Spatial visualization of DNA in solution.

An image analysis method is presented which allows for the reconstruction of the three-dimensional path of filamentous objects from two of their projections. Starting with stereo pairs, this method is used to trace the trajectory of DNA molecules embedded in vitreous ice and leads to a faithful representation of their three-dimensional shape in solution. This computer-aided reconstruction is superior to the subjective three-dimensional impression generated by observation of stereo pairs of micrographs because it enables one to look at the reconstructed molecules from any chosen direction and distance and allows quantitative analysis such as determination of distances, curvature, persistence length, and writhe of DNA molecules in solution.

Apparent versus true gene expression changes of three hypoxia-related genes in autopsy derived tissue and the importance of normalisation.

The aim of our work was to show how a chosen normal-isation strategy can affect the outcome of quantitative gene expression studies. As an example, we analysed the expression of three genes known to be upregulated under hypoxic conditions: HIF1A, VEGF and SLC2A1 (GLUT1). Raw RT-qPCR data were normalised using two different strategies: a straightforward normalisation against a single reference gene, GAPDH, using the 2(-ΔΔCt) algorithm and a more complex normalisation against a normalisation factor calculated from the quantitative raw data from four previously validated reference genes. We found that the two different normalisation strategies revealed contradicting results: normalising against a validated set of reference genes revealed an upregulation of the three genes of interest in three post-mortem tissue samples (cardiac muscle, skeletal muscle and brain) under hypoxic conditions. Interestingly, we found a statistically significant difference in the relative transcript abundance of VEGF in cardiac muscle between donors who died of asphyxia versus donors who died from cardiac death. Normalisation against GAPDH alone revealed no upregulation but, in some instances, a downregulation of the genes of interest. To further analyse this discrepancy, the stability of all reference genes used were reassessed and the very low expression stability of GAPDH was found to originate from the co-regulation of this gene under hypoxic conditions. We concluded that GAPDH is not a suitable reference gene for the quantitative analysis of gene expression in hypoxia and that validation of reference genes is a crucial step for generating biologically meaningful data.

A qualitative continuous model of cellular auxin and brassinosteroid signaling and their crosstalk.

Motivation: Hormone pathway interactions are crucial in shaping plant development, such as synergism between the auxin and brassinosteroid pathways in cell elongation. Both hormone pathways have been characterized in detail, revealing several feedback loops. The complexity of this network, combined with a shortage of kinetic data, renders its quantitative analysis virtually impossible at present.Results: As a first step towards overcoming these obstacles, we analyzed the network using a Boolean logic approach to build models of auxin and brassinosteroid signaling, and their interaction. To compare these discrete dynamic models across conditions, we transformed them into qualitative continuous systems, which predict network component states more accurately and can accommodate kinetic data as they become available. To this end, we developed an extension for the SQUAD software, allowing semi-quantitative analysis of network states. Contrasting the developmental output depending on cell type-specific modulators enabled us to identify a most parsimonious model, which explains initially paradoxical mutant phenotypes and revealed a novel physiological feature.

A new poly(ortho ester)-based drug delivery system as an adjunct treatment in filtering surgery.

PURPOSE: Pharmacologic modulation of wound healing after glaucoma filtering surgery remains a major clinical challenge in ophthalmology. Poly(ortho ester) (POE) is a bioerodible and biocompatible viscous polymer potentially useful as a sustained drug delivery system that allows the frequency of intraocular injections to be reduced. The purpose of this study was to determine the efficacy of POE containing a precise amount of 5-fluorouracil (5-FU) in an experimental model of filtering surgery in the rabbit. METHODS: Trabeculectomy was performed in pigmented rabbit eyes. An ointmentlike formulation of POE containing 1% wt/wt 5-FU was injected subconjunctivally at the site of surgery, during the procedure. Intraocular pressure (IOP), bleb persistence, and ocular inflammatory reaction were monitored until postoperative day 30. Quantitative analysis of 5-FU was performed in the anterior chamber. Histologic analysis was used to assess the appearance of the filtering fistula and the polymer's biocompatibility. RESULTS: The decrease in IOP from baseline and the persistence of the filtering bleb were significantly more marked in the 5-FU-treated eyes during postoperative days 9 through 28. Corneal toxicity triggered by 5-FU was significantly lower in the group that received 5-FU in POE compared with a 5-FU tamponade. Histopathologic evaluation showed that POE was well tolerated, and no fibrosis occurred in eyes treated with POE containing 5-FU. CONCLUSIONS: In this rabbit model of trabeculectomy, the formulation based on POE and containing a precise amount of 5-FU reduced IOP and prolonged bleb persistence in a way similar to the conventional method of a 5-FU tamponade, while significantly reducing 5-FU toxicity.

