication chez la tomate, et participe au contrôle de l'expansion cellulaire. / In this study, we have isolated 4 cDNAs encoding putative proteins activating the APC/C in tomato: SlCCS52A, SlCCS52B, SlCDC20-1, and SlCDC20-2. Data obtained by RT-qPCR and in situ hybridization revealed different expression profiles in tissues but also during the development of tomato fruit, suggesting that different activator proteins could provide the spatio-temporal modulation of the APC/C activity in tomato. In addition, the SlCCS52A transcripts accumulate especially in the fruit during the cell expansion phase, while transcripts of the SlCCS52B gene are rather present during the early stages of development, characterized by a high activity of cell divisions. To clarify the role of SlCCS52A and SlCCS52B in cell cycle control and endocycle in tomato, we performed a functional analysis of these genes. Reducing the expression of SlCCS52A leads to reduced fruit size and cell size, accompanied by a decrease in the level of ploidy. The overexpression of this gene alters the kinetics of fruit development. The establishment of endocycle is delayed, but the increase in ploidy is faster and the relative growth of the fruit is much more important then. Finally, the reduced expression of SlCCS52B leads to an increased expression of SlCCS52A in fruit, suggesting the existence of compensatory mechanisms. All these results show that SlCCS52A is involved in the establishment of endoreduplication in tomato, and participates in the control of cell expansion.

Aging is a universal biological phenomenon in all living cells. Questions regarding how cells age are beginning to be answered. Thus, great biological interest and practical importance leading to interventions rest on uncovering the molecular mechanism of aging. This would ultimately delay the aging process while maintaining the physical and mental strengths of youth. The conservation of metabolic and signaling pathways between yeast and humans is remarkably high, leading to the expectation that aging mechanisms are also common across evolutionary boundaries. By utilizing the budding yeast, <i>Saccharomyces cerevisiae</i>, one of the best characterized model systems for studying aging, the span in knowledge between yeast and human aging can possibly be bridged. <p>Evidence is accumulating that a genetic program exists for lifespan determination. Model organisms expressing mutations in single specific genes live longer with increased resistance to stress and cancer development. Mutations that accelerate aging in yeast affect the activity of the APC (Anaphase-Promoting Complex). Our finding that the APC is critical for longevity provides us with a potential central mechanism controlling lifespan determination. The APC is required for mitotic progression and genomic stability in presumably all eukaryotes by targeting regulatory proteins, such as cyclin B (Clb2p in yeast) for degradation. The key feature defining the APC as a central mediator of lifespan is the fact that multiple signaling pathways regulate APC activity and many of these pathways influence lifespan. For example, Snf1 and PKA have antagonistic effects on the APC and on lifespan. Thus, it is intriguing to speculate that the APC may link these signaling pathways to downstream targets controlling longevity. <p>Our hypothesis states that the APC targets a protein that reduces lifespan for ubiquitin-dependent degradation. The results from our two-hybrid screen utilizing Apc5p as bait are consistent with this hypothesis, as Fob1p was isolated as an Apc5p binding partner. The FOB1 gene is located on chromosome IV and the well-known molecular function of FOB1 is the creation of a unidirectional block in replication of rDNA. Fob1p binds to the rDNA locus and overall stalls progression of the replication fork, which increases rDNA recombination and the production of toxic extrachromosomal rDNA circles (ERCs). The FOB1 deletion (fob1∆) mutant confers reduced rDNA recombination, and an increased lifespan of more than 50% compared to WT (wild type) cells.<p>In this study, we expanded on the molecular mechanisms controlling lifespan through a genetic approach, and found that Fob1p was targeted by the APC for degradation in order to prolong lifespan. By utilizing the yeast two-hybrid approach, we confirmed the Apc5p-Fob1p interaction, and determined that the C-terminal half of Fob1p was required for the interaction with Apc5p. BLAST search analysis revealed sequence similarity with the Fob1p C-terminus that was shared with many other proteins from yeast to humans. We speculate that this shared domain may serve as an APC interaction domain employed across evolutionary boundaries. A genetic interaction analysis revealed the influence of FOB1 on the APC, and the cell. For example, deletion of FOB1 increased lifespan in apc5CA and apc10∆ mutant cells and partially suppressed the temperature sensitive (ts) growth of apc10∆ cells. On the other hand, increased FOB1 expression reduced the lifespan of WT and cells and was toxic to apc mutants, particularly the more severe apc mutants, apc10∆ and cdc16-1. Interestingly, overexpression of SIR2, which prolongs lifespan and acts antagonistically with Fob1p, was toxic to WT cells, but suppressed apc5CA ts defects, especially when FOB1 was deleted. These observations suggest that accumulation of Fob1p is harmful to yeast cells, especially when the APC is compromised. This notion was borne out when a cell cycle and steady state analysis of Fob1p revealed that Fob1p was an unstable protein, which was stabilized in apc5CA cells. Taken together, the work presented in this thesis supports a model whereby Fob1p is targeted for degradation by the APC in order to prolong lifespan in yeast. In conclusion, the extreme evolutionarily conserved nature of the APC and the Fob1p C-terminal sequence homology observed in human proteins strongly suggests that the mechanism discovered here could be directing human lifespan.

