Molecular mechanisms and effects of a rasgap-derived peptide on metastatic progression

Cancer is the second leading cause of mortality worldwide. Cancer progression leads to metastasis formation, which accounts for more than ninety percent of cancer-related death. Metastases are more difficult to be surgically removed because of their invasive behavior and shape. In addition, during their transformation journey, they become more and more resistant to anticancer drugs. Significant improvements have been achieved in therapy against cancer in recent years but targeting the metastatic cascade remains the Achilles heel of the cure against cancer. A First step in the metastatic process is the escape of cancer cells from the primary tumor site. This involves an increase in cell motility and the concomitant ability to clear a path through the extracellular matrix. From a therapeutic point of view, inhibition of cell migration is a logical approach to develop anti-metastatic drugs. Our lab previously developed a cell permeable peptide derived from a caspase-3-generaied fragment of the RasGAP protein called TAT-RasGAP317-326. This peptide efficiently and specifically sensitizes cancer cells to chemotherapy- and radiotherapy-induced ceil death, which allows decreasing the anticancer drug doses and eventually their associated side- effects. In the present study we discovered that TAT-RasGAP317.326 also increases cell adhesion which was associated with inhibition of cell migration and invasion into the extracellular matrix. The ability of TAT-RasGAP317.326 to increase ceil adhesion involves the dramatic depolymerization of actin cytoskekton together with redistribution of focal adhesions. We found that the inhibitory effects on migration were mediated by a RhoGAP tumor and metastasis suppressor cailed DLC1 (Deleted in Liver Cancer 1). Moreover. DEC 1 was found to be a direct RasGAP-interacting protein and this interaction requires the RasGAP tryptophan 317 residue, the very first RasGAP residue of TAT-RasGAP317.326. We then evaluated the roie of RasGAP fragments in the in vivo metastatic cascade. We found that breast cancer cells overexpressing the parental RasGAP fragment, to which the TAT-RasGAP317.326 peptide belongs, have a markedly decreased ability to form lung metastases. Unfortunately, we were not able to recapitulate these an ti-metastatic effects when TAT-RasGAP317.326 was injected. However, we later understood that this was due to the fact that TAT-RasGAP317.326 was not properly delivered to the primary tumors. Further work, aimed at better understanding of how TAT-RasGAP317.326 functions, revealed that the ten amino acid TAT-RasGAP317.326 peptide could, be narrowed down to a three amino acid TAT-RasGAP317.329 peptide while keeping its sensitizer activity. In parallel, investigations on the RasGAP-DLCl binding indicated that the arginine linger of the DLC1 GAP domain is required for this interaction, which suggests that TAT-RasGAP317.326 modulates the GAP activity of DLC1. Additional work should be performed to fully elucidate its mechanism of action and render TAT-RasGAP317.326 usable as a tool to fight cancer on two fronts, by improving chemotherapy and preventing metastatic progression. - Le cancer est la deuxième cause de mortalité dans le monde. La formation de métastases est la dernière étape de la progression cancéreuse et représente plus du nonante pour cent des morts induites par le cancer. De par leur morphologie et comportement invasifs, ii est difficile d'avoir recours à la chirurgie pour exciser des métastases. De plus, les cellules cancéreuses en progression deviennent souvent de plus en plus résistantes aux drogues anticancéreuses. Ces dernières années, des avancements significatifs ont contribué à l'amélioration de la lutte contre le cancer. Néanmoins, pouvoir cibler spécifiquement la cascade métastatique demeure cependant le talon d'Achille des thérapies anticancéreuses. Une première étape dans ie processus métastatique est l'évasion des cellules cancéreuses du site de la tumeur primaire. Ceci requiert une augmentation de la motiliié cellulaire couplée à la capacité de se frayer un chemin au sein de la matrice extracelluiaire. D'un point de vue thérapeutique, inhiber la migration cellulaire est une approche attrayante. Notre laboratoire a développé un peptide, nommé TAT-RasGAP317.326 dérivé d'un fragment qui est lui-même le résultat du clivage de la protéine RasGAP par la caspase-3. Ce peptide est capable de pénétrer les cellules cancéreuses et de les sensibiliser spécifiquement à la mort induite par la radiothérapie et la chimiothérapie. La finalité des effets de ce peptide est de pouvoir diminuer les doses des traitements anti-cancéreux et donc des effets secondaires qu'ils engendrent. Dans cette étude, nous avons découvert que TAT-RasGAP317.326 augmente l'adhésion des cellules et inhibe la migration cellulaire ainsi que l'invasion des cellules à travers une matrice extracellulaire. La capacité de TAT-RasGAP317.326 à induire l'adhésion repose sur ia dépolymérisation du cytosquelette d'actine associée à une redistribution des points d'ancrage cellulaire. Nous avons découvert que l'inhibition de ia migration par TAT-RasGAP317.326 nécessitait la présence d'un suppresseur de tumeur et de métastases appelé DLC1 (Deleted in Liver Cancer l), qui par ailleurs s'avère aussi être une protéine RhoGAP. De plus, nous avons aussi trouvé que DLC1 était un partenaire d'interaction de RasGAP et que cette interaction s'effectuait via l'acide aminé tryptophane 317 de RasGAP. qui s'avère être le premier acide aminé du peptide TAT-RasGAP317.326. Nous avons ensuite évalué le rôle joué par certains fragments de RasGAP dans le processus de métastatisation. Dans ce contexte, des cellules de cancer du sein qui sur-expriment un fragment de RasGAP contenant la séquence TAT-RasGAP317.326 ont vu leur potentiel métastatique diminuer drastiquerment. Malheureusement, aucun effet anti-métastatique n'a été obtenu après injection de TAT-RasGAP317.326 dans les souris. Cependant, nous avons réalisé rétrospectivement que TAT-RasGAP317.326 n'était pas correctement délivré à la tumeur primaire, ce qui nous empêche de tirer des conclusions sur le rôle anti-métastatique de ce peptide. La suite de cette étude visant à mieux comprendre comment TAT-RasGAP317.326 agit, a mené à la découverte que les dix acides aminés de TAT-RasGAP317.326 pouvaient être réduits à trois acides aminés, TAT-RasGAP317.329, tout en gardant l'effet sensibilisateur à la chimiothérapie. En visant à élucider le mode d'interaction entre RasGAP et DLC1, nous avons découvert qu'un acide aminé nécessaire à l'activité GAP de DLC1 était requis pour lier RasGAP, ce qui laisse présager que TAT-RasGAp317.32c, module i'activité GAP de DLC1. Des travaux supplémentaires doivent encore être effectués pour complètement élucider les mécanismes d'action de TAT-RasGAP317.326 et afin de pouvoir l'utiliser comme un outil pour combattre le cancer sur deux fronts, en améliorant les chimiothérapies et en inhibant la formation de métastases.

