New genetic models and mesenchymal cells as tools to ameliorate anti-tumor immunity

Résumé Le but final de ce projet est d'utiliser des cellules T ou des cellules souches mésenchymateuses modifiées génétiquement afin de surexprimer localement les deux chémokines CXCL13 et CCL2 ensemble ou chacune séparément à l'intérieur d'une tumeur solide. CXCL13 est supposé induire des structures lymphoïdes ectopiques. Un niveau élevé de CCL2 est présumé initier une inflammation aiguë. La combinaison des deux effets amène à un nouveau modèle d'étude des mécanismes régulateur de la tolérance périphérique et de l'immunité tumorale. Les connaissances acquises grâce à ce modèle pourraient permettre le développement ou l'amélioration des thérapies immunes du cancer. Le but premier de ce travail a été l'établissement d'un modèle génétique de la souris permettant d'exprimer spécifiquement dans la tumeur les deux chémokines d'intérêt à des niveaux élevés. Pour accomplir cette tâche, qui est en fait une thérapie génétique de tumeurs solides, deux types de cellules porteuses potentielles ont été évaluées. Des cellules CD8+ T et des cellules mésenchymateuses de la moelle osseuse transférées dans des receveurs portant une tumeur. Si on pouvait répondre aux besoins de la thérapie génétique, indépendamment de la thérapie immune envisagée, on posséderait là un outil précieux pour bien d'autres approches thérapeutiques. Plusieurs lignées de souris transgéniques ont été générées comme source de cellules CD8+ T modifiées afin d'exprimer les chémokines d'intérêt. Dans une approche doublement transgénique les propriétés de deux promoteurs spécifiques de cellules T ont été combinées en utilisant la technologie Cre-loxP. Le promoteur de granzyme B confère une dépendance d'activation et le promoteur distal de lck assure une forte expression constitutive dès que les cellules CD8+ T ont été activées. Les transgènes construits ont montré une bonne performance in vivo et des souris qui expriment CCL2 dans des cellules CD8+ T activées ont été obtenues. Ces cellules peuvent maintenant être utilisées avec différents protocoles pour transférer des cellules T cytotoxiques (CTL) dans des receveurs porteur d'une tumeur, permettant ainsi d'évaluer leur capacité en tant que porteuse de chémokine d'infiltrer la tumeur. L'établissement de souris transgéniques, qui expriment pareillement CXCL13 est prévu dans un avenir proche. L'évaluation de cellules mésenchymateuses de la moelle osseuse a démontré que ces cellules se greffent efficacement dans le stroma tumoral suite à la co-injection avec des cellules tumorales. Cela représente un outil précieux pour la recherche, vu qu'il permet d'introduire des cellules manipulées dans un modèle tumoral. Les résultats confirment partiellement d'autres résultats rapportés dans un modèle amélioré. Cependant, l'efficacité et la spécificité suggérées de la migration systémique de cellules mésenchymateuses de la moelle osseuse dans une tumeur n'ont pas été observées dans notre modèle, ce qui indique, que ces cellules ne se prêtent pas à une utilisation thérapeutique. Un autre résultat majeur de ce travail est l'établissement de cultures de cellules mésenchymateuses de la moelle osseuse in vitro conditionnées par des tumeurs, ce qui a permis à ces cellules de s'étendre plus rapidement en gardant leur capacité de migration et de greffe. Cela offre un autre outil précieux, vu que la culture in vitro est un pas nécessaire pour une manipulation thérapeutique. Abstract The ultimate aim of the presented project is to use genetically modified T cells or mesenchymal stem cells to locally overexpress the two chemokines CXCL13 and CCL2 together or each one alone inside a solid tumor. CXCL13 is supposed to induce ectopic lymphoid structures and a high level of CCL2 is intended to trigger acute inflamation. The combination of these two effects represents a new model for studying mechanisms that regulate peripheral tolerance and tumor immunity. Gained insights may help developing or improving immunotherapy of cancer. The primary goal of the executed work was the establishment of a genetic mouse model that allows tumor-specific expression of high levels of the two chemokines of interest. For accomplishing this task, which represents gene therapy of solid tumors, two types of potentially useful carrier cells were evaluated. CD8+ T cells and mesenchymal bone marrow cells to be used in adoptive cell transfers into tumor-bearing mice. Irrespectively of the envisaged immunotherapy, satisfaction of so far unmet needs of gene therapy would be a highly valuable tool that may be employed by many other therapeutic approaches, too. Several transgenic mouse lines were generated as a source of CD8+ T cells modified to express the chemokines of interest. In a double transgenic approach the properties of two T cell-specific promoters were combined using Cre-loxP technology. The granzyme B promoter confers activation-dependency and the lck distal promoter assures strong constitutive expression once the CD8+ T cell has been activated. The constructed transgenes showed a good performance in vivo and mice expressing CCL2 in activated CD8+ T cells were obtained. These cells can now be used with different protocols for adoptively transferring cytotoxic T cells (CTL) into tumor-bearing recipients, thus allowing to study their capacity as tumor-infiltrating chemokine carrier. The establishment of transgenic mice likewisely expressing CXCL13 is expected in the near future. In addition, T cells from generated single transgenic mice that have high expression of an EGFP reporter in both CD4+ and CD8+ cells can be easily traced in vivo when setting up adoptive transfer conditions. The evaluation of mesenchymal bone marrow cells demonstrated that these cells can efficiently engraft into tumor stroma upon local coinjection with tumor cells. This represents a valuable tool for research purposes as it allows to introduce manipulated stromal cells into a tumor model. Therefore, the established engraftment model is suited for studying the envisaged immunotherapy. These results confirm to some extend previously reported results in an improved model, however, the suggested systemic tumor homing efficiency and specificity of mesenchymal bone marrow cells was not observed in our model indicating that these cells may not be suited for therapeutic use. Another major result of the presented work is the establishment oftumor-conditioned in vitro culture of mesenchymal bone marrow cells, which allowed to more rapidly expand these cells while maintaining their tumor homing and engrafting capacities. This offers another valuable tool as in vitro culture is a necessary step for therapeutic manipulations.

