Dr. John Palisano, professor of biology, describes the following ongoing research projects that students can participate in under his direction.
Undergraduate students working in my laboratory have been engaged in three different research projects over the last couple of years. Several students have been investigating the origin of confronting cisternae (CC), a membrane that is found in selected fetal and tumor cells. Several other students are using molecular biology techniques to investigate the changes in bacterial diversity that accompany land use changes on the Cumberland Plateau. I have also recently initiated an investigation of the response of amoeba to electrical fields with Dr. Frank Hart in the physics department and welcome student participation in this investigation.
The Origin of Confonting Cisternae. The objective of this research is to determine if the fragmented membranes that are observed in mitotic cells by immunofluorescence microscopy (IFM) are indeed confronting cisternae (CC). Because lamin B is a protein that is found only in the membrane surrounding the nucleus of the cell and because CC are thought to arise from the nuclear envelope (NE), a monoclonal antibody that recognizes the NE-specific protein, lamin B, is used to determine the location of both NE and CC in dividing cells. While my undergraduate research students and I (see abstracts listed in my CV) have successfully utilized a fluorescently labeled monoclonal antibody to lamin B, it is not possible to determine positively if these fragments are NE fragments or CC with light (immunofluorescence) microscopy techniques. This is because NE and CC are smaller than the limits of the resolution of light microscopy. Scientists can label membranes with fluorescent molecules and visualize where the fluorochrome is because there is a large accumulation of fluorochrome; however, the technique does not allow one to identify which membrane is being labeled, making positive identification impossible. Now that we have successfully demonstrated that we can visualize NE and CC under a fluorescent microscope employing immunofluorochromes, we are using iRNA to knock down the production of lamin B to determine if cells deficient in lamin B are incapable of forming NE following mitosis in an attempt to learn the role that CC play in the formation of NE in selective fetal and tumor cells.
Bacterial Biodiversity with Land Use Change. Several of my students and I have become interested in exploring the microbial diversity that exists on the Cumberland Plateau and to determine what changes in the soil microorganisms occur as land-use change rapidly changes on the Plateau. With this goal in mind, we are utilizing modern molecular biology techniques to learn which bacteria are in the soil and how the population of bacteria changes with land-use change.
Briefly, this investigation involves isolating all the DNA found in the soil at a given location and using the molecular biology techniques known as polymerase chain reaction (PCR) to pull out the DNA of bacteria from all the other DNA found in soil (like worm, insect, fungal, etc.). This is done by using a set of primers (short fragments of DNA that recognize specific genes in the genome of the targeted organism), in this case, that are specific for the 16S rDNA gene of bacteria that encodes for the 16S rRNA. It has been demonstrated through evolutionary DNA and RNA studies that 16S rRNA is a highly conversed (has undergone little change over the eons) molecule because it is essential for protein synthesis. Furthermore, the minor changes that have occurred over time in 16S rDNA gene can be used to show evolutionary relatedness of organisms and predict when major taxa of organisms evolved for pre-existing ones.
Response of Amoeba to Electrical Fields. I recently initiated a study with Dr. Frank Hart who is in the physics department at Sewanee. We are studying the response of Amoeba proteus to DC and AC electrical fields because we are observing directional response changes that occur faster than the currently accepted explanation can explain. While it has been hypothesized that the directional change of amoeba to changing electrical fields is due to the migration of electrically charged receptor in the plasma membrane, we believe that the changes in cellular migration that we are observing in this unicellular protozoan are due to some other factor.
Upon completion of the investigation of the response of amoeba to electrical fields, we will initiate a study of the response of macrophages to electrical fields. Scientists have shown that there are small, but measurable, electrical fields around wound sites in animals; however, no one has adequately investigated this phenomenon. We want to study how macrophages, which are involved in wound healing, respond to these electrical fields to determine if manipulation of the electrical field around a wound can be used to facilitate the healing process. Furthermore, immature macrophages, known as monocytes that are found in the blood stream, have been implicated in the formation of plaques during healing in the arteries. It would be interesting to see if this plaque formation can be inhibited by manipulating the electrical field at the wound that initiates the plaque.