Whole-transciptome analysis of [psi+] budding yeast via cDNA microarrays

Introduction: Prions of yeast present a novel analytical challenge in terms of both initial characterization and in vitro manipulation as models for human disease research. Presently, few robust analysis strategies have been successfully implemented which enable the efficient study of prion behavior in vivo. This study sought to evaluate the utilization of conventional dual-channel cDNA microarrays for the surveillance of transcriptomic regulation patterns by the [PSI+] yeast prion relative to an identical prion deficient yeast variant, [psi-]. Methods: A data analysis and normalization workflow strategy was developed and applied to cDNA array images, yielded quality-regulated expression ratios for a subset of genes exhibiting statistical congruence across multiple experimental repetitions and nested hybridization events. The significant gene list was analyzed using classical analytical approaches including several clustering-based methods and singular value decomposition. To add biological meaning to the differential expression data in hand, functional annotation using the Gene Ontology as well as several pathway-mapping approaches was conducted. Finally, the expression patterns observed were queried against all publicly curated microarray data performed using S. cerevisiae in order to discover similar expression behavior across a vast array of experimental conditions. Results: These data collectively implicate a low-level of overall genomic regulation as a result of the [PSI+] state, where the maximum statistically significant degree of differential expression was less than ±1 Log2(FC) in all cases. Notwithstanding, the [PSI+] differential expression was localized to several specific classes of structural elements and cellular functions, implying under homeostatic conditions significant up or down regulation is likely unnecessary but possible in those specific systems if environmental conditions warranted. As a result of these findings additional work pertaining to this system should include controlled insult to both yeast variants of differing environmental properties to promote a potential [PSI+] regulatory response coupled with co-surveillance of these conditions using transcriptomic and proteomic analysis methodologies

Using microarray analysis to find novel genes involved in somitogenesis

During the development of vertebrates the body is subdivided into repeated units, the somites. They are generated from the presomitic mesoderm (PSM) and are flanking the notochord on both sides. For this coordinated process components of the Delta-Notch signalling pathway are important. Novel genes involved in somitogenesis were searched using microarray-technology. This technique had to be established in the lab and this was done using a cDNA library, which was prepared from embryos of mixed embryogenesis stages. The basic protocols were established like printing, labelling and hybridisation. The usage of spiked in controls was established as well. The controls were genes from the bacteriophage lambda. Later the cDNA library was used to compare the expression of wt and fss embryos. The fss mutant is linked to the gene Tbx24, which is no component of the Delta-Notch pathway, but belongs to the fused somite type mutants. To compare the other fused somite type embryos against wt the zebrafish oligo library from Sigma-Genosys was used. The other mutants are des, bea and aei. These mutants are defective in components of the Delta-Notch pathway, des in notch1a, bea in deltaC and aei in deltaD. As these components are involved in the starting of the cascade and because of this some rescue mechanisms might occur the mediator of the pathway Su(H) was used as well. For this gene morpholino knock down embryos were produced. The PSM consists of several compartments, namely a posterior, an intermediate and an anterior compartment. The PSM of the zebrafish was not dissected in these parts, as it is very small. The PSM of the mouse is bigger and therefore it was cut in an anterior and a posterior part, where the anterior part contains the intermediate and the anterior compartment. For this approach the mouse oligo library from Sigma-Genosys was used. The candidate genes found in these screens had to be validated by another method. The in situ hybridisation was chosen, as this allows the localisation of the expression and validation. For the mouse PSM screen this method was not used. Here the literature was scanned to find the expression pattern of the genes found. Doing this validated two candidates from this approach. The genes found in the zebrafish screens were validated using the in situ hybridisation. Genes with an interesting expression pattern will be further characterised, i.e. their functional role in somitogenesis will be elucidated

Evaluating cell-mimicking giant unilamellar vesicles as simplified biological models using single molecule methods