Breast cancer suppressor candidate-1 (BCSC-1) is a melanoma tumor suppressor that down regulates MITF.

Understanding the molecular aberrations involved in the development and progression of metastatic melanoma (MM) is essential for a better diagnosis and targeted therapy. We identified breast cancer suppressor candidate-1 (BCSC-1) as a novel tumor suppressor in melanoma. BCSC-1 expression is decreased in human MM, and its ectopic expression in MM-derived cell lines blocks tumor formation in vivo and melanoma cell proliferation in vitro while increasing cell migration. We demonstrate that BCSC-1 binds to Sox10, which down regulates MITF, and results in a switch of melanoma cells from a proliferative to a migratory phenotype. In conclusion, we have identified BCSC-1 as a tumor suppressor in melanoma and as a novel regulator of the MITF pathway.

Embryonic development of NMRI mice: relationship between the weight, age and ossification of embryos.

The time and the order of the appearance of the ossification centres were found to be similar in C3H and NMRI mice. Bodyweight comparisons confirmed these results. Location in the right as opposed to the left uterine horn, or in the upper, middle or lower part, was not found to influence the weight of the embryo. Significant differences in the weight of embryos within the same litter were used in investigating the sequence of ossification in embryos. This should prove useful in comparative morphology and teratology.

GLUT3 is induced during epithelial-mesenchymal transition and promotes tumor cell proliferation in non-small cell lung cancer.

BACKGROUND: Alterations in glucose metabolism and epithelial-mesenchymal transition (EMT) constitute two important characteristics of carcinoma progression toward invasive cancer. Despite an extensive characterization of each of them separately, the links between EMT and glucose metabolism of tumor cells remain elusive. Here we show that the neuronal glucose transporter GLUT3 contributes to glucose uptake and proliferation of lung tumor cells that have undergone an EMT. RESULTS: Using a panel of human non-small cell lung cancer (NSCLC) cell lines, we demonstrate that GLUT3 is strongly expressed in mesenchymal, but not epithelial cells, a finding corroborated in hepatoma cells. Furthermore, we identify that ZEB1 binds to the GLUT3 gene to activate transcription. Importantly, inhibiting GLUT3 expression reduces glucose import and the proliferation of mesenchymal lung tumor cells, whereas ectopic expression in epithelial cells sustains proliferation in low glucose. Using a large microarray data collection of human NSCLCs, we determine that GLUT3 expression correlates with EMT markers and is prognostic of poor overall survival. CONCLUSIONS: Altogether, our results reveal that GLUT3 is a transcriptional target of ZEB1 and that this glucose transporter plays an important role in lung cancer, when tumor cells loose their epithelial characteristics to become more invasive. Moreover, these findings emphasize the development of GLUT3 inhibitory drugs as a targeted therapy for the treatment of patients with poorly differentiated tumors.

The molecular signature of the stroma response in prostate cancer-induced osteoblastic bone metastasis highlights expansion of hematopoietic and prostate epithelial stem cell niches.

