Cephalobellus lobulata n. sp. (Oxyurida:Thelastomatidae) a parasite of Neocurtilla claraziana Saussure (Orthoptera: Gryllotalpidae) from Argentina

Cephalobellus lobulata n. sp. (Oxyurida: Thelastomatidae) a parasite of the mole cricket Neocurtilla claraziana Saussure (Orthoptera: Gryllotalpidae) found in Argentina is described and illustrated. It is characterized by a short buccal cavity armed with three teeth, a striated cuticle with the first annule wide with four lobes and the second annule divided in twelve lobes. The male have three pairs of preanal papillae and two pairs of postanal papillae.

Establishment and characterization of a new continuous cell line from Lutzomyia longipalpis (Diptera: Psychodidae) and its susceptibility to infections with arboviruses and Leishmania chagasi

Embryonic tissue explants of the sand fly Lutzomyia longipalpis (Lutz & Neiva 1912) the main vector of Leishmania chagasi (Cunha and Chagas), were used to obtain a continuous cell line (Lulo). The tissues were seeded in MM/VP12 medium and these were incubated at 28ºC. The first subculture was obtained 45 days after explanting and 96 passages have been made to date. Lulo is composed of epithelioid cells, showed a 0.04 generations/hour exponential growth rate and population doubling time at 24.7 h. The cell line isoenzymatic profiles were determined by using PGI, PGM, MPI and 6-PGDH systems, coinciding with patterns obtained from the same species and colony's pupae and adults. The species karyotype characteristics were recognized (2n = 8), in which pair 1 is subtelocentric and pairs 2, 3 and 4 are metacentric. Lulo was free from bacterial, fungal, mycoplasmic and viral infection. Susceptibility to five arbovirus was determined, the same as Lulo interaction with Leishmania promastigotes.

Determinació de la regió del genoma o gens implicats en la síntesi del polièster extracel•lular present en la variant llisa de Mycobacterium vaccae mitjançant transformació

Estudi realitzat a partir d’una estada a la Universidad de Zaragoza, Espanya, entre novembre del 2007 i abril del 2008. Mycobacterium vaccae és un micobacteri ambiental de creixement ràpid molt estudiat pel seu interès com a possible ús immunoterapéutic en el tractament de la tuberculosis i altres malalties. M.vaccae a l’igual que altres micobacteris presenta dues morfologies colonials: llisa i rugosa. M.vaccae ATCC15483T té originàriament una morfologia llisa. Quant aquest es cultiva en medi sòlid a 30ºC apareixen espontàniament variants rugoses estables que no reverteixen a llises. El motiu pel qual aquest procés té lloc no es coneix, encara que s’ha descrit en Mycobacterium smegmatis i en Mycobacterium avium que els lípids de la paret cel•lular es troben involucrats en aquest canvi de morfologia colonial. L’anàlisi dels contingut en lípids i glicolípids de la paret cel•lular de les dos variants morfològiques de M.vaccae, ens ha indicat que les soques llises presenten un compost extracel•lular que no es troba en les rugoses i que mitjançant l’anàlisi estructural d’aquest compost ha sigut identificat com un polièster extracel•lular de cadena llarga. El present estudi s’ha centrat en determinar els gens implicats en la síntesis d’aquest compost. Per a realitzar aquest anàlisi genètic s’ha construit una llibreria de mutants per transposició de la soca llisa de M. vaccae mitjançant un plàsmid ts/sac i un transposó. S’han obtingut colònies de morfologia rugosa on el plàsmid s’ha insertat en la zona del genoma que codifica per aquest compost extracel•lular. Aquests nous mutants s’han analitzat mitjançant tècniques moleculars (PCR, Southern y seqüenciació). A mès, s’ha construit una llibreria genòmica amb DNA de la soca llisa en plàsmids replicatius de micobacteris derivats de pAL5000 i s’ha transformat la soca rugosa seleccionant per a un fenotip llis estudiant els gens que complementen.

Mouse interleukin-2 receptor alpha gene expression. Delimitation of cis-acting regulatory elements in transgenic mice and by mapping of DNase-I hypersensitive sites.

