Lentiviral vectors efficiently transduce the EGFP gene into cardiomyocytes in vivo : immunohistology and Elisa quantification of the transgene

RESUME Les maladies cardio-vasculaires représentent la cause la plus importante de mortalité et de morbidité dans les pays occidentaux. La thérapie génique offre une nouvelle approche au traitement de ces maladies. L'expression de gènes protecteurs dans le myocarde par des technologies de transfert génique peut améliorer la fonction ventriculaire lors de l'insuffisance cardiaque ou stimuler la formation de nouveaux vaisseaux dans la maladie coronarienne. Etant donné qu'une majorité des maladies cardiaques sont des maladies chroniques, l'expression durable du gène thérapeutique introduit dans le coeur est souhaitable dans de nombreux cas. Malheureusement, l'utilité des vecteurs de transfert génique les plus utilisés en thérapie génique cardiovasculaire est limitée par une performance faible (ADN plasmidique) et une courte durée d'expression (adénovirus). Récemment, des vecteurs de transfert génique dérivés des lentivirus, une sous-famille des rétrovirus, ont retenu l'attention de la communauté scientifique en raison de leur capacité à exprimer des gènes à long terme. Contrairement aux vecteurs rétroviraux traditionnels, les vecteurs lentiviraux transduisent des gènes même dans des cellules qui ne se divisent pas, ce qui est le cas des cardiomyocytes adultes. Ces vecteurs présentent un profil de biosécurité comparable à celui des vecteurs rétroviraux traditionnels. Nous avons donc décidé de tester l'utilité des vecteurs lentiviraux pour le transfert génique dans des cardiomyocytes de rat adulte in vitro et in vivo. Plusieurs versions de vecteurs lentiviraux contenant différent promoteurs ont été construites. Ces vecteurs contenant le gène marqueur EGFP (enhanced green fluorescent protein) ont été testés dans des cardiomyocytes de rat in vitro, ainsi que dans des coeurs de rat in vivo. Le but de ces expériences était de déterminer la durée de l'expression du transgène après injection intramyocardique chez le rat. Pour ce faire, nous avons développé une technique ELISA pour détecter la protéine EGFP dans des extraits de tissu cardiaque. Les résultats ont montré que la protéine EGFP était encore présente à des niveaux significatifs jusqu'à dix semaines après l'injection de vecteurs lentiviraux, alors que l'expression transgénique obtenue avec un vecteur adénoviral traditionnel a été plus limitée dans le temps. Ces résultats démontrent la capacité des vecteurs lentiviraux à exprimer des gènes d'intérêt de manière performante et stable dans le cœur de rat adulte in vivo. SUMMARY Cardiovascular diseases are the first cause of morbidity and mortality in Western countries. Gene therapy offers a new approach to these diseases. Expression of therapeutic genes in the myocardium by gene transfer technologies can improve ventricular function in heart failure and stimulate neovascularization in coronary disease. Chronic heart diseases likely require sustained expression of the therapeutic gene within the heart itself. Unfortunately, the most commonly used vectors in cardiovascular gene therapy, i.e. plasmid DNA and recombinant adenovirus vectors, are limited by poor DNA uptake and transient transgene expression, respectively. Recently, lentivirus-derived vectors have attracted much interest because of their ability to achieve long-term transgene expression. In contrast to traditional retroviral vectors, lentiviral vectors are also able to transduce non- dividing cells, while presenting a comparable biosafety profile. Adult cardiomyocytes are terminally differentiated cells that do not divide under normal conditions. For these reasons, we have decided to evaluate the efficiency of lentiviral vectors for gene-transduction of adult cardiomyocytes both in vitro and in vivo. We constructed various types of lentiviral vectors containing various promoters. Vectors encoding EGFP as a reporter gene were tested in rat cardiomyocytes in vitro and in rat hearts in vivo. The aim of the experiments involved in this thesis work was to determine the duration of the expression of the transgene after rat intramyocardial injection using a quantitative assay. Therefore, an ELISA technique was set up to measure the EGFP protein in rat heart tissue extracts. Our results showed that the EGFP protein was still present at significant levels at ten weeks after lentiviral vector injection, whereas the duration of expression with adenoviral vectors was shorter. These results demonstrate that lentiviral vectors efficiently deliver genes and achieve sustained transgene expression in adult rat cardiomyocytes in vivo.