The reciprocal interaction between cancer cells and the tissue-specific stroma is critical for primary and metastatic tumor growth progression. Prostate cancer cells colonize preferentially bone (osteotropism), where they alter the physiological balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, and elicit prevalently an osteoblastic response (osteoinduction). The molecular cues provided by osteoblasts for the survival and growth of bone metastatic prostate cancer cells are largely unknown. We exploited the sufficient divergence between human and mouse RNA sequences together with redefinition of highly species-specific gene arrays by computer-aided and experimental exclusion of cross-hybridizing oligonucleotide probes. This strategy allowed the dissection of the stroma (mouse) from the cancer cell (human) transcriptome in bone metastasis xenograft models of human osteoinductive prostate cancer cells (VCaP and C4-2B). As a result, we generated the osteoblastic bone metastasis-associated stroma transcriptome (OB-BMST). Subtraction of genes shared by inflammation, wound healing and desmoplastic responses, and by the tissue type-independent stroma responses to a variety of non-osteotropic and osteotropic primary cancers generated a curated gene signature ("Core" OB-BMST) putatively representing the bone marrow/bone-specific stroma response to prostate cancer-induced, osteoblastic bone metastasis. The expression pattern of three representative Core OB-BMST genes (PTN, EPHA3 and FSCN1) seems to confirm the bone specificity of this response. A robust induction of genes involved in osteogenesis and angiogenesis dominates both the OB-BMST and Core OB-BMST. This translates in an amplification of hematopoietic and, remarkably, prostate epithelial stem cell niche components that may function as a self-reinforcing bone metastatic niche providing a growth support specific for osteoinductive prostate cancer cells. The induction of this combinatorial stem cell niche is a novel mechanism that may also explain cancer cell osteotropism and local interference with hematopoiesis (myelophthisis). Accordingly, these stem cell niche components may represent innovative therapeutic targets and/or serum biomarkers in osteoblastic bone metastasis.

The effect of COX-2 inhibitors on the formation of heterotopic bone in a rat model : O1323

Aims: 1) to create a new and reproducible animal model to produce heterotopic ossification (HO) 2) to be able to exactly quantify the amount of HO using a microCT scan and 3) to prove the hypothesis that COX-2 inhibitors are efficacious in the prevention of HO. Methods: We developed a IACUC-approved Lewis rat model, in which the ventral side of the right femur was scraped to mechanically disrupt the periosteum. By clamping the vastus intermedius ischemic injury to the muscle was produced to enhance HO. Finally homologous bone marrow from a donor rat was placed on the anterior surface of the femur. Half of the study group (8 rats) received chow mixed with a COX-2 inhibitor, while the other half received normal chow. After 6 weeks the animals were sacrificed, the femurs removed and imaged by microCT. Grading of HO was based on the thickness of ectopic bone as evaluated in a blinded fashion by 3 independent observers. Results: All animals developed bilateral HO. Rats treated with COX-2 inhibitors developed significantly less ectopic bone than the control group rats. Conclusions: The results suggest that we have created a very reliable, reproducible model to form ectopic bone in rats. Using the microCT we can precisely quantify the amount of HO. We have been able to show that COX-2 inhibitors significantly decrease the amount of HO formation and are thus a good alternative to non-specific NSAIDs with their potential serious side effects on the gastrointestinal tract and on hemo-stastis.

Chlamydia: signes d'appel, diagnostic et traitement [Chlamydia: diagnostic and treatment].

Chlamydia are obligate intracellular bacteria. Three species are considered human pathogens. Chlamydophila pneumoniae is one of the most common agents of atypical community-acquired pneumonia. Chlamydophila psittaci causes psittacosis, a severe zoonotic pneumonia transmitted by birds. Finally, Chlamydia trachomatis is the etiologic agent of trachoma and urogenital infections. The latter are commonly asymptomatic or paucisymptomatic. Thus, they may remain undiagnosed for years, leading to serious late complications such as salpingitis, ectopic pregnancy and infertility. Currently, the diagnosis of chlamydial infections is essentially based on molecular methods. Treatment should use an antibiotic with good intracellular bioavailability such as tetracycline, macrolides and new generation fluoroquinolones.

In-depth analysis of hyaline fibromatosis syndrome frameshift mutations at the same site reveal the necessity of personalized therapy.