Evaluation of respiratory and dermal occupational exposures to monoethanolamine during cleaning

Professional cleaning is a basic service occupation with a wide variety of tasks carried out in all kind of different sectors and workplaces by a large workforce. One important risk for cleaning workers is the exposure to chemical substances that are present in cleaning products.Monoethanolamine was found to be often present in cleaning products such as general purpose cleaners, bathroom cleaners, floor cleaners and kitchen cleaners. Monoethanolamine can injure the skin, and exposure to monoethanolamine was associated to asthma even when the air concentrations were low. It is a strong irritant and known to be involved in sensitizing mechanisms. It is very likely that the use of cleaning products containing monoethanolamine gives rise to respiratory and dermal exposures. Therefore there is a need to further investigate the exposures to monoethanolamine for both, respiratory and dermal exposure.The determination of monoethanolamine has traditionally been difficult and analytical methods available are little adapted for occupational exposure assessments. For monoethanolamine air concentrations, a sampling and analytical method was already available and could be used. However, a method to analyses samples for skin exposure assessments as well as samples of skin permeation experiments was missing. Therefore one main objective of this master thesis was to search an already developed and described analytical method for the measurement of monoethanolamine in water solutions, and to set it up in the laboratory. Monoethanolamine was analyzed after a derivatisation reaction with o-pthtaldialdehyde. The derivated fluorescing monoethanolamine was then separated with high performance liquid chromatography and detection took place with a fluorescent detector. The method was found to be suitable for qualitative and quantitative analysis of monoethanolamine. An exposure assessment was conducted in the cleaning sector to measure the respiratory and dermal exposures to monoethanolamine during floor cleaning. Stationary air samples (n=36) were collected in 8 companies and samples for dermal exposures (n=12) were collected in two companies. Air concentrations (Mean = 0.18 mg/m3, Standard Deviation = 0.23 mg/m3, geometric Mean = 0.09 mg/m3, Geometric Standard Deviation = 3.50) detected were mostly below 1/10 of the Swiss 8h time weighted average occupational exposure limit. Factors that influenced the measured monoethanolamine air concentrations were room size, ventilation system and the concentration of monoethanolamine in the cleaning product and amount of monoethanolamine used. Measured skin exposures ranged from 0.6 to 128.4 mg/sample. Some cleaning workers that participated in the skin exposure assessment did not use gloves and had direct contact with the solutions containing the cleaning product and monoethanolamine. During the entire sampling campaign, cleaning workers mostly did not use gloves. Cleaning workers are at risk to be regularly exposed to low air concentrations of monoethanolamine. This exposure may be problematic if a worker suffers from allergic reactions (e.g. Asthma). In that case a substitution of the cleaning product may be a good prevention measure as several different cleaning products are available for similar cleaning tasks. Currently there are no occupational exposure limits to compare the skin exposures that were found. To prevent skin exposures, adaptations of the cleaning techniques and the use of gloves should be considered. The simultaneous skin and airborne exposures might accelerate adverse health effects. Overall the risks caused by exposures to monoethanolamine are considered as low to moderate when the cleaning products are used correctly. Whenever possible, skin exposures should be avoided. Further research should consider especially the dermal exposure routes, as very high exposures might occur by skin contact with cleaning products. Dermatitis but also sensitization might be caused by skin exposures. In addition, new biomedical insights are needed to better understand the risks of the dermal exposure. Therefore skin permeability experiments should be considered.