Estudio sobre evaluación de la usabilidad móvil y propuesta de un método para tests de usabilidad cuantitativos basado en técnicas de eyetracking

Este trabajo se divide en tres partes: contextualización, estado del arte en evaluación de la usabilidad en dispositivos móviles y propuesta y validación de un método que combina eyetracker de sobremesa y dispositivos móviles. El trabajo culmina con un estudio experimental con un doble propósito: realizar un primer estudio de la validez del método y analizar empíricamente cómo sacarle el máximo rendimiento tratando en todo momento de equipararlo al uso real de un dispositivo físico.

Supervised localimage feature detection

This thesis is about detection of local image features. The research topic belongs to the wider area of object detection, which is a machine vision and pattern recognition problem where an object must be detected (located) in an image. State-of-the-art object detection methods often divide the problem into separate interest point detection and local image description steps, but in this thesis a different technique is used, leading to higher quality image features which enable more precise localization. Instead of using interest point detection the landmark positions are marked manually. Therefore, the quality of the image features is not limited by the interest point detection phase and the learning of image features is simplified. The approach combines both interest point detection and local description into one phase for detection. Computational efficiency of the descriptor is therefore important, leaving out many of the commonly used descriptors as unsuitably heavy. Multiresolution Gabor features has been the main descriptor in this thesis and improving their efficiency is a significant part. Actual image features are formed from descriptors by using a classifierwhich can then recognize similar looking patches in new images. The main classifier is based on Gaussian mixture models. Classifiers are used in one-class classifier configuration where there are only positive training samples without explicit background class. The local image feature detection method has been tested with two freely available face detection databases and a proprietary license plate database. The localization performance was very good in these experiments. Other applications applying the same under-lying techniques are also presented, including object categorization and fault detection.

Efeitos das culturas de milho (Zea mays), feijão (Phaseolus vulgaris) e caupi (Vigna unguiculata) na agregação de valor ao cultivo da bananeira 'Terra', em Teolândia, litoral sul da Bahia

São apresentados resultados pertinentes às culturas do milho (Z. mays), feijão (P. vulgaris) e caupi (V. unguiculata) consorciados com bananeira 'Terra' (Musa AAB) em Teolândia, Litoral Sul da Bahia, em cultivo simultâneo, no estabelecimento da bananeira e sucessivo logo após a colheita do primeiro. Os espaçamentos utilizados foram o 4,0 x 2,0 x 3,0 m, 4,0 x 2,0 x 2,6m e 4,0 x 2,0 x 2,8m em fileira dupla, o que permitiu melhor proveito da área pelas culturas intercalares. O milho, ou o feijão, ou o caupi ocupou apenas as ruas entre as fileiras duplas de bananeiras, ficando livres as ruas formadas pelas fileiras duplas, devido ao seu sombreamento pela bananeira ser mais rápido. O objetivo deste trabalho foi avaliar o desempenho das culturas do milho, feijão e caupi na agregação de valor à bananeira 'Terra', quando consorciadas com esta cultura. Nos melhores tratamentos (T9, T6, T8 e T4), o índice de rentabilidade-IR (receita total ÷ custos) alcançou valores expressivos, de 3,36 até 6,68, resultando em efeitos altamente positivos em decorrência das tecnologias e insumos utilizados, o que vai permitir a agregação de um elevado valor à bananeira e, conseqüentemente, uma boa remuneração ao produtor. Estes resultados indicam que, para cada real investido, houve um retorno de R$ 3,36 e R$ 6,68, respectivamente. O IR 0,91 (T2), relativo à cultura do milho, implica que houve prejuízo, o que significa que a receita líquida deste tratamento vai reduzir e não incrementar a receita líquida proporcionada pela cultura da bananeira.