Hyaline fibromatosis syndrome is an autosomal recessive disease caused by mutations in ANTXR2, a gene involved in extracellular matrix homeostasis. Sixty percent of patients carry frameshift mutations at a mutational hotspot in exon 13. We show in patient cells that these mutations lead to low ANTXR2 mRNA and undetectable protein levels. Ectopic expression of the proteins encoded by the mutated genes reveals that a two base insertion leads to the synthesis of a protein that is rapidly targeted to the ER-associated degradation pathway due to the modified structure of the cytosolic tail, which instead of being hydrophilic and highly disordered as in wild type ANTXR2, is folded and exposes hydrophobic patches. In contrast, one base insertion leads to a truncated protein that properly localizes to the plasma membrane and retains partial function. We next show that targeting the nonsense mediated mRNA decay pathway in patient cells leads to a rescue of ANTXR2 protein in patients carrying one base insertion but not in those carrying two base insertions. This study highlights the importance of in-depth analysis of the molecular consequences of specific patient mutations, which even when they occur at the same site can have drastically different consequences.

Roles of PPARβ and PPARγ in mouse placental development

RESUME Les gènes des PPARs jouent des rôles importants dans la régulation du métabolisme énergétique, lipidique et glucidique. Le présent travail, caractérise et analyse les défauts placentaires responsables de la mort embryonnaire des souris mutantes pour PPARβ et pour PPARγ, entre le jour E9.5 et E10.5. Les placentas issus d'embryons PPARP présentent un sévère retard de croissance, alors que les placentas mutants PPARγ montrent de graves défauts vasculaires. Nous montrons que les placentas issus d'embryons PPARβ-/-, au jour E9.5 présentent une réduction prononcée de la couche de cellules géantes, associée à une diminution des niveaux de protéines exprimées par les cellules géantes, tel que le placenta lactogène-I et la « proliferin ». Par ailleurs, nous montrons que le traitement d'un lignée trophoblastique par un ligand spécifique de PPARP augmente considérablement leur différentiation en cellules géantes. Cette différentiation dépendante de la voie de signalisation P13-kinase, s'accompagne d'une élévation de l'expression de l'ADRP, une protéine de structure associée aux vésicules lipidiques. Ainsi nous démontrons que PPAR5 est un régulateur majeur de la différentiation des cellules géantes, lesquelles sont primordiales aussi bien pour l'établissement de la structure placentaire, que pour la fonction endocrine. Par contre, les placentas PPARγ-/- présentent un défaut de vascularisation. Le niveau d'une protéine anti-angiogénique, la « proliferin-related protein », est très basse et ne peut pas contre-balancer l'élévation normale de la protéine pro-angiogénique « proliferin ». La formation des vaisseaux se trouve alors altérée. Ainsi, PPARγ constitue un régulateur majeur de l'activité anti-angiogénique. En conclusion, ce travail fournit de nouveaux éléments sur le rôle complémentaires de PPARβet PPARγ dans les événements complexes qui régissent le développement placentaire. SUMMARY Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors involved in energy homeostasis and growth. Herein, we characterize the placental defects that cause embryonic lethality around E9.5/E10.5 in PPARβ- and in PPARγ-deficient mouse lines. Most but not all PPARβ-null mutants die around E9.5/E10.5 with severe growth retardation. The placentas from PPARβ-/- embryos at E9.5 exhibit a strongly reduced giant cell layer, associated with reduced levels of proteins expressed by giant cells such as Placental lactogen-I and Proliferin. Ectopic treatment of a rat trophoblast cell line with PPARβ ligand markedly accelerated PI3 kinase-dependent giant cell differentiation. In addition, we demonstrate that ADRP, a pen-related lipid droplet-bound protein, is up-regulated by PPARβ in differentiated Rcho-1 cells. These results indicate that PPARβ is a crucial regulator of the differentiation secondary giant cells, which play a major role in the establishment of the placental structure as well as an important endocrine function. In contrast, the main alteration of the PPARγ-null placentas concerns the vasculogenesis. We show that in these placentas, the level of the anti-angiogenic proliferin-related protein is very low, and cannot balance the normal elevation of the pro-angiogenic proliferin expression, leading to the defective placental vessel formation. Consistently, the dramatic increase of PPARγ expression in late stage of gestation in wild-type mice is likely a major regulator of the anti-angiogenic activity, particularly important at the end of the pregnancy. This work emphasizes the important and complementary roles of PPARβ and PPARγ in mouse placental development and provides new tools for understanding the complex regulatory events that governs placental development and function. Understanding the function of PPARβ and PPARγ are of crucial interest with respect to human placental development and associated pathologies.

