Lymphatic vessel function in atherosclerosis

L'athérosclérose est une maladie inflammatoire chronique caractérisée par l'accumulation de cholestérol dans la paroi artérielle et associée à une réponse immunitaire anormale dans laquelle les macrophages jouent un rôle important. Récemment, il a été démontré que les vaisseaux lymphatiques jouent un rôle primordial dans le transport inverse du cholestérol (Martel et al. JCI 2013). L’objectif global de mon stage de maîtrise a été de mieux caractériser la dysfonction lymphatique associée à l’athérosclérose, en étudiant de plus près l’origine physiologique et temporelle de ce mauvais fonctionnement. Notre approche a été d’étudier, depuis l’initiation de l’athérosclérose jusqu’à la progression d’une lésion athérosclérotique tardive, la physiologie des deux constituants principaux qui forment les vaisseaux lymphatiques : les capillaires et collecteurs lymphatiques. En utilisant comme modèle principal des souris Ldlr-/-; hApoB100+/+, nous avons pu démontrer que la dysfonction lymphatique est présente avant même l’apparition de l’athérosclérose, et que cette dysfonction est principalement associée avec un défaut au niveau des vaisseaux collecteurs, limitant ainsi le transport de la lymphe des tissus périphériques vers le sang. De plus, nous avons démontré pour la première fois l’expression du récepteur au LDL par les cellules endothéliales lymphatiques. Nos travaux subséquents démontrent que ce défaut de propulsion de la lymphe pourrait être attribuable à l’absence du récepteur au LDL, et que la dysfonction lymphatique observée précocement dans l’athérosclérose peut être limitée par des injections systémiques de VEGF (vascular endothelial growth factor) –C. Ces résultats suggèrent que la caractérisation fonctionnelle de la capacité de pompage des vaisseaux collecteurs serait une condition préalable à la compréhension de l'interaction entre la fonction du système lymphatique et la progression de l'athérosclérose. Ultimement, nos travaux nous ont amené à considérer de nouvelles cibles thérapeutiques potentielles dans la prévention et le traitement de l’athérosclérose.

A survey of VANET technologies

Recent advancement in wireless communication technologies and automobiles have enabled the evolution of Intelligent Transport System (ITS) which addresses various vehicular traffic issues like traffic congestion, information dissemination, accident etc. Vehicular Ad-hoc Network (VANET) a distinctive class of Mobile ad-hoc Network (MANET) is an integral component of ITS in which moving vehicles are connected and communicate wirelessly. Wireless communication technologies play a vital role in supporting both Vehicle to Vehicle (V2V) and Vehicle to Infrastructure (V2I) communication in VANET. This paper surveys some of the key vehicular wireless access technology standards such as 802.11p, P1609 protocols, Cellular System, CALM, MBWA, WiMAX, Microwave, Bluetooth and ZigBee which served as a base for supporting both Safety and Non Safety applications. It also analyses and compares the wireless standards using various parameters such as bandwidth, ease of use, upfront cost, maintenance, accessibility, signal coverage, signal interference and security. Finally, it discusses some of the issues associated with the interoperability among those protocols.

Biochemical characterization of serine transport in Leishmania (Leishmania) amazonensis

In addition to its role as a protein component in Leishmania, serine is also a precursor for the synthesis of both phosphatidylserine, which is a membrane molecule involved in parasite invasion and inactivation of macrophages, and sphingolipids, which are necessary for Leishmania to differentiate into its infective forms. We have characterized serine uptake in both promastigote and amastigote forms of Leishmania (Leishmania) amazonensis. In promastigotes, kinetic data show a single, saturable transport system, with a Km of 0.253 +/- 0.01 mM and a maximum velocity of 0.246 +/- 0.04 nmol/min per 107 cells. Serine transport increased linearly with temperature in the range from 20 degrees C to 45 degrees C, allowing the calculation of an activation energy of 7.09 kJ/mol. Alanine, cysteine, glycine, threonine, valine and ethanolamine competed with the substrate at a ten-fold excess concentration. Serine uptake was dependent on pH, with an optimum activity at pH 7.5. The characterization of the serine transport process in amastigotes revealed a transport system with a similar Km, energy of activation and pH response to that found in promastigotes, suggesting that the same transport system is active in both insect vector and mammalian host Leishmania stages. This could constitute an evolutionary mechanism that guarantees the provision of such an essential molecule during host change events, such as differentiation into amastigotes and macrophage invasion, as well as to ensure that the parasite maintains the infection in the mammalian host. (C) 2008 Elsevier B.V. All rights reserved.