Selection of peptides and synthesis of pentameric peptabody molecules reacting specifically with ErbB-2 receptor.

The HER-2/ErbB-2 oncoprotein is overexpressed in human breast and ovarian adenocarcinomas and is clearly associated with the malignant phenotype. Although no specific ligand for this receptor has been positively identified, ErbB-2 was shown to play a central role in a network of interactions with the related ErbB-1, ErbB-3 and ErbB-4 receptors. We have selected new peptides binding to ErbB-2 extracellular domain protein (ECD) by screening 2 newly developed constrained and unconstrained random hexapeptide phage libraries. Out of 37 phage clones, which bound specifically to ErbB-2 ECD, we found 6 constrained and 10 linear different hexapeptide sequences. Among the latter, 5 consensus motifs, all with a common methionine and a positively charged residue at positions 1 and 3, respectively, were identified. Furthermore, 3 representative hexapeptides were fused to a coiled-coil pentameric recombinant protein to form the so-called peptabodies recently developed in our laboratory. The 3 peptabodies bound specifically to the ErbB-2 ECD, as determined by enzyme-linked immunosorbent assay and BIAcore analysis and to tumor cells overexpressing ErbB-2, as shown by flow cytometry. Interestingly, one of the free selected linear peptides and all 3 peptabodies inhibited the proliferation of tumor cells overexpressing ErbB-2. In conclusion, a novel type of ErbB-2-specific ligand is described that might complement presently available monoclonal antibodies.