Physiopathologie du coeur embryonnaire soumis à l'anoxie-réoxygénation: rôle protecteur du NO et des canaux KATP

RESUME Introduction : Dans le coeur adulte, l'ischémie et la reperfusion entraînent des perturbations électriques, mécaniques, biochimiques et structurales qui peuvent causer des dommages réversibles ou irréversibles selon la sévérité de l'ischémie. Malgré les récents progrès en cardiologie et en chirurgie foetales, la connaissance des mécanismes impliqués dans la réponse du myocarde embryonnaire à un stress hypoxique transitoire demeure lacunaire. Le but de ce travail a donc été de caractériser les effets chrono-, dromo- et inotropes de l'anoxie et de la réoxygénation sur un modèle de coeur embryonnaire isolé. D'autre part, les effets du monoxyde d'azote (NO) et de la modulation des canaux KATP mitochondriaux (mito KATP) sur la récupération fonctionnelle postanoxique ont été étudiés. La production myocardique de radicaux d'oxygène (ROS) et l'activité de MAP Kinases (ERK et JNK) impliquées dans la signalisation cellulaire ont également été déterminées. Méthodes : Des coeurs d'embryons de poulet âgés de 4 jours battant spontanément ont été placés dans une chambre de culture puis soumis à une anoxie de 30 min suivie d'une réoxygénation de 60 min. L'activité électrique (ECG), les contractions de l'oreillette, du ventricule et du conotroncus (détectées par photométrie), la production de ROS (mesure de la fluorescence du DCFH) et l'activité kinase de ERK et JNK dans le ventricule ont été déterminées au cours de l'anoxie et de la réoxygénation. Les coeurs ont été traités avec un bloqueur des NO synthases (L-NAME), un donneur de NO (DETA-NONOate), un activateur (diazoxide) ou un inhibiteur (5-HD) des canaux mitoKATP un inhibiteur non-spécifique des PKC (chélérythrine) ou un piégeur de ROS (MPG). Résultats : L'anoxie et la réoxygénation entraînaient des arythmies (essentiellement d'origine auriculaire) semblables à celles observées chez l'adulte, des troubles de la conduction (blocs auriculo-ventriculaires de 1er, 2ème et 3ème degré) et un ralentissement marqué du couplage excitation-contraction (E-C) ventriculaire. En plus de ces arythmies, la réoxygénation déclenchait le phénomène de Wenckelbach, de rares échappements ventriculaires et une sidération myocardique. Aucune fibrillation, conduction rétrograde ou activité ectopique n'ont été observées. Le NO exogène améliorait la récupération postanoxique du couplage E-C ventriculaire alors que L'inhibition des NOS la ralentissait. L'activation des canaux mito KATP augmentait la production mitochondriale de ROS à la réoxygénation et accélérait la récupération de la conduction (intervalle PR) et du couplage E-C ventriculaire. La protection de ce couplage était abolie par le MPG, la chélérythrine ou le L-NAME. Les fonctions électrique et contractile de tous les coeurs récupéraient après 30-40 min de réoxygénation. L'activité de ERK et de JNK n'était pas modifiée par L'anoxie, mais doublait et quadruplait, respectivement, après 30 min de réoxygénation. Seule l'activité de JNK était diminuée (-60%) par l'activation des canaux mitoKATP. Cet effet inhibiteur était partiellement abolit par le 5-HD. Conclusion: Dans le coeur immature, le couplage E-C ventriculaire semble être un paramètre particulièrement sensible aux conditions d'oxygénation. Sa récupération postanoxique est améliorée par l'ouverture des canaux mitoKATP via une signalisation impliquant les ROS Ies PKC et le NO. Une réduction de l'activité de JNK semble également participer à cette protection. Nos résultats suggèrent que les mitochondries jouent un rôle central dans la modulation des voies de signalisation cellulaire, en particulier lorsque les conditions métaboliques deviennent défavorables. Le coeur embryonnaire isolé représente donc un modèle expérimental utile pour mieux comprendre les mécanismes associés à une hypoxie in utero et pour améliorer les stratégies thérapeutiques en cardiologie et chirurgie foetales. ABSTRACT Physiopathology of the anoxic-reoxygenated embryonic heart: Protective role of NO and KATP channel Aim: In the adult heart, the electrical, mechanical, biochemical and structural disturbances induced by ischemia and reperfusion lead to reversible or irreversible damages depending on the severity and duration of ischemia. In spite of recent advances in fetal cardiology and surgery, little is known regarding the cellular mechanisms involved in hypoxia-induced dysfunction in the developing heart. The aim of this study was to precisely characterize the chrono-, dromo- and inotropic disturbances associated with anoxia-reoxygenation in an embryonic heart model. Furthermore, the roles that nitric oxide (NO), reactive oxygen species (ROS), mitochondrial KATP, (mito KATP) channel and MAP Kinases could play in the stressed developing heart have been investigated. Methods: Embryonic chick hearts (4-day-old) were isolated and submitted in vitro to 30 min anoxia followed by 60 min reoxygenation. Electrical (ECG) and contractile activities of atria, ventricle and conotruncus (photometric detection), ROS production (DCFH fluorescence) and ERK and JNK activity were determined in the ventricle throughout anoxia-reoxygenation. Hearts were treated with NO synthase inhibitor (L-NAME), NO donor (DETA-NONOate), mitoKATP channel opener (diazoxide) or blocket (5-HD), PKC inhibitor (chelerythrine) and ROS scavenger (MPG). Results: Anoxia and reoxygenation provoked arrhythxnias (mainly originating from atrial region), troubles of conduction (st, 2nd, and 3rd degree atrio-ventricular blocks) and disturbances of excitation-contraction (E-C) coupling. In addition to these types of arrhythmias, reoxygenation triggered Wenckebach phenomenon and rare ventricular escape beats. No fibrillations, no ventricular ectopic beats and no electromechanical dissociation were observed. Myocardial stunning was observed during the first 30 min of reoxygenation. All hearts fully recovered their electrical and mechanical functions after 30-40 min of reoxygenation. Exogenous NO improved while NOS inhibition delayed E-C coupling recovery. Mito KATP, channel opening increased reoxygenation-induced ROS production and improved E-C coupling and conduction (PR) recovery. MPG, chelerythrine or L-NAME reversed this effect. Reoxygenation increased ERK and JNK activities land 4-fold, respectively, while anoxia had no effect. MitoKATP channel opening abolished the reoxygenation-induced activation of JNK but had no effect on ERK activity. This inhibitory effect was partly reversed by mitoKATP channel blocker but not by MPG. Conclusion: In the developing heart, ventricular E-C coupling was found to be specially sensitive to hypoxia-reoxygenation and its postanoxic recovery was improved by mitoKATP channel activation via a ROS-, PKC- and NO-dependent pathway. JNK inhibition appears to be involved in this protection. Thus, mitochondria can play a pivotal role in the cellular signalling pathways, notably under critical metabolic conditions. The model of isolated embryonic heart appears to be useful to better understand the mechanisms underlying the myocardial dysfunction induced by an in utero hypoxia and to improve therapeutic strategies in fetal cardiology and surgery.

