Biochemical markers

The purpose of this study was to determine if maximal aerobic exercise promotes
BDNF expression in obese individuals. Plasma levels and the expression of BDNF in
PBMCs were examined. 22 participants (10 obese, 12 non-obese) completed a V02max
treadmill test and blood was obtained pre, post, and 1 and 2 hours into exercise recovery.
Plasma and PBMCs were isolated and analyzed for BDNF via ELISA and Western blot
techniques. A significant effect for time was observed for plasma BDNF (P= <0.00 1 ).
Additionally, A significant group-by-time interaction was found from pre-to-RIH for
BDNF expression in PBMCs (P= 0.046). Further, significant correlations were found
between BMI and waist circumference (r= .91, P< 0.001), WHR (r= .51, P= 0.002) and
Pre-to-RlH ratio (r=0.58, P=0.008). Young obese subject's BDNF response to maximal
exercise was consistent with previous studies. Post-exercise BDNF expressed in PBMCs
were significantly higher than rest suggesting immunological-neuroprotective
interactions in the CNS.

Harman's theory of aging proposes that a buildup of damaging reactive oxygen species (ROS) is one of the primary causes of the deleterious symptoms attributed to aging. Cellular defenses in the form of antioxidants have evolved to combat ROS and reverse damage; one such group is the methionine sulfoxide reductases (Msr), which function to reduce oxidized methionine. MsrA reduces the S enantiomer of methionine sulfoxide, Met-S-(o), while MsrB reduces the R enantiomer, Met-R-(o). The focus of this study was to investigate how the absence of one or both forms of Msr affects locomotion in Drosophila using both traditional genetic mutants and more recently developed RNA interference (RNAi) strains. Results indicate that lack of MsrA does not affect locomotion. However, lack of MsrB drastically reduces rates of locomotion in all age classes. Furthermore, creation of an RNAi line capable of knocking down both MsrA and MsrB in progeny was completed.

Briareum asbestinum, a soft coral, is a rich source of diterpenoid natural products. The secondary metabolites of B. asbestinum fall into four classes : asbestinins, briarellins, briareolate esters, and briaranes. Briareolate esters have been shown to possess biological activity and were previously only reported from Tobago. Our group recently isolated briareolate esters from a specimen collected off the coast of Boca Raton, Florida. To determine whether location has an impact on the chemistry produced by the organism, a method to discern between chemotypes was sought. Several techniques including thin layer chromatography (TLC), high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and sclerite analysis were employed, with NMR being the most successful method. By utilizing both 1H and COSY NMR experiments, it is possible to differentiate between the chemotypes of B. asbestinum. Application of this method allowed analysis of chemical variability with respect to location.

Stroke is the third leading cause of mortality in the United States, and so far, no clinical interventions have been proved truly effective in stroke treatment. Stroke my result in hypoxia, glutamate release and oxidative stress, etc. The purpose of this dissertation study is to evaluate the neuroprotective effects of four drugs (taurine, G-CSF sulindac and DETC-MeSO) on PC12 cell line or primary cortical neuronal cell culture, and to understand the protective mechanisms underlying in three stroke-related models : hypoxia, excessive glutamtate and oxidative stress. In the first part of this dissertation, we studied the neuroprotection of taurine against oxidative stress induced by H2O2 in PC12 cells. Our results show that extracellular taurine exerts a neuroprotective function by restoring the expression of Bcl-2 and downregulation of the three Endoplasmic Reticulum (ER) stress markers : GRP78, Bim and CHOP/GADD153, suggesting that ER stress can be provoked by oxidative stress and can be suppressed by taurine. In the second part, glutamate excitotoxicity-induced ER stress was studied with dose and time as variables in primary cortical neurons. The results demonstrate that glutamate excitotoxicity leads to the activation of three ER stress pathways (PERK, ATF6 and IRE1) by initiating PERK first, ATF6 second and IRE1 pathway last. The third part of this dissertation studied the robust and beneficial protection of taurine in cortical neurons under hypoxia/reoxygenation or glutamate toxicity condition. We found that taurine suppresses the up-regulation of GRP778, Bim, caspase-12 and GADD153/CHOP induced by excessive glutamate or hypoxia/reoxygenation, suggesting that taurine may exert a protective function against hypoxia/regeneration by reducing the ER stress.

The Effect of Sulindac was studied on an animal model of ischemic stroke. Sulindac, a non steroid anti inflammatory drug (NSAID) could protect cell death due to hypoxia/reoxygenation. This drug was given 2 days before and 24 hrs after ischemia until animals were sacrificed on 3rd or 11th day. Infarct size was measured for these animals. Sulindac induced Hsp 27 in ischemic penumbra and core on Day 3 & 11 with uncoated nylon suture which shows its cell-survival and anti-apoptotic activity. Also, it increased expression of cell survival markers such as Akt, Bcl2 & Grp 78 in ischemic penumbra and core. With silicon suture it reduced expression of Hsp 27 in ischemic penumbra and core, alleviating cell stress and having pro-survival and anti-stress effects. In conclusion sulindac may have excellent potential as neuro protective agent against oxidative stress in cerebral ischemia.

