This dissertation was aimed to describe significant changes on the level of messenger RNA upon the concentration modification of a feed component in animal diets. In addition, the gene expression analysis with high-density microarrays was established with differently fed animals. The book describes the detailed setup of the experiment from the animal diet over the tissue and sample preparation to the analysis of the gene expression data and the detection of regulatory transcription factors. The thesis gives important tips and tricks for genomics in animal nutrition with living animals. For the selenium experiment Sel-Plex®(Alltech Inc.), a selenized yeast, was supplemented to the broiler diet. Subsequently to the gene expression analysis in the liver, several altered protein activities and fatty acid concentration changes could be proven. In the second and third project alterations on the gene expression level could be shown upon the addition of mycotoxin-contaminated wheat and the myctoxin-binder Mycosorb® (Alltech Inc.). The differently expressed genes were assigned to previous results and several causal genes and transcription factors of detrimental effects were identified.
Fasciolosis is a liver disease caused by Fasciola hepatica or F. gigantica that causes significant economic loss in cattles, other animal species and man. The appearance of Fasciola hepatica populations that are resistant to common flukicidal drugs make a need for the development of anti-liver fluke vaccines. Though we selected a target Fasciola gene that expresses (rFhp) protein, in which its native form shows significant induction to lymphoproliferative response of Peripheral blood mononuclear cells. Then we amplified the gene through Polymerase chain reaction process using ?gt11 specific primers. This gene was cloned through TOPO (TA) cloning vector in XL10-Gold competent cells, then positive colonies that contain rTOPO were used for the excision of the gene to be expressed in expression vectors. ECORI digestions of the purified plasmids were done to detect the existence of the target insert. The results showed the excision of Fh?400 from recombinant PQE32 vector and its size determined at 500bp. Expression and screening of small cultures was done and a 6xHis-tagged protein was purified & stained on SDS-PAGE that appeared at about 14.5 kDa.
Although gene expression studies have been performed on Escherichia coli using starvation models, there has been no study of long-term changes in gene expression due to the difficulty of isolating RNA of sufficient yield and quality at late time-points. Furthermore, there is evidence that these starvation models are not physiologically relevant compared to aging cells in batch culture. This document describes the development of a methodology for isolating RNA from aging and starving bacterial cells using organic extraction. Reverse-transcription quantitative PCR is then utilized to measure changes in gene expression for the purpose of corroborating previous microarray studies. Suggestions for isolating RNA from even older cultures are discussed as well as the limits of statistical analysis of gene expression in aging cell populations. A literature review of the small number of studies attempting to analyze starvation-induced and temporal gene expression in bacteria is also included.
Molecular biology of the placenta is a fascinating research subject to investigate the mechanisms underlying fetal growth abnormalities and developmental disorders. Analysis of Placental Gene Expression Profile in Abnormal Fetal Growth provides a new insight into the gene dysregulation and pathway changes in the placenta of pregnancies affected by fetal growth restriction and macrosomia. This book reviews the physiology of the placenta and its divers roles in maintaining a healthy fetal development. There is a summary of the aetiology of abnormal fetal growth and its adverse effects on the future of infants. The main focus of this book is presenting the findings achieved by microarray gene expression analysis in the placentas collected from growth restricted and macrosomic newborns, introducing some candidate genes for screening purposes. In addition, molecular function of dysregulated genes and affected biological pathways have been described in detail.
The current work aimed at achieving a better understanding about the effect of stress factors on plant gene expression. In the first part a search for genes up-regulated in response to drought/osmotic stress was carried out by using a drought-tolerant wheat (Triticum aestivum L.) cultivar. Through a subtraction molecular approach differentially expressed genes were selected that could be further used in strategies for improving drought tolerance in crops. The second part of the work was focused on the regulation of alfalfa (Medicago sativa L.) B-type cyclin-dependent kinase (Medsa;CDKB2;1). A fragment from the kinase upstream gene region was cloned and shown to be sufficient to assure a cell cycle-dependent and G2/M-phase-specific gene expression, similarly to the endogenous CDKB2;1 kinase. Wounding and ethylene activated CDKB2;1 expression in a non cell cycle-dependent manner, which revealed the complex integration of a cell cycle phase-specific gene into the wound stress response. The study contributes to elucidation of the mechanism of environmental impact on plant development through transcription.