Differences in osteogenic and apoptotic genes between osteoporotic and osteoarthritic patients.

BACKGROUND Osteoporosis is a metabolic disorder characterized by a reduction in bone mass and deterioration in the microarchitectural structure of the bone, leading to a higher risk for spontaneous and fragility fractures.The main aim was to study the differences between human bone from osteoporotic and osteoarthritic patients about gene expression (osteogenesis and apoptosis), bone mineral density, microstructural and biomechanic parameters. METHODS We analyzed data from 12 subjects: 6 with osteoporotic hip fracture (OP) and 6 with hip osteoarthritis (OA), as the control group. All subjects underwent medical history, analytical determinations, densitometry, histomorphometric and biochemical study. The expression of 86 genes of osteogenesis and 86 genes of apoptosis was studied in pool of bone samples from patients with OP and OA by PCR array. RESULTS We observed that most of the genes of apoptosis and osteogenesis show a decrease in gene expression in the osteoporotic group in comparison with the osteoarthritic group. The histomorphometric study shows a lower bone quality in the group of patients with hip fractures compared to the osteoarthritic group. CONCLUSIONS The bone tissue of osteoporotic fracture patients is more fragile than the bone of OA patients. Our results showed an osteoporotic bone with a lower capacities for differentiation and osteoblastic activity as well as a lower rate of apoptosis than osteoarthritic bone. These results are related with structural and biochemical parameters.

