Towards automated test and validation of SIP solutions

IP networks are currently the major communication infrastructure used by an increasing number of applications and heterogeneous services, including voice services. In this context, the Session Initiation Protocol (SIP) is a signaling protocol widely used for controlling multimedia communication sessions such as voice or video calls over IP networks, thus performing vital functions in an extensive set of public and enter- prise solutions. However, the SIP protocol dissemination also entails some challenges, such as the complexity associated with the testing/validation processes of IMS/SIP networks. As a consequence, manual IMS/SIP testing solutions are inherently costly and time consuming tasks, being crucial to develop automated approaches in this specific area. In this perspective, this article presents an experimental approach for automated testing/validation of SIP scenarios in IMS networks. For that purpose, an automation framework is proposed allowing to replicate the configuration of SIP equipment from the pro- duction network and submit such equipment to a battery of tests in the testing network. The proposed solution allows to drastically reduce the test and validation times when compared with traditional manual approaches, also allowing to enhance testing reliability and coverage. The automation framework comprises of some freely available tools which are conveniently integrated with other specific modules implemented within the context of this work. In order to illustrate the advantages of the proposed automated framework, a real case study taken from a PT Inovação customer is presented comparing the time required to perform a manual SIP testing approach with the one time required when using the proposed auto- mated framework. The presented results clearly corroborate the advantages of using the presented framework.

Design and validation of a biomechanical bioreactor for cartilage tissue culture

Specific tissues, such as cartilage undergo mechanical solicitation under their normal performance in human body. In this sense, it seems necessary that proper tissue engineering strategies of these tissues should incorporate mechanical solicitations during cell culture, in order to properly evaluate the influence of the mechanical stimulus. This work reports on a user-friendly bioreactor suitable for applying controlled mechanical stimulation - amplitude and frequency - to three dimensional scaffolds. Its design and main components are described, as well as its operation characteristics. The modular design allows easy cleaning and operating under laminar hood. Different protocols for the sterilization of the hermetic enclosure are tested and ensure lack of observable contaminations, complying with the requirements to be used for cell culture. The cell viability study was performed with KUM5 cells.

Identification and characterization of deforestation hot spots in Venezuela using MODIS satellite images

Land cover changes over time as a result of human activity. Nowadays deforestation may be considered one of the main environmental problems. The objective of this study was to identify and characterize changes to forest cover in Venezuela between 2005-2010. Two maps of deforestation hot spots were generated on the basis of MODIS data, one using digital techniques and the other by means of direct visual interpretation by experts. These maps were validated against Landsat ETM+ images. The accuracy of the map obtained digitally was estimated by means of a confusion matrix. The overall accuracy of the maps obtained digitally was 92.5%. Expert opinions regarding the hot spots permitted the causes of deforestation to be identified. The main processes of deforestation were concentrated to the north of the Orinoco River, where 8.63% of the country's forests are located. In this region, some places registered an average annual forest change rate of between 0.72% and 2.95%, above the forest change rate for the country as a whole (0.61%). The main causes of deforestation for the period evaluated were agricultural and livestock activities (47.9%), particularly family subsistence farming and extensive farming which were carried out in 94% of the identified areas.

Implementation and validation of a strain rate dependent anisotropic continuum model for masonry

A newly developed strain rate dependent anisotropic continuum model is proposed for impact and blast applications in masonry. The present model adopted the usual approach of considering different yield criteria in tension and compression. The analysis of unreinforced block work masonry walls subjected to impact is carried out to validate the capability of the model. Comparison of the numerical predictions and test data revealed good agreement. Next, a parametric study is conducted to evaluate the influence of the tensile strengths along the three orthogonal directions and of the wall thickness on the global behavior of masonry walls.

Embedding, evolution, and validation of model-driven spreadsheets

This paper proposes and validates a model-driven software engineering technique for spreadsheets. The technique that we envision builds on the embedding of spreadsheet models under a widely used spreadsheet system. This means that we enable the creation and evolution of spreadsheet models under a spreadsheet system. More precisely, we embed ClassSheets, a visual language with a syntax similar to the one offered by common spreadsheets, that was created with the aim of specifying spreadsheets. Our embedding allows models and their conforming instances to be developed under the same environment. In practice, this convenient environment enhances evolution steps at the model level while the corresponding instance is automatically co-evolved.Finally,wehave designed and conducted an empirical study with human users in order to assess our technique in production environments. The results of this study are promising and suggest that productivity gains are realizable under our model-driven spreadsheet development setting.