The alpha chain of the interleukin-2 receptor (IL-2R alpha) is a key regulator of lymphocyte proliferation. To analyze the mechanisms controlling its expression in normal cells, we used the 5'-flanking region (base pairs -2539/+93) of the mouse gene to drive chloramphenicol acetyltransferase expression in four transgenic mouse lines. Constitutive transgene activity was restricted to lymphoid organs. In mature T lymphocytes, transgene and endogenous IL-2R alpha gene expression was stimulated by concanavalin A and up-regulated by IL-2 with very similar kinetics. In thymic T cell precursors, IL-1 and IL-2 cooperatively induced transgene and IL-2R alpha gene expression. These results show that regulation of the endogenous IL-2R alpha gene occurs mainly at the transcriptional level. They demonstrate that cis-acting elements in the 5'-flanking region present in the transgene confer correct tissue specificity and inducible expression in mature T cells and their precursors in response to antigen, IL-1, and IL-2. In a complementary approach, we screened the 5' end of the endogenous IL-2R alpha gene for DNase-I hypersensitive sites. We found three lymphocyte specific DNase-I hypersensitive sites. Two, at -0.05 and -5.3 kilobase pairs, are present in resting T cells. A third site appears at -1.35 kilobase pairs in activated T cells. It co-localizes with IL-2-responsive elements identified by transient transfection experiments.

Disseny i optimització de membranes compòsit amb xarxes metal•loorgàniques per a nanofiltració i separacions de gasos

Projecte de recerca elaborat a partir d’una estada a la Katholieke Universiteit Leuven, Belgium, entre 2007 i 2009. Aquest projecte descriu la síntesi i aplicació de nous tipus de membranes compòsit basades en xarxes metal•loorgàniques (MOFs). Aquestes es van seleccionar tenint en compte les seves propietats estructurals per tal de discriminar les espècies a separar en funció de la seva mida molecular. Les membranes obtingudes s'han aplicat satisfactòriament tant en separacions líquides, concretament en nanofiltració resistent a dissolvents (SRNF), i en separació de parells de gasos com CO2/CH4, CO2/N2 i H2/CO2. Els resultats obtinguts posen de manifest l'obtenció de membranes sense defectes i amb rendiments prometedors, en la majoria dels casos, amb permeabilitats i selectivitats superiors a membranes purament polimèriques. Tanmateix s'ha desenvolupat un nou equipament d'alt rendiment (HT) per a separacions de gasos que inclou un mòdul que permet realitzar 16 experiments simultàniament. Els resultats obtinguts amb el nou equip són comparables amb els obtinguts amb mòduls convencionals, i alhora presenten una millor reproduïbilitat. Finalment, s'ha establert un nou mètode per a obtenir membranes per a SRNF, que han estat aplicades en processos de separació de catalitzadors homogenis en dissolvents polars apròtics i s'han caracteritzat emprant la tècnica d'espectroscòpia d'annihilació de positrons, que ha permès establir per primer cop una relació entre les propietats estructurals de les membranes a nivell molecular i el seu rendiment en les aplicacions anteriors.

Incidencia y gravedad de la enfermedad por citomegalovirus en pacientes trasplantados renales que han recibido tratamiento anticipado o profilaxis con valganciclovir oral

Antecedentes: La infección por CMV es la más frecuente que sufren los receptores de trasplante de órgano sólido (TOS) en los primeros meses del trasplante. Son abundantes las estrategias de prevención y tratamiento de la enfermedad por CMV que se han desarrollado. Métodos: El objetivo fue evaluar la incidencia y severidad de la enfermedad por CMV en 387 receptores de trasplante renal que recibieron tratamiento profiláctico (grupo de alto riesgo, n = 251) o realizaron seguimiento de PCR de CMV por ser grupo de bajo riesgo (n = 136) y recibieron tratamiento anticipado (n = 17) con valganciclovir oral (900 mg/día) cuando presentaron & 1000 copias/ml. Resultados: Presentaron enfermedad por CMV, 2 (11,76%) pacientes del grupo de riesgo estándar, descrita como enfermedad leve y 20 (7,96%) pacientes del grupo de alto riesgo, descrita como enfermedad leve en 15 casos y como enfermedad grave en 5 casos. Aquellos pacientes que desarrollaron la enfermedad, respondieron al tratamiento con ganciclovir endovenoso durante 14 días seguido de valganciclovir oral hasta completar tres meses. Conclusión: El tratamiento profiláctico con valganciclovir oral para la prevención de enfermedad o infección por CMV solamente es necesario en los receptores de TOS de alto riesgo.