Polymorphisms of toll-like receptor 2 and 4 genes in Chagas disease

The aim of this study was to test the possible implication of toll-like receptor 2 (TLR2) and TLR4 gene polymorphisms in determining the susceptibility to Chagas' disease. Genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism in 475 individuals from Colombia, 143 seropositive with chagasic cardiomyopathy, 132 seropositive asymptomatic and 200 seronegative. The TLR2 arginine to glutamine substitution at residue 753(Arg753Gln) polymorphism was absent in the groups analyzed. The TLR4 Asp299Gly and Thr399Ile polymorphisms are in linkage disequilibrium and we observed a very low frequency of these polymorphisms in our study population (2.6% and 1.8% respectively). The overall TLR2 and TLR4 alleles and genotype distribution in seronegative and seropositive were not significantly different. We compared the frequencies between asymptomatic patients and those with chagasic cardiomyopathy and we did not observe any significant differences in the distribution of alleles or genotypes. In summary, this study corroborates the low frequency of TLR2 and TLR4 polymorphisms observed in other populations and suggest that these do not play an important role in Chagas' disease. The validation of these findings in independent cohorts is needed to firmly establish a role for TLR2 and TLR4 variants in Chagas' disease.

α-N-acetylglucosamine-linked O-glycans of sialoglycoproteins (Tc-mucins) from Trypanosoma cruzi Colombiana strain

Trypanosoma cruzi sialoglycoproteins (Tc-mucins) are mucin-like molecules linked to a parasite membrane via a glycosylphosphatidylinositol anchor. We previously determined the structures of Tc-mucin O-glycan domains from several T. cruzi strains and observed significant differences among them. We now report the amino acid content and structure of Tc-mucin O-glycan chains from T. cruzi Colombiana, a strain resistant to common trypanocidal drugs. Amino acid analysis demonstrated the predominance of threonine residues (42%) and helped to identify the O-glycans as belonging to a Tc-mucin family that contain a ²-galactofuranose (²-Galf) residue attached to an α-N-acetylglucosamine (α-GlcNAc) O-4, with the most complex glycan, a pentasaccharide-GlcNAc-ol with a branched trigalactopyranose chain, on the GlcNAc O-6. The presence of ²-Galf on O-glycans from T. cruzi Colombiana mucins supports the use of glycosylation as a phylogenetic marker for the classification of Colombiana in the T. cruzi I group.

Structural analysis of TCR-ligand interactions studied on H-2Kd-restricted cloned CTL specific for a photoreactive peptide derivative.

To study the interaction of the TCR with its ligand, the complex of a MHC molecule and an antigenic peptide, we modified a TCR contact residue of a H-2Kd-restricted antigenic peptide with photoreactive 4-azidobenzoic acid. The photoreactive group was a critical component of the epitope recognized by CTL clones derived from mice immunized with such a peptide derivative. The majority of these clones expressed V beta 1-encoded beta chains that were paired with J alpha TA28-encoded alpha chains. For one of these TCR, the photoaffinity labeled sites were mapped on the alpha chain as a J alpha TA28-encoded tryptophan and on the beta chain as a residue of the C' strand of V beta 1. Molecular modeling of this TCR suggested the presence of a hydrophobic pocket that harbors this tryptophan as well as a tyrosine on the C' strand of V beta 1 between which the photoreactive side chain inserts. It is concluded that this avid binding principle may account for the preferential selection of V beta 1 and J alpha TA28-encoded TCR.

Analysis of the cytolytic T lymphocyte response of melanoma patients to the naturally HLA-A*0201-associated tyrosinase peptide 368-376.

