Papers by Luiza Handschuh
BioMed Research International, 2015
High-throughput sequencing technologies, such as the Illumina Hi-seq, are powerful new tools for ... more High-throughput sequencing technologies, such as the Illumina Hi-seq, are powerful new tools for investigating a wide range of biological and medical problems. Massive and complex data sets produced by the sequencers create a need for development of statistical and computational methods that can tackle the analysis and management of data. The data normalization is one of the most crucial steps of data processing and this process must be carefully considered as it has a profound effect on the results of the analysis. In this work, we focus on a comprehensive comparison of five normalization methods related to sequencing depth, widely used for transcriptome sequencing (RNA-seq) data, and their impact on the results of gene expression analysis. Based on this study, we suggest a universal workflow that can be applied for the selection of the optimal normalization procedure for any particular data set. The described workflow includes calculation of the bias and variance values for the control genes, sensitivity and specificity of the methods, and classification errors as well as generation of the diagnostic plots. Combining the above information facilitates the selection of the most appropriate normalization method for the studied data sets and determines which methods can be used interchangeably.
Molecular Genetics and Genomics, 2015
Heat shock inhibits NF-κB signaling, yet the knowledge about its influence on the regulation of N... more Heat shock inhibits NF-κB signaling, yet the knowledge about its influence on the regulation of NF-κB-dependent genes is limited. Using genomic approaches, i.e., expression microarrays and ChIP-Seq, we aimed to establish a global picture for heat shock-mediated impact on the expression of genes regulated by TNFα cytokine. We found that 193 genes changed expression in human U-2 osteosarcoma cells stimulated with cytokine (including 77 genes with the κB motif in the proximal promoters). A large overlap between sets of genes modulated by cytokine or by heat shock was revealed (86 genes were similarly affected by both stimuli). Binding sites for heat shock-induced HSF1 were detected in regulatory regions of 1/3 of these genes. Furthermore, pre-treatment with heat shock affected the expression of 2/3 of cytokine-modulated genes. In the largest subset of co-affected genes, heat shock suppressed the cytokine-mediated activation (antagonistic effect, 83 genes), which genes were associated with the canonical functions of NF-κB signaling. However, subsets of co-activated and co-repressed genes were also revealed. Importantly, pre-treatment with heat shock resulted in the suppression of NF-κB binding in the promoters of the cytokine-upregulated genes, either antagonized or co-activated by both stimuli. In conclusion, we confirmed that heat shock inhibited activation of genes involved in the classical cytokine-mediated functions of NF-κB. On the other hand, genes involved in transcription regulation were over-represented in the subset of genes upregulated by both stimuli. This suggests the replacement of NF-κB-mediated regulation by heat shock-mediated regulation in the latter subset of genes, which may contribute to the robust response of cells to both stress conditions.
PloS one, 2014
Small non-coding RNAs (sncRNAs) are indispensable for proper germ cell development, emphasizing t... more Small non-coding RNAs (sncRNAs) are indispensable for proper germ cell development, emphasizing the need for greater elucidation of the mechanisms of germline development and regulation of this process by sncRNAs. We used deep sequencing to characterize three families of small non-coding RNAs (piRNAs, miRNAs, and tRFs) present in Sus scrofa gonads and focused on the small RNA fraction present in both male and female gonads. Although similar numbers of reads were obtained from both types of gonads, the number of unique RNA sequences in the ovaries was several times lower. Of the sequences detected in the testes, 2.6% of piRNAs, 9% of miRNAs, and 10% of tRFs were also present in the ovaries. Notably, the majority of the shared piRNAs mapped to ribosomal RNAs and were derived from clustered loci. In addition, the most abundant miRNAs present in the ovaries and testes are conserved and are involved in many biological processes such as the regulation of homeobox genes, the control of cel...
Acta biochimica Polonica, 2011
Two-color DNA microarrays are commonly used for the analysis of global gene expression. They prov... more Two-color DNA microarrays are commonly used for the analysis of global gene expression. They provide information on relative abundance of thousands of mRNAs. However, the generated data need to be normalized to minimize systematic variations so that biologically significant differences can be more easily identified. A large number of normalization procedures have been proposed and many softwares for microarray data analysis are available. Here, we have applied two normalization methods (median and loess) from two packages of microarray data analysis softwares. They were examined using a sample data set. We found that the number of genes identified as differentially expressed varied significantly depending on the method applied. The obtained results, i.e. lists of differentially expressed genes, were consistent only when we used median normalization methods. Loess normalization implemented in the two software packages provided less coherent and for some probes even contradictory resu...
