There are emerging lines of evidence to suggest that infectious complications in gastrointestinal cancer patients undergoing curative resection may associate with cancer recurrence, but the exact mechanism is not well understood. Toll-like receptor 4 (TLR4) is the sole receptor for lipopolysaccharide (LPS), a gram-negative bacterial antigen involved in such infectious complications. Previously, TLR4 expression has been found on several cancer types including prostate and lung; in this study, we characterized TLR4 expression for several esophageal and colorectal cell lines, and evaluated the effects of LPS on tumor proliferation, tumor cytokine production, and tumor adhesion. Even though LPS has no direct impact on tumor proliferation, LPS stimulation increased IL-6 production in HKESC-2 esophageal cancer cells. LPS-TLR4 signaling through p38 MAP kinase was necessary for LPS induced cancer cell attachment to fibronectin, an important composition of liver extracellular matrix. LPS-treated colon cells also showed an increase adherence to liver sinusoids. Both inhibition of p38 map kinase and TLR4 antagonist significantly prevented LPS induced adhesion to fibronectin. This study has provided some of the mechanisms responsible for inflammation associated cancer recurrence in patients who undergo curative surgical resection. These data suggest that LPS-TLR4 signaling in gastrointestinal cancer cells increases their metastatic potential and TLR4 blockade may have a therapeutic value in the prevention of cancer metastasis. / Des données récentes suggèrent que le développement de complications infectieuses à la suite d'une résection curative chez les patients atteints d'un cancer gastro-intestinal peut être associé à une récidive du cancer, cependant le mécanisme exact n'est pas bien compris. Toll-like receptor 4 (TLR4) est le seul récepteur connu pour le lipopolysaccharide (LPS), un antigène provenant des bactéries à Gram négatif impliquées dans de telles complications infectieuses. Précédemment, l'expression de TLR4 a été détectée pour plusieurs types de cancers incluant la prostate et le poumon. Dans le cadre de cette étude, nous avons caractérisé l'expression de TLR4 pour de multiples lignées cellulaires œsophagiennes et colorectales, ainsi qu'évalué l'effet du LPS sur la prolifération tumorale, la production de cytokine tumorale, et l'adhésion tumorale. Malgré le fait que le LPS n'ait aucun effet sur la prolifération cellulaire tumorale, la stimulation par LPS augmente la production de IL-6 chez les cellules cancéreuses HKESC-2 provenant de l'œsophage. Le signalement intracellulaire LPS-TLR4 par p38 MAP Kinase est nécessaire pour l'attachement à la fibronectin des cellules cancéreuses traitées avec LPS, une composante importante de la matrice extracellulaire hépatique. Les cellules provenant du colon traitées avec LPS ont démontrées une augmentation de leur adhésion aux sinusoïdes hépatiques. Cette étude a fournie quelques-uns des mécanismes responsables de la récurrence cancéreuse associée à l'inflammation chez les patients subissant une résection chirurgicale curative. Ces résultats suggèrent que la signalisation par LPS-TLR4 dans les cancers gastro-intestinaux augmente leur potentiel métastatique et que le blocage de TLR4 ait possiblement un effet thérapeutique dans la prévention des métastases cancéreuses.

