Genetic and biochemical characterization

Ornithine decarboxylase

Regulation of chlorophyll biosynthesis, gene expression and organisation

Meat quality

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) using NADPH as a cofactor. Since THF is an essential factor for nucleotide biosynthesis, inhibition of this enzyme in bacteria with folate analogs such as trimethoprim results in bacterial cell death. Plasmid encoded R67 DHFR confers resistance to trimethoprim and is both sequentially and structurally unrelated to any known chromosomal version of the enzyme. R67 DHFR is a 34,000 Da. homo-tetramer containing a rare 222 axis of symmetry in the center of its active site pore. The active site pore is contacted by residues belonging to each of the four subunits. The enzyme can nonspecifically bind 2 NADPH’s, 2 DHF’s, or, in the case of the productive complex, 1 NADPH and 1 DHF. R67 DHFR employs the endo transition state as opposed to the exo transition state used by E. coli DHFR. In this study molecular dynamics approach using the CHARMM program is employed to study the dynamics of the enzyme and energetics of the hydride transfer step catalyzed by R67 DHFR in silico. Structural and dynamic properties of four different mutants are also examined.

Crohn's disease is a chronic inflammatory bowel disease of unknown origin that continues to affect millions of people worldwide. Mycobacterium avium subsp. paratuberculosis (MAP) has been a suspected etiological agent of Crohn’s disease for nearly two decades. Recently, our laboratory was able to achieve a test sensitivity of 95.2% (n=21) by using a flow cytometry method (FCM) to detect anti-MAP antibodies in Johne’s diseased cattle. Here we investigate whether FCM can differentiate Crohn’s patients’ antibody titers from healthy human control antibody levels. As a second research goal, we investigated other suspected bacteria and serum-differentiating yeast using the FCM. In brief, bacteria/yeast were incubated with serum samples and washed to remove nonspecific antibodies. Antibodies bound to the surface of bacteria/yeast were then labeled with fluorescently-tagged secondary antibody and this binding evaluated by flow cytometry. Serum samples from 37 Crohn’s patients and 37 healthy human controls were tested for antibody (IgG and IgA) binding to bacteria/yeast. When targeting antibodies directed towards MAP, no significant difference was observed between the two populations (ANOVA, alpha=0.05). FCM was also unable to detect an antibody response for either group when directed against a cell-wall-deficient form of MAP, which has been reported to be near infected tissues of Crohn’s diseased patients. However, a differential antibody response to two yeasts that included S. cerevisiae and C. albicans was detected when looking for both IgG and IgA. This differentiation using yeast agrees with previous studies that have shown Crohn’s diseased subjects to have significantly higher antibody titers to this yeast than do healthy controls. This suggests that the flow cytometry method could be useful in the future for distinguishing Crohn’s subjects from healthy controls.

The thresholding problem is important in today’s data-rich research scenario. A threshold is a well-defined point in the data distribution beyond which the data is highly likely to have scientific meaning. The selection of threshold is crucial since it heavily influences any downstream analysis and inferences made there from. A legitimate threshold is one that is not arbitrary but scientifically well grounded, data-dependent and best segregates the information-rich and noisy sections of data. Although the thresholding problem is not restricted to any particular field of study, little research has been done. This study investigates the problem in context of network-based analysis of transcriptomic data. Six conceptually diverse algorithms – based on number of maximal cliques, correlations of control spots with genes, top 1% of correlations, spectral graph clustering, Bonferroni correction of p-values and statistical power – are used to threshold the gene correlation matrices of three time-series microarray datasets and tested for stability and validity. Stability or reliability of the first four algorithms towards thresholding is tested upon block bootstrapping of arrays in the datasets and comparing the estimated thresholds against the bootstrap threshold distributions. Validity of thresholding algorithms is tested by comparison of the estimated thresholds against threshold based on biological information. Thresholds based on the modular basis of gene networks are concluded to perform better both in terms of stability as well as validity. Future challenges to research the problem have been identified. Although the study utilizes transcriptomic data for analysis, we assert its applicability to thresholding across various fields.

Vitronectin is a multifunctional glycoprotein involved in regulation of such processes as hemostasis, cell migration, immunity, and tumor metastasis. Many of its functions require interaction with various binding partners. One such partner, plasminogen activator inhibitor-type 1 (PAI-1), belongs to the serine protease inhibitor (serpin) superfamily of proteins. PAI-1 helps regulate hemostasis by affecting blood clot breakdown, and helps regulate cell migration and tissue remodeling by affecting extracellular matrix digestion. Our laboratory is pursuing structural information on vitronectin and the complex formed between vitronectin and PAI-1. To this end, the work presented in this thesis focuses on two aims: characterizing the interaction of vitronectin and PAI-1 with metal ions, and developing reagents to be used in structural studies of vitronectin. We have used electron paramagnetic resonance (EPR) spectroscopy to study metal binding by vitronectin and PAI-1. We have tested vitronectin and PAI-1 for binding of Mn2+ and Cu2+, and also established the feasibility of studying protein-Cu2+ interaction in solution by EPR, using bovine serum albumin (BSA) and Cu2+. In another set of experiments, we have used activity assays to assess the effect of metal ions on PAI-1 activity and on stabilization of PAI-1 by vitronectin. Regarding reagents, we have expressed and purified active, perdeuterated PAI-1, which can be used in complex with vitronectin in neutron scattering and NMR experiments. We characterized several monoclonal antibodies to vitronectin, and identified one suitable for use in co-crystallization screens. These accomplishments will aid in development of a more detailed picture of the interaction between vitronectin and PAI-1.

The recent completion of the sequencing of several species of the <em>Shewanella</em> genus provides a unique opportunity for comparative genomics studies. We chose the first 10 fully sequenced Shewanella genomes to investigate the evolution of signal transduction proteins (ST). ST is a universal and highly regulated system, and as a very well-studied system provides an excellent starting point for investigation. Furthermore, Shewanella have been shown to have a large number of two-component systems and diguanylate cyclases relative to their genome size. In this study we investigate the evolution of signal transduction across several Shewanella strains by utilizing a domainlevel approach for determining homology and orthology of the parent proteins. Proteins were broken down into their constituent domains and domain sized sequences and compared using a reciprocal best BLAST hit approach to determine homology between all of the species. Analysis of homologous domains and proteins revealed several levels of conservation and a core group of signal transduction proteins common to all members. Further analysis of domain homology provided putative annotations of previously unrecognized sequences and highlighted deficiencies in specific Pfam domain models. Analysis of paralogous domains and proteins showed agreement with 16s rRNA based estimates of evolution, although the position of S. oneidensis MR-1 was novel.

The Boise Basin Eperimental Forest was chosen as the study area. Virgin stands typical of the ponderosa pine type of Central Idaho were found within its boundaries. The area had been divided into compartments and some mapped for forest types, age classes and topography. The mapped areas were used as the sampling units and ranged in elevation from 4900 to 5600 feet. Description of forest conditions and aspects Three forest conditions which could be easily identified in the field were chosen as sampling areas: open-brush areas, sapling-pole stands, and mature-overmature timber.