Local-global splitting for spatiotemporal-adaptive multiscale methods

We present a novel spatiotemporal-adaptive Multiscale Finite Volume (MsFV) method, which is based on the natural idea that the global coarse-scale problem has longer characteristic time than the local fine-scale problems. As a consequence, the global problem can be solved with larger time steps than the local problems. In contrast to the pressure-transport splitting usually employed in the standard MsFV approach, we propose to start directly with a local-global splitting that allows to locally retain the original degree of coupling. This is crucial for highly non-linear systems or in the presence of physical instabilities. To obtain an accurate and efficient algorithm, we devise new adaptive criteria for global update that are based on changes of coarse-scale quantities rather than on fine-scale quantities, as it is routinely done before in the adaptive MsFV method. By means of a complexity analysis we show that the adaptive approach gives a noticeable speed-up with respect to the standard MsFV algorithm. In particular, it is efficient in case of large upscaling factors, which is important for multiphysics problems. Based on the observation that local time stepping acts as a smoother, we devise a self-correcting algorithm which incorporates the information from previous times to improve the quality of the multiscale approximation. We present results of multiphase flow simulations both for Darcy-scale and multiphysics (hybrid) problems, in which a local pore-scale description is combined with a global Darcy-like description. The novel spatiotemporal-adaptive multiscale method based on the local-global splitting is not limited to porous media flow problems, but it can be extended to any system described by a set of conservation equations.

A comparative study on IgG-ELISA, IgM-IFT and Kato-Katz methods for epidemiological purposes in a low endemic area for schistosomiasis

The high sensitivity and the possibility of automation of the enzyme-linked-immunosorbent-assay (ELISA) has indicated this technique as one of the most useful serological test for epidemiological studies. In the present study, an ELISA for detection of IgG antibodies against adult worm antigens (IgG-ELISA) was investigated for epidemiological purposes, in a rural area of the municipality of Itariri (São Paulo, Brazil). Blood on filter paper (1,180 samples) from about 650 schoolchildren were submitted to ELISA and the data compared to the results of the parasitological method of Kato-Katz and also to the IgM-IFT (immunofluorescence test for IgM antibodies to gut associated antigens). The prevalence rates respectively of 8.5%, 43.0%, and 56.2% by the Kato-Katz, IgG-ELISA, and IgM-IFT methods suggest the poor sensitivity of the parasitological method for detection of Schistosoma mansoni eggs in individuals with low worm burden, situation commonly observed in low endemic areas. These results can partially explain the poor degree of agreement between the IgG-ELISA and the Kato-Katz, as suggested by the Kappa index of 0.170. Otherwise, the Kappa index of 0.675 showed substantial agreement between the two serological tests. Some discrepancy of results between the two serological techniques must be better investigated.

Improved Methods for the Diagnosis of African Trypanosomosis

The diagnosis of trypanosomosis in animals with low parasitaemia is hampered by low diagnostic sensitivity of traditional detection methods. An immunodiagnostic method based on a direct sandwich enzyme-linked immunosorbent assay (ELISA), using monoclonal antibodies, has been examined in a number of African laboratories for its suitability for monitoring tsetse control and eradication programmes. Generally, the direct sandwich ELISAs for the detection of trypanosomal antigens in serum samples have proved to be unsatisfactory with respect to diagnostic sensitivity when compared with traditional parasitological methods such as the dark ground/phase contrast buffy-coat technique. Consequently, antigen-detection systems exploiting various other direct, indirect and sandwich ELISA systems and sets of reagents are being developed to improve diagnosis. In addition, an existing indirect ELISA for the detection of antibodies has been improved and is being evaluated in the field in order to detect cattle that are or have been recently infected with trypanosomes. Developments and advantages of other diagnostic techniques, such as dip-stick assay and tests based on the polymerase chain reaction are also considered.