The human tyrosinase gene codes for two distinct antigens that are recognized by HLA-A*0201-restricted CTLs. For one of them, tyrosinase peptide 368-376, the sequence identified by mass spectrometry in melanoma cell eluates differs from the gene-encoded sequence as a result of posttranslational modification of amino acid residue 370 (asparagine to aspartic acid). Here, we used fluorescent tetrameric complexes ("tetramers") of HLA-A*0201 and tyrosinase peptide 368-376 (YMDGTMSQV) to characterize the CD8+ T-cell response to this antigen in lymphoid cell populations from HLA-A2 melanoma patients. Taking advantage of the presence of significant numbers of tetramer-positive CD8+ T cells in tumor-infiltrated lymph node cells from a melanoma patient, we derived polyclonal and monoclonal tyrosinase peptide 368-376-specific CTLs by tetramer-guided flow cytometric sorting. These CTLs efficiently and specifically lysed HLA-A*0201- and tyrosinase-positive melanoma cells. As assessed with tyrosinase peptide variants, the fine antigen specificity of the CTLs was quite diverse at the clonal level. Flow cytometric analysis of PBMCs stained with tetramers showed that tyrosinase peptide 368-376-specific CD8+ T cells were hardly detectable in peripheral blood of melanoma patients. However, significant numbers of such cells were detected after short-term stimulation of CD8+ lymphocytes with tyrosinase peptide 368-376 in 6 of 10 HLA-A2 melanoma patients. Taken together, these findings emphasize the significant contribution of the natural tyrosinase peptide 368-376 to the antigenic specificities recognized by the tumor-reactive CTLs that may develop in HLA-A2 melanoma patients.

Molecular analysis of non-melanoma skin cancer

SUMMARY:Cylindroma, trichoepithelioma and spiradenoma are benign tumors of hair follicle. They are caused by mutations and loss of heterozygosity in the CYLD gene. CYLD is a ubiquitously expressed, but the tumors are restricted to skin, suggesting that the tumorigenesis is influenced by skin-specific regulators and probably by mutations in other genes. The objectives of the thesis were to analyze the molecular mechanisms leading to the aforementioned tumors. In the first project, we have identified five new mutations in CYLD gene in tive families affected with different combinations of these skin appendage tumors. F our of these mutations caused the introduction of a premature stop codon in CYLD protein sequence, but one was a missense mutation changing aspartic acid 681 into glycine (D68lG), in patients exhibiting multiple trichoepitheliomas. CYLD is a deubiquitinase which can downregulate NF-κB and INK pathways through the deubiquitination of TRAF2, for example. We showed that the CYLD-D681G mutant was unable to remove polyubiquitin chains from TRAF2. We also proved that CYLD-D68lG could not inhibit TRAP 2- or TNFα- mediated NF-κB or INK activations in 293T cells. These results underlined the importance of the D68l residue for the enzymatic activity of CYLD. TRAP-interacting protein (TRIP), which is a E3-Ubiquitin ligase, is a partner of CYLD. In the second project of the thesis, we studied the function of TRIP in the epidermis. We found that TRIP was a nucleolar protein in cultured human primary keratinocytes (HEK) and HeLa cells, and was detected in the midbody of HeLa cells. Moreover, TRIP expression was shown to be downregulated through a PKC-dependent mechanism before induction of keratinocyte differentiation. We also proved that TRIP was upregulated in basal cell carcinomas. Furthermore, TRIP was found to be important for keratinocyte survival and proliferation through the regulation of the Gl/S transition. Our results suggest that TRIP may be involved in keratinocyte tumorigenesis.RÉSUMÉ :Les cylindromes, trichoépithéliomes et spiradénomes sont des tumeurs bénignes du follicule pileux causées par des mutations et une perte d'hétérozygotie du gène CYLD. CYLD est ubiquitaire mais les tumeurs sont limitées à la peau, suggérant que la tumorigénèse est influencée par des protéines spécifiques de la peau et par des mutations dans d'autres gènes. Les objectifs de la thèse étaient d'2malyser les mécanismes moléculaires aboutissant à la formation de ces tumeurs. Dans le premier projet, cinq nouvelles mutations du gène CYLD ont été identifiées chez cinq familles présentant différentes combinaisons des tumeurs citées ci- dessus. Quatre de ces mutations causaient I' introduction d'un codon stop prématuré dans la séquence protéique, mais une était une mutation «misser1se» changeant l'aspartate 681 en résidu glycine (D68lG) chez des patients présentant des trichoépithéliomes multiples. CYLD est une déubiquitinase qui inhibe les voies de signalisation de NF-κB et JNK, en déubiquitinant notamment TRAF2. Nous avons montré que la protéine mutante CYLD- D68lG ne pouvait pas cliver la chaîne de poly-ubiquitines liée à TRAF2. CYLD-D68lG était aussi incapable d'inhiber l'activation de NF-κB ou de JNK induite par TRAF2 ou TNF-o dans les cellules 293T. Ces résultats ont donc souligné l'impo1tance du résidu D68l pour l'activité de CYLD. «TRAF-interacting protein (TRIP)», qui est une «E3-ubiquitin-ligase», est un partenaire de CYLD. Dans le second proj et de la thèse, nous avons étudié la fonction de TRIP dans l'épidenne. Nous avons montrépque TRIP était nucléolaire dans les cellules HeLa et les kératinocytes primaires humains en culture et était détectée dans le «midbody» des cellules HeLa. Nous avons prouvé que l'ARNm de TRIP était diminué avant l'induction de la différentiation des kératinocytes, par un mécanisme dépendent de la protéine kinase C, tandis qu'il était augmenté dans les carcinomes baso-cellulaires. Nous avons aussi montré que TRIP influençait la prolifération et la survie des kératinocytes en régulant la transition G1/S, Nos résultats suggèrent que TRIP est peut-être impliquée dans la tumorigénèse des kératinocytes. 7

