No description available.

<p>Thrombospondin-1 (TSP-1) is a multifunctional extracellular matrix glycoprotein that is released from platelets α-granule to regulate angiogenesis process. TSP-1 is well-known as an inhibitory factor of angiogenesis that binds to angiogenesis stimulating factors, for example fibroblast growth factor 2 (FGF-2), vascular endothelial growth factor (VEGF) and hepatocyte growth factor/scatter factor (HGF/SF), to inhibit angiogenesis. We have cloned TSP-1 domains separately to allow studying of their function and effect on proliferation of human umbilical vein endothelial cells (HUVECs). We used an <em>Escherichia coli</em> expressionsvektor including poly histidin-tags and lac-promoter for induction of the seven successfully cloned domains by IPTG and arabinose. Our result shows that we have very low expression and induction of our protein in the <em>E.coli</em> by IPTG and arabinose, which is most likely due to complications associated with expressing a human protein in a prokaryotic system.</p>

<p>Metagenomics has been successfully used to discover novel enzymes from uncultured microorganisms in the environment. In this study, metagenomic DNA from a Malawian hot spring soil sample was used to construct a fosmid library. This metagenomic library comprised of more than 10000 clones with an average insert size of 30 kb, representing more than 3.0 x 108 bp of metagenomic DNA (equivalent to approximately 100 bacterial genomes). The library was screened for cellulase activity using a Congo red plate assay to detect zones of carboxymethylcellulose hydrolysis. This yielded 15 positive fosmid clones, of which five were further characterised for activity and thermostability using the 3, 5-dinitrosalicylic assay. Two of the five fosmids (XP008C2 and XP026G5) were selected for DNA pyrosequencing. The full sequence of the XP008C2 (29800bp) fosmid insert is presented in this study and genes thereon were chosen for further study.</p>

Thrombospondin-1 (TSP-1) is a multifunctional extracellular matrix glycoprotein that is released from platelets α-granule to regulate angiogenesis process. TSP-1 is well-known as an inhibitory factor of angiogenesis that binds to angiogenesis stimulating factors, for example fibroblast growth factor 2 (FGF-2), vascular endothelial growth factor (VEGF) and hepatocyte growth factor/scatter factor (HGF/SF), to inhibit angiogenesis. We have cloned TSP-1 domains separately to allow studying of their function and effect on proliferation of human umbilical vein endothelial cells (HUVECs). We used an Escherichia coli expressionsvektor including poly histidin-tags and lac-promoter for induction of the seven successfully cloned domains by IPTG and arabinose. Our result shows that we have very low expression and induction of our protein in the E.coli by IPTG and arabinose, which is most likely due to complications associated with expressing a human protein in a prokaryotic system.

The North American elk or wapiti is an indigenous species to Canada. Understanding of the reproductive physiology of elk is limited, as little research has been conducted in this field as compared to domestic farmed species. In order to make available the tools to study reproductive physiology of the elk this thesis describes the cloning and expression of elk pituitary glycoprotein hormone cDNAs. The common gonadotropin a-subunit, and FSH, LH and TSH b-subunit elk cDNAs were amplified by reverse transcription and polymerase chain reaction (RT-PCR). There was a high degree of nucleotide similarity between the elk a and b subunits when compared with reported sequences from other species. The cDNAs for the pituitary glycoprotein hormone genes were used as probes to investigate seasonal expression in the female elk pituitary gland. Steady state levels of the common a-subunit mRNA was observed regardless of the reproductive season, but a significant increase in expression occurred during the breeding season. The FSH and LH b-subunit genes were expressed at low levels in pituitary glands of animals during presumed anestrous and pregnancy, but levels considerably increased during estrus. In contrast, levels of TSH b-subunit mRNA were similar regardless of the reproductive status. The FSH cDNAs were also transfected into a Chinese hamster ovary (CHO) mammalian expression system, aimed at the production of recombinant elk FSH. Transfected CHO cell lines were screened for expression of a- and FSH b-subunit mRNA by Northern blot. Activity of FSH was equivalent to ~100 mIU/ml of recombinant human FSH (Gonal-FTM), identified by FSH receptor signaling in an in vitro cell based assay. In conclusion, this work represents an advance towards understanding the molecular basis of seasonal reproduction in elk. This information and the availability of elk recombinant FSH will be useful for the application of advanced reproductive technologies required for the rapid expansion of healthy, disease resistant, and genetically superior animals, which are important for domestic production and wildlife management.