Community ecology of the metazoan parasites of white croaker, Micropogonias furnieri (Osteichthyes: Sciaenidae), from the coastal zone of the State of Rio de Janeiro, Brazil

One hundred specimens of white croakers, Micropogonias furnieri (Desmarest 1823) (Osteichthyes: Sciaenidae) collected from Pedra de Guaratiba (23°01'S, 43°38'W), State of Rio de Janeiro, Brazil, from September 1997 to August 1999, were necropsied to study their parasites. The majority of the fish (95%) were parasitized by metazoan. Twenty-eight species of parasites were collected. The nematodes were the 40.5% of the total number of parasites specimens collected. Dichelyne elongatus was the most dominant species. Lobatostoma ringens, Pterinotrematoides mexicanum, Corynosoma australe, D. elongatus, and Caligus haemulonis showed a positive correlation between the host's total length and parasite prevalence and abundance. The monogenean P. mexicanum had differences in the prevalence and abundance in relation to sex of the host. The mean diversity in the infracommunities of M. furnieri was H=0.499±0.411, with correlation with the host's total length and without differences in relation to sex of the host. One pair of ectoparasites showed positive covariation, and two pairs of endoparasites showed positive association and covariation between their prevalences and abundances, respectively. Negative association or covariations were not found. The dominance of endoparasites in the croakers parasite infracommunities reinforced the differences found in sciaenids from the South American Pacific Ocean, in which the ectoparasites are dominant.

C-banding and FISH in chromosomes of the blow flies Chrysomya megacephala and Chrysomya putoria (Diptera, Calliphoridae)

The blow flies Chrysomya putoria and C. megacephala have 2n=12 chromosomes, five metacentric pairs of autosomes and an XX/XY sex chromosome pair. There are no substantial differences in the karyotype morphology of these two species, except for the X chromosome which is subtelocentric in C. megacephala and metacentric in C. putoria and is about 1.4 times longer in C. putoria. All autosomes were characterized by the presence of a C band in the pericentromeric region; C. putoria also has an interstitial band in pair III. The sex chromosomes of both species were heterochromatic, except for a small region at the end of the long arm of the X chromosome. Ribosomal genes were detected in meiotic chromosomes by FISH and in both species the NOR was located on the sex chromosomes. These results confirm that C. putoria was the species introduced into Brazil in 1970s, and not C. chloropyga as formerly described.

Brain cell karyotype of the phlebotomine sand fly Lutzomyia shannoni (Dyar) (Diptera: Psychodidae)

The brain cell karyotype of New World sand fly Lutzomyia shannoni was described. This species has four pairs of chromosomes, 2N=8, with one pair of heteromorphic chromosomes.

Optimisation de la cryo microscopie électronique pour les études des minicercles d'ADN