The goal of my project was to develop cell-based assays to investigate the functions of the p110 CUX1 homeodomain transcription factor during cell cycle progression. I observed that constitutive expression of p110 CUX1 stimulates cell proliferation. Using fluorescence-activated cell sorting (FACS) analysis and BrdU incorporation, I demonstrated that p110 CUX1 can accelerate entry into S phase whether cells were coming out of quiescence, were allowed to resume progression through the cell cycle after synchronization at the G1/S or were simply enriched in G2/M using counter-flow centrifugal elutriation. Consistent with these observations, mouse embryo fibroblasts (MEFs) derived from homozygous cux1-/- mice proliferated more slowly and spent more time in G1 than wild type MEFs. I identified the cyclin E2 and cyclin A2 genes as some of the downstream targets of p110 CUX1 that mediate its stimulatory effect on the G1/S transition. Constitutive expression of p110 CUX1 in some, but not all, cell lines led to the emergence of cells with a tetraploid DNA content. Whereas the induction of tetraploidy in normal cells causes cell cycle arrest or cell death, cells expressing p110 CUX1 were able to undergo bipolar division in spite of the presence of more than two centrosomes. Using various experimental approaches, I demonstrated that p110 CUX1 contributes to the establishment of a transcriptional program that allows cells to sustain a robust spindle assembly checkpoint. Tetraploid cells eventually evolved to become aneuploid and tumorigenic. Similarly, over 80% of mammary tumor cells from CUX1 transgenic mice exhibit a near-tetraploid DNA content. I therefore propose that one mechanism by which elevated p110 CUX1 expression contributes to tumor development is through its effect on the spindle assembly checkpoint. In this manner, p110 CUX1 enables the survival of cells that harbor supernumerary chromosome and/or centrosome numbers and have the capacity to generate a large num / L'objectif initial de mon projet consistait à mettre au point divers tests permettant d'étudier le rôle du facteur de transcription à homéodomaine p110 CUX1 au cours du cycle cellulaire. J'ai d'abord observé que p110 CUX1 stimule la prolifération cellulaire lorsque exprimé de façon soutenue. À partir d'analyses par cytométrie en flux et de marquages au BrdU, j'ai démontré que l'expression de p110 CUX1 suscite une accélération de l'entrée en phase S. Cet effet s'est manifesté dans trois situations : à partir de la phase de quiescence (G0), à partir d'une synchronisation en G1/S et à partir de la phase G2/M lors d'un enrichissement par centrifugation à contre-courant (élutriation). En accord avec ces résultats, les fibroblastes murins isolé à partir d'embryons Cux-/- proliférèrent plus lentement par rapport aux fibroblastes Cux+/+ à cause d'une prolongation de la phase G1. J'ai identifié les gènes cycline E2 et cycline A2 comme deux cibles transcriptionnelles de p110 CUX1 pouvant expliquer son effet sur la transition G1/S. J'ai observé qu'il se développait une sous-population tétraploïde dans certaines lignées cellulaires exprimant p110 CUX1. Alors que la tétraploïdie cause généralement un arrêt de la prolifération ou la mort cellulaire dans les cellules normales, j'ai noté que l'expression de p110 CUX1 permettait aux cellules tétraploïdes d'avoir une division bipolaire normale malgré la présence de plusieurs centrosomes. J'ai démontré que cet effet était dû à la capacité de p110 CUX1 d'induire la transcription de plusieurs gènes permettant une activation efficace et prolongée du point de contrôle mitotique. J'ai établi un lien entre l'instabilité génomique des cellules tetraploïdes exprimant p110 CUX1 et l'acquisition de caractéristiques tumorales. De plus, j'ai noté que plus de 80% des cellules de tumeurs mammaires provenant de souris transgéniques CUX1 sont quasi tétraploïdes. Je propose donc un nou

TGF-β signalling regulates various cellular activities throughout development and adulthood. Disruptions in the TGF-β signalling cascade are associated with several human diseases. SMAD2 is one of the principal effectors of TGF-β signalling. It interacts with various transcription factors, and is extensively regulated by post-translational modifications, such as ubiquitination. However, much remains unknown about the regulation of SMAD2 function. Using a proteomic screen, I identified ubiquitin-specific peptidase 11 (USP11) as a novel SMAD2 interactor. I confirmed their mutual interaction, and showed that USP11 specifically interacts with the linker domain of SMAD2, but did not appear to regulate either its stability or ubiquitination pattern. USP11 knockdown decreased TGF-β-mediated gene promoter-reporter activity, whereas USP11 over-expression potentiated it. USP11 knockdown did not, however, affect endogenous gene expression after 2 h TGF-β treatment, as determined by microarray analysis and quantitative reverse transcription PCR. I conclude that although USP11 did not regulate the stability of SMAD2, it might be involved in the regulation of other, currently unresolved, aspects of TGF-β signalling. / La signalisation de TGF-β contrôle des processus cellulaires variés au cours du développement et de l'âge adulte. Des perturbations dans la cascade TGF-β sont associées à plusieurs maladies humaines. SMAD2 est l'un des principaux effecteurs de cette cascade et sa fonction est modulée par des modifications post-traductionnelles comme l'ubiquitination. Cependant, il reste encore plusieurs interrogations à propos de sa modulation. En utilisant une approche protéomique, j'ai identifié la peptidase spécifique de l'ubiquitin -11 (USP11) comme un nouveau partenaire de SMAD2. J'ai confirmé leur interaction et démontré qu'USP11 interagit avec le domaine de liaison de SMAD2, mais n'affecte pas sa stabilité ou son ubiquitination. La suppression d'USP11 par siRNA a diminué l'activité de gène rapporteur de TGF-β, mais n'a pas affecté l'expression des gènes endogènes, comme déterminé à l'aide de puce à ADN et par qPCR. En conclusion, même si USP11 ne contrôle pas la stabilité de SMAD2, il peut être impliqué dans la régulation de la signalisation TGF-β à un autre niveau.