The North American elk or wapiti is an indigenous species to Canada. Understanding of the reproductive physiology of elk is limited, as little research has been conducted in this field as compared to domestic farmed species. In order to make available the tools to study reproductive physiology of the elk this thesis describes the cloning and expression of elk pituitary glycoprotein hormone cDNAs. The common gonadotropin a-subunit, and FSH, LH and TSH b-subunit elk cDNAs were amplified by reverse transcription and polymerase chain reaction (RT-PCR). There was a high degree of nucleotide similarity between the elk a and b subunits when compared with reported sequences from other species. The cDNAs for the pituitary glycoprotein hormone genes were used as probes to investigate seasonal expression in the female elk pituitary gland. Steady state levels of the common a-subunit mRNA was observed regardless of the reproductive season, but a significant increase in expression occurred during the breeding season. The FSH and LH b-subunit genes were expressed at low levels in pituitary glands of animals during presumed anestrous and pregnancy, but levels considerably increased during estrus. In contrast, levels of TSH b-subunit mRNA were similar regardless of the reproductive status. The FSH cDNAs were also transfected into a Chinese hamster ovary (CHO) mammalian expression system, aimed at the production of recombinant elk FSH. Transfected CHO cell lines were screened for expression of a- and FSH b-subunit mRNA by Northern blot. Activity of FSH was equivalent to ~100 mIU/ml of recombinant human FSH (Gonal-FTM), identified by FSH receptor signaling in an in vitro cell based assay. In conclusion, this work represents an advance towards understanding the molecular basis of seasonal reproduction in elk. This information and the availability of elk recombinant FSH will be useful for the application of advanced reproductive technologies required for the rapid expansion of healthy, disease resistant, and genetically superior animals, which are important for domestic production and wildlife management.

The advent of somatic cell nuclear transfer in cattle has provided the opportunity for researchers to generate genetically identical animals as well as animals that possess precise genetic modifications for agriculture and biomedical purposes. However, in spite of the revolutionary impact this technology presents, problems remain which hinder the production of healthy animals on a consistent basis. Research on cloned mice implicates improper reprogramming of epigenetic modifications and genomic imprinting for the low pregnancy rates and high incidence of abnormalities that are manifested in cloned animals; however, a systematic and comprehensive analysis of nuclear reprogramming in cloned cattle remains undone. The purpose of this research is to assess and characterize the patterns of genomic imprinting in normal and nuclear transfer derived bovine fetuses. To facilitate the identification of imprinted genes in the bovine, a Bos gaurus/Bos taurus interspecies model has been incorporated to maximize the genetic heterozygosity that exists between the alleles of putative imprinted genes for allelic discrimination and parental inheritance. The sequence of twenty-six genes, previously reported as imprinted in mice and humans, was analyzed in Bos gaurus (Gaur) and Bos taurus (Angus) cattle for the presence of single nucleotide polymorphisms (SNP). SNPs were detected in the Gene trap locus 2 (GTL2), Insulin like growth factor 2 (IGF2), Wilms tumor 1 (WT1) and the X chromosome inactivation specific transcript (XIST). Allelic expression analysis in interspecies hybrids indicated maternal genomic imprinting at the IGF2 and XIST loci, paternal genomic imprinting at the GTL2 locus and no imprinting at the WT1 locus. Analysis in cloned hybrids indicated fidelity of allelic expression at the IGF2 and GTL2 loci, however disruption of imprinting was observed at the XIST locus in the placenta of clones. These results are the largest identification of imprinted genes in the bovine and the first identification of the disruption of an imprinted gene in an animal derived from somatic cell nuclear transfer.

A crude extract of axenically grown amoebae was used as antigen in order to develop an AB microELISA for the detection and quantitation of E. histolytica-specific IgG antibodies. This ELISA was used to screen individual sera of patients suffering from invasive amoebiasis (n=47)and control individuals(n=33). Significant titers of E-histolytica- specific IgG antibodies were present only in sera of patients suffering from invasive amoebiasis. The AB-microELISA had a sensitivity of 98% and a specificity of 97%. Immunoblot analysis of the crude E. histolytica extract indicated the presence of several antigenic proteins. One of the common antigenic proteins recognized by the individual patients' sera had a molecular weight of 160.170 kDa Sera of five patienta with high titers of E. histotytica-specific IgG antibodies were used to prepare a serum pool. This pooled serum was used for immunoscreening of an E. histolyitca cDNA expression library prepared in the phage vector l ZAP-II. A strongly immunoreactive phage done was identified, from which he recombinant phagemid was released by in vivo excision for characterization of the cDNA insert.

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 82-89).

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 151-164). Available also in a digital version from Dissertation Abstracts.