Aging is a universal biological phenomenon in all living cells. Questions regarding how cells age are beginning to be answered. Thus, great biological interest and practical importance leading to interventions rest on uncovering the molecular mechanism of aging. This would ultimately delay the aging process while maintaining the physical and mental strengths of youth. The conservation of metabolic and signaling pathways between yeast and humans is remarkably high, leading to the expectation that aging mechanisms are also common across evolutionary boundaries. By utilizing the budding yeast, <i>Saccharomyces cerevisiae</i>, one of the best characterized model systems for studying aging, the span in knowledge between yeast and human aging can possibly be bridged. <p>Evidence is accumulating that a genetic program exists for lifespan determination. Model organisms expressing mutations in single specific genes live longer with increased resistance to stress and cancer development. Mutations that accelerate aging in yeast affect the activity of the APC (Anaphase-Promoting Complex). Our finding that the APC is critical for longevity provides us with a potential central mechanism controlling lifespan determination. The APC is required for mitotic progression and genomic stability in presumably all eukaryotes by targeting regulatory proteins, such as cyclin B (Clb2p in yeast) for degradation. The key feature defining the APC as a central mediator of lifespan is the fact that multiple signaling pathways regulate APC activity and many of these pathways influence lifespan. For example, Snf1 and PKA have antagonistic effects on the APC and on lifespan. Thus, it is intriguing to speculate that the APC may link these signaling pathways to downstream targets controlling longevity. <p>Our hypothesis states that the APC targets a protein that reduces lifespan for ubiquitin-dependent degradation. The results from our two-hybrid screen utilizing Apc5p as bait are consistent with this hypothesis, as Fob1p was isolated as an Apc5p binding partner. The FOB1 gene is located on chromosome IV and the well-known molecular function of FOB1 is the creation of a unidirectional block in replication of rDNA. Fob1p binds to the rDNA locus and overall stalls progression of the replication fork, which increases rDNA recombination and the production of toxic extrachromosomal rDNA circles (ERCs). The FOB1 deletion (fob1∆) mutant confers reduced rDNA recombination, and an increased lifespan of more than 50% compared to WT (wild type) cells.<p>In this study, we expanded on the molecular mechanisms controlling lifespan through a genetic approach, and found that Fob1p was targeted by the APC for degradation in order to prolong lifespan. By utilizing the yeast two-hybrid approach, we confirmed the Apc5p-Fob1p interaction, and determined that the C-terminal half of Fob1p was required for the interaction with Apc5p. BLAST search analysis revealed sequence similarity with the Fob1p C-terminus that was shared with many other proteins from yeast to humans. We speculate that this shared domain may serve as an APC interaction domain employed across evolutionary boundaries. A genetic interaction analysis revealed the influence of FOB1 on the APC, and the cell. For example, deletion of FOB1 increased lifespan in apc5CA and apc10∆ mutant cells and partially suppressed the temperature sensitive (ts) growth of apc10∆ cells. On the other hand, increased FOB1 expression reduced the lifespan of WT and cells and was toxic to apc mutants, particularly the more severe apc mutants, apc10∆ and cdc16-1. Interestingly, overexpression of SIR2, which prolongs lifespan and acts antagonistically with Fob1p, was toxic to WT cells, but suppressed apc5CA ts defects, especially when FOB1 was deleted. These observations suggest that accumulation of Fob1p is harmful to yeast cells, especially when the APC is compromised. This notion was borne out when a cell cycle and steady state analysis of Fob1p revealed that Fob1p was an unstable protein, which was stabilized in apc5CA cells. Taken together, the work presented in this thesis supports a model whereby Fob1p is targeted for degradation by the APC in order to prolong lifespan in yeast. In conclusion, the extreme evolutionarily conserved nature of the APC and the Fob1p C-terminal sequence homology observed in human proteins strongly suggests that the mechanism discovered here could be directing human lifespan.

