Stabilization of troponin I for applications in clinical chemistry and forensic medicine. Cardiac troponin I: A time of death marker

Cardiac troponin I (cTnI) is one of the most useful serum marker test for the determination of myocardial infarction (MI). The first commercial assay of cTnI was released for medical use in the United States and Europe in 1995. It is useful in determining if the source of chest pains, whose etiology may be unknown, is cardiac related. Cardiac TnI is released into the bloodstream following myocardial necrosis (cardiac cell death) as a result of an infarct (heart attack). In this research project the utility of cardiac troponin I as a potential marker for the determination of time of death is investigated. The approach of this research is not to investigate cTnI degradation in serum/plasma, but to investigate the proteolytic breakdown of this protein in heart tissue postmortem. If our hypothesis is correct, cTnI might show a distinctive temporal degradation profile after death. This temporal profile may have potential as a time of death marker in forensic medicine. The field of time of death markers has lagged behind the great advances in technology since the late 1850's. Today medical examiners are using rudimentary time of death markers that offer limited reliability in the medico-legal arena. Cardiac TnI must be stabilized in order to avoid further degradation by proteases in the extraction process. Chemically derivatized magnetic microparticles were covalently linked to anti-cTnI monoclonal antibodies. A charge capture approach was also used to eliminate the antibody from the magnetic microparticles given the negative charge on the microparticles. The magnetic microparticles were used to extract cTnI from heart tissue homogenate for further bio-analysis. Cardiac TnI was eluted from the beads with a buffer and analyzed. This technique exploits banding pattern on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by a western blot transfer to polyvinylidene fluoride (PVDF) paper for probing with anti-cTnI monoclonal antibodies. Bovine hearts were used as a model to establish the relationship of time of death and concentration/band-pattern given its homology to human cardiac TnI. The final concept feasibility was tested with human heart samples from cadavers with known time of death. ^

Theoretical study of chloroperoxidase catalyzed chlorination of beta-cyclopentanedione and role of water in the chlorination mechanism

Chloroperoxidase (CPO) is a potential biocatalyst for use in asymmetric synthesis. The mechanisms of CPO catalysis are therefore of interest. The halogenation reaction, one of several chemical reactions that CPO catalyzes, is not fully understood and is the subject of this dissertation. The mechanism by which CPO catalyzes halogenation is disputed. It has been postulated that halogenation of substrates occurs at the active site. Alternatively, it has been proposed that hypochlorous acid, produced at the active site via oxidation of chloride, is released prior to reaction, so that halogenation occurs in solution. The free-solution mechanism is supported by the observation that halogenation of most substrates often occurs non-stereospecifically. On the other hand, the enzyme-bound mechanism is supported by the observation that some large substrates undergo halogenation stereospecifically. The major purpose of this research is to compare chlorination of the substrate β-cyclopentanedione in the two environments. One study was of the reaction with limited hydration because such a level of hydration is typical of the active site. For this work, a purely quantum mechanical approach was used. To model the aqueous environment, the limited hydration environment approach is not appropriate. Instead, reaction precursor conformations were obtained from a solvated molecular dynamics simulation, and reaction of potentially reactive molecular encounters was modeled with a hybrid quantum mechanical/molecular mechanical approach. Extensive work developing parameters for small molecules was pre-requisite for the molecular dynamics simulation. It is observed that a limited and optimized (active-site-like) hydration environment leads to a lower energetic barrier than the fully solvated model representative of the aqueous environment at room temperature, suggesting that the stable water network near the active site is likely to facilitate the chlorination mechanism. The influence of the solvent environment on the reaction barrier is critical. It is observed that stabilization of the catalytic water by other solvent molecules lowers the barrier for keto-enol tautomerization. Placement of water molecules is more important than the number of water molecules in such studies. The fully-solvated model demonstrates that reaction proceeds when the instantaneous dynamical water environment is close to optimal for stabilizing the transition state.

Disturbance and recovery following catastrophic grazing: studies of a successional chronosequence in a seagrass bed

In August 1997, a large aggregation of the common sea urchin, Lytechinus variegatus, was discovered moving southward through a lush and productive seagrass monoculture of Syringodium filiforme in the Florida Keys, FL. Sea urchin densities at the grazing front were greater than 300 individuals m−2 which resulted in the overgrazing of seagrasses and a complete denuding of all vegetation from this area. The steady rate of the grazing front migration permitted the estimation of the time since disturbance for any point behind this grazing front allowing the use of a chronosequence approach to investigate the processes early on in succession of these communities. In May 1999, six north-south parallel transects were established across the disturbed seagrass communities and into the undisturbed areas south of the grazing front. Based on the measured rates of the migration of the grazing front, we grouped 60 sites into five categories (disturbed, recently grazed, active grazing front, stressed and undisturbed). The large scale loss of seagrass biomass initiated community-wide cascading effects that significantly altered resource regimes and species diversity. The loss of the seagrass canopy and subsequent death and decay of the below-ground biomass resulted in a de-stabilization of the sediments. As the sediments were eroded into the water column, turbidity significantly increased, reducing light availability and significantly reducing the sediment nitrogen pool and depleting the seed bank. The portion of the chronosequence that has had the longest period of recovery now consists of a mixed community of seagrass and macroalgae, as remnant survivors and quick colonizers coexist and jointly take advantage of the open space.

Diatom-based paleolimnological reconstruction of regional climate and local land-use change from a protected sinkhole lake in southern Florida, USA

Despite their sensitivity to climate variability, few of the abundant sinkhole lakes of Florida have been the subject of paleolimnological studies to discern patterns of change in aquatic communities and link them to climate drivers. However, deep sinkhole lakes can contain highly resolved paleolimnological records that can be used to track long-term climate variability and its interaction with effects of land-use change. In order to understand how limnological changes were regulated by regional climate variability and further modified by local land-use change in south Florida, we explored diatom assemblage variability over centennial and semi-decadal time scales in an ~11,000-yr and a ~150-yr sediment core extracted from a 21-m deep sinkhole lake, Lake Annie, on the protected property of Archbold Biological Station. We linked variance in diatom assemblage structure to changes in water total phosphorus, color, and pH using diatom-based transfer functions. Reconstructions suggest the sinkhole depression contained a small, acidic, oligotrophic pond ~11000–7000 cal yr BP that gradually deepened to form a humic lake by ~4000 cal yr BP, coinciding with the onset of modern precipitation regimes and the stabilization of sea-level indicated by corresponding palynological records. The lake then contained stable, acidophilous planktonic and benthic algal communities for several thousand years. In the early AD 1900s, that community shifted to one diagnostic of an even lower pH (~5.6), likely resulting from acid precipitation. Further transitions over the past 25 yr reflect recovery from acidification and intensified sensitivity to climate variability caused by enhanced watershed runoff from small drainage ditches dug during the mid-twentieth Century on the surrounding property.