One of the main drivers within the field of bottom-up synthetic biology is to develop artificial chemical machines, perhaps even living systems, that have programmable functionality. Bottom-up methods take an engineering approach to biology, with multiple downstream applications. Such applications that require very specific quantities of product e.g., antibodies, drug, or vaccines, necessitate the design of vesicles with total and precise control of a vesicle’s molecular machinery. There exists a wide range of toolkits to produce and engineer artificial cells with an ever-increasing array of functionality and capability. However, little attention has been given to the quality control of vesicle production; so, to date, there is a paucity of techniques that are able to measure their molecular constituents precisely upon formation and with absolute quantification. The work outlined here presents the development of a microfluidic-based methodology that is able to characterise artificial cells at the single vesicle level with single-molecule resolution. High content fluorescence microscopy was used to assess the properties of Giant unilamellar vesicles (GUVs) and their statistics at the population level. Since this technique is semi-quantitative, the relative concentration of protein across the GUV population was determined however the absolute concentration of protein within each GUV was not. To overcome this, a lab-on-a-chip approach was taken to determine the precise number of biomolecules within each GUV and across the population of GUVs. The pulldown-5 array single-molecule high-throughput (PASH) chip was developed and capable of determining the encapsulation efficiency of protein within GUVs produced by phase transfer of an inverted emulsion. Using this approach, it was possible to determine the variability of the encapsulation efficiency between GUVs across a population as well as between different populations while testing batch-to-batch variation. The encapsulation efficiency measured to be 11.4 ± 6.2% across all tested parameters. This has consequences for the use of GUVs as precise biological models as well as for their development in a variety of biotherapeutic applications. To determine whether GUVs with specific concentrations of biomolecules could be produced by phase transfer, changes in the concentration of the seeding materials and reagents were investigated; while inefficiencies in encapsulation could be overcome, the variation in concentration could not. The results presented in this thesis help to understand the limitation of the phase transfer technique applied to systems biology research. In biological systems, measuring changes in gene expression at the transcriptomic and proteomic level is important. When used to develop simplified models of protein expression, these results indicate that vesicles produced using phase transfer may only be confidently used to produce 10-fold changes in encapsulant protein. It is possible to distinguish different GUV populations with a precision down to a two-fold change in encapsulant protein but with significant population overlap. The understanding of the limitations of encapsulation efficiency provides insight into the potential relevance of such systems for a variety of therapeutic applications such as smart drug delivery.Open Acces

Signalling Pathways Involved in Adult Heart Formation Revealed by Gene Expression Profiling in Drosophila

Drosophila provides a powerful system for defining the complex genetic programs that drive organogenesis. Under control of the steroid hormone ecdysone, the adult heart in Drosophila forms during metamorphosis by a remodelling of the larval cardiac organ. Here, we evaluated the extent to which transcriptional signatures revealed by genomic approaches can provide new insights into the molecular pathways that underlie heart organogenesis. Whole-genome expression profiling at eight successive time-points covering adult heart formation revealed a highly dynamic temporal map of gene expression through 13 transcript clusters with distinct expression kinetics. A functional atlas of the transcriptome profile strikingly points to the genomic transcriptional response of the ecdysone cascade, and a sharp regulation of key components belonging to a few evolutionarily conserved signalling pathways. A reverse genetic analysis provided evidence that these specific signalling pathways are involved in discrete steps of adult heart formation. In particular, the Wnt signalling pathway is shown to participate in inflow tract and cardiomyocyte differentiation, while activation of the PDGF-VEGF pathway is required for cardiac valve formation. Thus, a detailed temporal map of gene expression can reveal signalling pathways responsible for specific developmental programs and provides here substantial grasp into heart formation

WNT-DEPENDENT REGENERATIVE FUNCTION IS INDUCED IN LEUKEMIA-INITIATING AC133BRIGHT CELLS

The Cancer Stem Cell model supported the notion that leukemia was initiated and maintained in vivo by a small fraction of leukemia-initiating cells (LICs). Previous studies have suggested the involvement of Wnt signaling pathway in Acute Myeloid Leukemia (AML) by the ability to sustain the development of LICs. A novel hematopoietic stem and progenitor cell marker, monoclonal antibody AC133, recognizes the CD34bright CD38- subset of human acute myeloid leukemia cells, suggesting that it may be an early marker for the LICs. During the first part of my phD program we previously evaluated the ability of leukemic AC133+ fraction, to perform engraftment following to xenotransplantation in immunodeficient mouse model Rag2-/-\u3b3c-/-. The results showed that the surface marker AC133 is able to enrich for the cell fraction that contains the LICs. In consideration of our previously reported data, derived from the expression profiling analysis performed in normal (n=10) and leukemic (n=33) human long-term reconstituting AC133+ cells, we revealed that the ligand-dependent Wnt signaling is induced in AML through a diffuse expression and release of WNT10B, a hematopoietic stem cells regenerative-associated molecule. In situ detection performed on bone marrow biopsies of AML patients, showed the activation of the Wnt pathway, through the concomitant presence of the ligand WNT10B and of the active dephosphorylated \u3b2-catenin form, suggesting an autocrine / paracrine-type ligand-dependent activation mechanism. In consideration of the link between hematopoietic regeneration and developmental signaling, we transplanted primary AC133+ AML A46 cells into developing zebrafish. This biosensor model revealed the formation of ectopic structures by activation of dorsal organizer markers that act downstream of the Wnt pathway. These results suggested that the misappropriating Wnt associated functions can promote pathological stem cell-like regeneration responsiveness. The analyses performed in situ retained information on the cellular localization, enabling determination of the activity status of individual cells and allowing the tumor environment view. Taking this issue into consideration, during the second part of my phD program, I set up the application of a new in situ method for localized detection and genotyping of individual transcripts directly in cells and tissues. The mRNA in situ detection technique is based on padlock probes ligation and target priming rolling circle amplification allowing the single nucleotide resolution in heterogenous tissues. The mRNA in situ detection performed on bone marrow biopsies derived from AML patients, showed a diffuse localization pattern of WNT10B molecule in the tissue. Conversely, only the AC133bright cell population shows the Wnt signaling activation signature represented by the cytoplasmatic accumulation and nuclear translocation of the active form of \u3b2-catenin. In spite of this, we previously evidenced that the regenerative function of WNT signaling pathway is defined by the up-regulation of WNT10B, WNT10A, WNT2B and WNT6 loci, we identified the WNT10B as a major locus associated with the regenerative function and over-expressed by all AML patients. By the molecular evaluation of the WNT10B transcript, we isolated an aberrant splicing variant (WNT10BIVS1), that identify Non Core-Binding Factor Leukemia (NCBFL) class and whose potential role is discussed. Moreover, we demonstrate that the function of "leukemia stem cell", present in the cell population enriched for the marker AC133bright, is strictly related to regenerative function associated with WNT signaling, defining the key role of WNT10B ligand as a specific molecular marker for leuchemogenesis. This thesis defines the new suitable approaches to characterize the leukemia-initiating cells (LICs) and suggest the role of WNT10B as a new suitable target for AML

