Promoter elements prediction with bioinformatics means is very promising direction in molecular biology. Such predictions allow to clarify the mechanisms of gene regulation and to increase the success of the “wet” experiments. The study was aimed to predict motifs for the 20 genes expressed in the thermosensory AFD neurons of the C.elegans and ortholog species. As theoretical basis of investigation was accepted theory about conservation of functionally important non-coding regions between ortholog genes. Some of such conserved regions can serve as the putative promoter elements. For the investigation the set of ortholog genes of the C.elegans, C.briggsae and C.remani was used. During the work suitable bioinformatics programs and the set of promoter criteria were worked out. Predicted promoters with the different level of conservation were compared to the each other and with the known regulatory elements. Some of the promoter elements (CAGCTG, CAGGTG) represent sequences are highly conserved from worms to the human others are similar to the known worm motifs (skn-1). Genes with the similar putative elements were combined in the groups with the possible common mechanisms of the gene regulation. The investigation of the putative promoter elements requires the development of new programs,methods and criteria of the evaluation. This would be appropriate strategy for the further motifs and non-coding DNA studying.

Cancer vaccines targeted against tumour cells seek to mimic immune responses against viral infections. Given the unique properties of B cells that allow them to present antigens proficiently, activated B cells can be used in a vaccine setting to launch effective anti-tumour T cell responses. Activation induced cell death, however, presents a major hurdle in the generation of large numbers of B cells. Since apoptosis is mediated by oxidative stress, uric acid was used as an antioxidant, enabling increased growth of normal B cells. Further investigation in TK6 cells revealed that uric acid was mediating its effects extracellularly and initiating signaling pathways that culminated in the activation of ERK and JNK proteins and production of IL10, which was necessary, but not sufficient, in mediating the growth effects of uric acid. The results outlined in this study implicate uric acid as a novel growth factor for activated B cells.

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

Thesis (M. S.)--Oklahoma State University, 2004. / Vita. Includes bibliographical references (p.61-65).

Thesis (M. S.)--Oklahoma State University, 2005. / Vita. Includes bibliographical references (p.79).

Plants produce a large diverse array of organic compounds that may function in protection against pathogens. Diverse antifungal compounds were reported to exist in barley (Hordeum vulgare L.); the indole alkaloid, gramine, and the pathogenesis-related proteins are some of them. Both the N-methyltransferase that is involved in gramine biosynthesis and PR-17a were studied in barley upon infestation by the bird cherry-oat aphid (Rhopalosiphum padi). The effect of infestation by R. padi on induction of PR-17a and N-methyltransferase was investigated in different barley lines, susceptible and resistant. The gene expression of PR-17a was down-regulated in the susceptible cv. Golf and to some extent up-regulated at the first days in var. Lina and then down-regulated. The PR-17a was induced by the aphid infestation in the resistant line CI16145; the gene expression was stronger in the infested plants than in the controls. The different responses in resistant and susceptible lines indicate that the induced PR-17a may play a role in the resistance against aphid infestation. PR-17a was up-regulated systemically in the base in barley after infestation by R. padi. In the susceptible varieties Lina and Golf, the accumulation of N-methyltransferase did not increase with time from 1 day to 7 days after infestation, as determined by western blots with antibody raised against NMT from barley. The NMT-gene was down-regulated after 7 days infestation in both variety Lina and Golf both locally in the first leaf and in the base. Barley line CI16145 had no accumulation of NMT as was seen by western blotting. There was no induction of NMT in barley upon aphid infestation.

The goal of this project is to express both the normal CYP26 B1 and the spliced CYP26 B1 from human in Escherichia coli (E.coli) cells for further crystallization. This will be achieved by cloning in the DNA fragments into the Champion pET SUMO vector that is later transformed into E.coli cells. The CYP26 B1 contains a hydrophobic helix at the N-terminal of the protein, making both protein expression and crystallization difficult. Two variants of both full-length CYP26 B1 and the spliced variant will therefore be made, one with the trans-membrane helix present and one without the helix. The SUMO-vector will produce a fusion protein that will make CYP26 B1 more hydrophilic and improve the purification of the two proteins.

There is ample evidence that both diabetes as well as obesity leads to various metabolic disturbances that leads to oxidative stress. Oxidative stress has been shown  to  be  associated  with  congenital  malformations  of  which  neural  tube defects  and  cardiac  malformations  are  more  common.  The  cellular  and molecular  mechanisms  through  which  oxidative  stress  induces  these  defects during the developmental stage are not well known. Previous work in this field suggests   that   oxidative   stress   results   in   lipid   peroxidation   and   altered expression  of  genes  that  have  key  roles  in  the  developmental  processes.  The present study aimed to investigate gene alterations in embryos from pregnant diabetic  or  obese  rats.  Embryos  and  adipose  tissue  obtained  from  the  locally bred  diabetic  and  obese  Sprague-Dawley  inbred  rat  strain  were  subjected  to Total  RNA  extraction  and  were  quantified  using  Real  time  PCR  for  relative gene expressions analysis. The present study showed that maternal diabetes as well  as  obesity  diminishes  the  antioxidative  defense  mechanisms  by  down regulating the gene expressions of the key reactive oxygen species scavenging enzymes   copper   zinc   superoxide   dismutase   and   manganese   superoxide dismutase  in  day  10  rat  embryos.  There  was  also  altered  embryonic  gene expression  for  several  developmental  genes  due  to  maternal  diabetes  at gestational day 11 and 13 in rat embryos.

The presented work describes construction and analysis of recombinantly produced forms of Aleuria aurantia lectin (AAL). The binding properties of the produced AAL forms were studied using techniques such as tryptophan fluorescence, hemagglutination analysis, ELISA and surface plasmon resonance analysis. Lectins are proteins that are ubiquitous in nature with the ability to bind specifically to different types of carbohydrates. The physiological function of different lectins is not always known, but they are involved in many recognition events at molecular and cellular levels. In research, lectins are widely used for structural and functional studies of complex carbohydrates, and they are also used to detect changes in the carbohydrate pattern on glycoproteins in different diseases. With the use of recombinant technology it is now possible to refine properties of lectins such as decreasing the valency and alter specificity and affinity. This may be a way of constructing more suitable reagents for use in diagnostic glycosylation analysis assays. AAL has been extensively used in different types of research for its ability to bind the monosaccharide fucose and to fucose-containing oligosaccharides. It is composed of two identical subunits where each subunit contains five binding sites for fucose. AAL was expressed recombinantly (rAAL) and its properties was investigated. These studies reveled that one of the binding sites in rAAL had unusually high affinities towards fucose and fucosecontaining oligosaccharides with Kd-values in the nanomolar range. This binding site is not detected in AAL that have been exposed to fucose during its purification, and therefore we proposed that this site may be blocked with free fucose in commercial preparations of AAL. Normally lectin-oligosaccharide interactions are considered to be of weak affinity, so the finding of a high affinity site was interesting for the future study of recombinant forms of AAL. The next step was to produce recombinant AAL forms with decreased valency. This was done using site-directed mutagenesis. First a monomeric form of AAL (mAAL) was constructed and then a monovalent form of AAL, containing only one fucose-binding site (S2-AAL) was constructed. Both of these forms had retained ability to bind fucose. The binding characteristics of mAAL were similar to that of rAAL, but mAAL showed decreased hemagglutinating activity. S2-AAL showed a lower binding affinity to fucosylated oligosaccharides and did not bind to sialylated fuco-oligosaccharides such as sialyl-LewisX. This study shows that molecular engineering techniques could be important tools for development of reliable and specific diagnostic and biological assays for carbohydrate analysis.

No description available.