Chromosomal divergence and evolutionary inferences in Rhodniini based on the chromosomal location of ribosomal genes

In this study, we used fluorescence in situ hybridisation to determine the chromosomal location of 45S rDNA clusters in 10 species of the tribe Rhodniini (Hemiptera: Reduviidae: Triatominae). The results showed striking inter and intraspecific variability, with the location of the rDNA clusters restricted to sex chromosomes with two patterns: either on one (X chromosome) or both sex chromosomes (X and Y chromosomes). This variation occurs within a genus that has an unchanging diploid chromosome number (2n = 22, including 20 autosomes and 2 sex chromosomes) and a similar chromosome size and genomic DNA content, reflecting a genome dynamic not revealed by these chromosome traits. The rDNA variation in closely related species and the intraspecific polymorphism in Rhodnius ecuadoriensis suggested that the chromosomal position of rDNA clusters might be a useful marker to identify recently diverged species or populations. We discuss the ancestral position of ribosomal genes in the tribe Rhodniini and the possible mechanisms involved in the variation of the rDNA clusters, including the loss of rDNA loci on the Y chromosome, transposition and ectopic pairing. The last two processes involve chromosomal exchanges between both sex chromosomes, in contrast to the widely accepted idea that the achiasmatic sex chromosomes of Heteroptera do not interchange sequences.

Controlling cytokinesis in the fission yeast Schizosaccharomyces pombe : the role of dma1; and ubc8

ABSTRACT The fission yeast Schizosaccharomyces pombe is a single celled eukaryote that has proved to be an excellent model system for the study of cell cycle control. S. pombe cells are rod shaped and grow mainly by elongation at their tips. They divide by formation of medially-placed cell wall, or septum, which cleaves the cell in two. Once the cell commits itself to mitosis the site of division is determined by formation of an acto-myosin based contractile ring at the cell cortex. The ring is assembled in stages throughout mitosis and contracts at the end of anaphase, coincident with spindle disassembly. The contraction, but not the assembly, of the ring requires the signal transduction network called the septation initiation network or SIN. The core components of the SIN are three protein kinases (cdc7p, sidl p and sid2p) and their regulatory subunits (spg1 p, cdcl4p and moblp, respectively). Signalling is dependent upon the nucleotide status of the GTPase spgl p, which is regulated by a two-component GAP protein, cdc16p-byr4p. Signalling is thought to emanate from the spindle pole body, where core SIN components are anchored to a scaffold comprised of sid4p and cdc11p. Activation of the SIN requires the protein kinase plolp, which also has additional roles in mitosis. SIN signalling is tightly regulated to assure the proper co-ordination of mitosis and cytokinesis. Ectopic activation of the SIN in interphase can uncouple septum formation from mitosis, while deregulated SIN signalling leads to formation of cells with multiple septa that do not cleave. Regulators of SIN activity are therefore of considerable interest. This study has concentrated upon two of these, dma1 and ubc8. I have demonstrated that dmal becomes essential when SIN signalling is activated. This leads me to propose a tripartite model for regulation of the SIN during the mitotic cell cycle. Increased expression of dma1 inhibits SIN signalling and prevents cell division. To identify potential targets and mediators of this, multicopy suppressors of dma1 toxicity were identified. One of these, ubc8, is the subject of this thesis. Genetic and molecular analyses are consistent with the view that ubc8p acts as an inhibitor of the SIN Localisation of ubc8p indicates that it is a nuclear protein. The ubc8 gene is not essential, but in its absence cells are unable to prevent septum formation if progression through mitosis is impaired. These data suggest that it may be an effector of the spindle assembly checkpoint. Together, these data shed new light upon the mechanisms by which cytokinesis is regulated in S. pombe. RESUME La levure Schizosaccharomyces pombe est un eucaryote unicellulaire qui est un bon système d'étude du cycle cellulaire. Les cellules de S. pombe sont en forme de bâtonnets et poussent par allongement aux deux bouts. Elles se divisent en formant une paroi au milieu de la cellule, qui s'appelle un septum et qui sépare la cellule en deux. Une fois que la cellule est engagée dans la mitose, le site de clivage est déterminé par la formation d'un anneau contractile d'acto-myosine au niveau du cortex cellulaire. Cet anneau est séquentiellement assemblé au cours de la mitose et se contacte à la fin de l'anaphase, au moment où le fuseau mitotique et désassemblé. La contraction, mais non pas l'assemblage, de l'anneau dépend d'un réseau de signalisation appelé septation initiation netvvork' ou SIN. Les composants centraux du SIN sont trois kinases (cdc7, sidi et sid2) ainsi que leurs sous-unités régulatrices (spgl, cdc14 et mob1, respectivement). La signalisation dépend du nucléotide rattaché à la GTPase spgl qui est régulée par une GAP comprenant deux sous-unités cdc16 et byr4. La signalisation est présumée provenir du pôle du fuseau où les composants centraux du SIN sont ancrés grâce à un échafaudage comprenant sid4 et cdcl 1. La signalisation est étroitement régulée pour assurer une bonne coordination entre mitose et cytokinèse. Une activation ectopique du SIN en interphase peut découpler la formation du septum de la mitose, engendrant des cellules à multiples septa qui ne sont pas clivés. C'est pourquoi les régulateurs du SIN sont d'un intérêt considérable. Cette étude se concentre autour de deux ces régulateurs, dma1 et ubc8. J'ai montré que dma1 devient essentiel quand la signalisation du SIN est activée. Ceci m'amène à proposer un modèle en trois parties pour la régulation du SIN durant la mitose. Une expression élevée de dma1 inhibe la signalisation du SIN et empêche la division cellulaire. Afin d'identifier des substrats ou médiateurs potentiels de la toxicité de dma1, des supresseurs en copies multiples ont été identifiés. Un de ces supresseurs, ubc8, constitue le deuxième sujet de cette thèse. Les études génétiques et moléculaires suggèrent un rôle inhibiteur du SIN par ubc8. Ubc8p est une protéine nucléaire, non essentielle, mais en son absence les cellules ne peuvent pas restreindre la fomation du septum, lorsque la progression de la mitose est perturbée. Les données suggèrent que ubc8 pourrait être un effecteur de point de contrôle de l'assemblage du fuseau mitotique. Prises dans leur ensemble, ces données apportent un nouvel éclairage sur les mécanismes de régulation de la cytokinèse dans S. pombe.