No description available.

Post-translational modification of proteins with ubiquitin regulates many aspects of cell physiology, including protein degradation. A uniform polyubiquitin chain that is linked through Lys48 has been widely accepted as central for recognition and destruction by the 26S proteasome. Work in more recent years has demonstrated that the repertoire of proteolytic signals may encompass chains of other linkage types, including Lys11-linked ubiquitin chains and short assemblies of mixed linkage. In this dissertation I examine whether catalysis mediated by the Anaphase-Promoting Complex/Cyclosome (APC/C) is dependent on polyubiquitination and whether the proteolytic machinery exerts a requirement for specific ubiquitin linkages to efficiently degrade cyclin B1. In chapter II, I describe a novel method in which Xenopus cell-cycle extracts are made largely dependent on exogenous ubiquitin by inhibiting ubiquitin recycling, allowing us to evaluate the relative contribution of distinct ubiquitin linkages in APC/C-mediated ubiquitination and degradation. Utilizing this approach, in chapter III, I found that the conjugation of single ubiquitin moieties to multiple lysine residues in cyclin promotes efficient degradation of cyclin B1 in mitotic Xenopus extracts. Lysine11-ubiquitin chain-formation becomes essential to proteasomal targeting only when the number of available lysine residues in cyclin B1 is restricted. Analysis in a reconstituted system revealed that APC/C catalyzes multiple monoubiquitination with rapid kinetics and species bearing four or more monoubiquitins on distinct lysines are recognized by ubiquitin receptors. These multiply monoubiquitinated species are rapidly degraded by purified proteasomes. In chapter IV, I examine the role of distinct E2 enzymes in APC/C-dependent proteolysis. I demonstrate that the chain-extending E2 UBE2S and long Lys11-linked ubiquitin assemblies are dispensable for cyclin B1 degradation, but become increasingly important with restriction of the number of ubiquitination sites. Our findings support a model where through attachment of monoubiquitin to multiple lysine residues, and possibly elaboration of some short chains, UBCH10, or possibly members of the UBC4/5 family, cooperate with the APC/C to generate a robust proteolytic signal on cyclin B1.