Expert system to calculate the cofficient of friction : an approach to enhance traffic safety

Friction plays a key role in causing slipperiness as a low coefficient of friction on the road may result in slippery and hazardous conditions. Analyzing the strong relation between friction and accident risk on winter roads is a difficult task. Many weather forecasting organizations use a variety of standard and bespoke methods to predict the coefficient of friction on roads. This article proposes an approach to predict the extent of slipperiness by building and testing an expert system. It estimates the coefficient of friction on winter roads in the province of Dalarna, Sweden using the prevailing weather conditions as a basis. Weather data from the road weather information system, Sweden (RWIS) was used. The focus of the project was to use the expert system as a part of a major project in VITSA, within the domain of intelligent transport systems

Swarm control designs applied to a non-ideal load transportation system

In this paper, a load transport system in platforms is considered. It is a transport device and is modelled as an inverted pendulum built on a car driven by a DC motor. The motion equations were obtained by Lagrange's equations. The mathematical model considers the interaction between the DC motor and the dynamic system. The dynamic system was analysed and a Swarm Control Design was developed to stabilize the model of this load transport system. ©2010 IEEE.

Bridging infrastructural gaps in Central America: prospects and potential for maritime transport

Includes bibliography

Intermolecular interactions of the malate synthase of Paracoccidioides spp

Background: The fungus Paracoccidioides spp is the agent of paracoccidioidomycosis (PCM), a pulmonary mycosis acquired by the inhalation of fungal propagules. Paracoccidioides malate synthase (PbMLS) is important in the infectious process of Paracoccidioides spp because the transcript is up-regulated during the transition from mycelium to yeast and in yeast cells during phagocytosis by murine macrophages. In addition, PbMLS acts as an adhesin in Paracoccidioides spp. The evidence for the multifunctionality of PbMLS indicates that it could interact with other proteins from the fungus and host. The objective of this study was to identify and analyze proteins that possibly bind to PbMLS (PbMLS-interacting proteins) because protein interactions are intrinsic to cell processes, and it might be possible to infer the function of a protein through the identification of its ligands. Results: The search for interactions was performed using an in vivo assay with a two-hybrid library constructed in S. cerevisiae; the transcripts were sequenced and identified. In addition, an in vitro assay using pull-down GST methodology with different protein extracts (yeast, mycelium, yeast-secreted proteins and macrophage) was performed, and the resulting interactions were identified by mass spectrometry (MS). Some of the protein interactions were confirmed by Far-Western blotting using specific antibodies, and the interaction of PbMLS with macrophages was validated by indirect immunofluorescence and confocal microscopy. In silico analysis using molecular modeling, dynamics and docking identified the amino acids that were involved in the interactions between PbMLS and PbMLS-interacting proteins. Finally, the interactions were visualized graphically using Osprey software. Conclusion: These observations indicate that PbMLS interacts with proteins that are in different functional categories, such as cellular transport, protein biosynthesis, modification and degradation of proteins and signal transduction. These data suggest that PbMLS could play different roles in the fungal cell. © 2013 de Oliveira et al.; licensee BioMed Central Ltd.