Los retos de la educación intercultural: Cataluña-Quebec

La educación multicultural y la intercultural, generalmente, suponen ideologías y prácticas diferentes, pero en otras ocasiones bajo estos conceptos se esconden realidades parecidas. Fijándonos en los discursos y las actuaciones llevadas a cabo en Cataluña y en Quebec, ambas políticas con la etiqueta de educación intercultural (Cataluña hacia donde creen que se debe avanzar y Quebec que lleva en este camino algunos años), pretendemos poner de manifiesto algunos de los retos que ambos contextos han tenido, tienen o tendrán que afrontar. De esta forma el texto se divide en tres partes: la primera describe la evolución catalana realizando, al final, un breve análisis de las relaciones entre los agentes educativos; la segunda presenta los pasos realizados desde los años sesenta en Quebec, con una explicación previa de su posición en comparación con Canadá multiculturalista; y, por último, las conclusiones apuntan a los citados retos de futuro que deberán tomarse en consideración.

The role of peptidoglycan in chlamydial cell division: towards resolving the chlamydial anomaly.

Chlamydiales are obligate intracellular bacteria including some important pathogens causing trachoma, genital tract infections and pneumonia, among others. They share an atypical division mechanism, which is independent of an FtsZ homologue. However, they divide by binary fission, in a process inhibited by penicillin derivatives, causing the formation of an aberrant form of the bacteria, which is able to survive in the presence of the antibiotic. The paradox of penicillin sensitivity of chlamydial cells in the absence of detectable peptidoglycan (PG) was dubbed the chlamydial anomaly, since no PG modified by enzymes (Pbps) that are the usual target of penicillin could be detected in Chlamydiales. We review here the recent advances in this field with the first direct and indirect evidences of PG-like material in both Chlamydiaceae and Chlamydia-related bacteria. Moreover, PG biosynthesis is required for proper localization of the newly described septal proteins RodZ and NlpD. Taken together, these new results set the stage for a better understanding of the role of PG and septal proteins in the division mechanism of Chlamydiales and illuminate the long-standing chlamydial anomaly. Moreover, understanding the chlamydial division mechanism is critical for the development of new antibiotics for the treatment of chlamydial chronic infections.

Reproductive morphology in Mediterranean Seirospora giraudyi (Ceramiales, Rhodophyta)

This paper presents a detailed description of the reproductive characters of Mediterranean Seirospora giraudyi based on fresh material collected in the northwestern coast of Spain. Vegetative cells are uninucleate. The plant is monoecious. Spermantangial parent’s cells are clustered on modified dwarf determinate filaments, usually situated on adaxial surfaces of branches. One to four spermatia are formed by elongation and proximal divisions of the spermatangial parent cells. Spermatium with a nucleus situated ina mec. The thallus is procarpic. The four-celled carpogonial branch is initially L-shaped, and it is situated on a periaxial supporting fertile axial cell. The mature carpogonial branch is U-shaped and the supporting cell and second periaxial cell enlarge and divide transversely to reproduce a pair uninicleate auxiliary cell. The nucleus in the ferlilized carpogonium divides twice and the carpogonium cleaves vertically into two cells that, turn, cut off a pair of uninucleate connecting cells that fuse with the auxiliary cells on opposite sides; the diploid nuclei in the connecting cells divide at the site of fusion and one of the nuclei enters the auxiliary cell white the other is extruded. Each auxiliary cell gives to a terminal primary gonimolobe initials. Gonimolobes form lax chains of carposporangia. As the gonimoblasts mature, both lobes of the foot cell which is situated on the supporting cell elongate the upper one secondary connecting with the supporting cell, and the lower one with the fertile axial cell. The gonimoblasts are subtended at maturity by one to several clusters of involucral flaments. Seirospora is currently placed in the tribe Euptiloteae; however the reproductive character of S.giraudyi is dentical to those described for the Cañllithamnieae. Molecular studies are needed to confirm the taxonomic position of S.giraudyi as well as that of the other species placed Seirospora

Vulnerability as a heuristic for interdisciplinary research: Assessing the thematic and methodological structure of empirical life-course studies