Made available in DSpace on 2016-08-17T18:39:45Z (GMT). No. of bitstreams: 1 4808.pdf: 1657962 bytes, checksum: abb30e9c4dc48bb4625a333ce6600cb8 (MD5) Previous issue date: 2012-02-28 / Eosinophils are a hematopoietic cell originated from precursor cells found in bone marrow, whose differentiation and proliferation is regulated by cytokines such as GM-CSF, IL-3 and IL-5. When activated, eosinophils are capable of phagocytosis of small particles and bacteria, but their main form of activity in the inflammatory process is the release of toxic proteins, cytokines, enzymes, lipid mediators and reactive oxygen products. The increase in eosinophil is an important feature in many diseases such as allergy and parasitic infections. Provided APC (Antigen-Presenting Cells), eosinophils are considered similar to the CD (Dendritic Cells) in its potential to activate naïve T cells and may have potential as efficient as the CD in stimulating lung T cells in the upper airways in the model inflammation. The APC are defined by being able to take, processing and presenting antigen such as CD, macrophages, B lymphocytes and possibly eosinophils. The surface expression of APC is characterized by coestimatórias molecules CD80 (B7-1) and CD86 (B7-2) and also by MHCII. The proposed model for this evaluation was to Visceral Larva Migrans syndrome (VLMS) caused by Toxocara canis, one of the most frequent helminth in young dogs. One of the main consequences of this infection is the marked increase in circulating and tissue eosinophils. Eosinophilia has been associated with parasitic diseases particularly when the parasite invades or promotes tissue damage at mucosal surfaces In the present study we evaluated the expression of MHC II and CD80 and CD86 molecules coestimulatórias in eosinophils in VLMS. Our results showed that the molecules studied were expressed in eosinophils in the blood of mice infected with Toxocara canis compared with the control group. Correlating an intense eosinophil still during the course of the disease with increased IL-5 in the infected group. Suggests that during the course of Toxocara canis, eosinophils can exhibit behavior of an APC, increasing the expression of MHCII molecules coestimulatorias and possibily amplifyng the immune response in this model. / O eosinófilo é uma célula hematopoiética, originada a partir de células precursoras presentes na medula óssea, cuja diferenciação e proliferação são reguladas por citocinas como GMCSF, IL-3 e IL-5. Quando ativados, os eosinófilos são capazes de realizar fagocitose de pequenas partículas e bactérias, mas sua principal forma de atuação no processo inflamatório consiste na liberação de proteínas tóxicas, citocinas, enzimas, mediadores lipídicos e produtos reativos de oxigênio. O aumento no número de eosinófilos é uma característica importante em diversas doenças como a alergia e as infecções parasitárias. Na condição de APC (Células Apresentadoras de Antígenos), os eosinófilos são considerados similares as CD (Células Dendríticas) em seu potencial para ativar células T naïve, podendo ter potencial tão eficiente quanto as CD pulmonares em estimular células T nas vias aéreas superiores no modelo da inflamação. As APC são definidas por serem capazes de ingerir, processar e apresentar o antígeno como: CD, macrófagos, linfócitos B e possivelmente os eosinófilos. A expressão na superfície da APC é caracterizada por moléculas coestimatórias CD80 (B7-1) e CD86 (B7-2) e ainda pelo MHCII. O modelo proposto para esta avaliação foi a Síndrome da Larva Migrans Visceral (SLMV) causada pelo Toxocara canis, um dos helmintos mais freqüentes em cães jovens. Uma das principais consequências desta infecção é o aumento marcante de eosinófilos circulantes e teciduais. A eosinofilia tem sido associada com doenças parasitárias particularmente quando o parasita invade os tecidos ou promove danos na superfície das mucosas. No presente estudo avaliamos a expressão de MHC II e moléculas coestimulatórias CD80 e CD86 em eosinófilos na SLMV. Nossos resultados mostraram que as moléculas analisadas foram expressas em eosinófilos no sangue de camundongos infectados com Toxocara canis quando comparado com o grupo controle. Correlacionando ainda uma intensa eosinofilia durante o curso da doença com o aumento de IL-5 no grupo infectado. Sugere que, durante o curso da infecção pelo Toxocara canis, eosinófilos podem apresentar comportamento de uma APC, aumentando a expressão de moléculas coestimulatórias e MHCII e possivelmente amplificando a resposta imune nesse modelo.

Elevated deoxycholic acid (DCA) and mutations in the Adenomatous Polyposis Coli (APC) tumor suppressor gene have been associated with increased risk of colorectal cancer (CRC). Chronic inflammation has also been associated with increased risk of CRC. It is unclear if DCA mediates inflammation in the normal or transformed colonic mucosa. The status of APC was manipulated in human CRC cell lines to study the role of DCA mediated inflammation. The chemotactic cytokine, CXCL8, was used as a marker of inflammation. Addition of DCA to the HT29-parental cell line with mutant-APC increased the steady state mRNA and protein levels of CXCL8. Conversely, addition of DCA to the HT29-APC cell line with wild type-APC was protective for increased steady state RNA and protein levels of CXCL8. DCA activated transcription factors which had binding regions in the CXCL8 5’-promoter. To elucidate the mechanism of induction, the 5’-promoter of CXCL8 was investigated. DCA increased promoter-reporter activity of the CXCL8 gene in HT29-parental cell line but wild type-APC blocked this effect. Chromatin immunoprecipitation (ChIP) revealed that DCA activated transcription factors, AP-1 and NF-κB were bound to the 5’-promoter of CXCL8. The transcription factor, β-catenin, was also bound to the 5’-promoter of CXCL8. Phenotypic effects were measured. Increased CXCL8 lead to matrix metalloproteinase-2 (MMP-2) production and increased invasion by HT29-parental cells on laminin coated filters. The DCA-mediated invasion was blocked by antibody directed against CXCL8 and wild type- APC. Therefore DCA-mediated inflammation occurs in transformed colonic epithelium and increases the invasive phenotype of CRC cells by CXCL8.