Polyphasic approach including MALDI-TOF MS/MS analysis for identification and characterisation of Fusarium verticillioides in Brazilian corn kernels

Fusarium verticillioides is considered one of the most important global sources of fumonisin contamination in food and feed. Corn is one of the main commodities produced in the Northeastern Region of Brazil. The present study investigated potential mycotoxigenic fungal strains belonging to the F. verticillioides species isolated from corn kernels in 3 different Regions of the Brazilian State of Pernambuco. A polyphasic approach including classical taxonomy, molecular biology, MALDI-TOF MS and MALDI-TOF MS/MS for the identification and characterisation of the F. verticillioides strains was used. Sixty F. verticillioides strains were isolated and successfully identified by classical morphology, proteomic profiles of MALDI-TOF MS, and by molecular biology using the species-specific primers VERT-1 and VERT-2. FUM1 gene was further detected for all the 60 F. verticillioides by using the primers VERTF-1 and VERTF-2 and through the amplification profiles of the ISSR regions using the primers (GTG)5 and (GACA)4. Results obtained from molecular analysis shown a low genetic variability among these isolates from the different geographical regions. All of the 60 F. verticillioides isolates assessed by MALDI-TOF MS/MS presented ion peaks with the molecular mass of the fumonisin B1 (721.83 g/mol) and B2 (705.83 g/mol)

Development and Validation of Predictive Models of Cardiac Mortality and Transplantation in Resynchronization Therapy

AbstractBackground:30-40% of cardiac resynchronization therapy cases do not achieve favorable outcomes.Objective:This study aimed to develop predictive models for the combined endpoint of cardiac death and transplantation (Tx) at different stages of cardiac resynchronization therapy (CRT).Methods:Prospective observational study of 116 patients aged 64.8 ± 11.1 years, 68.1% of whom had functional class (FC) III and 31.9% had ambulatory class IV. Clinical, electrocardiographic and echocardiographic variables were assessed by using Cox regression and Kaplan-Meier curves.Results:The cardiac mortality/Tx rate was 16.3% during the follow-up period of 34.0 ± 17.9 months. Prior to implantation, right ventricular dysfunction (RVD), ejection fraction < 25% and use of high doses of diuretics (HDD) increased the risk of cardiac death and Tx by 3.9-, 4.8-, and 5.9-fold, respectively. In the first year after CRT, RVD, HDD and hospitalization due to congestive heart failure increased the risk of death at hazard ratios of 3.5, 5.3, and 12.5, respectively. In the second year after CRT, RVD and FC III/IV were significant risk factors of mortality in the multivariate Cox model. The accuracy rates of the models were 84.6% at preimplantation, 93% in the first year after CRT, and 90.5% in the second year after CRT. The models were validated by bootstrapping.Conclusion:We developed predictive models of cardiac death and Tx at different stages of CRT based on the analysis of simple and easily obtainable clinical and echocardiographic variables. The models showed good accuracy and adjustment, were validated internally, and are useful in the selection, monitoring and counseling of patients indicated for CRT.

Identification and characterization of novel molecular mechanisms involved in initiation and progression of myelination