MicroRNAs (miRNAs) may serve as diagnostic and predictive biomarkers for cancer. The aim of this study was to identify novel cancer biomarkers from miRNA datasets, in addition to those already known. Three published miRNA cancer datasets (liver, breast, and brain) were evaluated, and the performance of the entire feature set was compared to the performance of individual feature filters, an ensemble of those filters, and a support vector machine (SVM) wrapper. In addition to confirming many known biomarkers, the main contribution of this study is that seven miRNAs have been newly identified by our ensemble methodology as possible important biomarkers for hepatocellular carcinoma or breast cancer, pending wet lab confirmation. These biomarkers were identified from miRNA expression datasets by combining multiple feature selection techniques (i.e., creating an ensemble) or by the SVM-wrapper, and then classified by different learners. Generally speaking, creating a subset of features by selecting only the highest ranking features (miRNAs) improved upon results generated when using all the miRNAs, and the ensemble and SVM-wrapper approaches outperformed individual feature selection methods. Finally, an algorithm to determine the number of top-ranked features to include in the creation of feature subsets was developed. This algorithm takes into account the performance improvement gained by adding additional features compared to the cost of adding those features.

Sulindac is a known NSAID that has also been shown to have anti-cancer activity that is not related to its ability to inhibit COX 1 and 2. During the past 15 years there have been a large number of studies attempting to elucidate its mechanism of action. Our laboratory has shown that sulindac can both protect normal cells and enhance the killing of cancer cells under oxidative stress from H2O2 and TBHP. However, except for mitochondrial dysfunction and ROS production, the mechanism by which sulindac sensitized the cancer cells to oxidative stress remains unknown. Results of this research project suggest that the effect of sulindac and oxidative stress not only involves mitochondrial ROS production, but also aspects of the preconditioning response. In normal cells this leads to survival by a preconditioning pathway, likely involving PKCε. . However, cancer cells react by initiating a pathway leading to apoptosis involving PKCδ.

The bald eagle, Haliaeetus leucocephalus, population declined dramatically in the early 20th century reducing the population from tens of thousands of birds within the lower 48 states, to <450 pairs of birds, effectively inducing a population bottleneck. The overall population has recovered and was removed from the endangered species list in 2007. This study investigates whether such overall population statistics are appropriate descriptors for this widespread species. I investigated the genetic differentiation between three populations of bald eagles from Alaska, North Florida and Florida Bay using both mitochondrial and nuclear DNA loci to determine whether discrete subpopulations comprise the broad range. Significant FST values, for both mtDNA and microsatellites, were found between both Florida populations and Alaska, but not within Florida populations. Results indicate that there is strong population structure, rejecting the null hypothesis of a panmictic population. Future conservation efforts should focus on subpopulations rather than the overall population.

The methionine sulfoxide reductase (Msr) enzymes catalyze the reduction of methionine sulfoxide (Met(O)) to methionine. The Msr enzymes protect cells against oxidative stress and may have a role in aging. The MsrA family of enzymes reduces stereospecifically the S epimer of free and protein-bound Met(O) while the MsrB family reduces the R epimer of Met(O) in proteins. It has been generally accepted, primarily from studies on MsrA, that the biological reductant for the Msr enzymes is thioredoxin (Trx), although high levels of dithiothreitol (DTT) can be used as the reductant in vitro. In contrast, certain MsrB enzymes show less than 10% of the activity with Trx as compared to DTT. This raises the possibility that in animal cells Trx may not be the direct hydrogen donor for the MsrB enzymes. Studies with bovine liver extracts have shown that thionein, the apoprotein of metallothionein, can function as a reductant for the Msr proteins. Certain selenium compounds such as selenocystamine and selenocystine can also serve as potent reducing agents for the Msr enzymes. Since an increased activity of Msr enzymes can reduce the level of oxidative damage in tissues, compounds that could activate Msr may have therapeutic potential. A high-throughput screening assay has been developed to screen large chemical libraries to find activators of MsrA, as well as specific inhibitors that could be useful research tools. This study will be done in collaboration with The Scripps Florida Research Institute. Sulindac was originally developed as a non-steroidal anti-inflammatory drug but has also shown efficacy in the treatment of certain cancers. The S epimer of sulindac is known to be reduced by MsrA, but the enzymes responsible for reduction of the R epimer are not known.

In light of exceptionally delayed reproductive senescence exhibited by a 64 year old female chimpanzee (Pan troglodytes) housed in Florida, endocrinal analyses meant to determine the state of her current reproductive viability were conducted. Urine was collected from the study subject for a period of 88 days spaced within an interim of roughly 6 months and the specimens were sent to the Hominoid Reproductive Ecology Laboratory for assessment. Additional data was collected from three control females in order to provide a basis of comparison against the hormonal markers present in the geriatric study animal. Results indicate that the geriatric female does not presently appear to be cycling, but nor does she exhibit signs of complete reproductive cessation. This could signify that Pan troglodytes adheres to a pattern of reproductive aging not necessarily shared by Homo sapiens, which has further implications for the evolutionary trajectory of menopause in the human female.