Serine protease inhibitors (serpins) are a large superfamily of proteins that regulate critical proteolytic pathways by inhibiting serine proteases. Tick-encoded serpins are thought to play a vital role in the feeding process. To determine the relationship of Amblyomma americanum serpin 6 (S6) to tick feeding regulation, this study attempted to define the biological significance of this molecule through transcription and protein expression profiles, biochemical characterization of recombinant s6 (rS6), and the effects of in vivo post-transcriptional gene silencing on blood meal acquisition and fecundity. Transcriptional analysis revealed that S6 mRNA is ubiquitously expressed in unfed and partially fed ticks through the initial 5 days of the feeding period. S6 mRNA abundance in dissected tick organs showed a 3.7, 3.4, and 1.7- fold upregulation from 24 h to 96 h in the salivary gland (SG), midgut (MG) and the carcass (CA) remnant after removal of SG, MG respectively before downregulating at 120 h. Native S6 protein is downregulated in response to tick feeding, with correlation between transcription and protein expression profiles only consistent from the unfed to 48 h. Similarly, S6 protein expression in dissected female tick tissues is reduced as feeding progresses, with S6 being identified in SG, MG, ovary (OV), and CA from 24 h until 72 h. Biochemical characterization of S6 was not achieved, as rS6 did not form an irreversible complex when incubated with chymotrypsin or trypsin. Although complete silencing of S6 and S6/S17 mRNA was achieved, post-transcriptional gene knockdown had no effect on tick feeding efficiency or fecundity. These findings have been discussed in regards to the development of a vaccine against A. americanum and necessary future studies have been suggested for further characterization and assessment of biological significance.

Oligomers of hard and soft segments of unsaturated polyesters were synthesized in two steps. For the hard segment, isophthalic acid was reacted with 1,2-propanediol first, then maleic anhydride was added for further esterification. For the soft segment, diethylene glycol was used to replace 1,2-propanediol. In the previous study, the excess amount of glycol was 20% in weight. In this study, glycol was 10 and 5 wt% in excess, respectively. Decreasing the excess amount of glycol from 20 to 5 wt%, the number average molecular weight of both hard and soft segments increased about 30-40% from 1000 g/mol, and the degree of isomerization of maleic acid changed from 40.5 to 57.3% for the oligomers of the soft segment. The hard and soft segments synthesized in this study were blended in weight ratios from 0 to 100 % in an interval of 20 %, and then cured with styrene for further mechanical testing. The micro-tensile strength of cured soft and hard segments increased 1.3 and 8 times, respectively, in this study compared with that of specimens prepared under the condition of 20 wt% in excess of glycol. Therefore, the criteria of inhibitors can also be achieved by varying the excess amount of glycols. To evaluate the nitroglycerine migration and the erosion rate, only two (60% hard segment and 40% soft segment) of the formulas which passed the criteria of mechanical properties were investigated by replacing lithophone with magnesium hydroxide. In the case of 10 wt% in excess of glycol, the migration of nitroglycerine at infinite time (M

Nitric oxide (NO) is produced in different mammalian tissues by nitric oxide synthase (NOS), which has three isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). All NOS isoforms contain two domains, an oxygenase domain and a reductase domain. NO is an important transmitter of information between cells in many physiological processes; however, overproduction of this molecule may lead to health problems. Therefore, selective inhibition of NOS isoforms has useful therapeutic potential for treatment of certain diseases that can appear because of the pathological overproduction of nitric oxide. Producing useful isoform selective-inhibitors that bind to the active site in the oxygenase domain has proven to be difficult when based solely on the structure of these enzymes. Biophysical studies in combination with structural properties should provide better insights into isoform-specific inhibitor development. The first step of this study was to produce and purify truncated versions of NOS isozymes consisting of the oxygenase domain as they contain the active site of the enzyme. As a result of differences between humans and other mammals in the amino acids found in the second and third shells/layers surrounding the active site, all the experiments were performed with genes coding for human proteins. The major result of this project was the development of an Escherichia coli (E. coli) expression system to produce large amounts of pure protein. This system will allow for the testing of inhibitors that bind to the active site of NOS enzymes.

This thesis reports the development of potent and selective inhibitors of m-calpain for the treatment of cataract. SJA6017 has been proven to prevent lens opacity in rat and has been our lead compound. A series of Val-Leu peptidyl aldehyde inhibitors (33a-e, 33g, 33i and 35) have been designed, synthesized, and tested for therapeutic potential as cataract inhibitors. Chapter 1 is an introduction to calpain and the diseases associated with it's over activation. A review of the literature on calpain inhibition is given. Structure activity relationship (SAR) theory is presented. The techniques that have been applied in our research group to drug design include molecular modeling, synthesis, assay and animal studies which are all briefly discussed. The importance of a -strand conformation for an inhibitor to bind to calpain is discussed. Chapter 2 describes the synthesis of m-calpain inhibitors. This comprises the preparation of the Val-Leu dipeptide core 29, Val-Leu dipeptidyl alcohols 31a-g and 31i, and the synthesis of dipeptidyl aldehydes 33a-e, 33g, 33i and 35. The choice of coupling regents and conditions in the coupling reactions is investigated. Sulfur trioxide pyridine oxidation for the conversion of Val-Leu dipeptidyl alcohols to aldehydes is discussed. The molecular modeling and biological assay results are presented.

Univ., Diss.--Münster, 2005.

Univ., Diss.--Regensburg, 1996.

Univ., Diss., 2010--Würzburg.

Würzburg, Univ., Diss., 2009.

Regensburg und Paris, Univ., Diss., 2009.

No description available.