Schwann cells synthesize a large amount of membrane that form a specialized structure called myelin that surrounds axons and facilitate the transmission of electrical signal along neurons in peripheral nervous system (PNS). Previous studies demonstrated that both Schwann cell differentiation and de-differentiation (in the situation of a nerve injury or demyelinating disease) are regulated by cell-intrinsic regulators including several transcription factors. In particular, the de-differentiation of mature Schwann cells is driven by the activation of multiple negative regulators of myelination including Sox2, c-Jun, Notch and Pax3, all usually expressed in immature Schwann cells and suppressed at the onset of myelination. In order to identify new regulators of myelination involved in the development of the PNS, we analyzed the gene-expression profiling data from developing PNS and from three models of demyelinating neuropathies. This analysis led to the identification of Sox4, a member of the Sox family of transcription factors, as a potential candidate. To characterize the molecular function of Sox4 in PNS, we generated two transgenic lines of mice, which overexpress Sox4 specifically in Schwann cells. Detailed analysis of these mice showed that the overexpression of Sox4 in Schwann cells causes a delay in progression of myelination between post-natal day 2 (P2) and P5. Our in vitro analysis suggested that Sox4 cDNA can be overexpressed while the protein translation is tightly regulated. Interestingly, we observed that Sox4 protein is stabilized in nerves of the CMT4C mouse, a model of the human neuropathy. We therefore crossed Sox4 transgenic mice with CMT4C mice and we observed that Sox4 overexpression exacerbated the neuropathy phenotype in these mice. While recognized as being crucial for the normal function of both neurons and myelinating glial cells, the processes that regulate the beginning of myelination and the nature of the neuro-glial cross-talk remains mostly unknown. In order to gain insight into the molecular pathways involved in the interactions between neurons and associated glial cells, we developed a neuron-glia co-culture system based on microfluidic chambers and successfully induced myelination in this system by ascorbic acid. Importantly, we observed that in addition to acting on Schwann cells, ascorbic acid also modulate neuronal/axonal NRG1/ErbB2-B3 signalling. The experimental setting used in our study thus allowed us to discover a novel phenomena of propagation for myelination in vitro. The further characterization of this event brought us to identify other compounds able to induce myelination: ADAMs secretases inhibitor GM6001 and cyclic-AMP. The results generated during my thesis project are therefore not only important for the advancement of our understanding of how the PNS works, but may also potentially help to develop new therapies aiming at improvement of PNS myelination under disease conditions. - Les cellules de Schwann synthétisent une grande quantité de membrane formant une structure spécialisée appelée myéline qui entoure les axones et facilite la transmission du signal électrique le long des neurones du système nerveux périphérique (SNP). Des études antérieures ont démontré que la différenciation et la dédifférenciation des cellules de Schwann (dans la situation d'une lésion nerveuse ou d'une maladie démyélinisante) sont régulées par des régulateurs cellulaires intrinsèques, incluant plusieurs facteurs de transcription. En particulier, la dédifférenciation des cellules de Schwann matures est contrôlée par l'activation de plusieurs régulateurs négatifs de la myélinisation dont Sox2, c-Jun, Notch et Pax3, tous habituellement exprimés dans des cellules de Schwann immatures et supprimés au début de la myélinisation. Afin d'identifier de nouveaux régulateurs de myélinisation impliqués dans le développement du SNP, nous avons analysé le profil d'expression génique durant le développement du SNP ainsi que dans trois modèles de neuropathies démyélinisantes. Cette analyse a mené à l'identification de Sox4, un membre de la famille des facteurs de transcription Sox, comme étant un candidat potentiel. Dans le but de caractériser la fonction moléculaire de Sox4 dans le SNP, nous avons généré deux lignées transgéniques de souris qui surexpriment Sox4 spécifiquement dans les cellules de Schwann. L'analyse détaillée de ces souris a montré que la surexpression de Sox4 dans les cellules de Schwann provoque un retard dans la progression de la myélinisation entre le jour postnatal 2 (P2) et P5. Notre analyse in vitro a suggéré que l'ADNc de Sox4 peut être surexprimé alors que la traduction des protéines est quand à elle étroitement régulée. De façon intéressante, nous avons observé que la protéine Sox4 est stabilisée dans les nerfs des souris CMT4C, un modèle de neuropathie humaine. Nous avons donc croisé les souris transgéniques Sox4 avec des souris CMT4C et avons observé que la surexpression de Sox4 exacerbe le phénotype de neuropathie chez ces souris. Bien que reconnus comme étant cruciaux pour le fonctionnement normal des neurones et des cellules gliales myélinisantes, les processus qui régulent le début de la myélinisation ainsi que la nature des interactions neurone-glie restent largement méconnus. Afin de mieux comprendre les mécanismes moléculaires impliqués dans les interactions entre les neurones et les cellules gliales leur étant associés, nous avons développé un système de co-culture neurone-glie basé sur des chambres microfluidiques et y avons induit avec succès la myélinisation avec de l'acide ascorbique. Étonnamment, nous avons remarqué que, en plus d'agir sur les cellules de Schwann, l'acide ascorbique module également la voie de signalisation neuronale/axonale NRG1/ErbB2-B3. Le protocole expérimental utilisé dans notre étude a ainsi permis de découvrir un nouveau phénomène de propagation de la myélinisation in vitro. La caractérisation plus poussée de ce phénomène nous a menés à identifier d'autres composés capables d'induire la myélinisation: L'inhibiteur de sécrétases ADAMs GM6001 et l'AMP cyclique. Les résultats obtenus au cours de mon projet de thèse ne sont donc pas seulement importants pour l'avancement de notre compréhension sur la façon dont le SNP fonctionne, mais peuvent aussi potentiellement aider à développer de nouvelles thérapies visant à l'amélioration de la myélinisation du SNP dans des conditions pathologiques.