Use of stains to detect fingermarks

Detection of fi ngermarks at a crime scene or on related items is of prime interest for forensicinvestigators, mainly for identifi cation purposes. Most of the fi ngermarks are invisible to thenaked eye, however. The application of detection techniques is required to establish visual contrastbetween the secretion residue and the underlying substrate. We give here a review of thefi eld related to the concept of using stains to detect fi ngermarks. A distinction has been madebetween the physically driven classical detection techniques, the chemically driven ones, andthose based on nanostructured materials, an emerging fi eld in forensic science.

The β-sliding clamp directs the localization of HdaA to the replisome in Caulobacter crescentus.

The initiation of chromosome replication is tightly regulated in bacteria to ensure that it takes place only once per cell cycle. In many proteobacteria, this process requires the ATP-bound form of the DnaA protein. The regulatory inactivation of DnaA (RIDA) facilitates the conversion of DnaA-ATP into replication-inactive DnaA-ADP, thereby preventing overinitiation. Homologues of the HdaA protein, together with the β-clamp of the DNA polymerase (DnaN), are required for this process. Here, we used fluorescence resonance energy transfer experiments to demonstrate that HdaA interacts with DnaN in live Caulobacter crescentus cells. We show that a QFKLPL motif in the N-terminal region of HdaA is required for this interaction and that this motif is also needed to recruit HdaA to the subcellular location occupied by the replisome during DNA replication. An HdaA mutant protein that cannot colocalize or interact with DnaN can also not support the essential function of HdaA. These results suggest that the recruitment of HdaA to the replisome is needed during RIDA in C. crescentus, probably as a means to sense whether chromosome replication has initiated before DnaA becomes inactivated. In addition, we show that a conserved R145 residue located in the AAA+ domain of HdaA is also needed for the function of HdaA, although it does not affect the interaction of HdaA with DnaN in vivo. The AAA+ domain of HdaA may therefore be required during RIDA after the initial recruitment of HdaA to the replisome by DnaN.

Efficient in vivo induction of CTL by cell-associated covalent H-2Kd-peptide complexes.