Changes in human lives are studied in psychology, sociology, and adjacent fields as outcomes of developmental processes, institutional regulations and policies, culturally and normatively structured life courses, or empirical accounts. However, such studies have used a wide range of complementary, but often divergent, concepts. This review has two aims. First, we report on the structure that has emerged from scientific life course research by focusing on abstracts from longitudinal and life course studies beginning with the year 2000. Second, we provide a sense of the disciplinary diversity of the field and assess the value of the concept of 'vulnerability' as a heuristic tool for studying human lives. Applying correspondence analysis to 10,632 scientific abstracts, we find a disciplinary divide between psychology and sociology, and observe indications of both similarities of-and differences between-studies, driven at least partly by the data and methods employed. We also find that vulnerability takes a central position in this scientific field, which leads us to suggest several reasons to see value in pursuing theory development for longitudinal and life course studies in this direction.

Tuotannonohjauksen kehittäminen liittimiä valmistavassa yrityksessä

Diplomityön tarkoituksena oli kehittää ja yksinkertaistaa tuotannonohjausta. Teoriaosassa selvitettiin erilaisia tuotannonohjaustekniikoita sekä vertailtiin niitä toisiinsa. Käytännön osassa perehdyttiin ensin toimitusajan varmistamisen ja tuotannonsuunnittelun rutiineihin. Niitä analysoimalla etsittiin kehityskohteita. Toimitusvarmuutta analysoimalla löydettiin järjestelmän kapeikko. Läpäisyaikoja analysoimalla tutustuttiin kuinka toimitusvastaavien tuotannon kuormittamisen erot vaikuttavat läpäisyaikoihin. ABC-analyysillä löydettiin meisto- ja pinnoitusosastoa kuormittavimmat puolivalmisteet. Järjestelmän pullonkaulana on pintakäsittely ja liikkuvana pullonkaulana meisto. Kehityskohteita arvioitiin kriittisten menestystekijöiden perusteella. Kehitettävien kohteiden lopullinen valinta tehtiin yhdessä työn ohjausryhmän kanssa. Kiireisimmät kehityskohteet olivat uuden tuotannonohjausmallin rakentaminen, tuotteiden ja puolivalmisteiden jakaminen eri ohjaustavoille, Excel-taulukoiden käytön laajentaminen ja monipuolistaminen sekä tuotannon osittainen visualisointi. Näiden toteuttaminen aloitettiin työn aikana ja sitä tullaan vielä jatkamaan.

Factors Related to Study Success at Lappeenranta University of Technology

Tutkimuksen avulla selvitettiin opintomenestykseen liittyviä tekijöitä Lappeenrannan teknillisessä korkeakoulussa (LTKK). Tutkimus liittyi opetuksen ja oppimisen kehitystyöhön tuotantotalouden osastolla. Tutkimuksen viitekehyksen muodosti oppimistuloksia selittävä malli, joka on laadittu Tynjälän (1999) kokoaman mallin perusteella. Tutkimuksen perusjoukko muodostui LTKK:n läsnä olevista perusopiskelijoista lukuun ottamatta jatko- ja vaihto-opiskelijoita. Opiskelijat jaettiin ositetulla otannalla ryhmiin, joissa suoritettiin yksinkertainen satunnaisotanta. Otoskoko oli 645 opiskelijaa. Tiedonkeruumenetelmänä oli Internet-kysely. Aineisto analysoitiin useiden kvantitatiivisten ja kvalitatiivisten menetelmien avulla. Tutkimuksen tuloksia voidaan pitää luotettavina ja tutkimuksen avulla saatiin tärkeää ja hyödyllistä tietoa opintomenestyksestä ja oppimisprosesseista. Tulosten perusteella merkittävimmät oppimistuloksiin positiivisesti liittyvät tekijät ovat syväsuuntautunut opiskelustrategia ja luottaminen omiin kykyihin, ja negatiiviset tekijät ovat oppimisen itsesäätelyn puute, omien kykyjen epäily ja pintasuuntautunut opiskelustrategia. Merkitysorientoituneet, itsesäätelykykyiset opiskelijat menestyivät LTKK:ssa parhaiten.

