 |
Research Advisor & Department
Patrick Stover - Nutritional Sciences
Name of Project:
Role of cSHMT in adult neurogenesis and age-related cognitive decline
Abstract:
In the cell, folate is present in the form of tetrahydrofolate (THF) and serves as a cofactor that activates and carries one-carbon units—much in the same way that ATP carries phosphate groups. THF is involved in reactions that include the synthesis of thymidylate, purines, and SAM, an important cofactor in a plethora of methylation reactions. Recently, it has been shown that deficiencies in folate metabolism reduce neuronal proliferation in the adult hippocampus—a process thought to be important in memory formation; however, the mechanism behind this reduction remains unknown. To begin addressing this paucity of information, we examined cytoplasmic serine hydroxymethyltransferase (cSHMT) expression in the brain because cSHMT is an enzyme that regulates folate metabolism by preferentially shuttling one-carbon units toward thymidylate synthesis in a cell-specific manner. In situ hybridization experiments in our lab localized cSHMT to regions of constitutive neurogenesis, including the hippocampus. Currently, I am working on examining the effects of depressed cSHMT expression and depressed dietary folate levels on both neuronal proliferation and differentiation using immunohistochemical-labeling procedures. Preliminary findings suggest that decreased cSHMT reduces proliferation in the hippocampus, but these results must still be confirmed.
|
|
Research Advisor & Department
John Bunge - Social Statistics
Name of Project:
Estimating Microbial Richness Using Statistical and Stochastic Modeling
Abstract:
The purpose of my project was to estimate biodiversity of microorganisms based on multiple samples of data sent to me by researchers in New Zealand who had taken all the microbial sequences from Genbank. Then the sequences where broken down into 46 samples and they were given to me to analyze using the statistical software package SAS to convert this array like data into a format that the program DENSITY, which is for capture-recapture work could use. We had about 10,000 observed species in a typical data set. The array like data that I had before consisted of an array of 0’s and 1’s, a "1" means that the given species (row) was observed in a given sample (column). The ultimate goal of the research was to estimate the number of uNobserved species in the sample, such a species would have all- zero rows but would not show up in the sample at all. Overall the research has progressed reasonably well despite the set backs I have had due to my inexperience with SAS, and my lack of programming knowledge in general. The results of the unobserved species in the samples thus far have been very preliminary and more analysis will be done when all the samples from Genbank 2008 are uploaded, then the same type of analysis when be performed in 2008 when Genbank is even larger than it is now. I was not very involved in the statistical analysis of the data, as it was secondary data and thus produced by some other people and given to me to convert into a usable form that the various peaces of software could understand. My primary research involved simply converting the data into various formats for the software to use involving SAS programming and developing SAS programs to automate the data analysis and conversion process. I have my data results for the population size of the observed species, but given my inexperience with the statistical methods and having dealt with secondary data from another party, I cannot say with certainty weather the estimation model was accurate in estimating the number of unobserved species in the 46 samples.
|
 |
Research Advisor & Department
Rob Thorne - Physics
Name of Project:
Studies of Protein Activity and Cell Membrane Injury after Freeze-Thaw, and Optimizing Recovery Utilizing Different Techniques
Abstract:
Cryopreservation is a powerful technique to store and preserve tissues, cells and proteins for future use. It is useful in preserving genetic material, preventing microbial contamination, reducing risk of genetic drift and morphological changes, and for indefinite storage of genetic material of bioengineered lines and of endangered species.
Cryopreservation faces several challenges that hinder achieving high recovery percentage of the cryopreserved biological material. These challenges might be on the scale of tissues and cells (i.e. mechanical damage because of growing ice crystals) or on the scale of molecules (chemical and electrostatic effect because of increase in solute concentration, disrupted electrostatic interaction and pH shift, all due to withdrawal of water from solution to crystallize). Proteins often get denatured during the process of cryopreservation, particularly by the chemical and electrostatic effect.
However, these challenges could be minimized and recovery percentage could be maximized through carefully adjusting several factors like cooling rate, thawing rate, cryoprotectants and buffers used.
In this project, cryopreservation of protein solutions was investigated in an attempt to develop a protocol that leads to maximize recovery after freeze-thaw cycle. The effect of cooling and thawing rates, and the effect of the geometry of the protein solution containers -which affects the temperature gradient through the protein solution-were investigated. The work has been done with LDH (lactate dehydrogenase, from rabbit muscle).