A novel procedure is presented describing the induction of antigen-specific cytolytic T lymphocytes (CTL) in vivo, that uses as immunogen syngeneic Concanavalin A stimulated spleen cells expressing H-2Kd (Kd) molecules photocrosslinked with a photoreactive peptide derivative. The Kd restricted Plasmodium berghei circumsporozoite (PbCS) peptide 253-260 (YIPSAEKI) was conjugated with photoreactive iodo-4-azidosalicylic acid (IASA) at the NH2-terminus and with 4-azidobenzoic acid (ABA) at the TCR contact residue Lys259 to make IASA-YIPSAEK(ABA)I. Selective photoactivation of the IASA group allowed specific photoaffinity labeling of cell-associated Kd molecules. Optimal peptide derivative binding to Kd molecules of concanavalin A stimulated spleen cells was obtained upon 4-6 h incubation at 26 degrees C in the presence of human beta 2 microglobulin. Photocrosslinking prevented the rapid dissociation of cell-associated Kd-peptide derivative complexes at 37 degrees C. The photoaffinity labeled cells were injected i.p. into syngeneic recipients. After 10 days, the peritoneal exudate lymphocytes were harvested and in vitro stimulated with peptide derivative pulsed P815 mastocytoma cells. The resulting bulk cultures displayed high cytolytic activity that was specific for IASA-YIPSAEK(ABA)I and YIPSAEK(ABA)I. In contrast, peritoneal exudate lymphocytes from mice inoculated with concanavalin A blasts that were pulsed, but not photocrosslinked, with IASA-YIPSAEK(ABA)I expressed only marginal levels of IASA-YIPSAEK(ABA)I-specific cytolytic activity. This immunization strategy, using neither adjuvants nor potentially hazardous transfected/transformed cells, is safe and should be universally applicable.

The zinc finger protein TcZFP2 binds target mRNAs enriched during Trypanosoma cruzi metacyclogenesis

Trypanosomes are parasitic protozoa in which gene expression is primarily controlled through the regulation of mRNA stability and translation. This post-transcriptional control is mediated by various families of RNA-binding proteins, including those with zinc finger CCCH motifs. CCCH zinc finger proteins have been shown to be essential to differentiation events in trypanosomatid parasites. Here, we functionally characterise TcZFP2 as a predicted post-transcriptional regulator of differentiation in Trypanosoma cruzi. This protein was detected in cell culture-derived amastigotes and trypomastigotes, but it was present in smaller amounts in metacyclic trypomastigote forms of T. cruzi. We use an optimised recombinant RNA immunopreciptation followed by microarray analysis assay to identify TcZFP2 target mRNAs. We further demonstrate that TcZFP2 binds an A-rich sequence in which the adenosine residue repeats are essential for high-affinity recognition. An analysis of the expression profiles of the genes encoding the TcZFP2-associated mRNAs throughout the parasite life cycle by microarray hybridisation showed that most of the associated mRNAs were upregulated in the metacyclic trypomastigote forms, also suggesting a role for TcZFP2 in metacyclic trypomastigote differentiation. Knockdown of the orthologous Trypanosoma brucei protein levels showed ZFP2 to be a positive regulator of specific target mRNA abundance.