αβ-TCR+CD3+CD4-CD8-NK1.1- double negative (DN) T cells comprise 1-5% of T lymphocytes in mice and humans. Previous studies have demonstrated that DN T cells can suppress auto-, allo- and xeno-immune responses in an antigen-specific fashion. However, the mechanisms by which DN T cells regulate immune responses remain elusive. Whether DN T cells can regulate antigen presenting cells has not been investigated previously. The focus of this thesis is to determine the consequences of DN T cells interaction with antigen presenting cells (APCs) and the underlying mechanisms. In this thesis, using a murine skin transplantation model, we found that donor B cells, but not dendritic cells (DCs), are the major surviving donor APCs in recipients following donor lymphocyte infusion. Infusing donor B, but not non-B, cells resulted in significantly enhanced donor-specific skin allograft survival. Mice that had received donor B cells showed higher expression of activation markers on antigen-specific DN T cells. B cells could present alloantigen to DN T cells and prime DN T cell proliferation in an antigen-specific fashion. Activated DN T cells were not able to down regulate the expression of CD80 or CD86 on LPS-activated B cells, but they could kill activated allogeneic as well as syngeneic B cells via a perforin-dependent pathway in vitro. In addition, DN T cells expressed high levels of CTLA4 and were capable of down regulating CD80 and CD86 expressed on antigen-expressing mature DCs through CTLA4. DN T cells killed both immature and mature allogeneic DCs, as well as antigen-loaded syngeneic DCs, in an antigen-specific manner in vitro and in vivo, mainly through the Fas-FasL pathway. Taken together, the data presented in this thesis demonstrate, for the first time, that DN T cells are potent regulators of APCs and further clarify the mechanisms of DN T cell-mediated immune suppression. These findings provide novel insights for DN T cells to be developed as a potent immune suppression treatment for a variety of diseases.

αβ-TCR+CD3+CD4-CD8-NK1.1- double negative (DN) T cells comprise 1-5% of T lymphocytes in mice and humans. Previous studies have demonstrated that DN T cells can suppress auto-, allo- and xeno-immune responses in an antigen-specific fashion. However, the mechanisms by which DN T cells regulate immune responses remain elusive. Whether DN T cells can regulate antigen presenting cells has not been investigated previously. The focus of this thesis is to determine the consequences of DN T cells interaction with antigen presenting cells (APCs) and the underlying mechanisms. In this thesis, using a murine skin transplantation model, we found that donor B cells, but not dendritic cells (DCs), are the major surviving donor APCs in recipients following donor lymphocyte infusion. Infusing donor B, but not non-B, cells resulted in significantly enhanced donor-specific skin allograft survival. Mice that had received donor B cells showed higher expression of activation markers on antigen-specific DN T cells. B cells could present alloantigen to DN T cells and prime DN T cell proliferation in an antigen-specific fashion. Activated DN T cells were not able to down regulate the expression of CD80 or CD86 on LPS-activated B cells, but they could kill activated allogeneic as well as syngeneic B cells via a perforin-dependent pathway in vitro. In addition, DN T cells expressed high levels of CTLA4 and were capable of down regulating CD80 and CD86 expressed on antigen-expressing mature DCs through CTLA4. DN T cells killed both immature and mature allogeneic DCs, as well as antigen-loaded syngeneic DCs, in an antigen-specific manner in vitro and in vivo, mainly through the Fas-FasL pathway. Taken together, the data presented in this thesis demonstrate, for the first time, that DN T cells are potent regulators of APCs and further clarify the mechanisms of DN T cell-mediated immune suppression. These findings provide novel insights for DN T cells to be developed as a potent immune suppression treatment for a variety of diseases.

Septins are evolutionarily conserved proteins with roles in chromosome congression and segregation, cytokinesis and microtubule destabilization. Septins form homo- and hetero-oligomeric complexes, which are thought to act as dynamic scaffolds. We identified SEPT2/9/11/10 as novel interacting partners of adenomatous polyposis coli (APC), a bona fide tumor suppressor. Since septins and APC have similar roles and knockdown phenotypes, I sought to determine if they work together to perform their cellular functions. I showed that APC co-immunoprecipitates with endogenous septins in colon cancer cell lines. Using siRNA, I found that SEPT2 and APC may function within the same pathway to regulate DNA congression and segregation. Co-depleting SEPT9 with APC slightly alleviates the chromosome congression and segregation defects caused by siAPC alone. siSEPT9 increased abscission times, which was rescued by co-depleting APC. Future studies should elucidate the significance of the rescue data obtained upon APC and SEPT9 co-depletion and APC’s interactions with SEPT10/11.