Evaluation of three different DNA extraction methods from blood samples collected in dried filter paper in Plasmodium subpatent infections from the Amazon region in Brazil

Asymptomatic Plasmodium infection is a new challenge for public health in the American region. The polymerase chain reaction (PCR) is the best method for diagnosing subpatent parasitemias. In endemic areas, blood collection is hampered by geographical distances and deficient transport and storage conditions of the samples. Because DNA extraction from blood collected on filter paper is an efficient method for molecular studies in high parasitemic individuals, we investigated whether the technique could be an alternative for Plasmodium diagnosis among asymptomatic and pauciparasitemic subjects. In this report we compared three different methods (Chelex®-saponin, methanol and TRIS-EDTA) of DNA extraction from blood collected on filter paper from asymptomatic Plasmodium-infected individuals. Polymerase chain reaction assays for detection of Plasmodium species showed the best results when the Chelex®-saponin method was used. Even though the sensitivity of detection was approximately 66% and 31% for P. falciparum and P. vivax, respectively, this method did not show the effectiveness in DNA extraction required for molecular diagnosis of Plasmodium. The development of better methods for extracting DNA from blood collected on filter paper is important for the diagnosis of subpatent malarial infections in remote areas and would contribute to establishing the epidemiology of this form of infection.

MEMBRANE FRACTIONS FROM Strongyloides venezuelensis IN THE IMMUNODIAGNOSIS OF HUMAN STRONGYLOIDIASIS

Strongyloides venezuelensis is a parasitic nematode of rodents frequently used to obtain heterologous antigens for the immunological diagnosis of human strongyloidiasis. The aim of this study was to evaluate membrane fractions from S. venezuelensis for human strongyloidiasis immunodiagnosis. Soluble and membrane fractions were obtained in phosphate saline (SS and SM) and Tris-HCl (TS and TM) from filariform larvae of S. venezuelensis. Ninety-two serum samples (n = 92) were obtained from 20 strongyloidiasis patients (Group I), 32 from patients with other parasitic diseases (Group II), and 40 from healthy individuals (Group III), and were analyzed by enzyme-linked immunosorbent assay (ELISA). Soluble fractions (SS and TS) showed 90.0% sensitivity and 88.9% specificity, whereas the membrane fractions (SM and TM) showed 95.0% sensitivity and 94.4% specificity. The present results suggest the possible use of membrane fractions of S. venezuelensis as an alternative antigen for human strongyloidiasis immunodiagnosis.

IMMUNODIAGNOSIS OF HUMAN STRONGYLOIDIASIS: USE OF SIX DIFFERENT ANTIGENIC FRACTIONS FROM Strongyloides venezuelensis PARASITIC FEMALES

SUMMARY The aim of this study was to evaluate six different antigenic fractions from Strongyloides venezuelensis parasitic females for the immunodiagnosis of human strongyloidiasis. Soluble and membrane fractions from S. venezuelensis parasitic females were prepared in phosphate-buffered saline (SSF and SMF, respectively), Tris-HCl (TSF and TMF, respectively), and an alkaline buffer (ASF and AMF, respectively). Serum samples obtained from patients with strongyloidiasis or, other parasitic diseases, and healthy individuals were analyzed by enzyme-linked immunosorbent assay (ELISA). Soluble fractions SSF, TSF, and ASF showed 85.0%, 75.0%, and 80.0% sensitivity and 93.1%, 93.1%, and 87.5% specificity, respectively. Membrane fractions SMF, TMF, and AMF showed 80.0%, 75.0%, and 85.0% sensitivity, and 95.8%, 90.3%, and 91.7% specificity, respectively. In conclusion, the present results suggest that the fractions obtained from parasitic females, especially the SSF and SMF, could be used as alternative antigen sources in the serodiagnosis of human strongyloidiasis.

