Bloomsburg University of Pennsylvania

Dr. Mark A. Tapsak

Office 203 HSC – Research Lab 249 Hartline

Course Materials – Research Interests

Introduction

There is no substitute for a good research experience during your education. Although research is a requirement for the ACS certified Chemistry degree, all science majors should engage in some form of independent undergraduate research for a variety of reasons. Research can help you:

· Solidify the concepts from your classes - it provides an example of how scientific information and theory is applied to the real world.

· Sharpen your laboratory skills with both equipment and notebooks.

· Decide on a career path - as compared to lab classes, the experience is much closer to what you will do daily in industrial jobs and/or graduate school.

· Familiarize yourself with the science faculty at Bloomsburg and build relationships that can result in great reference letters.

· Build your resume with good experience with the potential of authorship on publications.

For Chemistry majors, the normal track for performing undergraduate research begins after you have completed Introduction to Scientific Literature, Chem 281, during the spring of your second year. This class introduces you to the use of the library and scientific journals for the acquisition of information for research, proposals, and presentations.

Credit is given for research performed during the academic year, provided that you enroll in the appropriate course, Introduction to Research, Chemistry 492 and Chemical Research, Chemistry 493. These courses are essentially ongoing research under the guidance of a faculty member. The earliest that you can take Chemistry 492 is during the spring semester of your third year.

However, this does not mean that you have to wait until your third year to get involved in research at Bloomsburg! First and second year students are encouraged to volunteer their time for research. This is an excellent opportunity to find out your true interests and skills. In addition, research experience can also help you perform better in upper level laboratory courses that require techniques that you may have already have mastered. The remainder of this document will highlight some of the research that will be pursued in my laboratory. Please feel free to stop by for more detailed information.

Project Title – Measurement of Creatine in Blood, Urine and Muscle

Project Objective

The overall goal of this work is to develop a fast, accurate and cost effective electronic sensor for creatine and creatinine in blood, urine and muscle samples. The initial objective of this work will be to develop a membrane-based amperometric sensor and validate its performance with creatine in human plasma samples.

Background

Approximately 95% of the body’s creatine is stored in skeletal muscle, where it exists in free (i.e. creatine) and phosphorylated (i.e. phosphocreatine) form. During intense physical activity, phosphocreatine donates its phosphate to adenosine diphosphate to produce adenosine triphosphate (ATP), so muscle contractions can be sustained. Brief, intense, physical activities such as climbing stairs or lifting a heavy weight rely heavily on the creatine-phosphocreatine energy system.

It has been demonstrated that ingestion of creatine supplements by young healthy individuals of about 20 g/d for 5 days results in approximately a 25% increase in total muscle creatine. The greater level of muscle creatine available for energy production results in an improved ability to perform intense exercise tasks, and in fact, 70% of studies examining the effects of creatine supplementation on exercise performance demonstrate an ergogenic (increased strength, decreased fatigue) effect. Additionally, individuals who ingest creatine may experience a marked increase in body mass or lean body mass.

A great deal of information regarding the absorption, retention, and metabolism of creatine can be gathered from blood, urine and muscle samples. Unfortunately, measurement methods for creatine and creatinine suffer from complexity, both in terms of equipment and labor. Methods such as HPLC,[1,2] and NMR[3] have been successfully been used but require expensive analytical equipment. Without this equipment, investigators are forced to use commercial colorimetric kits that require relatively large sample volumes for measurement replication. Typically, these samples must be collected over long time intervals and frozen for later evaluation in a lab.[4] The user must prep these samples using good analytical techniques and precise sample handling. Regrettably, the sum of these limitations leads researchers to be skeptical of reported creatine and creatinine data.

While the measurement of creatine and creatinine has been accomplished by other researchers using an oxidase enzyme,[5,6] a reusable membrane-based sensor has not been developed for use in this assay. When such a technology is developed, it will enjoy several practical advantages over the state of the art.