Estudi de la relació estructura-funció de les proteïnes CPT1

En aquest treball s’ha analitzat la relació estructura-funció dels enzims CPT1, o Carnitina palmitoïltransferasa 1, que catalitza la reacció de transesterificació dels àcids grassos de cadena llarga a acil-carnitines, per tal que puguin accedir a la matriu mitocondrial i ser oxidats. Aquest enzim es troba estrictament regulat per malonil-CoA, primer intermediari de la síntesi d’àcids grassos, establint-se així una regulació coordinada entre la formació i la degradació de grasses. S’han estudiat els tres isotips de CPT1 descrits fins al moment: CPT1A, CPT1B i CPT1C. Mitjançant l’expressió heteròloga de mutants de CPT1A de rata i CPT1B de porc en el llevat P. pastoris, s’ha estudiat l’efecte sobre la inhibició per malonil-CoA de petits canvis en la seva estructura, per tal de trobar una relació entre la seva funció enzimàtica i la disposició conformacional de la proteïna. Segons els resultats obtinguts, el residu Glu590 de CPT1A de rata estaria impedint la unió de l’inhibidor, mentre que el residu Met593 estaria afavorint aquesta unió. Els estudis amb l’enzim CPT1B de porc demostraren l’existència d’un determinant positiu per la sensibilitat al malonil-CoA en els primers 18 residus de la proteïna, i definiren la posició Glu17 com la responsable de l’alta afinitat a la carnitina i la baixa sensibilitat a la inhibició per malonil-CoA (8). Es clonà i caracteritzà la regió promotora del gen de CPT1C humana, amb la intenció d’analitzar la funcionalitat de putatius elements de resposta identificats in silico. Cap dels elements estudiats resultà ser funcional in vivo. A més, es demostrà que la manca d’activitat catalítica de la proteïna no és deguda a l’extensió C-terminal que presenta respecte els isotips A i B, tot i presentar un alt percentatge d’identitat de seqüència. S’ha amplificat una isoforma humana de CPT1C (Pubmed Acc. Num. AK299866), corresponent a la regió carboxiterminal de la proteïna, que es pretén utilitzar per obtenir el primer cristall de la part soluble d’una proteïna CPT1.     

Efecto de la microestructura en el comportamiento mecánico de aceros de herramienta para trabajo en frío

El estudio desarrollado en la tesis doctoral profundiza en las relaciones existentes entre la microestructura y el comportamiento a rotura y fatiga de tres tipos de Aceros de Herramienta para trabajo en Frío, dos obtenidos por colada y un tercero mediante pulvimetalúrgia. En primer lugar se evaluaron los aspectos microestructurales relacionados con las dos vias de procesamiento empleadas, además del efecto del cambio en la composición química. Asimismo se documentó la influencia del trabajado en caliente sobre la isotropía microestructural. Considerando las caracteristicas microestructurales se evaluó el comportamiento a rotura en tres orientaciones referidas al sentido de la deformación en caliente. Para ello , se empleó la Mecánica de la Fractura Elástica Lineal como marco analítico. Los resultados fueron tratados siguiendo la estadística de Weibull. Para el caso de los aceros colados se implementa un analisis de curva-R para explicar el crecimiento subcrítico de grietas observado experimentalmente. El estudio del comportamiento a fatiga incluyó la determinación del limite a fatiga para todas la orientaciones microestructurales escogidas y el estudio de la cinetica de propagación de fisuras físicamente pequeñas para la orientación con mejor respuesta mecánica.

The use of proteomics to identify markers associated with metastasis in "Leishmania Viannia" species the role of tryparedoxin peroxidase