Identificação e análise da expressão de genes relacioanados com tolerância à seca em soja através de microarranjos de DNA e PCR em tempo real

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeito da desintegrina recombinante DisBa-01 incorporada em micelas ou livre em células portando ou não a integrina αvβ3

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Selenium and sulfur: mitigation in plant stresses

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Selenium and sulfur: mitigation in plant stresses

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Variation in peach fruit susceptibility to Monilinia laxa and gene expression

Brown rot caused by Monilinia laxa and Monilinia fructigena is considered one of the most important diseases affecting Prunus species. Although some losses can result from the rotten fruits in the orchard, most of the damage is caused to fruits during the post-harvest phase. Several studies reported that brown rot incidence during fruit development highly varies; it was found that at a period corresponding to the the pit hardening stage, fruit susceptibility drastically decreases, to be quickly restored afterwards. However the molecular basis of this phenomenon is still not well understood. Furthermore, no difference in the rot incidence was found between wound and un-wound fruits, suggesting that resistance associated more to a specifc biochemical response of the fruit, rather than to a higher mechanical resistance. So far, the interaction Monilinia-peach was analyzed through chemical approaches. In this study, a bio-molecular approach was undertaken in order to reveal alteration in gene expression associated to the variation of susceptibility. In this thesis three different methods for gene expression analysis were used to analyze the alterations in gene expression occurring in peach fruits during the pit hardening stage, in a period encompassing the temporary change in Monilinia susceptibility: real time PCR, microarray and cDNA AFLP techniques. In 2005, peach fruits (cv.K2) were weekly harvested during a 19-week long-period, starting from the fourth week after full bloom, until full maturity. At each sampling time, three replicates of 5 fruits each were dipped in the M.laxa conidial suspension or in distilled water, as negative control. The fruits were maintained at room temperature for 3 hours; afterwards, they were peeled with a scalpel; the peel was immediately frozen in liquid nitrogen and transferred to -80 °C until use. The degree of susceptibility of peach fruit to the pathogen was determined on 3 replicates of 20 fruits each, as percentage of infected fruits, after one week at 20 °C. Real time PCR analysis was performed to study the variation in expression of those genes encoding for the enzymes of the phenylpropanoid pathway (phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), cinnamate 4-hydroxylase (C4H), leucoanthocyanidine reductase (LAR), hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase (HQT) and of the jasmonate pathway, such as lipoxygenase (LOX), both involved in the production of important defense compounds. Alteration in gene expression was monitored on fruit samples of a period encompassing the pit hardening stage and the corresponding temporary resistance to M.laxa infections, weekly, from the 6thto the 12th week after full bloom (AFB) inoculated with M. laxa or mock-inoculated. The data suggest a critical change in the expression level of the phenylpropanoid pathway from the 7th to the 8th week AFB; such change could be directly physiologically associated to the peach growth and it could indirectly determine the decrease of susceptibility of peach fruit to Monilinia rot during the subsequent weeks. To investigate on the transcriptome variation underneath the temporary loss of susceptibility of peach fruits to Monilinia rot, the microarray and the cDNA AFLP techniques were used. The samples harvested on the 8th week AFB (named S, for susceptible ones) and on the 12th week AFB (named R, for resistant ones) were compared, both inoculated or mock-inoculated. The microarray experiments were carried out at the University of Padua (Dept. of Environmental Agronomy and Crop Science), using the μPEACH1.0 microarray together with the suited protocols. The analysis showed that 30 genes (corresponding to the 0.6% of the total sequences (4806) contained in the μPeach1.0 microarray) were found up-regulated and 31 ( 0.6%) down regulated in RH vs. SH fruits. On the other hand, 20 genes (0.4%) were shown to be up-regulated and 13 (0.3%) down-regulated in the RI vs. SI fruit. No genes were found differentially expressed in the mock-inoculated resistant fruits (RH) vs. the inoculated resistant ones (RI). Among the up-regulated genes an ATP sulfurylase, an heat shock protein 70, the major allergen Pru P1, an harpin inducing protein and S-adenosylmethionine decarboxylase were found, conversely among the down-regulated ones, cinnamyl alcohol dehydrogenase, an histidine- containing phosphotransfer protein and the ferritin were found. The microarray experimental results and the data indirectly derived, were tested by Real Time PCR analysis. cDNA AFLP analysis was also performed on the same samples. 339 transcript derived fragments considered significant for Monilinia resistance, were selected, sequenced and classified. Genes potentially involved in cell rescue and defence were well represented (8%); several genes (12.1%) involved in the protein folding, post-transductional modification and genes (9.2%) involved in cellular transport were also found. A further 10.3% of genes were classified as involved in the metabolism of aminoacid, carbohydrate and fatty acid. On the other hand, genes involved in the protein synthesis (5.7%) and in signal transduction and communication (5.7%) were found. Among the most interesting genes found differentially expressed between susceptible and resistant fruits, genes encoding for pathogenesis related (PR) proteins were found. To investigate on the association of Monilinia resistance and PR biological function, the major allergen Pru P1 (GenBank accession AM493970) and its isoform (here named Pru P2), were expressed in heterologous system and in vitro assayed for their anti-microbial activity. The ribonuclease activity of the recombinant Pru P1 and Pru P2 proteins was assayed against peach total RNA. As the other PR10 proteins, they showed a ribonucleolytic activity, that could be important to contrast pathogen penetration. Moreover Pru P1 and Pru P2 recombinant proteins were checked for direct antimicrobial activity. No inhibitory effect of Pru P1 or Pru P2 was detected against the selected fungi.