Maize is an important cereal crop belonging to family, Poaceae. It is one of the staple food crop and widely cultivated crop throughout the world. It is the only cereal which can be used as food at various stages of its plant development. The potential of heterosis is just beginning to be exploited in developing countries through expansion of hybrid seeds. It has the highest potential of per day carbohydrate productivity. Thus, the father of green revolution, the renowned Nobel Laureate, Dr. Norman E. Borlaug, believes that “after the last two decades saw the revolution in rice and wheat, the next few decades will be known as maize era”. Shull (1908 and 1911) gave the original concept for production and growing of single cross hybrids. The recent trends even in the developing and under developed countries single cross hybrids are more popular due to their higher yield under favourable environment and uniformity in expression. Hence, there is a greater scope for the exploitation of heterosis through single cross hybrids, than double cross hybrids.
Nowadays, parthenogenesis has attracted wide attention due to the potential of deriving pluripotent lines. Embryonic stem cells (ESCs), typically derived from the inner cell mass of the mammalian blastocyst, are in fact of fundamental value for developmental research and in both cell and tissue replacement therapy. In this context, a better understanding of the molecular events occurring during the various developmental stages is essential. This book address the in vitro developmental competence of preimplantation ovine embryos produced by in vitro fertilization (IVF) and Parthenogenetic activation (PA) and the expression pattern of panel of genes related to antioxidant protection (Peroxiredoxins- PRDX 1-6), pluripotency (OCT 4 and NANOG) and stress response (HSP 90?) in both immature and mature oocytes and at various stages of preimplantation ovine embryos produced by in vitro fertilization and Parthenogenetic activation.
Aflatoxins (AFs) are naturally occurring secondary metabolites produced principally by Aspergillus flavus and Aspergillus parasiticus in food and feed commodities worldwide. Contaminations of compound feeds by AFs do not only affect animal health, but the economy as well. It is for this purpose that a study was carried out to establish the quality of South African feeds with respect to AF-producing fungi, establish a correlation between levels of AFs and determinant gene (nor-1) responsible for producing these toxins.
Large number of primordial follicles present in mammalian ovaries at birth, grow during the course of reproductive life primarily in size but also in complexity of the follicular structure. Among all the primordial follicles present during birth, only 1% of follicles reach the stage of ovulation, while the rest undergo atresia. In order to rescue these ovarian primordial follicles that undergo atresia, in-vitro culture of these follicles to ovulatory stage is being attempted in different species. But the fertilization rates of oocytes obtained from the cultured preantral follicles were not satisfactory probably due to the synthesis of insufficient quantities of regulatory factors and / or paracrine signals. In order to verify this hypothesis, various gene expression pattern studies have been attempted in in-vivo and in-vitro cultured preantral and early antral follicles in sheep.
Breast cancer is the most common type of cancer among females, both in incidence and death. As meaningful biological understanding of the disease is confounded by the existence of various molecular groups and sub-groups, the challenge for targeted drug development may lie in understanding the molecular mechanisms of various sub-groups in breast cancer. This book describes the gene expression profiling approach for deeper understanding of the dynamics of breast cancer progression and prognosis.
The Psychobiology of Gene Expression – Neuroscience & Neurogenesis in Hypnosis & the Healing Arts
Follicular lymphoma (FL) represents the most common low-grade Non-Hodgkin''s lymphoma. FLs are of B cell origin, generally incurable, and frequently transform to high-grade diffuse large B cell lymphoma (DLBCL). Transformation is generally correlated with poor prognosis and the molecular mechanisms involved are largely unknown. This book gives an introduction to the field of carcinogenesis with focus on the events leading to Non-Hodgkin''s lymphoma and presents the results of a study aimed at identifying novel genes differentially expressed in patient-matched biopsies of FL and DLBCL to increase the understanding of disease progression. A general discussion of the methods applied, including the subtraction methodology cDNA Representational Difference Analysis (cDNA RDA), and the findings from this study are included. This book should be of interest to anyone who wants to get an understanding of the processes causing normal cells to transform into cancer cells and how studies of gene expression can highlight these issues.
In the post-genomic era, one of the most important goals for the community of plant biologists is to take full advantage of the knowledge generated by the Arabidopsis thaliana genome project, and to employ state-of-the-art functional genomics techniques to assign function to each gene. This will be achieved through a complete understanding of what all cellular components do, and how they interact with one another to produce a phenotype. This book discusses the findings about the function of proteins belonging to the Arabidopsis SABATH family, by applying a combination of genomics tools, including genome-wide expression analysis and gas-chromatography coupled with mass spectrometry-based metabolite profiling. These results combined with available biochemical information, provide a better understanding of the physiological role of SABATH methyltransferases, further insights of secondary plant metabolism and deeper knowledge of the consequences of modulating the expression of SABATH methyltransferases, both at the gene expression and metabolite levels.