Light microscopic immunoperoxidase staining of rat testes with polyclonal anti-Sm and monoclonal anti-Y$ sb{12}$ localized U1, U2, U4/U6 and U5 (spliceosome) snRNPs predominantly to nuclei of germ cells up to step 10 spermatids. The absence of reactivity after step 11 is concomitant with nuclear condensation and transcriptional arrest (Monesi, 1964). Nuclear and cytoplasmic reactivity in pachytene spermatocytes reached a peak at stage XII, coincident with maximal rates of RNA synthesis (Monesi, 1964). Quantitative EM immunogold labeling of Lowicryl embedded testicular sections confirmed the light microscope observations. Immunogold speckles were observed along nuclear chromatin. The chromatoid bodies of spermatids and spermatocytes and the intermitochondrial material of spermatocytes were found to be additional sites of snRNP localization. This co-localization suggests that these dense cytoplasmic structures are related. Anti-U1 snRNP antibodies applied to frozen sections co-localized with spliceosome snRNPs. Anti-U3 snRNP antibodies applied to frozen sections stained nucleoli of germ cells where pre-rRNA is usually spliced.

In response to stresses such as the accumulation of misfolded proteins, the Endoplasmic Reticulum (ER) triggers cytosolic and nuclear signalling events, which are integrated by the cell to result in adaptation or apoptosis. The activation of stress-responsive kinases (SAPKs) and the mitogen-activated kinases (MAPKs) are known to have a key influence cell fate determination. We developed a technique to study the regulation of these kinases by the ER in response to drugs inducing protein misfolding. We isolated ER like compartments with a relatively high degree of purity using magnetic beads coated with anti-calnexin antibodies and vie reconstituted in a cell free system the ER signalling induced by different stresses. We demonstrate here that ER-like compartments isolated from cells treated with azetidine-2 carboxylic acid or Tunicamycin differentially activates ERK-1, JNK-1 and p38 in vitro. Moreover, we show that the molecular adaptors Shc and Nck are involved in the ER-mediated regulation of these kinases.

There are a number of isoforms in the PLA2 superfamily, including secretory PLA2s (sPLA2) cytosolic PLA2s (cPLA 2), and calcium independent PLAS (iPLA2beta-short, beta-long, and gamma). In membranous nephropathy, glomerular epithelial cell (GEC) injury by complement C5b-9 leads to morphological changes in GEC and proteinuria, in association with cPLA2alpha activation. The present study addresses the role of iPLA2 in GEC injury. Complement-mediated release of [3H] arachidonic acid was most significantly augmented in GEC overexpressing iPLA2gamma (GEC-iPLA2gamma) as compared with GEC-Neo control. The accelerated AA release was inhibited by the iPLA2-directed catalytic inhibitor bromoenol lactone (BEL). For comparison, GEC-iPLA2gamma also amplified [3H]AA release after incubation of GEC with H2O2, or chemical anoxia followed by re-exposure to glucose, whereas stable clones overexpressing iPLA2gamma only amplified [3H]AA release after incubation with H2O2. Complement-mediated cytotoxicity (measured by release of lactate dehydrogenase) was attenuated significantly in GEC-iPLA2gamma, as compared with GEC-Neo, and the cytoprotective effect of iPLA2gamma was reversed by BEL and partially by Indomethacin. In keeping with previous results, incubation of GEC-cPLA2alpha with complement increased free [3H]AA. This increase in [ 3H]AA was blocked by BEL, although BEL did not block cPLA2alpha activity in cell extracts in vitro. Thus, in addition to cPLA2alpha, iPLA2gamma may be involved in complement-mediated release of AA. Moreover, activation of cPLA2alpha by complement appears to be, at least in part, dependent on iPLA2gamma. Modulation of iPLA2gamma activity may provide a new approach to reducing GEC injury.

