The aim of the current project is the development, validation and introduction to karotyping practice of a novel DNA diagnostic technology called Multiplex Amplifiable Probe Hybridization ? DNA-based Chromosomal Analysis (MAPH-DCA), for the detection of structural chromosomal abnormalities in case of mental retardation. The technology allows to identify chromosomal abnormalities beyond the resolution of current cytogenetic techniques. During the first step of planned project, four sets of unique, well-localized, non-polymorphic, non-repetitive probes with different resolution covering 500, 1000, 2000, and 3000 loci, respectively, and all human chromosomes will be designed. These probes will be amplified by PCR, cloned and probe banks constructed. Secondly, MAPH-DCA probe mixtures will be made, where each probe contains universal primer sequences on both ends allowing for later amplification. DNA microarrays carrying the same probes but without universal primers will also be prepared. For karyotyping MAPH-DCA reactions will be performed and results quantified using rehybridization on DNA microarrays, which allows parallel analysis of all used loci. The result is a chromosomal analysis completed within 48 hours, which is much faster and cheaper than other cytogenetic methods currently applied. The new technology will be used in prenatal, postnatal and cancer chromosomal analysis as well as for various research purposes to solve the reasons of mental retardation and to understand the molecular basis of intelligence development.
   

Prion diseases are fatal neurodegenerative diseases, including Creutzfeldt-Jacob disease in humans, scrapie in sheep and bovine spongiform encephalophaty or "mad cow" disease, characterized by neuronal loss and accumulation of abnormal prion protein (PrPSc). The mechanisms leading to neurodegeneration and cell death are not known, but it has been recently shown that there is an elevated iron content and iron-induced oxidative stress in the brains of scrapie-infected animals. The aim of the current research project is to study iron regulation and oxidative stress in scrapie-infected neuroblastoma cells, and possible involvement of the changed iron metabolism in the pathogenesis of prion diseases. We are specifically interested in the characterization of the expression and regulation of NifS, an enzyme involved in the biogenesis of cellular iron-sulfur proteins, in scrapie infected cells since the dysregulation of iron-sulfur protein biosynthesis has been shown to lead to abnormal iron sensing in mitochondria and increased oxidative stress. The applicant has earlier cloned the nifs cDNA from human cells and shown that the single mRNA of nifs gives rise to at least two forms of NifS proteins localized to mitochondria and cytosol or nucleus. Studies in yeast cells have shown that the down-regulation of NifS expression results in abnormal high iron levels in mitochondria, lower iron-sulfur cluster biosynthesis and elevated oxidative stress in cells. Our preliminary studies have shown that scrapie-infected mouse N2a neuroblastoma cells express about 2-fold lower amounts of NifS protein as compared to the wild-type cells. Therefore, studying mechanisms leading to the dysregulation of NifS biosynthesis and iron metabolism in prion-infected neuroblastoma cells would help us to better understand the processes leading to the pathogenesis of prion diseases and could lead to the design of drugs.
   

Chronic inflammatory diseases that involve the airways are inevitably accompanied by restructuring processes in the airway mucosa, where the qualitative and quantitative changes in extracellular matrix (ECM) impair the perspective of restitution and result in a steep and irreversible decline in lung function. This process, termed as airway remodeling, involves a significant increase in expression of certain ECM proteins (tenascin, laminins, collagens). To date, no systematic comparative studies are available on the expression and regulation of tenascin (Tn), collagens, and laminins (Lns) in human airway structural cells at the level of both protein and mRNA that approaches to simulate an inflammation generated by oxidative stress (OS) or proinflammatory mediators (cysteinyl leukotrienes, histamine), as well as to investigate the effect of anti-inflammatory cytokines such as IL-10 or antioxidants. The main goals of the project are: 1) to define the expression level of the ECM proteins, which play a significant role in the process of human airway remodeling (Tn, Ln chains alpha-2, beta-2, and gamma-2, and collagen type III), in human airway structural cell lines (bronchial epithelial cells, fibroblasts, and smooth muscle cells); 2) to clarify the changes in the expression levels of these ECM proteins during the inflammatory processes by studying the effects of OS and inflammatory mediators (cysteinyl leukotrienes, histamine) and that of the growth factor TGF-beta to the synthesis of the above mentioned ECM constituents in the airway structural cells; 3) to study the ability of antioxidants and IL-10 to modulate the expression of the ECM proteins in pre-stimulated airway structural cells with the aim to find new aspects to hinder airway remodeling in inflammatory airway diseases. As a result, more information will be obtained about the possibilities of influencing the deposition of the ECM proteins in the process of airway remodeling, as well as about the issue of reversibility of airway fibrosis that, in turn, provides opportunity for evidence-based application of new treatment strategies.
   

Aim of the project: to develop novel, DNA-chip based methods for genetic diagnosis of inherited diseases that can be applied prenatally, neonatally or in preimplantation. As a result, it should be possible to screen all pregnant women, i.e. their fetuses for more frequent genetic diseases. The greater number of genetic diseases to be screened in the general population can effectively influence the quality of life on the level of a single family as well as the society as a whole. Successful in vitro fertilization requires careful selection of embryos to be transferred. Apart from aneuploidy, the embryo's ability to implant can be hampered by genomic activation that could be monitored using novel DNA array (chip) technology.
   

A new method for whole-genome genotyping is being developed, which will be applicable for screening of human populations. The results of this screening will be applied for association studies to find new disease genes. The method is based on enzymatic primer extension enabling to use ca 300.000 different SNP analysis on a single DNA chip. The main goal is the development of high-density DNA chip based on SNP-s by solving several fundamental scientific problems like the manufacturing and optimization of high-density DNA chip and development of soft- and hardware for chip analysis. First year results will be the development SNP-based DNA chip for single genes analysis and optimization of reaction conditions.
   

A number of polypeptide factors not included in structural ribosomal proteins are associated with the ribosome, and they can be separated by treating the "crude ribosome" fraction with stronger ionic forces ("ribosome wash"). The aim of the current grant application is to separate these eukaryotic proteins by 2-dimensional protein electrophoresis, to analyse them by mass spectrometric methods, and, using the obtained information, to isolate the ESTs, cDNAs and genes corresponding to the previously unknown proteins. Thereafter we concentrate on studying the function of these proteins, try to find connections with diseases and try to improve the existing picture about translation regulation.

PROTEINURIC DISEASES - EC 5 Framework Programme grant #QLG1-2000-00619, "Nephrin in Protenuric Diseases. Development of diagnostic, prognostic and treatment modalities"
   

European Science Foundation "APEX on DNA microarrays; applications in SNP analysis, mutation detection and DNA resequencing", part of ESF Programme on Integrated Approach for Functional Genomics."