Recombinant " Physcomitrella patens " as a sensitive tool to study heat sensing and heat-shock signal transduction in plants

SUMMARY When exposed to heat stress, plants display a particular set of cellular and molecular responses, such as chaperones expression, which are highly conserved in all organisms. In chapter 1, I studied the ability of heat shock genes to become transiently and abundantly induced under various temperature regimes. To this aim, I designed a highly sensitive heat-shock dependent conditional gene expression system in the moss Physcomitrella patens, using the soybean heatinducible promoter (hsp17.3B). Heat-induced expression of various reporter genes was over three orders of magnitude, in tight correlation with the intensity and duration of the heat treatments. By performing repeated heating/cooling cycles, a massive accumulation of recombinant proteins was obtained. Interestingly, the hsp17.3B promoter was also activated by specific organic chemicals. Thus, in chapter 2, I took advantage of the extreme sensitivity of this promoter to small temperature variations to further address the role of various natural and organic chemicals and develop a plant based-bioassay that can serve as an early warning indicator of toxicity by pollutants and heavy metals. A screen of several organic pollutants from textile and paper industry showed that chlorophenols as well as sulfonated anthraquinones elicited a heat shock like response at noninducing temperatures. Their effects were synergistically amplified by mild elevated temperatures. In contrast to standard methods of pollutant detection, this plant-based biosensor allowed to monitor early stress-responses, in correlation with long-term toxic effect, and to attribute effective toxicity thresholds for pollutants, in a context of varying environmental cues. In chapter 3, I deepened the study of the primary mechanism by which plants sense mild temperature variations and trigger a cellular signal leading to the heat shock response. In addition to the above described heat-inducible reporter line, I generated a P. patens transgenic line to measure, in vivo, variations of cytosolic calcium during heat treatment, and another line to monitor the role of protein unfolding in heat-shock sensing and signalling. The heat shock signalling pathway was found to be triggered by the plasma membrane, where temperature up shift specifically induced the transient opening of a putative high afimity calcium channel. The calcium influx triggered a signalling cascade leading to the activation of the heat shock genes, independently on the presence of misfolded proteins in the cytoplasm. These results strongly suggest that changes in the fluidity of the plasma membrane are the primary trigger of the heatshocksignalling pathway in plants. The present thesis contributes to the understanding of the basic mechanism by which plants perceive and respond to heat and chemical stresses. This may contribute to developing appropriate better strategies to enhance plant productivity under the increasingly stressful environment of global warming. RÉSUME Les plantes exposées à des températures élevées déclenchent rapidement des réponses cellulaires qui conduisent à l'induction de gènes codant pour les heat shock proteins (HSPs). En fonction de la durée d'exposition et de la vitesse à laquelle la température augmente, les HSPs sont fortement et transitoirement induites. Dans le premier chapitre, cette caractéristique aété utilisée pour développer un système inductible d'expression de gènes dans la mousse Physcomitrella patens. En utilisant plusieurs gènes rapporteurs, j'ai montré que le promoteur du gène hsp17.3B du Soja est activé d'une manière. homogène dans tous les tissus de la mousse proportionnellement à l'intensité du heat shock physiologique appliqué. Un très fort taux de protéines recombinantes peut ainsi être produit en réalisant plusieurs cycles induction/recovery. De plus, ce promoteur peut également être activé par des composés organiques, tels que les composés anti-inflammatoires, ce qui constitue une bonne alternative à l'induction par la chaleur. Les HSPs sont induites pour remédier aux dommages cellulaires qui surviennent. Étant donné que le promoteur hsp17.3B est très sensible à des petites augmentations de température ainsi qu'à des composés chimiques, j'ai utilisé les lignées développées dans le chapitre 1 pour identifier des polluants qui déclenchent une réaction de défense impliquant les HSPs. Après un criblage de plusieurs composés, les chlorophénols et les antraquinones sulfonés ont été identifiés comme étant activateurs du promoteur de stress. La détection de leurs effets a été réalisée seulement après quelques heures d'exposition et corrèle parfaitement avec les effets toxiques détectés après de longues périodes d'exposition. Les produits identifiés montrent aussi un effet synergique avec la température, ce qui fait du biosensor développé dans ce chapitre un bon outil pour révéler les effets réels des polluants dans un environnement où les stress chimiques sont combinés aux stress abiotiques. Le troisième chapitre est consacré à l'étude des mécanismes précoces qui permettent aux plantes de percevoir la chaleur et ainsi de déclencher une cascade de signalisation spécifique qui aboutit à l'induction des gènes HSPs. J'ai généré deux nouvelles lignées afin de mesurer en temps réel les changements de concentrations du calcium cytosolique ainsi que l'état de dénaturation des protéines au cours du heat shock. Quand la fluidité de la membrane augmente après élévation de la température, elle semble induire l'ouverture d'un canal qui permet de faire entrer le calcium dans les cellules. Ce dernier initie une cascade de signalisation qui finit par activer la transcription des gènes HSPs indépendamment de la dénaturation de protéines cytoplasmiques. Les résultats présentés dans ce chapitre montrent que la perception de la chaleur se fait essentiellement au niveau de la membrane plasmique qui joue un rôle majeur dans la régulation des gènes HSPs. L'élucidation des mécanismes par lesquels les plantes perçoivent les signaux environnementaux est d'une grande utilité pour le développement de nouvelles stratégies afin d'améliorer la productivité des plantes soumises à des conditions extrêmes. La présente thèse contribue à décortiquer la voie de signalisation impliquée dans la réponse à la chaleur.

