Research in The Roper Group
Biological systems have been fine tuned over millions of years to be both highly regulated and highly efficient. For example, signaling processes within single cells can be influenced by small variations in concentrations, locations, and modifications of specific biomolecules. To help understand the biological processes that control normal function (and thereby give us an insight into what goes wrong during a disease state), the technology used to study these biological processes are becoming faster, smaller, and integrated with new methodology.
The overall goal in the Roper laboratory is the development of analytical methods that are faster, can provide new information, are more accurate, or are more efficient than current techniques. We will then apply these techniques to obtain new information on specific biological problems with, of course, the main goal a treatment or cure of the biological problem, but more realistically, a better understanding of the biological problem.
One biological problem that we are interested in is cellular communication. Multiple cell types throughout the body communicate to one another to ensure regulated biological functions. For instance, regulation of blood sugar is coordinated by communication between the pancreas, liver, muscle, adipose, and brain. To communicate, a multitude of small molecules, peptides, and proteins are secreted from these tissues. These secreted messengers can be thought of as words, sentences, paragraphs, etc emanating from the various tissues to communicate their biological state. We would like to develop analytical methods which allow us to "eavesdrop" on these communication processes. Understanding the communication patterns between cells will enable a fundamental understanding of biological processes and may allow us to control the communication in the future. The biological problem we are most interested in is the Metabolic Syndrome. Below you will find more information on aspects of our research.
Multi-color Immunoassays
Capillary electrophoresis (CE) immunoassays have been used for high speed monitoring of cellular secretion. Unfortunately, this methodology has only been applied to a single analyte at a time so that the rapid temporal relationships between multiple analytes has not been observed. One reason for the inability of CE immunoassays to monitor multiple analytes is that the peaks corresponding to the higher concentration analyte will overwhelm any analytes at lower concentrations. Therefore, it is difficult to quantify the analyte found at lower concentrations.
We are utilizing a multi-color detection format to monitor each analyte at different wavelengths. In this way, although spatial separation may not be achieved, spectral resolution enables independent quantitation.
IR-Mediated PCR for Generation of Aptamers
This movie is of infrared-mediated polymerase chain reaction (PCR). The PCR process, which won Kary Mullis the Nobel Prize in 1993, is an enzymatic process that amplifies specific regions of DNA. Click here for more information on PCR, but to understand the movie just know that three temperatures must be reached for the amplification process to occur: 94 °C, 58 °C, 72 °C. We are using a tungsten lamp to heat our PCR solution so that when the lamp turns on, water inside the microfluidic device absorbs the IR radiation and heats to 94 °C. The lamp then turns off and the temperature goes down to 58 °C, etc. A thermocouple placed in the channel senses the temperature and the computer then turns on or off the lamp depending if the temperature is too low or too high, respectively. The program we use to control the temperature is a bit more complicated than that a PID controller is used to ensure that our temperatures are stable.
We use this IR-mediated PCR to generate aptamers in a short period of time. Aptamers are short pieces of oligonucleotides that bind to specific target sequences. The process of generating aptamers is known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment) and was developed in the early 1990's. SELEX is like PCR in that it is a cyclical process; however, in SELEX, an oligonucleotide sequence is selected corresponding to a shape that binds to the target molecule. There are two main steps that are repeated in SELEX: 1. binding of the oligonucleotide to the target molecule 2. amplification of the bound oligomers by PCR. It is in this 2nd step where we use the IR-mediated PCR to greatly reduce the overall time of our SELEX process.
Metabolic Syndrome
Metabolic Syndrome is the term given to a cluster of diseases in an individual, such as type 2 diabetes1, hypertension2, and cardiovascular disease3. This syndrome is often associated with obese individuals, although mice genetically-engineered to have no fat4 and humans that have little fat5, also display some of these same diseases indicating that adipocytes, or fat cells, play a major role in the generation of this disease.6 Due to the prevalence of overweight individuals across the world7, the levels of metabolic syndrome are increasing, unfortunately, the role adipocytes play in this disease have not been fully investigated. Therefore, the analytical methods developed in the Roper laboratory will be applied to the study of aspects of the Metabolic Syndrome.
1. Lazar, M. A. “How obesity causes diabetes: Not a tall tale” Science 2005, 307, 373-375. PDF
2. Ehrhart-Bornstein, M.; Lamounier-Zepter, V.; Schraven, A.; Langenbach, J.; Willenberg, H. S.; Barthel, A.; Hauner, H.; McCann, S. M.; Scherbaum, W. A.; Bornstein, S. R. “Human adipocytes secrete mineralocorticoid-releasing factors” Proc. Nat. Acad. Sci. USA 2003, 100, 14211-14216. PDF
3. Matsuzawa, Y.; Funahashi, T.; Kihara, S.; Shimomura, I. “Adiponectin and metabolic syndrome” Arterioscler. Thromb. Vasc. Biol. 2004, 24, 29-33. PDF
4. Gavrilova, O.; Marcus-Samuels, B.; Graham, D.; Kim, J. K.; Shulman, G. I.; Castle, A. L.; Vinson, C.; Eckhaus, M.; Reitman, M. L. “Surgical implantation of adipose tissue reverses diabetes in lipoatrophic mice” J. Clin. Invest. 2000, 105, 271-278. PDF
5. Petersen, K. F.; Oral, E. A.; Dufour, S.; Befroy, D.; Ariyan, C.; Yu, C.; Cline, G. W.; DePaoli, A. M.; Taylor, S. I.; Gorden, P.; Shulman, G. I. “Leptin reverses insulin resistance and hepatic steatosis in patients with severe lipodystrophy” J. Clin. Invest. 2002, 109, 1345-1350. PDF
6. Saltiel, A. R. "You are what you secrete" Nat. Med. 2001, 7, 887-888. PDF
7. “Obesity: preventing and managing the global epidemic” Report of a WHO Consultation. Geneva, World Health Organization, 2000 (WHO Technical Report Series, No. 894). PDF
IR-Mediated PCR (Quicktime Movie 560KB)
IR-Mediated PCR (Windows Media 2.8 MB)