ATMAD : robust image analysis for Automatic Tissue MicroArray De-arraying

International audienceBackground. Over the last two decades, an innovative technology called Tissue Microarray (TMA),which combines multi-tissue and DNA microarray concepts, has been widely used in the field ofhistology. It consists of a collection of several (up to 1000 or more) tissue samples that are assembledonto a single support – typically a glass slide – according to a design grid (array) layout, in order toallow multiplex analysis by treating numerous samples under identical and standardized conditions.However, during the TMA manufacturing process, the sample positions can be highly distorted fromthe design grid due to the imprecision when assembling tissue samples and the deformation of theembedding waxes. Consequently, these distortions may lead to severe errors of (histological) assayresults when the sample identities are mismatched between the design and its manufactured output.The development of a robust method for de-arraying TMA, which localizes and matches TMAsamples with their design grid, is therefore crucial to overcome the bottleneck of this prominenttechnology.Results. In this paper, we propose an Automatic, fast and robust TMA De-arraying (ATMAD)approach dedicated to images acquired with bright field and fluorescence microscopes (or scanners).First, tissue samples are localized in the large image by applying a locally adaptive thresholdingon the isotropic wavelet transform of the input TMA image. To reduce false detections, a parametricshape model is considered for segmenting ellipse-shaped objects at each detected position.Segmented objects that do not meet the size and the roundness criteria are discarded from thelist of tissue samples before being matched with the design grid. Sample matching is performed byestimating the TMA grid deformation under the thin-plate model. Finally, thanks to the estimateddeformation, the true tissue samples that were preliminary rejected in the early image processingstep are recognized by running a second segmentation step.Conclusions. We developed a novel de-arraying approach for TMA analysis. By combining waveletbaseddetection, active contour segmentation, and thin-plate spline interpolation, our approach isable to handle TMA images with high dynamic, poor signal-to-noise ratio, complex background andnon-linear deformation of TMA grid. In addition, the deformation estimation produces quantitativeinformation to asset the manufacturing quality of TMAs

Comparative transcriptomic analysis of Porphyromonas gingivalis biofilm and planktonic cells

<p>Abstract</p> <p>Background</p> <p><it>Porphyromonas gingivalis </it>in subgingival dental plaque, as part of a mature biofilm, has been strongly implicated in the onset and progression of chronic periodontitis. In this study using DNA microarray we compared the global gene expression of a <it>P. gingivalis </it>biofilm with that of its planktonic counterpart grown in the same continuous culture.</p> <p>Results</p> <p>Approximately 18% (377 genes, at 1.5 fold or more, <it>P</it>-value < 0.01) of the <it>P. gingivalis </it>genome was differentially expressed when the bacterium was grown as a biofilm. Genes that were down-regulated in biofilm cells, relative to planktonic cells, included those involved in cell envelope biogenesis, DNA replication, energy production and biosynthesis of cofactors, prosthetic groups and carriers. A number of genes encoding transport and binding proteins were up-regulated in <it>P. gingivalis </it>biofilm cells. Several genes predicted to encode proteins involved in signal transduction and transcriptional regulation were differentially regulated and may be important in the regulation of biofilm growth.</p> <p>Conclusion</p> <p>This study analyzing global gene expression provides insight into the adaptive response of <it>P. gingivalis </it>to biofilm growth, in particular showing a down regulation of genes involved in growth and metabolic activity.</p

DNA microarray experimental design and software based data normalization and analysis

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