RESUME : Bien que les propriétés physiques de la structure de l'ADN aient été intensivement étudiées pendant plus de 50 ans il y a encore beaucoup de questions importantes qui attendent des réponses. Par exemple, qu'arrive-t-il à la structure de la double hélice d'ADN nue (sans protéines liées) lorsqu'elle est fortement courbée, de la même manière que dans les nucléosomes? Cet ADN nu est-il facilement plié (il reste dans le régime élastique) ou réduit-il la contrainte de flexion en formant des sites hyperflexibles «kinks» (il sort du régime élastique en cassant l'empilement des paires de bases à certains endroits) ? La microscopie électronique peut fournir une réponse à cette question par visualisation directe des minicercles d'ADN de la longueur d'un tour de nucléosome (environ 90 paires de bases). Pour que la réponse soit scientifiquement valide, on doit observer les molécules d'ADN lorsqu'elles sont en suspension dans la solution d'intérêt et sans que des colorations, produits chimiques ou fixatifs n'aient été ajoutés, étant donné que ceux-ci peuvent changer les propriétés de l'ADN. La technique de la cryo-microscopie électronique (cryo-EM) développée par le groupe de Jacques Dubochet au début des années 80, permet la visualisation directe des molécules d'ADN suspendues dans des couche minces vitrifiées de solutions aqueuses. Toutefois, le faible contraste qui caractérise la cryo-EM combinée avec la très petite taille des minicercles d'ADN rendent nécessaire l'optimisation de plusieurs étapes, aussi bien dans la préparation des échantillons que dans le processus d'acquisition d'images afin d'obtenir deux clichés stéréo qui permettent la reconstruction 3-D des minicercles d'ADN. Dans la première partie de ma thèse, je décris l'optimisation de certains paramètres pour la cryoEM et des processus d'acquisition d'image utilisant comme objets de test des plasmides et d'autres molécules d'ADN. Dans la deuxième partie, je .décris comment j'ai construit les minicercles d'ADN de 94 bp et comment j'ai introduit des modifications structurelles comme des coupures ou des lacunes. Dans la troisième partie, je décris l'analyse des reconstructions des rninicercles d'ADN. Cette analyse, appuyée par des tests biochimiques, indique fortement que des molécules d'ADN sont capables de former de petites molécules circulaires de 94 bp sans dépasser les limites d'élasticité, indiquant que les minicercles adoptent une forme circulaire régulière où la flexion est redistribuée le long la molécule. ABSTRACT : Although physical properties of DNA structure have been intensively studied for over 50 years there are still many important questions that need to be answered. For example, what happens to protein-free double-stranded DNA when it is strongly bent, as in DNA forming nucleosomes? Is such protein-free DNA smoothly bent (i.e. it remains within elastic limits of DNA rigidity) or does it release its bending stress by forming sharp kinks (i.e. it exits the elastic regime and breaks the stacking between neighbouring base-pairs in localized regions)? Electron microscopy can provide an answer to this question by directly visualizing DNA minicircles that have the size of nucleosome gyres (ca 90 bp). For the answer to be scientifically valid, one needs to observe DNA molecules while they are still suspended in the solution of interest and no staining chemicals or fixatives have been added since these can change the properties of the DNA. CryoEM techniques developed by Jacques Dubochet's group beginning in the 1980's permit direct visualization of DNA molecules suspended in cryo-vitrified layers of aqueous solutions. However, a relatively weak contrast of cryo-EM preparations combined with the very small size of the DNA minicircles made it necessary to optimize many of the steps and parameters of the cryo-EM specimen preparation and image acquisition processes in order to obtain stereo-pairs of images that permit the 3-D reconstruction of the observed DNA minicircles. In the first part of my thesis I describe the optimization of the cryo-EM preparation and the image acquisition processes using plasmid size DNA molecules as a test object. In the second part, I describe how I formed the 94 by DNA minicircles and how I introduced structural modifications like nicks or gaps. In the third part, I describe the cryo-EM analysis of the constructed DNA minicircles. That analysis, supported by biochemical tests, strongly indicates that DNA minicircles as small as 94 by remain within the elastic limits of DNA structure, i.e. the minicircles adopt a regular circular shape where bending is redistributed along the molecules.

Attractiveness of black Shannon trap for phlebotomines

A white Shannon-type trap was used for captures of female sand flies in the search for natural infection with flagellates, however, due to its low productivity and as a large number of phlebotomines settled on the researchers' black clothes, we decided to compare the relative attractiveness of black and white Shannon-type traps for sand flies. Several pairs of black and white traps were placed side by side in front of caves in four areas in the Serra da Bodoquena, Bonito county, State of Mato Grosso do Sul, Brazil, for a total of 12 observations and 44 h of capture. The experiment resulted in 889 phlebotomines captured, 801 on the black and 88 on the white trap, representing 13 species. The hourly Williams' means were 8.67 and 1.24, respectively, and the black/white ratio was 7.0:1.0. Lutzomyia almerioi, an anthropophilic species closely associated with caves, was predominant (89%). Only two other species, Nyssomyia whitmani and Psathyromyia punctigeniculata, also anthropophilic, were significantly attracted to the black rather than to the white trap (chi2 test; p <= 0.01). The difference between the diversity index of the two traps was not significant at level 0.05. The black trap in these circumstances was much more productive than the white, especially for anthropophilic species.

Artifacts in the analysis of thioridazine and other neuroleptics.

Although the sensitivity to light of thioridazine and its metabolites has been described, the problem does not seem to be widely acknowledged. Indeed, a survey of the literature shows that assays of these compounds under light-protected conditions have been performed only in a few of the numerous analytical studies on this drug. In the present study, thioridazine, its metabolites, and 18 other neuroleptics were tested for their sensitivity to light under conditions used for their analysis. The results show that light significantly affects the analysis of thioridazine and its metabolites. It readily causes the racemization of the isomeric pairs of thioridazine 5-sulphoxide and greatly decreases the concentration of thioridazine. This sensitivity to light varied with the medium used (most sensitive in acidic media) and also with the molecule (in order of decreasing sensitivity: thioridazine > mesoridazine > sulforidazine). Degradation in neutral or basic media was slow, with the exception of mesoridazine in a neutral medium. Twelve other phenothiazines tested, as well as chlorprotixene, a thioxanthene drug, were found to be sensitive to light in acidic media, whereas flupenthixol and zuclopenthixol (two thioxanthenes), clozapine, fluperlapine, and haloperidol (a butyrophenone) did not seem to be affected. In addition to being sensitive to light, some compounds may be readily oxidized by peroxide-containing solvents.