Respiratory electron transport activity in plankton: determinants and detection

[ES]La presente tesis, se centra en el estudio del Sistema de Transporte de Electrones (ETS) en organismos del plancton marino, los factores que lo influencian la interpretación de estas mediciones y su detección mediante espectrofotometría y espectrofluorometría, en muestras oceánicas naturales y en cultivos de organismos marinos. Se pudo establecer, la biomasa, la respiración (R) y la respiración potencial (ɸ), en tres transectos en los océanos Índico y Atlántico Norte Sur. A su vez, se determino el estado fisiológico, en tres tamaños del zooplancton, midiendo la relación R/ɸ. Se exploró los efectos de la inanición sobre la R y la variación con respecto a la ɸ en el zooplancton

Rekonstitution des Fumaratsensors DcuS in Liposomen und Transport von Fumarat und Succinat in Escherichia coli

Das Zweikomponentenregulationssystem DcuSR kontrolliert die Expression der wichtigsten fumaratinduzierten Gene in Escherichia coli. Die Gene dcuB und dctA, die fürDicarboxylatcarrier kodieren, sowie das Fumaratreduktase-Operon (frd), sind Zielgene für DcuSR. DcuS ist eine membranständige Sensorkinase mit einer großen periplasmatischen Domäne. NMR-spektroskopische Untersuchungen dieser Domäne zeigen Alpha-Helices und Beta-Faltblätter. Für die Fumaratbindung wichtige Aminosäuren wurden durch Mutagenese identifiziert. Gereinigtes DcuS wurde in Liposomen rekonstituiert. In Anwesenheit von ATP wird DcuS autophosphoryliert. Der Phosphatrest kann dann auf DcuR übertragen werden und beweist somit die Aktivität dieses in vitro Testsystems.Der Transport von C4-Dicarboxylaten erfolgt unter anaeroben Bedingungen durch die sekundären Carrier DcuA, DcuB und DcuC. Es konnte ein weiteres Protein (DcuD) identifiziert werden, das hohe Sequenzähnlichkeit zu DcuC aufweist. Eine dcuD-Mutante zeigte keinen Phänotyp und überproduziertes DcuD konnte den Ausfall der anderen Dcu-Carrier nicht kompensieren. DcuD ist damit ein kryptisches Mitglied der Dcu-Carrierfamilie. Unter aeroben Bedingungen katalysiert DctA den Transport von C4-Dicarboxylaten. Dennoch können dctA-Mutanten noch mit Succinat wachsen. Die Diffusionsrate von Succinat durch Membranen wurde bestimmt. Sie ist um Größenordnungen niedriger als der Transport in der Mutante. Bei dem DctA unabhängigen Transportsystem handelt es sich um einen H+/Succinat2-Symporter, der bei saurem pH aktiv ist und viele Eigenschaften eines Monocarboxylatcarriers aufweist.