Generation of cell polarity in plants links endocytosis, auxin distribution and cell fate decisions.

Dynamically polarized membrane proteins define different cell boundaries and have an important role in intercellular communication-a vital feature of multicellular development. Efflux carriers for the signalling molecule auxin from the PIN family are landmarks of cell polarity in plants and have a crucial involvement in auxin distribution-dependent development including embryo patterning, organogenesis and tropisms. Polar PIN localization determines the direction of intercellular auxin flow, yet the mechanisms generating PIN polarity remain unclear. Here we identify an endocytosis-dependent mechanism of PIN polarity generation and analyse its developmental implications. Real-time PIN tracking showed that after synthesis, PINs are initially delivered to the plasma membrane in a non-polar manner and their polarity is established by subsequent endocytic recycling. Interference with PIN endocytosis either by auxin or by manipulation of the Arabidopsis Rab5 GTPase pathway prevents PIN polarization. Failure of PIN polarization transiently alters asymmetric auxin distribution during embryogenesis and increases the local auxin response in apical embryo regions. This results in ectopic expression of auxin pathway-associated root-forming master regulators in embryonic leaves and promotes homeotic transformation of leaves to roots. Our results indicate a two-step mechanism for the generation of PIN polar localization and the essential role of endocytosis in this process. It also highlights the link between endocytosis-dependent polarity of individual cells and auxin distribution-dependent cell fate establishment for multicellular patterning.

The Wnt inhibitory factor 1 (WIF1) is targeted in glioblastoma and has a tumor suppressing function potentially by induction of senescence.

Gene expression-based prediction of genomic copy number aberrations in the chromosomal region 12q13 to 12q15 that is flanked by MDM2 and CDK4 identified Wnt inhibitory factor 1 (WIF1) as a candidate tumor suppressor gene in glioblastoma. WIF1 encodes a secreted Wnt antagonist and was strongly downregulated in most glioblastomas as compared with normal brain, implying deregulation of Wnt signaling, which is associated with cancer. WIF1 silencing was mediated by deletion (7/69, 10%) or epigenetic silencing by promoter hypermethylation (29/110, 26%). Co-amplification of MDM2 and CDK4 that is present in 10% of glioblastomas was associated in most cases with deletion of the whole genomic region enclosed, including the WIF1 locus. This interesting pathogenetic constellation targets the RB and p53 tumor suppressor pathways in tandem, while simultaneously activating oncogenic Wnt signaling. Ectopic expression of WIF1 in glioblastoma cell lines revealed a dose-dependent decrease of Wnt pathway activity. Furthermore, WIF1 expression inhibited cell proliferation in vitro, reduced anchorage-independent growth in soft agar, and completely abolished tumorigenicity in vivo. Interestingly, WIF1 overexpression in glioblastoma cells induced a senescence-like phenotype that was dose dependent. These results provide evidence that WIF1 has tumor suppressing properties. Downregulation of WIF1 in 75% of glioblastomas indicates frequent involvement of aberrant Wnt signaling and, hence, may render glioblastomas sensitive to inhibitors of Wnt signaling, potentially by diverting the tumor cells into a senescence-like state.