Identification and characterization of sex-linked proteins of Schistosoma mansoni

The proteins of adults worms (male and female) of two isolates (BH and RJ) of Shistosoma mansoni were extracted using Triton X-114 phase separation. The SDS-polyacrilamide gel electrophoresis profiles of the three phases (detergent, aqueous and insoluble proteins) obtained were compared after Coomassie blue and silver staining, surface radioiodination and Western blotting. No major differences were detected between the 2 isolates. Of the 25 or more proteins which partitioned into the detergent phase, only about 8 proteins could be surface radiodinated on live adult worms. A comparison was also made between the profiles of mael and females worms, isolated from bisexually infected mice. Two major female-specific and one male-specific band were detected by silver and/or Coomassie staining. The female bands, 32 KDa and 18 KDa, partitioned into the detergent and aqueous phase, respectively. The male-specific band of 42 KDa remained in the insoluble phase. Antigenic differences between male and females protins were detected by Western vlotting using a sera from infected Nectomys squamipes.

Identification and purification of two distinct complexes containing the five RAD51 paralogs.

Cells defective in any of the RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) are sensitive to DNA cross-linking agents and to ionizing radiation. Because the paralogs are required for the assembly of DNA damage-induced RAD51 foci, and mutant cell lines are defective in homologous recombination and show genomic instability, their defect is thought to be caused by an inability to promote efficient recombinational repair. Here, we show that the five paralogs exist in two distinct complexes in human cells: one contains RAD51B, RAD51C, RAD51D, and XRCC2 (defined as BCDX2), whereas the other consists of RAD51C with XRCC3. Both protein complexes have been purified to homogeneity and their biochemical properties investigated. BCDX2 binds single-stranded DNA and single-stranded gaps in duplex DNA, in accord with the proposal that the paralogs play an early (pre-RAD51) role in recombinational repair. Moreover, BCDX2 complex binds specifically to nicks in duplex DNA. We suggest that the extreme sensitivity of paralog-defective cell lines to cross-linking agents is owing to defects in the processing of incised cross links and the consequential failure to initiate recombinational repair at these sites.

The Amazonia Variant of Vibrio cholerae: Molecular Identification and Study of Virulence Genes

The pathogenic O1 Amazonia variant of Vibrio cholerae has been shown previously to have a cytotoxin acting on cultured Vero and Y-1 cells, and to lack important virulence factors such as the cholera toxin (Coelho et al. 1995a). This study extends the molecular analysis of the Amazonia strains, detecting the presence of the toxR gene, with a very similar sequence to that of the El Tor and classical biotypes. The outer membrane proteins are analyzed, detecting a variation among the group of Amazonia strains, with three different patterns found. As a by-product of this work a polymerase chain reaction fragment was sequenced, reading part of the sequence of the Lon protease of the Amazonia strains. This gene was not previously described in V. cholerae, but its sequence is present in the TIGR database specific for this species.

I. Identification and characterisation of epigenetically altered tumor supressor genes by proteomic and genomic approches / II. Studies of genes regualted by MeCP2 union to its promoter regions by proteomic and genmomic approches

DNA methylation has an important impact on normal cell physiology, thus any defects in this mechanism may be related to the development of various diseases In this project we are interested in identifying epigeneticaliy modified genes, in general controlled by processes related to the DNA methylation, by means of a new strategy combining protomic and genomic analyses. First, the two Dimensional-Difference Gel Electrophoresis (2-DIGE) protein analyses of extracts obtained from HCT-116 wt and double knockout for DNMT1 and DNMT3b (DKO) cells revealed 34 proteins overexpressed in the condition of DNMTs depletion. From five genes with higher transcript lavels in DKO cells, comparing with HCT-116 wt. oniy AKR1B1, UCHLl and VIM are melhylated in HCT-116. As expected. the DNA methvlation 1s lost in DKO cells. The rneth,vl ation of VIM and UCHLl promoters in some cancer samples has already been repaired, thus further studies has been focused on AKRlBI. AKR1B1 expression due lo DNA methyiaton of promoter region seems to occur specilfically in the colon cancer cell Iines. which was confirmed in the DNA rnethylation status and expression analyses. performed on 32 different cancer cell lines (including colon, breast, lymphoma, leukemia, neuroblastoma, glioma and lung cancer cell Iines) as well as normal colon and normal lymphocytes samples. AKRIBI expression after treatments with DNA demethvlating agent (AZA) was rescued in 5 coloncancer cell lines (including genetic regulation of the candidate gene. The methylation status of the rest of the genes identified in proteomic analysis was checked by methylation specific PCR (MSP) experiment and all appeared to be unmethylated. The similar research has been done also bv means of Mecp2-null mouse model For 14 selected candidate genes the analyses of expression leveis, methylation Status and MeCP2 interaction with promoters are currently being performed.