Acta biochimica Polonica, 2007
PR-10 proteins (pathogensis-related), ubiquitous within the plant kingdom, are usually encoded by... more PR-10 proteins (pathogensis-related), ubiquitous within the plant kingdom, are usually encoded by multigene families. To date we have identified 10 homologous pr-10 genes in a yellow lupine cDNA library. Here, the structure and expression of two newly identified yellow lupine pr-10 genes (LlYpr10-2b and LlYpr10-2f) are presented. Many potential regulatory sites were found in both gene promoters including common ones as well as those unique for each gene. However, promoter deletion analysis in transgenic tobacco plants revealed similar patterns of reporter gene (gus) expression. Shortened fragments of both gene promoters studied caused high GUS activity in leaves (along vascular bundles), stamen stigma, anthers and pollen grains. When conjugated with longer LlYpr-10.2 promoter fragments, GUS was additionally present in petal edges. Only a long fragment of the LlYpr10-2b gene promoter caused GUS expression in the stem. In yellow lupine the pr-10.2 genes are present in all studied orga...
Remote Instrumentation and Virtual Laboratories, 2010
Nowadays, there is no science domain that does not use specialized software, on-line tools, and c... more Nowadays, there is no science domain that does not use specialized software, on-line tools, and computational resources. Genomics is a new branch of science that developed rapidly in the last decade. As the genome research is very complex it must be supported by professional informatics. In a microarray field the following steps cannot be performed without computational work: design of probes, quantitative analysis of hybridization results, post processing, and finally data storage and management. Here, the general aspects of Virtual Laboratory systems are presented, together with perspectives of their implementation in genomics in order to automate and facilitate this area of research.
The International Journal of Biochemistry & Cell Biology, 2014
Heat Shock Factor 1 (HSF1) is the primary transcription factor responsible for the response to ce... more Heat Shock Factor 1 (HSF1) is the primary transcription factor responsible for the response to cellular stress, while HSF2 becomes activated during development and differentiation, including spermatogenesis. Although both factors are indispensable for proper spermatogenesis, activation of HSF1 by heat shock initiates apoptosis of spermatogenic cells leading to infertility of males. To characterize mechanisms assisting such heat induced apoptosis we studied how HSF1 and HSF2 cooperate during the heat shock response. For this purpose we used chromatin immunoprecipitation and the proximity ligation approaches. We looked for co-occupation of binding sites by HSF1 and HSF2 in untreated (32 • C) or heat shocked (at 38 • C or 43 • C) spermatocytes, which are cells the most sensitive to hyperthermia. At the physiological temperature or after mild hyperthermia at 38 • C, the sharing of binding sites for both HSFs was observed mainly in promoters of Hsp genes and other stress-related genes. Strong hyperthermia at 43 • C resulted in an increased binding of HSF1 and releasing of HSF2, hence co-occupation of promoter regions was not detected any more. The close proximity of HSF1 and HSF2 (and/or existence of HSF1/HSF2 complexes) was frequent at the physiological temperature. Temperature elevation resulted in a decreased number of such complexes and they were barely detected after strong hyperthermia at 43 • C. We have concluded that at the physiological temperature HSF1 and HSF2 cooperate in spermatogenic cells. However, temperature elevation causes remodeling of chromatin binding and interactions between HSFs are disrupted. This potentially affects the regulation of stress response and contributes to the heat sensitivity of these cells.
In this work the basic aspects of microarray data normalization are presented. Due to high level ... more In this work the basic aspects of microarray data normalization are presented. Due to high level of complexity of microarray experiments their results are usually distorted. The normalization process allows to eliminate bias and to make comparison between distinct microarrays reliable. The main types of normalization of two-color microarray data are reviewed and presented using R and Bioconductor tools.
Computational Methods in Science and Technology, 2010
... Clustering with the pvclust package is used for assessing the uncertainty in the hierarchical... more ... Clustering with the pvclust package is used for assessing the uncertainty in the hierarchical cluster analysis. ... What is important, pvclust provides two types of p-values: AU (Approximately Unbiased) p-value and BP (Bootstrap Probability) value. ...
Computational Methods in Science and Technology, 2009
Medical Oncology, 2013
Development of modern proteomic methods in recent years has opened also new perspectives in the i... more Development of modern proteomic methods in recent years has opened also new perspectives in the identification of new biomarkers which ensure more effective diagnosis, treatment monitoring and prediction of therapeutic outcome. We evaluated usefulness of comparative proteomics (MALDI-TOF) in two subtypes of acute myeloid leukemia (AML), M1 and M2, according to FAB classification. The bone marrow or blood cell proteomes were examined in 33 newly diagnosed patients before ''3 ? 7'' induction therapy, after treatment and when the disease relapsed. We found that bone marrow and peripheral mononuclear cells from healthy volunteers revealed a number of quantitative and qualitative differences between the two proteomes, reflecting differences in the maturational status of normal cells. Such differences were not detected in our AML M1/M2 patients. Additionally, we found 9 proteins, which are potential biomarkers differentiating between the AML patients and healthy volunteers.