RESUME En Amérique Centrale et en Amérique du Sud, la leishmaniose cutanéo-muqueuse (LCM) est provoquée par le protozoaire Leishmania du sous-genre Viannia dont font partie L. (V.) braziliensis, L. (V.) panamensis et L. (V.) guyanensis. Dans la LCM, après guérison apparente de la lésion primitive, des lésions secondaires peuvent apparaître dues à la migration de l'infection à partir du site d'inoculation vers les muqueuses de l'ororhino-pharynx. Ce type de dissémination, communément appelé métastase, peut se produire plusieurs années après la guérison de la lésion cutanée initiale, et est un facteur majeur contribuant à la morbidité associée à la LCM. L'expression reproductible de l'activité métastatique au sein de populations discrètes de leishmanies chez le hamster fournit un modèle expérimental permettant d'étudier le degré de virulence du parasite. Nous avons utilisé des clones de L. (V.) guyanensis présentant des phénotypes stables allant d'un caractère hautement métastatique (M+) à non-métastatique (M-) comme outils pour mettre en évidence des facteurs spécifiques liés à la métastase chez les leishmanies du Nouveau Monde. Des analyses protéomiques comparatives utilisant l'électrophorèse bidimensionnelle sur gel de polyacrylamide couplée à de la spectrométrie de masse ont permis l'identification de plusieurs formes de la tryparedoxine peroxidase (TXNPx) en tant que polypeptides associés au phénotype métastatique. TXNPx, une enzyme de la famille des peroxiredoxines (Prxs), protéines antioxydantes, fonctionne comme la dernière peroxydase d'une cascade d'oxydoréductases qui réduit le peroxyde d'hydrogène aux dépens de NADPH. Toutes les Prxs sont caractérisées par un (1-Cys Prx) ou par deux résidus cystéines (2-Cys Prx), respectivement placés dans un environnement structurel conservé de la protéine et sont centrales dans la réaction catalytique. Des immuno-empreintes (« immunoblotting ») ont révélé que TXNPx est présente sous forme dimérique dans les promastigotes (M+) alors que dans les promastigotes, (M-) TXNPx est présente sous forme monomérique et dimérique. Cette caractéristique spécifique de dimérisation pourrait expliquer les différentes activités enzymatiques observées entre les deux promastigotes (M+) et (M-) en présence de peroxyde d'hydrogène ainsi que leur différence de survie et de charge parasitaire à l'intérieur des macrophages. Par conséquent, le processus métastatique pourrait être lié à la capacité du parasite à échapper efficacement aux défenses microbicides de la cellule hôte. ABSTRACT In South and Central America, protozoan parasites of the Leishmania Viannia subgenus including L. (V.) braziliensis, L. (V.) guyanensis and L. (V). panamensis cause mucocutaneous leishmaniasis (MCL). In MCL, after apparent cure of the primary lesion, secondary lesions may appear in the nasopharyngeal tissues of the infected host due to dissemination of the infection from the inoculation site. This type of dissemination, known as metastasis, can occur several years after healing of the original cutaneous lesion, and is a major contributory factor to the morbidity associated with MCL. The reproducible expression of metastasis by discrete populations of Leishmania parasites in hamsters provides an experimental model to examine the expression of parasite virulence. We used laboratory clones of L. (V.) guyanensis with stable phenotypes ranging from highly metastatic (M+) to non-metastatic (M-) as tools for the discovery of specific factors associated with metastasis in New World Leishmania species. Comparative proteome analyses via 2D-electrophoresis (2-DE) coupled with mass spectrometry (MS) enabled the identification of various isoforms of tryparedoxin peroxidase (TXNPx) as polypeptides associated with the metastatic phenotype. TXNPx, an enzyme related to the antioxidant peroxiredoxin family (Prx) functions as the terminal peroxidase of a redox cascade that reduces hydroperoxides by NADPH. All Prxs are characterized by one (1-Cys Prx) or two cysteine residue(s) (2-Cys Prx), respectively, located in a conserved structural environment of the protein which are central for the catalytic reaction. Immunoblotting analysis revealed that, under non-reducing denaturing conditions, TXNPx is present in dimeric forms in (M+) promastigotes, whereas in (M-) promastigotes, both monomeric and dimeric forms are found. This specific dimerization feature may explain the different enzymatic activities of both (M+) and (M-) promastigote parasites in the presence of H2O2 and their difference in survival and parasite load inside macrophages. Therefore, the metastatic process could be related to the ability of the parasite to efficiently evade the microbicidal effect of the host cell.

Characterization of a cAMP-regulated enhancer-binding protein.

The transcription regulation of many hormone genes is modulated by intracellular second messengers such as cAMP. The cAMP response element binding protein, CREB, binds to the 8 base pair CRE enhancer, TGACGTCA, that is found in the 5'-flank of certain genes including those for somatostatin and the alpha-subunit of human chorionic gonadotropin. The recent characterization of CREB and CREB-related cDNA clones, combined with Southwesterns and Northern blot analyses, reveals a family of transcription factors that dimerize via a leucine zipper motif and bind to the CRE through positively charged basic regions. The CREB cDNA encoding a 327 residue protein is transcriptionally activated via phosphorylation by protein kinases, including the cAMP-dependent protein kinase-A.