Acid sphingomyelinase (ASM), a member of the saposin like protein (SAPLIP) family, is a lysosomal hydrolase that converts sphingomyelin to ceramide. The deficient activity of ASM causes a variant form (i.e., type A/B) of the inherited disorder Niemann-Pick disease. The lysosomal targeting mechanism of ASM has not been conclusively identified. Previous studies suggested that ASM could use another membrane-associated receptor as well as M6P receptor to target lysosomes. Sortilin, a type I transmembrane glycoprotein, belongs to a novel family of receptor proteins. Both the luminal domain and the cytoplasmic domain of sortilin show structural features typical of receptors involved in lysosomal or vacuolar targeting. Using a dominant-negative sortilin construct lacking the cytoplasmic tail, I proved that sortilin was involved in the lysosomal targeting of ASM. Confocal microscopy revealed that truncated sortilin partially inhibited the lysosomal targeting of ASM in COS 7 cells and completely abolished the lysosomal targeting of ASM in I-cells. Pulse-chase experiments also suggested that sortilin was involved in normal sorting of newly synthesized ASM. Over-expression of truncated sortilin accelerated and enhanced the secretion of ASM from COS 7 cells and I-cells. Co-immunoprecipitation assays further confirmed the interaction between sortilin and ASM. I also observed that the lysosomal transport of ASM was reduced to some extent in I-cell disease fibroblasts as compared to normal fibroblasts. In conclusion, both the M6P receptor and sortilin mediate lysosomal targeting of ASM.

The quaking viable mice have myelination defects and develop a tremor in their hind limbs 10 days after birth. This is caused by a deletion in the promoter/enhancer region of the quaking gene. The quaking gene is alternatively spliced, producing QUAKING isoforms that differ in their C-terminal 8--30 amino acid sequence. The QKI-5 isoform is nuclear, whereas the QKI-6 and QKI-7 isoforms are predominantly cytoplasmic. The QKI isoforms contain a single KH RNA-binding domain suggesting a role in RNA metabolism. Although the dysmyelinating phenotype of the mutant mouse suggests a role in myelination, the function of the protein still remains unknown. The goal of this thesis is to characterize the possible functions of the quaking protein. We first explored the ability of the QKI-7 isoform to induce apoptosis in fibroblast cells and confirmed this event in oligodendrocytes, where most of the QUAKING isoforms are present. We mapped the domain responsible for this event to the alternatively spliced C-terminus, which we named 'KILLER' sequence. Heterodimerization of the other QKI isoforms with QKI-7 causes its relocalization into the nucleus, suppressing its ability to induce apoptosis. Therefore we hypothesize that the balance between the different isoforms dictates whether the cell will live or die. We were also interested in finding a RNA binding substrate for this protein, therefore we looked at potential proteins that are altered in the quaking viable mouse. We found that the mRNA myelin basic protein (MBP) is in fact a target for the QKI proteins. We show that this interaction occurs through the 3'-UTR of MBP, in a 110 nucleotide region named QRE for Quaking Responsive Element. In addition, we have recreated a defect of the quaking viable phenotype by over-expressing QKI-5 into the brain of mice. This causes retention of the MBP mRNA into the nucleus of oligodendrocytes. As a result, MBP expression is significantly reduced. These observations

Apoptosis is a phenomenon conserved from flies to humans, crucial in development and in the maintenance of cellular homeostasis. Throughout the years various molecules have been identified as critical regulators of apoptotic signaling. Among these, the serine/threonine kinase Akt has been demonstrated to play a pivotal role in the regulation of cell survival by phosphorylating a broad range of effectors. This thesis looks at the regulation of anti and pro-apototic Akt substrates, and examines the survival-promoting activity of Akt in neural tumor cells and neurons. / In the first section of this thesis I demonstrated, using a recombinant adenovirus-based approach, that Akt is necessary for the NGF-dependent survival of differentiated neuronal-like PC12 (pheochromocytoma) cells and undifferentiated PC12 cells. Furthermore, I suggested that Akt possibly performs this function via the repression of the c-jun N-terminal kinase (JNK) pathway, upstream of JNK, as overexpression of kinase-inactive Akt activates JNK. / In the second section of this thesis, I identified mixed-lineage kinase-3 (MLK-3) as a substrate of Akt. I showed that Akt associates with the JIP/MLK-3/JNK scaffold complex, and that it inactivates MLK-3 by phosphorylating T477. This study strongly suggests that in certain cellular systems, Akt represses the JNK signaling pathway and apotosis via the inhibition of MLK-3. / Finally, in the third section, we discovered that Akt phosphorylates and interacts with the caspase inhibitor, X-linked inhibitor of apoptosis (XIAP). The phosphorylation of XIAP occurs on serine 87. We also showed that PI3-K/Akt synergize with XIAP to promote sympathetic neuron survival. / These studies contribute to the understanding of Akt's anti-apoptotic mechanisms and might also help design highly specific therapeutic approaches.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2006. / Source: Dissertation Abstracts International, Volume: 68-02, Section: B, page: 0707. Adviser: Jonathan Joseph Henry. Includes bibliographical references (leaves 121-149) Available on microfilm from Pro Quest Information and Learning.