Examples of project tasks

Task	Skills Used / Learned
Calibrate H₂O₂ sensors	Work with computer controlled electrochemical sensor, data collection with LabView software, data work-up with MS Excel.
Stock solution prep	Using analytical glassware, prepare stock solutions of H2O2, creatine, phosphate buffers, sarcosine, ascorbic acid etc.
Run colorimetric test kits	Use analytical glassware to run commercial clinical lab test kits for creatine, use spectrophotometer to measure concentrations.
Prepare polymer membranes for sensor	Develop polymer formulations and prepare sensor membranes.

Project Title – Development of Wound Responsive Biomaterials

Project Objective

Unfortunately, current controlled drug release systems do not account for biological variations or traumatic events to implantable devices beyond their initial healing periods. They simply deliver the biologically active agents with a known release rate versus time. In other words, these systems are not responsive to the environment that they are placed, i.e. host tissues. This project will begin to address these issues by developing the chemistry that facilitates a responsive release of drugs from a biomaterial with respect to pH. It is the object of this research to develop a drug-releasing silicone biomaterial that is responsive to the localized wound-healing environment by increasing the rate of drug delivery with a decrease in localized pH. Furthermore, the rate of drug delivery will decrease or even stop when the local pH returns to more neutral levels. In this way, a responsive delivery material will not only be useful for the initial implant period, but such a material would reactivate the release of bound drug upon reinjury of the implant site.

Background

It has long been recognized that the materials commonly used to construct implantable medical devices stimulate an inflammatory response. Upon implantation, all foreign material is detected as such and a set of responses is triggered in reaction to the wound and continuous presence of the implanted material. This response can be divided into two phases. The first phase consists of mobilization of mast cells and then infiltration of predominantly polymorphonuclear (PMN) cells. This phase is termed the acute inflammatory phase. Interestingly, it has been reported that during this time the localized pH can reach levels as low as six.[7-9]

Over the course of days to weeks, these cells are replaced by chronic cell types, thus begins the second phase of inflammation. Macrophage and lymphocyte cells predominate during this phase. Ultimately, permanent scar tissue is formed and is called a foreign body capsule (FBC). The types of cells, their general roles and their orders of appearance and disappearance in the FBC have been well characterized. During this second stage the local pH returns to more neutral physiologic levels.

Examples of project tasks

Task	Skills Used / Learned
Calibrate diffusion cell sensors	Using analytical glassware, prepare drug solutions and develop electrochemical sensor parameters for their detection.
Measure drug diffusion	Use calibrated electrochemical drug sensors and diffusion cells to measure transport of drugs through silicone membranes.
Synthesize pro-drug compounds	Using organic chemistry techniques, prepare pro-drug compounds that will model the drug attached to a silicone rubber.
Study pro-drug kinetics at variable pH	Expose purified pro-drugs to variable pH solutions and determine rate of cleavage to drug.

[1] Persky, A. M.; Hochhaus, G.; Brazeau, G. A. J. Chromatogr. 2003, 794, 157-165.

[2] Dunnett, M.; Harris, R. C.; Orme, C. E. Scand. J. Clin. Lab. Invest. 1991, 51, 137-141.

[3] Bottomley, P. A.; Lee, Y. H.; Weiss, R. G. Radiology 1997, 204, 403-410.

[4] Rawson, E. E.; Clarkson, P. M.; Price, T. B.; Miles, M. P. Acta. Physiol. Scand. 2002, 174, 57-65.

[5] Tombach, B; Schneider, J.; Matzkies, F.; Schaefer, R. M.; Chenitius, G. C. Clinica. Chimica. Acta. 2001, 312, 129-134.

[6] Erlenkotter, A.; Fobker, M.; Chemnitius, G. C. Anal. Bioanal. Chem. 2002, 372, 284-292.

[7] Varghese, M. C.; Balin, A. K.; Carter, D. M., et al. Arch Dermatol. 1986, 122, 52.

[8] Eisinger, M.; Lee, J. S.; Hefton, J. M., et al. Proc Natl Acad Sci. 1979, 76, 5340.

[9] Blank, I.H. J Invest Dermatol. 1939, 2, 75.