Lysosomaler Transport kationischer Aminosäuren durch Mitglieder der SLC7-Familie

Lysosomaler Transport kationischer Aminosäuren (KAS) stellt einen Rettungsweg in der Cystinose-Therapie dar. Ein solches Transportsystem wurde in humanen Hautfibroblasten beschrieben und mit System c benannt. Des Weiteren stellt lysosomales Arginin eine Substratquelle für die endotheliale NO-Synthase (eNOS) dar. Das von der eNOS gebildete NO ist ein wichtiges vasoprotektiv wirkendes Signalmolekül. Ziel war es daher, herauszufinden, ob Mitglieder der SLC7-Unterfamilie hCAT möglicherweise System c repräsentieren.rnIn dieser Arbeit konnte ich die lysosomale Lokalisation verschiedener endogener, sowie als EGFP-Fusionsproteine überexprimierter CAT-Isoformen nachweisen. Mittels Fluoreszenz-mikroskopie wurde festgestellt, dass die in U373MG-Zellen überexprimierten Fusionsproteine hCAT-1.EGFP sowie SLC7A14.EGFP mit dem lysosomalen Fluoreszenz-Farbstoff LysoTracker co-lokalisieren. Eine Lokalisation in Mitochondrien oder dem endoplasmatischem Retikulum konnte mit entsprechenden Fluoreszenz-Farbstoffen ausgeschlossen werden. Zusätzlich reicherten sich die überexprimierten Proteine hCAT-1.EGFP, hCAT-2B.EGFP und SLC7A14.EGFP in der lysosomalen Fraktion C aus U373MG-Zellen zusammen mit den lysosomalen Markern LAMP-1 und Cathepsin D an. Gleiches galt für den endogenen hCAT-1 in der lysosomalen Fraktion C aus EA.hy926- und U373MG-Zellen sowie für den SLC7A14 in den humanen Hautfibroblasten FCys5. Mit dem im Rahmen dieser Arbeit generierte Antikörper gegen natives SLC7A14 konnte erstmals die endogene Expression und Lokalisation von SLC7A14 in verschiedenen Zelltypen analysiert werden.rnObwohl eine Herunterregulation des hCAT-1 in EA.hy926-Endothelzellen nicht zu einer Reduktion der Versorgung der eNOS mit lysosomalem Arginin führte, ist eine Funktion von hCAT-1 im Lysosom wahrscheinlich. Sowohl die [3H]Arginin- als auch die [3H]Lysin-Aufnahme der Fraktion C aus U373MG-hCAT-1.EGFP war signifikant höher als in die Fraktion C aus EGFP-Kontrollzellen. Dies konnte ebenfalls für den hCAT-2B.EGFP gezeigt werden. Zusätzlich zeigten lysosomale Proben aus U373MG-hCAT-2B.EGFP-Zellen in der SSM-basierten Elektrophysiologie eine elektrogene Transportaktivität für Arginin. Das Protein SLC7A14.EGFP zeigte in keiner der beiden durchgeführten Transportstudien eine Aktivität. Dies war unerwartet, da die aus der Diplomarbeit stammende und im Rahmen dieser Dissertation erweiterte Charakterisierung der hCAT-2/A14_BK-Chimäre, die die „funktionelle Domäne“ des SLC7A14 im Rückgrat des hCAT-2 trug, zuvor den Verdacht erhärtet hatte, dass SLC7A14 ein lysosomal lokalisierter Transporter für KAS sein könnte. Diese Studien zeigten allerding erstmals, dass die „funktionelle Domäne“ der hCATs die pH-Abhängigkeit vermittelt und eine Rolle in der Substraterkennung spielt.rnZukünftig soll weiter versucht werden auch endogen eine Transportaktivität der hCATs für KAS im Lysosom nachzuweisen und das Substrat für das intrazellulär lokalisierte Waisen-Protein SLC7A14 zu finden. Eine mögliche Rolle könnte SLC7A14 als Transporter für Neurotransmitter spielen, da eine sehr prominente Expression im ZNS festgestellt wurde.rn