New perspectives in the use of ink evidence in forensic science

Resume : L'utilisation de l'encre comme indice en sciences forensiques est décrite et encadrée par une littérature abondante, comprenant entre autres deux standards de l'American Society for Testing and Materials (ASTM). La grande majorité de cette littérature se préoccupe de l'analyse des caractéristiques physiques ou chimiques des encres. Les standards ASTM proposent quelques principes de base qui concernent la comparaison et l'interprétation de la valeur d'indice des encres en sciences forensiques. L'étude de cette littérature et plus particulièrement des standards ASTM, en ayant a l'esprit les développements intervenus dans le domaine de l'interprétation de l'indice forensique, montre qu'il existe un potentiel certain pour l'amélioration de l'utilisation de l'indice encre et de son impact dans l'enquête criminelle. Cette thèse propose d'interpréter l'indice encre en se basant sur le cadre défini par le théorème de Bayes. Cette proposition a nécessité le développement d'un système d'assurance qualité pour l'analyse et la comparaison d'échantillons d'encre. Ce système d'assurance qualité tire parti d'un cadre théorique nouvellement défini. La méthodologie qui est proposée dans ce travail a été testée de manière compréhensive, en tirant parti d'un set de données spécialement créer pour l'occasion et d'outils importés de la biométrie. Cette recherche répond de manière convaincante à un problème concret généralement rencontré en sciences forensiques. L'information fournie par le criminaliste, lors de l'examen de traces, est souvent bridée, car celui-ci essaie de répondre à la mauvaise question. L'utilisation d'un cadre théorique explicite qui définit et formalise le goal de l'examen criminaliste, permet de déterminer les besoins technologiques et en matière de données. Le développement de cette technologie et la collection des données pertinentes peut être justifiées économiquement et achevée de manière scientifique. Abstract : The contribution of ink evidence to forensic science is described and supported by an abundant literature and by two standards from the American Society for Testing and Materials (ASTM). The vast majority of the available literature is concerned with the physical and chemical analysis of ink evidence. The relevant ASTM standards mention some principles regarding the comparison of pairs of ink samples and the evaluation of their evidential value. The review of this literature and, more specifically, of the ASTM standards in the light of recent developments in the interpretation of forensic evidence has shown some potential improvements, which would maximise the benefits of the use of ink evidence in forensic science. This thesis proposes to interpret ink evidence using the widely accepted and recommended Bayesian theorem. This proposition has required the development of a new quality assurance process for the analysis and comparison of ink samples, as well as of the definition of a theoretical framework for ink evidence. The proposed technology has been extensively tested using a large dataset of ink samples and state of the art tools, commonly used in biometry. Overall, this research successfully answers to a concrete problem generally encountered in forensic science, where scientists tend to self-limit the usefulness of the information that is present in various types of evidence, by trying to answer to the wrong questions. The declaration of an explicit framework, which defines and formalises their goals and expected contributions to the criminal and civil justice system, enables the determination of their needs in terms of technology and data. The development of this technology and the collection of the data is then justified economically, structured scientifically and can be proceeded efficiently.

Neyraiella distinctus n. sp. (Oxyurida, Blattophilidae) Parasite of Nymphs of Gryllodes laplatae Sauss (Orthoptera, Gryllidae) in Argentina

Neyraiella distinctus n. sp. was found parasitizing nymphs of the cricket Gryllodes laplatae Sauss in City Bell, Argentina. This species was characterized by having the excretory pore in the posterior end of the basal bulb, vulva protruding with one lip well developed in the 1/3 end of the body, anus of the female with wings, male with a single spicule without any sculpture, gubernaculum and bursa are absent, six pairs of genital papillae arranged in two preanal pairs, one adanal pair and three postanal pairs, and the tail appendage in both sexes was short and conic.