Caracterização estrutural de enzimas de molibdénio e suas chaperonas

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Recommendations for the detection of Leptospira in urine by PCR

In the present study PCR was applied to detect leptospires in human urine. Several approaches for sample processing were evaluated to optimize the detection of leptospires in urine mixed with this bacterium. Furthermore, some changes in the composition of the reaction mix were studied. No amplification was observed in acidic urine, therefore neutralization of the sample immediately after collection is strongly recommended. PBS gave better results than Tris or NaOH as neutralizing reagents. Freezing and thawing of samples before processing yielded negative results. Elimination of epithelial cells, leukocytes and crystals by centrifugation at 3,000 rpm at room temperature increased sensitivity. In addition, both the washing step after collecting leptospires by centrifugation and the inclusion of 0.1% bovine serum albumin in the reaction mix minimized the interference of other inhibitory compounds. These modifications were useful to improve the detection of Leptospira in urine by PCR.

Control of a white organic light emitting diode’s emission parameters using a single doped RGB active layer

White Color tuning is an attractive feature that Organic Light Emitting Diodes (OLEDs) offer. Up until now, there hasn’t been any report that mix both color tuning abilities with device stability. In this work, White OLEDs (W-OLEDs) based on a single RGB blend composed of a blue emitting N,N′-Di(1-naphthyl)-N,N′-diphenyl-(1,1′-biphenyl)-4,4′-diamine (NPB) doped with a green emitting Coumarin-153 and a red emitting 4-(Dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran (DCM1) dyes were produced. The final device structure was ITO/Blend/Bathocuproine (BCP)/ Tris(8-hydroxyquinolinato)aluminium (Alq3)/Al with an emission area of 0.25 cm2. The effects of the changing in DCM1’s concentration (from 0.5% to 1% wt.) allowed a tuning in the final white color resulting in devices capable of emitting a wide range of tunes – from cool to warm – while also keeping a low device complexity and a high stabilitty. Moreover, an explanation on the optoelectrical behavior of the device is presented. The best electroluminescense (EL) points toward 160 cd/m2 of brightness and 1.1 cd/A of efficiency, both prompted to being enhanced. An Impedance Spectroscopy (IS) analysis allowed to study both the effects of BCP as a Hole Blocking Layer and as an aging probe of the device. Finally, as a proof of concept, the emission was increased 9 and 64 times proving this structure can be effectively applied for general lighting.

Wonder Woman et la mythologie contemporaine des amazones

[Extrait] Notre point de départ est la constatation dans les différents médias, genres et arts de la culture populaire et médiatique de la grande quantité de personnages féminins qui défendent par la force la liberté de choisir leur destin: Wonder Woman, Lara Croft, Beatrix Kiddo (Kill Bill), Salt, Katniss Everdeen (The Hunger Games), Tris (Divergent), Lisbeth Salander (The Millenium Trilogy), Elektra, Yoko Tsuno, Marie des Dragons, Artémis Delambre (Les Pirates de Barataria), Isabellae, entre beaucoup d’autres (1). Toutes ces femmes combattent. La conjugaison des valeurs de la liberté et de la force – la force comme condition de la liberté – structure un certain profil féminin qui se réapproprie deux qualités, l’une physique, l’autre politique, qui constituent dans la tradition socioculturelle patriarcale une prérogative typiquement masculine. (...)

Spéciation of chromium in a chromium enriched yeast.

The sample under investigation in this project is an experimental chromium enriched yeast used as a possible additive in animal foodstuff, which was produced by growing yeast in the presence of chromium (III) chloride. Chromium on its own in not biologically active but chromium in the form of chromium enriched yeast is biologically active. The objective of this project was to show the complete absence of chromium(VI) from the sample. A literature survey describing previous work carried out on the speciation of Cr(VI) has been carried out. The principal methods of detection of Cr(VI) used in this project are Polarography, G.F.A.A. Spectroscopy, U.V. Spectroscopy and H.P.L.C. For each of the above methods a calibration curve was obtained and each method was applied to the yeast extract. The H.P.L.C. and U.V. spectroscopic method are specific for Cr(VI) but polarography and G.F.A.A. spectroscopy measure total chromium. Tris-NaOH buffer has been investigated for the extraction of chromium(VT). Problems associated with air oxidation of Cr(III) in alkaline solution have identified and procedures described for the suppression of air oxidation. Procedures are described for the application of the extraction procedure to the yeast extract and for the determination of Cr(VI) in the extract. Procedures are also described for the preconcentration of Cr(VI) on a HPLC column and for the application to the yeast extract. The rate of reduction of Cr(VI) by ascorbic acid is investigated and found to be first order with respect to ascorbic acid concentration. The reduction capacity of the yeast is also investigated and it was found that in acid solution the yeast will reduce Cr(VI) but in neutral or basic solution the reduction capacity is diminished. Conclusions regarding the objectives of the project are drawn and suggestions for further work are given.