Evaluation of detection methods for Virus, Viroids and Phytoplasmas affecting pear and apple

The RT-PCR technique for the detection of apple stem grooving virus (ASGV), apple stem pitting virus (ASPV), apple chlorotic leaf spot virus (ACLSV), apple mosaic virus (ApMV) and pear blister canker viroid (PBCV) was evaluated for health control of fruit plants from nurseries. The technique was evaluated in purified RNA and crude extracts and also in phloem collected in autumn and from young spring shoots. The results obtained for phytoplasma detection with ribosomal and non-ribosomal primers are also presented.

Effect of immunisation against angiotensin II with CYT006-AngQb on ambulatory blood pressure: a double-blind, randomised, placebo-controlled phase IIa study.

BACKGROUND: Hypertension can be controlled adequately with existing drugs such as angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers. Nevertheless, treatment success is often restricted by patients not adhering to treatment. Immunisation against angiotensin II could solve this problem. We investigated the safety and efficacy of CYT006-AngQb-a vaccine based on a virus-like particle-that targets angiotensin II to reduce ambulatory blood pressure. METHODS: In this multicentre, double-blind, randomised, placebo-controlled phase IIa trial, 72 patients with mild-to-moderate hypertension were randomly assigned with a computer-generated randomisation list to receive subcutaneous injections of either 100 mug CYT006-AngQb (n=24), 300 mug CYT006-AngQb (24), or placebo (24), at weeks 0, 4, and 12. 24-h ambulatory blood pressure was measured before treatment and at week 14. The primary outcomes were safety and tolerability. Analyses were done by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00500786. FINDINGS: Two patients in the 100 mug group, three in the 300 mug group, and none in the placebo group discontinued study treatment. All patients were included in safety analyses; efficacy analyses did not include the five dropouts, for whom no data were available at week 14. Five serious adverse events were reported (two in the 100 mug group, two in the 300 mug group, and one in the placebo group); none were deemed to be treatment related. Most side-effects were mild, transient reactions at the injection site. Mild, transient influenza-like symptoms were seen in three patients in the 100 mug group, seven in the 300 mug group, and none in the placebo group. In the 300 mug group, there was a reduction from baseline in mean ambulatory daytime blood pressure at week 14 by -9.0/-4.0 mm Hg compared with placebo (p=0.015 for systolic and 0.064 for diastolic). The 300 mug dose reduced the early morning blood-pressure surge compared with placebo (change at 0800 h -25/-13 mm Hg; p<0.0001 for systolic, p=0.0035 for diastolic). INTERPRETATION: Immunisation with CYT006-AngQb was associated with no serious adverse events; most observed adverse events were consistent with local or systemic responses similar to those seen with other vaccines. The 300 mug dose reduced blood pressure in patients with mild-to-moderate hypertension during the daytime, especially in the early morning. FUNDING: Cytos Biotechnology AG.

