Project description:Anthocyanins and flavonols are natural compounds that accumulate preferentially in grapevine flowers and fruits. They are among the most abundant flavonoids and play a very important role in grape and wine quality. In particular, they confer and stabilize colour and contribute to other organoleptic characteristics of the final product. Their complex profile in terms of concentration and relative abundance varies among cultivars and makes wine typicity. The synthesis of these compounds is mainly regulated at transcriptional level. Flavonoids accumulate at specific stages and in specific tissues during flower and berry development, as a consequence of the timely expression of the genes necessary for their synthesis. Although the general flavonoid pathway has been genetically and biochemically elucidated and the main determinants of colour have been identified, the molecular reasons of the fine variation among grape cultivars are still not completely understood. To shed light on this issue, extreme genotypes of a segregating population derived from the cross Syrah x Pinot Noir were characterized at transcriptional level. The transcriptome analysis along three berry developmental stages of these genotypes has allowed the identification of a large set of transcripts differentially modulated between the two groups. A population derived from M-bM-^@M-^XSyrahM-bM-^@M-^Y x M-bM-^@M-^XPinot NoirM-bM-^@M-^Y consisting of 170 F1 individuals plus the parental lines was characterized for the content of flavonols and anthocyanins in the skins of mature berries (38E-L; 18M-BM-0Brix). Based on the biochemical data, 4 high- and 4 low-flavonol producers (HFPs and LFPs: F1 16, F1 56, F1 63, F1 223 and F1 64, F1 256, F1 260, PN), two of which having also either very high or very low anthocyanin content (HAPs and LAPs: F1 63, F1 223 and F1 256, F1 260), were selected for gene expression analysis. A representative sample for each individual (one biological replicate) was collected at three berry developmental stages (hard green berry (33E-L, PV), veraison (35E-L, 50% coloured berries, VER) and maturity (38E-L, 18M-BM-0Brix, MAT)) during the 2007 season.

Project description:The classical sacrococcygeal chordoma tumor presents with a typical morphology of lobulated myxoid tumor tissue with cords, strands and nests of tumor cells consisting of small non-vacuolated cells, intermediate cells with a wide range of vacuolization and large heavily vacuolated (physaliferous) cells. Because of its rare incidence, lack of suited model systems and technical limitations analysis was only performed on bulk tumor mass neglecting its heterogeneous composition. We aimed at elucidating the differences between small non-vacuolated and large physaliferous cells on the genomic and transcriptomic level. Secondly, we intended to clarify whether the observed cell types are derived from genetically distinct clones or rather represent different phenotypes. Using the chordoma cell line MUG-Chor1 we monitored morphological changes via time lapse experiments. We isolated pure fractions of each phenotype by means of laser microdissection or micromanipulation allowing phenotype-specific analysis. Pools of 100 cells each were genetically profiled after whole genome amplification by array comparative genomic hybridization. For expression analysis 20 cells each were subjected to whole transcriptom amplification, forwarded to RNA microarray analysis and qRT-PCR. Time lapse analysis unveiled small non-vacuolated cells to develop into large physaliferous cells via intermediate cells containing an increasing amount of vacuoles. Furthermore, we showed small and large physaliferous cells to proliferate at the same rate but intermediate cells to be the most proliferating cell phenotype. Small non-vacuolated and large physaliferous cells showed identical copy number variations. Despite their obvious morphological disparities we detected only modest changes in over all gene expression. However, verification of candidate genes yielded significant up-regulation of ALG11 (700-fold), PPP2CB (18.6-fold), and UCHL3 (18.7-fold) in large physaliferous cells. Of two different cell types (large and small MUG-Chor1 cells) we analysed each in triplicates. In total 6 cell pools were analysed.

Project description:Thermal injury incites inflammatory responses that often transcend the local environment and lead to structural deficiencies in skin that give way to scar formation. We hypothesized that extensive perturbations within burned skin following thermal insult and during subsequent events of wound repair induce vast alterations in gene expression that likely serve as a wound and systemic healing deterrent. A high-throughput microarray experiment was designed to analyze genetic expression patterns and identify potential genes to target for therapeutic augmentation or silencing. The study compares gene expression from burn wound margins at various times following thermal injury to expression observed in normal skin. Utilizing this design, we report that the totality of gene expression alterations is indeed enormous. Further, we observed that the differential expression of many inflammatory and immune response genes appear to be continually up-regulated in burn wound margins seven days or more after initial thermal insult. As it is well established that the inflammatory process must abate for wound healing to proceed, the finding of ongoing local inflammation is cause for further investigation. To our knowledge, this is the first report of the gene expression alterations induced by thermal injury of human skin. As such, it provides a wealth of data to mine with the ultimate goal of better understanding the local pathophysiologic changes at the site of thermal injury that not only affect wound healing capacity, but may also contribute to systemic derangements within the burn patient. Keywords: time course, disease state analysis The study compares gene expression from burn wound margins at various times following thermal injury to expression observed in normal skin. All skin specimens were obtained in the operating room within minutes of being removed from the patient. Burn specimens were taken from wound margins. Harvested tissue at the burn wound margin maximized the capture of viable cells from the multiple lineages important to the healing process and minimized the inclusion of non-viable cells destroyed by full-thickness injury. After isolation, RNA samples were pooled equally by mass as to contain RNA from 5 tissue specimens for each array replicate.

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OCI Ly3 SP vs. nonSP cells

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