The distribution of agglutinins and lytic activity against Trypanosoma rangeli and erythrocytes in Rhodnius prolixus and Triatoma infestans tissue extracts and haemolymph

Haemolymph, heads, salivary glands, crops, midguts, hindguts, and Malpighian tubules from Rhodnius prolixus and Triatoma infestans were extracted in phosphate or Tris buffer saline with calcium, and tested for agglutination and lytic activities by microtitration against both vertebrateerythrocytes and cultured epimatigote forms of Trypanosoma rangeli. Haemagglutination activity against rabbit erythrocytes was found in the crop, midgut and hindgut extracts of T. infestans but only in the haemolymph of R. prolixus. Higher titres of parasite agglutinins were found in R. prolixus haemolymph than T. infestans, whilst the converse occurred for the tissue extracts. In addition, the extracts of T. infestans salivary glands, but not those of R. prolixus, showed a trypanolytic activity that was heat-inactivated and was not abolished by pre-incubation with any of the sugars or glycoproteins tested. T. infestans, which is refractory to infection by T. rangeli, thus appears to contain a much wider distribution of agglutinating and trypanolytic factors in its tissues than the more susceptible species, R. prolixus

Optimization and inhibition of the adherent ability of Plasmodium falciparum-infected erythrocytes

The vast majority of the 1-2 million malaria associated deaths that occur each year are due to anemia and cerebral malaria (the attachment of erythrocytes containing mature forms of Plasmodium falciparum to the endothelial cells that line the vascular beds of the brain). A "model" system"for the study of cerebral malaria employs amelanotic melanoma cells as the "target"cells in an vitro cytoadherence assay. Using this model system we determined that the optimum pH for adherence is 6.6 to 6.8, that high concentrations of Ca²* (50mM) result in increased levels of binding, and that the type of buffer used influences adherence (Bis Tris > MOPS > HEPES > PIPES). We also observed that the ability of infected erythrocytes to cytoadhere varied from (erythrocyte) donor to donor. We have produced murine monoclonal antibodies against P. falciparum-infected red cells which recognized modified forms of human band 3; these inhibit the adherence of infected erythrocytes to melanoma cells in a doso responsive fashion. Antimalarials (chloroquine, quinacrine, mefloquine, artemisinin), on the other hand, affected adherence in an indirect fashion i.e. since cytoadherence is due, in part to the presence of knobs on the surface of the infected erythrocyte, and knob formation is dependent on intracellular parasite growth, when plasmodial development is inhibited so is knob production, and consequently adherence is ablated.

Nivell global d'estrès en pares i mares de fills diagnosticats amb trastorn del desenvolupament en les primeres etapes evolutives

Aquest treball és un estudi descriptiu centralitzat en una població concreta i en un moment concret. La investigació estudia el nivell d’estrès global que tenen els pares i les mares de fills/es amb un trastorn del seu desenvolupament en les seves primeres edats maduratives i com afronten aquest procés. S’han estudiat pares i mares de nens i nenes d’entre 2 i 6 anys d’edat i que han estat atesos en el Centre de Desenvolupament Infantil d’Atenció Precoç (CDIAP) Tris-Tras de Vic (Barcelona).