Cell wall precursors are required to organize the chlamydial division septum.

Members of the Chlamydiales order are major bacterial pathogens that divide at mid-cell, without a sequence homologue of the FtsZ cytokinetic tubulin and without a classical peptidoglycan cell wall. Moreover, the spatiotemporal mechanisms directing constriction in Chlamydia are not known. Here we show that the MreB actin homologue and its conserved regulator RodZ localize to the division furrow in Waddlia chondrophila, a member of the Chlamydiales order implicated in human miscarriage. RodZ is recruited to the septal site earlier than MreB and in a manner that depends on biosynthesis of the peptidoglycan precursor lipid II by the MurA enzyme. By contrast, crosslinking of lipid II peptides by the Pbp3 transpeptidase disperses RodZ from the septum. Altogether, these findings provide a cytological framework for understanding chlamydial cytokinesis driven by septal cell wall synthesis.

La bretxa digital: gènere i jocs d'ordinador

La finalitat d"aquest article és reflexionar sobre una pràctica concreta que s"ha revelat creadora de desigualtat social en l"era digital: els jocs d"ordinador des de la perspectiva del gènere. Per això, es fa una revisió de la literatura existent per mostrar com, malgrat les bondats educatives de l"activitat lúdica, l"ús educatiu dels jocs d"ordinador és mínim. A més, es detallen diverses investigacions que mostren el biaix de gènere en diferents aspectes relacionats amb els jocs multimèdia.

Laringocele: aspecto ultra-sonográfico - relato de caso

Laringoceles são lesões relativamente raras definidas como dilatações anômalas dos sáculos dos ventrículos laríngeos. A classificação usual divide a laringocele em interna, externa e combinada ou mista. Laringoceles internas são as que se localizam medialmente à cartilagem tireóidea e geralmente causam compressão nas bandas ventriculares levando a rouquidão e sintomas compressivos na via aérea. As externas se estendem através da membrana tireóidea, apresentando-se como massas cervicais, e as mistas são as que ocupam as duas regiões, podendo causar ambos os sintomas. O diagnóstico é geralmente feito por tomografia computadorizada e/ou laringoscopia. Apresentamos um caso de laringocele mista em que o diagnóstico foi sugerido no exame de ultra-sonografia, num paciente encaminhado com história de massa cervical.

Dpp spreading is required for medial but not for lateral wing disc growth.

Drosophila Decapentaplegic (Dpp) has served as a paradigm to study morphogen-dependent growth control. However, the role of a Dpp gradient in tissue growth remains highly controversial. Two fundamentally different models have been proposed: the 'temporal rule' model suggests that all cells of the wing imaginal disc divide upon a 50% increase in Dpp signalling, whereas the 'growth equalization model' suggests that Dpp is only essential for proliferation control of the central cells. Here, to discriminate between these two models, we generated and used morphotrap, a membrane-tethered anti-green fluorescent protein (GFP) nanobody, which enables immobilization of enhanced (e)GFP::Dpp on the cell surface, thereby abolishing Dpp gradient formation. We find that in the absence of Dpp spreading, wing disc patterning is lost; however, lateral cells still divide at normal rates. These data are consistent with the growth equalization model, but do not fit a global temporal rule model in the wing imaginal disc.

Signaling in the Rhizosphere.

Signaling studies in the rhizosphere have focused on close interactions between plants and symbiotic microorganisms. However, this focus is likely to expand to other microorganisms because the rhizomicrobiome is important for plant health and is able to influence the structure of the microbial community. We discuss here the shaping of the rhizomicrobiome and define which aspects can be considered signaling. We divide signaling in the rhizosphere into three categories: (i) between microbes, (ii) from plants to microorganisms, and (iii) from microorganisms to plants. Signals act on diverse organisms including the plant. Mycorrhizal and rhizobial interkingdom signaling has revealed its pivotal role in establishing associations, and the recent discovery of signaling with non-symbiotic microorganisms indicates the important role of communication in shaping the rhizomicrobiome.