Evolution and sedimentology of a channel fill in the sandy braided South Saskatchewan River and its comparison to the deposits of an adjacent compound bar

The depositional stratigraphy of within-channel deposits in sandy braided rivers is dominated by a variety of barforms (both singular `unit' bars and complex `compound' bars), as well as the infill of individual channels (herein termed `channel fills'). The deposits of bars and channel fills define the key components of facies models for braided rivers and their within-channel heterogeneity, knowledge of which is important for reservoir characterization. However, few studies have sought to address the question of whether the deposits of bars and channel fills can be readily differentiated from each other. This paper presents the first quantitative study to achieve this aim, using aerial images of an evolving modern sandy braided river and geophysical imaging of its subsurface deposits. Aerial photographs taken between 2000 and 2004 document the abandonment and fill of a 1 3 km long, 80 m wide anabranch channel in the sandy braided South Saskatchewan River, Canada. Upstream river regulation traps the majority of very fine sediment and there is little clay (<1%) in the bed sediments. Channel abandonment was initiated by a series of unit bars that stalled and progressively blocked the anabranch entrance, together with dune deposition and stacking at the anabranch entrance and exit. Complete channel abandonment and subsequent fill of up to 3 m of sediment took approximately two years. Thirteen kilometres of ground-penetrating radar surveys, coupled with 18 cores, were obtained over the channel fill and an adjacent 750 m long, 400 m wide, compound bar, enabling a quantitative analysis of the channel and bar deposits. Results show that, in terms of grain-size trends, facies proportions and scale of deposits, there are only subtle differences between the channel fill and bar deposits which, therefore, renders them indistinguishable. Thus, it may be inappropriate to assign different geometric and sedimentological attributes to channel fill and bar facies in object-based models of sandy braided river alluvial architecture.

Genome-wide DNA methylation detection by MethylCap-seq and Infinium HumanMethylation450 BeadChips: an independent large-scale comparison.

Two cost-efficient genome-scale methodologies to assess DNA-methylation are MethylCap-seq and Illumina's Infinium HumanMethylation450 BeadChips (HM450). Objective information regarding the best-suited methodology for a specific research question is scant. Therefore, we performed a large-scale evaluation on a set of 70 brain tissue samples, i.e. 65 glioblastoma and 5 non-tumoral tissues. As MethylCap-seq coverages were limited, we focused on the inherent capacity of the methodology to detect methylated loci rather than a quantitative analysis. MethylCap-seq and HM450 data were dichotomized and performances were compared using a gold standard free Bayesian modelling procedure. While conditional specificity was adequate for both approaches, conditional sensitivity was systematically higher for HM450. In addition, genome-wide characteristics were compared, revealing that HM450 probes identified substantially fewer regions compared to MethylCap-seq. Although results indicated that the latter method can detect more potentially relevant DNA-methylation, this did not translate into the discovery of more differentially methylated loci between tumours and controls compared to HM450. Our results therefore indicate that both methodologies are complementary, with a higher sensitivity for HM450 and a far larger genome-wide coverage for MethylCap-seq, but also that a more comprehensive character does not automatically imply more significant results in biomarker studies.

Pathologies vasculaires lymphatiques: apport de la lympho-fluoroscopie [Lymphatic vascular pathologies: contribution of lympho-fluoroscopy].

Si l'examen clinique revêt une importance essentielle en lymphologie et exige des praticiens expérimentés, la lymphoscintigraphie et plus récemment la lympho-fluoroscopie au vert d'indocyanine constituent des moyens d'investigation précieux dans la prévention, le diagnostic et le traitement des pathologies vasculaires lymphatiques. L'intérêt de la lymphoscintigraphie réside dans l'analyse qualitative et quantitative de la migration des macromolécules par les vaisseaux lymphatiques et l'évaluation du secteur lymphatique profond. La lympho-fluoroscopie se distingue de la lymphoscintigraphie par l'obtention d'une cartographie détaillée des vaisseaux lymphatiques superficiels et d'images dynamiques en temps réel. Elle apporte à l'angiologue et au physiothérapeute des informations irremplaçables sur leur contractilité et la présence de dérivations compensatoires à privilégier lors du drainage lymphatique manuel. If clinical examination has an essential importance in lymphology disorders and requires experimented practitioners, lymphoscintigraphy and more recently green indocyanine lympho-fluoroscopy constitute precious complementary investigations in prevention, diagnosis, and treatment of lymphatic vascular pathologies. The lymphoscintigraphy interest lies in qualitative and quantitative analysis of macromolecules migration within lymphatic vessels and the deep lymphatic network. The lympho-fluoroscopy distinguishes itself from lymphoscintigraphy allowing real time superficial lymphatic vessels detailed mapping, gathering important information on their contractility, and the presence of compensatory derivations to be favored during manual lymphatic drainage to angiologist and physiotherapist.