A comparison of three DNA extractive procedures with Leptospira for polymerase chain reaction analysis

Three DNA extraction methods were evaluated in this study: proteinase K followed by phenol-chloroform; a plant proteinase (E6870) followed by phenol-chloroform; and boiling of leptospires in 0.1 mM Tris, pH 7.0 for 10 min at 100°C, with no phenol treatment. Every strain treated with proteinase K or E6870 afforded positive polymerase chain reaction (PCR) reaction. On the other hand, from five strains extracted by the boiling method, three did not feature the 849 bp band characteristic in Leptospira. We also evaluated by RAPD-PCR, DNAs from serovars isolated with proteinase K and proteinase 6870 with primers B11/B12. Each of the DNA samples provided PCR profiles in agreement with previous data. Moreover, the results with E6870 showed less background non-specific amplification, suggesting that removal of nucleases was more efficient with E6870. The limit for detection by PCR using Lep13/Lep14 was determined to be 10(2) leptospira, using the silver stain procedure.

Opposite effect of counterions on the persistence length of nicked and non-nicked DNA.

Using cryo-electron microscopy we reconstructed the three-dimensional trajectories adopted in cryovitrified solutions by double-stranded DNA molecules in which the backbone of one strand lacked a phosphate at regular intervals of 20 nucleotides. The shape of such nicked DNA molecules was compared with that of DNA molecules with exactly the same sequence but without any single-stranded scissions. Upon changing the salt concentration we observed opposite effects of charge neutralization on nicked and non-nicked DNA. In low salt solutions (10 mM Tris-HCl, 10 mM NaCl) the applied dense nicking caused ca 3.5-fold reduction of the DNA persistence length as compared with non-nicked DNA. Upon increasing the salt concentration (to 150 mM NaCl and 10 mM MgCl2) the persistence length of non-nicked DNA appreciably decreased while that of nicked DNA molecules increased by a factor of 2.

Proteolytic activity in the adult and larval stages of the human roundworm parasite Angiostrongylus costaricensis

Angiostrongylus costaricensis is a nematode that causes abdominal angiostrongyliasis, a widespread human parasitism in Latin America. This study aimed to characterize the protease profiles of different developmental stages of this helminth. First-stage larvae (L1) were obtained from the faeces of infected Sigmodon hispidus rodents and third-stage larvae (L3) were collected from mollusks Biomphalaria glabrata previously infected with L1. Adult worms were recovered from rodent mesenteric arteries. Protein extraction was performed after repeated freeze-thaw cycles followed by maceration of the nematodes in 40 mM Tris base. Proteolysis of gelatin was observed by zymography and found only in the larval stages. In L3, the gelatinolytic activity was effectively inhibited by orthophenanthroline, indicating the involvement of metalloproteases. The mechanistic class of the gelatinases from L1 could not be precisely determined using traditional class-specific inhibitors. Adult worm extracts were able to hydrolyze haemoglobin in solution, although no activity was observed by zymography. This haemoglobinolytic activity was ascribed to aspartic proteases following its effective inhibition by pepstatin, which also inhibited the haemoglobinolytic activity of L1 and L3 extracts. The characterization of protease expression throughout the A. costaricensis life cycle may reveal key factors influencing the process of parasitic infection and thus foster our understanding of the disease pathogenesis.

La question nationale en Europe du Sud-Est : genèse, émergence et développement de l'identité nationale albanaise au Kosovo et en Macédoine