Gene expression time series and multi-dose experiments have gained growing importance in the field of biostatistics throughout the beginning of the 21st century. In general, gene expression time series challenge the bioinformatician with few time points, a small number of replicates per time point and a huge number of genes in parallel consideration. The analysis of the time course data on a level of a priori defined gene sets is rather neglected in the literature. Although, this is common practice in case of simpler experimental designs (e.g. 2 groups). Three algorithms are introduced to estimate a gene set activation profile, which denoted significant enrichment with up (+), down (-) or no (o) differential expression for each considered gene set per time point (or dose) in the experiment. A subsequent smoothing step improves the reliability of the resulting FDR adjusted profiles. Four freely available time series experiments are used for the application on GO, KEGG, BioCarta, Reactome and BioCyc gene sets. Furthermore, two large and complex simulation studies were conducted to evaluate the smoothing methods and compare the activation profile algorithms.
Most eukaryotic protein-coding genes are split into exons and introns, and introns need to be spliced for the production of mature mRNA by pre-mRNA splicing. Pre-mRNA splicing is very important for eukaryotic gene expression, because it is not only a key step in producing mature mRNA, but can also affect transcription and translation. The purpose of this study is to investigate the relationship between gene expression and splicing efficiency since the relationship has not been studied systematically from a bioinformatic approach. In this thesis research, we focus on the question of how gene expression would constrain the evolution of three principal splicing signals: the donor splice site, the acceptor splice site, and the branchpoint sequence (BPS).
Gene therapy potentially represents one of the most important developments to occur in the field of medicine. Gene therapy is an experimental technique that uses genes to treat or prevent diseases. This technique may allow doctors to treat a disorder by inserting a gene into a patient’s cells instead of using drugs or surgery. As gene therapy has moved from the laboratory into the clinic, several issues have emerged as central to the development of this technology: gene identification, gene expression and gene delivery.
Polygalacturonase one of the hydrolytic enzymes involved in the degradation of pectin and cellulose content of the growing tissues in many horticultural plants. CaPG gene is responsible for the production of polygalacturonase in capsicum cultivars, under laid project is associated with the cloning of CaPG gene and construction of many appropriate vectors carries the aim gene in bacteria and other interested commodities. Expression vector termed as pVBG2307 is unique and novel vector offering more broader cloning site with greater than twenty restriction endonucleases.
DNA microarray technology has now possible to simultaneously monitor the expression levels of thousands of genes during important biological processes and across collections of related samples. However, the large number of genes and the complexity of biological networks greatly increase the challenges of comprehending and interpreting the resulting mass of data, which often consists of different types of measurements. An important step toward addressing this challenge is the use of robust clustering techniques, which is essential in the data mining process to reveal natural structures and identify interesting patterns in the underlying data in Bioinformatics.This book greatly attracts a broad range of scientists who are interested in DNA microarray data analysis, because it provides a practical method critically necessary for gene expression data clustering and discovery of some genes responsible for cancer disease. Currently, breast cancer is the most common type of cancer and often causes death among women in the world. The presentation of this book is easy and intelligible by the beginner and help who are undertaking researcher on gene expression data analysis in Bioinformatics
Most of cells in a given body have the same base template of DNA. This fact begs the question: How do different cell types come into existence? Years of research have shown that the answer to this question is differential gene expression. Based on innate tendencies, or environmental effects to which a cell or group of cells is subject to, it is able to "turn up" or "turn down" the expression of genes in a systematic manner to yield a phenotype that is amenable to its intended function. In this work, we examine the effects of chromatin on changes in the patterns of gene expression on a multi-cellular scale, and also examine gene expression patterns on the single cell level in an array of different cell types. We take a different approach to the representation of the unique patterns of gene expression found within each of the cell(s) examined, making the identification of a given cell type more facile upon visual examination
The present study was designed and carried out to investigate the role of connective tissue growth factor (CTGF) during eye development of CD1 mice at pre- and postnatal stage by using different technique as Real time PCr and Immunohistochemistry In conclusion CTGF is essential for life and absence of this gene lead to the death, Knockout homozygous CTGF -/- severe from a lot of abnormalties during prenatal stages until death. The over-expression CTGF transgenic mice with high concentration in line 6 showed a disappearance of the ciliary body processes, while the moderate over-expression CTGF of line 5 and low over-expression of line 1 showed abnormalities in the trabecular meshwork on the levels of immunohistochemistry that was accompanied with an increase in intraocular pressure and degeneration of optic nerve axons. So, we suggest that the over-expression of CTGF can cause the primary open angle glaucoma. The mice from line 1 and 5 can use as a model for primary open angle glaucoma diseases