Poster Abstract


Spring 2011 Biomedical Seminar Series

April 22

Luis Galdino | Selma Cuya | Jennifer Uyere


The Mechanism of Ice Recrystallization Inhibition by Antifreeze Proteins Type in Different Aqueous Solutions

Luiz Galdino
MBRS-RISE MS to PhD Scholar

Antifreeze proteins are polypeptides capable of inhibiting ice from growing, which allows the survival of organisms living in extremely cold enviroments. It does so by subduing the freezing process originating from an ice seed in aqueous solutions; it is able to depress the freezing point depending on the molar concentration and cease the freezing process allowing liquid state to exists without changing osmotic pressure, which would otherwise be lethal to these organims. The protein has been found in many different isoforms in a variety of organism from bacteria to fishes. Understanding its mechanistic approach can lead to antifreeze proteins' potential uses in the cryoindustry from conservation of organs for transplatation to freezing structural failure prevention to agriculture in subzero temperature environments. The current proposed study has many facets but ultimately is intended in better understanding the mechanism of ice growth inhibition by antifreeze proteins. Many theoretical developments have been proposed to account for such enzymatic activity but they have not succeeded in becoming the sole mechanism. The colligative effect of thermodynamics fails to curve fit the freezing point depression to the concentration of protein and the adsorption mechanism (Kelvin) effect suggests an original ice seed much larger to induce its freezing point lowering and preventing the solution from freezing from it than experimentally observed. Our lab has proposed an alternate theory. Named Adsorption Enhanced Colligative Effect, it combines the colligative effect and Langmuir's adsorption theory and has with acceptable success fit to its theoretical development data encountered in literature. Albeit successful, this theory has yet to be fully accepted and a major goal of the proposed thesis is to refute or reinforce the theory developed by our lab. An even more ambitious goal is to measure chemical potential values of adsorption using experimental techniques yet to be attempted. Successful data from such novel experiments would elucidate critical light into the mechanics of adsorption of antifreeze proteins to ice and its inhibitory mechanisms. Furthermore, thermal hysteresis, a critical concept in studying antifreeze proteins mechanics will be re-examined in a variety of aqueous solutions. Throughout these series of experimental and theoretical evaluations, a few interesting scientific questions will be attempted to be answered: (1) the reversability of antifreeze protein's adsorption to ice, (2) antifreeze proteins ability to prevent ice nucleation and (3) antifreeze proteins' ability to produce superheated ice crystals and the nature of such crystals. It will also be able to evaluate the crystal growth of natural ice and attempt to describe how each ice face grows. Ultimately, the proposed work will be able to elucidate significant questions surrounding the field of antifreeze proteins and concomitantly their mechanism of ice growth inhibition. It is hoped that the culmination of proposed study should add to the scientific community that tries to shed light on how antifreeze proteins work in order to use them in applications that shall improve society's quality of life.


Optimization of p53 activity in a zinc inducible p53 expressing cell line as a feasibility test for its use in identification of mRNA accumulation levels modified in common by MyoD and p53

Selma Cuya
MBRS-RISE MS to PhD Scholar

The tumor suppressor p53 is known to regulate cell cycle checkpoints and apoptosis and has been found to be required for initiation of skeletal muscle differentiation in cultured cells. It is not, however, required for myogenesis in vivo. To address this apparent conflict, we and others hypothesize that p53 acts as a backup for the myogenesis regulatory factor MyoD, requiring that p53 and MyoD share common activities. We planned to use microarray analysis to determine changes in levels of mRNA accumulation found in common in response to MyoD and p53, and thereby to identify a suite of genome-wide regulatory activities shared by p53 and MyoD in cultured cells. Four cell lines were derived by the Sharp Lab: a line null for MyoD that expresses p53 from a zinc-inducible transgene (ZIP-p53); a line null for p53 that expresses MyoD constitutively from a MyoD transgene; and two control lines that express neither MyoD nor p53. My objective was to maximize p53 activity in the p53-expressing line using nutlin, an inhibitor of p53 degradation, in an effort to make the p53 activity similar to what is observed in the model myogenic C2C12 cell line. To determine conditions at which p53 activity is highest, a p53 responsive promoter/luciferase reporter assay was done to measure p53 activity in the presence and absence of nutlin over a period of 24-72 hours. p53 activity was higher +nutlin at 48 and 72 hours, and increased over time. To determine whether p53 from the transgene activated endogenous genes as expected, we used q-RT-PCR to assay for increased expression of the p53- responsive genes p21, pRb, and MDM2. Unexpectedly, MDM2, p21, and pRb mRNA accumulation was similar in the p53-non-expressing and –expressing cells, suggesting either that the transgenic p53 is not active on endogenous genes or factors other than p53 are contributing to the increase of MDM2, p21, and pRb in these cells. Luciferase and qRT-PCR results both showed that p53 activity in the Zip-p53 cells never approached that in C2C12 cells. Based on these results, we cannot use the ZIP-p53 line to study the effect of p53 on gene expression. During the course of these experiments, another lab member analyzed hind limb sections I had stained with hemotoxylin and eosin, and found no difference in muscle development between near term MyoD null and MyoD/p53 double null mice, suggesting that p53 does not function as a backup for MyoD in vivo. Together these results have led us to conclude that the time required to derive and vet a different p53 expressing line is not justified. We will instead compare the data we have from our MyoD expressing line with genome-wide p53 regulatory data from the literature. This research is supported by the NIH MBRS-RISE M.S. to Ph.D. Grant # GM61331

 


Functional Analysis and Characterization of the YCK3 Gene in Comparison to the YCK2 Gene in C. albicans

Jennifer Uyere

MBRS-RISE MS to PhD

Scholar Candida albicans is an opportunistic pathogen that is part of the normal human flora, often causing serious life-threatening diseases in immunocompromised patients. The mechanisms by which C. albicans initiates candidiasis are unknown and treatments are extremely limited and ineffective. Previous transcriptional analysis in our laboratory have shown that the C. albicans casein kinase gene YCK2 is significantly upregulated during C. albicans-epithelial cell interaction. In addition, the gene YCK3 was identified through BLAST analysis to be 80% homologous to YCK2. In order to elucidate the functional relationship between YCK2 and YCK3 in C. albicans pathogenesis, we employed gene-deletion, knocking out the function of YCK2 in a yck3 mutant strain through histidine transformation. That is, we replaced the YCK2 gene with a HIS1 selection marker. Positive transformants were confirmed via whole cell PCR using YCK2-specific primers. The positive transformants on selective medium were then characterized using growth curve, germination, and antimicrobial peptide resistance analysis. The results showed no significant difference in the growth, germination, and antimicrobial peptide resistance of mutants relative to wildtype. Thus, future overexpression studies of YCK3 in yck2 mutant strains are necessary to reveal possible novel phenotypes, and to analyze whether the yck2 mutant phenotype can be rescued.

Back to seminars