NOTE METHODOLOGIQUE Avant d'entamer notre travail d'analyse, nous tenons à souligner d'emblée un certain nombre de remarques sur les obstacles affrontés et aux difficultés que nous avons rencontrées durant cette recherche. Plusieurs observations s'imposent quant aux concepts utilités, aux sources et travaux consultés et à la manière d'aborder la thématique nationale et nationaliste albanaise. Sachant que notre objectif a été de rompre avec le discours dominant sur le thème de l'identité nationale albanaise et de celui des travaux qui sont l'oeuvre, dans la plupart des cas, d'observateurs et d'acteurs à la fois, il fallait utiliser avec beaucoup de précaution les termes désignant aujourd'hui des groupes ethniques ou alors des entités territoriales contemporaines telles que le Kosovo ou la Macédoine. Pour la clarté de l'analyse, il était nécessaire d'utiliser certains concepts tels que l'ethnie, populations albanophones, albanaises ou proto-albanaises, toutefois, nous n'avons pas retenu le même sens que celui des acteurs. Lorsqu'on évoque ces populations, ce n'est pas le sens ethnique contemporain que nous retenons, mais celui qui pouvait prévaloir dans les contextes historiques auxquels nous nous sommes référés. Quant aux lieux et entités politiques d'aujourd'hui, nous avons choisi de recourir aux concepts tels qu'espace ou aire culturelle albanophone pour éviter de projeter dans le passé des catégories contemporaines comme le fait volontiers l'iconographie nationaliste. Enfin, par un souci d'impartialité, nous avons utilisé l'appellation des villes et des noms des figures historiques selon les contextes historiques abordés et avons précisé, entre parenthèses, l'appellation dans d'autres langues aussi. Concernant les sources et les travaux utilisés, comme nous venons de l'évoquer, la plupart d'eux sont émaillés par des considérations d'ordre idéologique et prennent clairement position soit en faveur de la position albanaise, soit de celle serbe, macédonienne ou autre. En fait, dans l'entreprise nationaliste, la définition d'un problème est un enjeu de luttes dans le temps et dans l'espace. Afin d'éviter de s'enliser dans le piège d'une lecture unilatérale des événements historiques, nous avons systématiquement utilisé des sources directes, croisé les sources d'information, sélectionné les publications utilisées selon leur rigueur scientifique et les références utilisées dans l'élaboration de leur argumentation. L'établissement d'une chronologie fiable a été une tâche difficile. En fait, comme nous l'avons rappelé dans notre introduction, peu d'ouvrages traitent de la question identitaire albanaise. En ce qui concerne la littérature albanaise, celle-ci est abondante, cependant, nous avons exclusivement utilisé des travaux universitaires qui ont le souci de la clarté et de l'objectivité et qui abordent la question albanaise sur la base des sources variées consultées (basées sur les archives officielles albanaise, serbe et internationale). Toutefois, nous avons d'une part relevé que certains travaux historiques utilisés ont été produits en Albanie durant la période du régime totalitaire d'Enver Hoxha. L'influence de ce régime dans la lecture de l'histoire ressort implicitement dans le choix des thèmes et des faits socio-historiques relatés par les auteurs. D'autre part, la littérature albanaise du Kosovo et de Macédoine et l'approche qu'elle effectue de la question nationale albanaise varie selon les contextes politiques. Ainsi, par exemple, les publications des années 1980 sur la Ligue de Prizren sont riches et fiables et poursuivent des objectifs autres que ceux visant à légitimer les revendications politiques albanaises. Quant aux travaux des auteurs serbes et macédoniens sur la question nationale albanaise, force est de constater qu'ils sont sous une forte influence nationaliste sur cette question. En fait, les travaux de Dimitrije Tucović, d'Aleksandar Matkovski et la publication dirigée par Nebojša Popov font exception à toute une production qui ne prend pas uniquement partie dans son jugement, mais qui a une attitude pour le moins problématique à l'égard des Albanais du Kosovo et de Macédoine. Compte tenu de ces constatations et de ces difficultés et afin de nous protéger des éventuelles approximations, nous avons systématiquement vérifié les faits socio-historiques relatés par la littérature historique occidentale qui portait sur Byzance, sur l'Empire ottoman ou alors sur la période plus contemporaine. Tout au long de notre recherche, nous avons privilégié certaines références des chercheurs (triés sur la base de leur connaissance de la question et des sources consultées) sur la région des Balkans pour l'établissement de notre chronologie (notamment ceux de Tahir Abdyli, de Skender Anamali, d'Ivo Banac, de Sadulla Brestovci, de Georges Castellan, d'Alain Ducellier, d'Ali Hadri, de Branko Horvat, de Kristo Frashëri, de Hivzi Islami, de Kristaq Prifti, de Noel Malcolm, d'Aleksandar Matkovski, de Pajazit Nushi, de Stefanaq Pollo, de Selami Pulaha, de Halim Purellku, de Skënder Rizaj, de Limon Rushiti, de Michel Roux, de Zija Shkodra, de Stavro Skendi de Dimitrije Tucović et de Miranda Vickers). Ces travaux nous ont été d'une grande utilité, même si les thématiques abordées étaient parfois complémentaires à notre objectif de recherche.