International Journal of Molecular Medicine, 2013
DNA microarrays, which are among the most popular genomic tools, are widely applied in biology an... more DNA microarrays, which are among the most popular genomic tools, are widely applied in biology and medicine. Boutique arrays, which are small, spotted, dedicated microarrays, constitute an inexpensive alternative to whole-genome screening methods. The data extracted from each microarray-based experiment must be transformed and processed prior to further analysis to eliminate any technical bias. The normalization of the data is the most crucial step of microarray data pre-processing and this process must be carefully considered as it has a profound effect on the results of the analysis. Several normalization algorithms have been developed and implemented in data analysis software packages. However, most of these methods were designed for whole-genome analysis. In this study, we tested 13 normalization strategies (ten for double-channel data and three for single-channel data) available on R Bioconductor and compared their effectiveness in the normalization of four boutique array datasets. The results revealed that boutique arrays can be successfully normalized using standard methods, but not every method is suitable for each dataset. We also suggest a universal seven-step workflow that can be applied for the selection of the optimal normalization procedure for any boutique array dataset. The described workflow enables the evaluation of the investigated normaliza-tion methods based on the bias and variance values for the control probes, a differential expression analysis and a receiver operating characteristic curve analysis. The analysis of each component results in a separate ranking of the normalization methods. A combination of the ranks obtained from all the normalization procedures facilitates the selection of the most appropriate normalization method for the studied dataset and determines which methods can be used interchangeably.
Journal of Proteomics, 2012
Acute myeloid leukemia (AML) is a severe, rapidly progressing disease triggered by blocking granu... more Acute myeloid leukemia (AML) is a severe, rapidly progressing disease triggered by blocking granulocyte or monocyte differentiation and maturation. Because of its heterogeneity, AML is divided into a number of subtypes. Unfortunately, so far very few correlations have been found between AML classification and its clinical course or patient response to treatment.
Acta Crystallographica Section D Biological Crystallography, 2005
Pathogenesis-related (PR) proteins of class 10 are abundant in higher plants. Some of these prote... more Pathogenesis-related (PR) proteins of class 10 are abundant in higher plants. Some of these proteins are induced under stress conditions as part of the plant defence mechanism. Other homologues are developmentally regulated and their expression varies in different plant organs. The PR-10 proteins are encoded by multigene families, have a weight of about 17 kDa and are found in the cytosol. In yellow lupin, nine different homologues have been identified and divided into two subclasses, LlPR-10.1 and LlPR-10.2. Within each subclass the sequence identity is about 75-91%, while across the subclasses it is only 59-60%. Here, the crystal structure of a yellow lupin PR-10 protein from the second subclass, LlPR-10.2A, is presented. The structure was solved by molecular replacement and refined to R = 0.205 using 1.9 A resolution data. The general fold of LlPR-10.2A resembles that of the other PR-10 proteins and consists of a long C-terminal alpha-helix surrounded by a seven-stranded antiparallel beta-sheet, with two shorter alpha-helices located between strands beta1 and beta2. The most variable part of the structure, the C-terminal helix, is strongly kinked towards the beta-sheet core in both LlPR-10.2A molecules present in the asymmetric unit. This unexpected feature reduces the size of the hydrophobic cavity observed in other PR-10 proteins that is reported to be the ligand-binding site. As in other PR-10 structures, a surface loop located near the entrance to the cavity shows very high structural conservation and stability despite the high glycine content in its sequence.
Acta Crystallographica Section A Foundations of Crystallography, 2006
The phylogenetic relationships between Tc1 transposable elements have previously been reported fo... more The phylogenetic relationships between Tc1 transposable elements have previously been reported for the genomes of some fish species. However, research in this field has been hindered by the low number of fish genome sequences available in databases. The application of a DNA microarray as a universal tool for Tc1 transposon sequence analysis in fish genomes is described here. A prototype oligonucleotide microarray was constructed and used to compare samples of genomic DNA isolated from selected fish species. These results, combined with earlier reported molecular analysis of Tc1 showed the usefulness of DNA microarray in the screening of transposon sequences.
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Papers by Luiza Handschuh