Comparing Multilevel Clustering Methods on Weighted Graphs : The Case of Worldwide Air Passenger Traffic 2000-2004

Specific properties emerge from the structure of large networks, such as that of worldwide air traffic, including a highly hierarchical node structure and multi-level small world sub-groups that strongly influence future dynamics. We have developed clustering methods to understand the form of these structures, to identify structural properties, and to evaluate the effects of these properties. Graph clustering methods are often constructed from different components: a metric, a clustering index, and a modularity measure to assess the quality of a clustering method. To understand the impact of each of these components on the clustering method, we explore and compare different combinations. These different combinations are used to compare multilevel clustering methods to delineate the effects of geographical distance, hubs, network densities, and bridges on worldwide air passenger traffic. The ultimate goal of this methodological research is to demonstrate evidence of combined effects in the development of an air traffic network. In fact, the network can be divided into different levels of âeurooecohesionâeuro, which can be qualified and measured by comparative studies (Newman, 2002; Guimera et al., 2005; Sales-Pardo et al., 2007).

Imaging molasse and quaternary sediments in Lake Geneva, Switzerland, with 3-D high-resolution seismic reflection methods: A case study

A high-resolution three-dimensional (3-D) seismic reflection survey was conducted in Lake Geneva, near the city of Lausanne, Switzerland, as part of a project for developing such seismic techniques. Using a single 48-channel streamer, the 3-D site with an area of 1200 m x 600 m was surveyed in 10 days. A variety of complex geologic structures (e.g. thrusts, folds, channel-fill) up to similar to150 m below the water bottom were obtained with a 15 in.(3) water gun. The 3-D data allowed the construction of an accurate velocity model and the distinction of five major seismic facies within the Lower Freshwater Molasse (Aquitanian) and the Quaternary sedimentary units. Additionally, the Plateau Molasse (PM) and Subalpine Molasse (SM) erosional surface, "La Paudeze" thrust fault (PM-SM boundary) and the thickness of Quaternary sediments were accurately delineated in 3-D.

Expression of the gene encoding the high-Km glucose transporter 2 by the early postimplantation mouse embryo is essential for neural tube defects associated with diabetic embryopathy.

AIMS/HYPOTHESIS: Excess glucose transport to embryos during diabetic pregnancy causes congenital malformations. The early postimplantation embryo expresses the gene encoding the high-Km GLUT2 (also known as SLC2A2) glucose transporter. The hypothesis tested here is that high-Km glucose transport by GLUT2 causes malformations resulting from maternal hyperglycaemia during diabetic pregnancy. MATERIALS AND METHODS: Glut2 mRNA was assayed by RT-PCR. The Km of embryo glucose transport was determined by measuring 0.5-20 mmol/l 2-deoxy[3H]glucose transport. To test whether the GLUT2 transporter is required for neural tube defects resulting from maternal hyperglycaemia, Glut2+/- mice were crossed and transient hyperglycaemia was induced by glucose injection on day 7.5 of pregnancy. Embryos were recovered on day 10.5, and the incidence of neural tube defects in wild-type, Glut2+/- and Glut2-/- embryos was scored. RESULTS: Early postimplantation embryos expressed Glut2, and expression was unaffected by maternal diabetes. Moreover, glucose transport by these embryos showed Michaelis-Menten kinetics of 16.19 mmol/l, consistent with transport mediated by GLUT2. In pregnancies made hyperglycaemic on day 7.5, neural tube defects were significantly increased in wild-type embryos, but Glut2+/- embryos were partially protected from neural tube defects, and Glut2-/- embryos were completely protected from these defects. The frequency of occurrence of wild-type, Glut2+/- and Glut2-/- embryos suggests that the presence of Glut2 alleles confers a survival advantage in embryos before day 10.5. CONCLUSIONS/INTERPRETATIONS: High-Km glucose transport by the GLUT2 glucose transporter during organogenesis is responsible for the embryopathic effects of maternal diabetes.

Chromatic pupil responses: preferential activation of the melanopsin-mediated versus outer photoreceptor-mediated pupil light reflex.

OBJECTIVE: To weight the rod-, cone-, and melanopsin-mediated activation of the retinal ganglion cells, which drive the pupil light reflex by varying the light stimulus wavelength, intensity, and duration. DESIGN: Experimental study. PARTICIPANTS: Forty-three subjects with normal eyes and 3 patients with neuroretinal visual loss. METHODS: A novel stimulus paradigm was developed using either a long wavelength (red) or short wavelength (blue) light given as a continuous Ganzfeld stimulus with stepwise increases over a 2 log-unit range. The pupillary movement before, during, and after the light stimulus was recorded in real time with an infrared illuminated video camera. MAIN OUTCOME MEASURES: The percent pupil contraction of the transient and sustained pupil response to a low- (1 cd/m(2)), medium- (10 cd/m(2)), and high-intensity (100 cd/m(2)) red- and blue-light stimulus was calculated for 1 eye of each subject. From the 43 normal eyes, median and 25th, 75th, 5th, and 95th percentile values were obtained for each stimulus condition. RESULTS: In normal eyes at lower intensities, blue light evoked much greater pupil responses compared with red light when matched for photopic luminance. The transient pupil contraction was generally greater than the sustained contraction, and this disparity was greatest at the lowest light intensity and least apparent with bright (100 cd/m(2)) blue light. A patient with primarily rod dysfunction (nonrecordable scotopic electroretinogram) showed significantly reduced pupil responses to blue light at lower intensities. A patient with achromatopsia and an almost normal visual field showed selective reduction of the pupil response to red-light stimulation. A patient with ganglion cell dysfunction owing to anterior ischemic optic neuropathy demonstrated global loss of pupil responses to red and blue light in the affected eye. CONCLUSIONS: Pupil responses that differ as a function of light intensity and wavelength support the hypothesis that selected stimulus conditions can produce pupil responses that reflect phototransduction primarily mediated by rods, cones, or melanopsin. Use of chromatic pupil responses may be a novel way to diagnose and monitor diseases affecting either the outer or inner retina.

Direct methods for detecting picorna-like virus from dead and alive Triatomine insects

In this work we report four different destructive and non-destructive methods for detecting picorna-like virus particles in triatomines. The methods are based on direct observation under transmission electron microscope and they consist of four ways to prepare samples of presumable infected material. The samples are prepared processing dead or alive insect parts, or even dry or fresh insect feces. The methods can be used as analytical or preparative techniques, for quantifying virus infection and checking virus integrity as well. In this work the four methods are applied in order to detect Triatoma virus (TrV) particles in T. infestans colonies.

Analysis of genetic diversity of Trypanosoma cruzi: an application of riboprinting and gradient gel electrophoresis methods

Analysis of restriction fragment length polymorphism (RFLP) profiles derived from digestion of polymerase chain reaction (PCR) products of the ribosomal 18S from Trypanosoma cruzi yields a typical `riboprint' profile that can vary intraspecifically. A selection of 21 stocks of T. cruzi and three outgroup taxa: T. rangeli, T. conorhini and Leishmania braziliensis were analysed by riboprinting to assess divergence within and between taxa. T. rangeli, T. conorhini and L. braziliensis could be easily differentiated from each other and from T. cruzi. Phenetic analysis of PCR-RFLP profiles indicated that, with one or two exceptions, stocks of T. cruzi could be broadly partitioned into two groups that formally corresponded to T. cruzi I and T. cruzi II respectively. To test if ribosomal 18S sequences were homogeneous within each taxon, gradient gel electrophoresis methods were employed utilising either chemical or temperature gradients. Upon interpretation of the melting profiles of riboprints and a section of the 18S independently amplified by PCR, there would appear to be at least two divergent 18S types present within T. cruzi. Heterogeneity within copies of the ribosomal 18S within a single genome has therefore been demonstrated and interestingly, this dimorphic arrangement was also present in the outgroup taxa. Presumably the ancestral duplicative event that led to the divergent 18S types preceded that of speciation within this group. These divergent 18S paralogues may have, or had, different functional pressures or rates of molecular evolution. Whether or not these divergent types are equally transcriptionally active throughout the life cycle, remain to be assessed.

Analysis of parity between protein-based electrophoretic methods for the characterization of oral Candida species

Electrophoretic studies of multilocus-enzymes (MLEE) and whole-cell protein (SDS-PAGE) were carried out in order to evaluate the parity between different methods for the characterization of five Candida species commonly isolated from oral cavity of humans by numerical taxonomy methods. The obtained data revealed that sodium dodecyl sulfate polyacrylamide gel electrophoresis is more efficient in grouping strains in their respective species while MLEE has much limited resolution in organizing all strains in their respective species-specific clusters. MLEE technique must be regarded for surveys in which just one species of Candida is involved.