Results showed that fast cooling (using liquid nitrogen, 77k) is better than slow cooling (using conventional freezer, -15°C). Results also showed that fast thawing rates in warm water (37°C) and warm oil (37°C) to be better (warm water is best) and slow thawing rates using conventional refrigerator (9°C) and air (25°C) to be worse (refrigerator is worst). Moreover, thin wall plastic tubing (0.083Ó ID, 0.006Ó thick) proved to be better than Eppendorf tube (0.175Ó ID, 0.04Ó thick) as containers for protein solutions in cryopreservation.
|
|
Research Advisor & Department
Pat Rivlin - Neurobiology and Behavior
Name of Project:
The Effect of Octopamine, a Homologue of the Mammalian Norepinephrine, in Drosophila Bang-sensitive Flies
Abstract:
Norepinephrine (NE), a hormone and a neurotransmitter, is known as an anti-convulsive drug in mammals. Octopamine (OA), which is the structural and the functional homologue of NE in flies, is suspected to have the same effect on seizures. OA is synthesized from TA by an enzyme called tyramine-beta-hydroxylase (T_h).
Our experiment consists of two parts. In the first part of this experiment, a genetic test will be performed to generate bang-sensitive - Tbh null recombinant mutants, which will be tested for seizure susceptibility by exposing them to a strong stimulus, like vortexing. Since these mutants will have a lower level of OA, due to the Tbh deficiency, we expect it to be more prone to seizures. On the second part of the experiment, eas and bss mutant flies were fed OA for two days and they were tested for seizure susceptibility after each day. Results of seizure duration and the different seizure phenotypes observed were compared to non-OA fed eas and bss mutants as well as Canton s-5 flies(wild type, +OA food and ÐOA food), eas and bss (-OA food). We expected the OA-fed bang-sensitive mutants to have lower seizure susceptibility than the control mutants. This was done to further understand the change observed in the first part of the experiment, as to whether it was due to an increase in TA or a decrease in OA. Another advantage of doing both studies is to see if TA acts as an antagonist to OA in effecting seizure susceptibility as it does in some other physiological functions.
From the results we obtained, we observed a significant decrease in helicoptering (a form of post seizure behavior) in easily-shocked mutants after two days of OA feeding. We found no significant difference in seizure durations between the experimental and the control groups.
|
 |
Research Advisor & Department
Lee Kraus - Molecular Biology and Genetics
Name of Project:
Regulation of Chromatin Structure by Poly(ADP-Ribose) Polymerase-1
Abstract:
Chromosomes exist in a highly compact state in the nucleus of a cell. The least compact form of chromatin, known as the 11nm fiber, is composed of DNA punctuated uniformly by nucleosomes. Each nucleosome is an octomer of histones H2A, H2B, H3, and H4. Binding of histone H1 to nucleosomes leads to the formation of the 30nm fiber, a more compact form of chromatin. Previously, poly(ADP-ribose) polymerase-1 (PARP-1) was shown to cause similar compaction of the 11nm fiber to the 30nm fiber. My objective is to study the mechanism of chromatin compaction by PARP-1. I have used electrophoretic mobility shift assays as well as chromatosome stop assays to examine the in vitro binding of PARP-1 to mononucleosomes. These assays as well as other in vitro binding assays will be used to study the hypothesis that during compaction, two individual PARP-1 molecules dimerize via the catalytic domain while the DNA binding domain is necessary for binding to DNA.
|
 |
Research Advisor & Department
Maureen Hanson - Molecular Biology and Genetics / Plant Science
Name of Project:
Studies of the Function and Expression of a Pentatricopeptide Repeat Protein in Zea mays
Abstract:
Higher plants, such as maize, rice, and Arabidopsis, have a large family (over 450 members) of pentatricopeptide repeat (PPR) proteins which, though nuclear-encoded, typically act in the organelles (mitochondria and chloroplasts). PPR proteins contain multiple copies of the 35 amino acid PPR motif, which are found in tandem1. Proteins with this motif have been shown to bind RNA and have been implicated in many diverse aspects of transcription and translation, such as RNA splicing and editing. Maize lines with defective genes that encode plastid-targeted RNA-binding proteins such as PPRs can be identified via the screening of the Photosynthetic Mutant Library (PML) 2. In particular, this project investigates the function and localization of a maize gene product, PPR103, the absence of which results in an albino mutant phenotype.
The aims of the project are twofold: to identify the particular nucleic acid substrate with which PPR103 interacts and to determine the precise function of PPR103. I plan to achieve the first goal of this study by using a specific anti-PPR103 antibody to co-immunoprecipitate the protein while it is bound with its native interaction partners. The RNA substrate can then be eluted from the complex and then identified by hybridization to a microarray that contains the entire chloroplast genome3. Once the RNA substrate is known, editing and splicing assays can be performed to elucidate the specific role of PPR103 in its maturation. I also plan to use biolistics to transiently express a fusion protein composed of the putative targeting signal of PPR103 and green fluorescent protein to confirm the predicted subcellular targeting of the protein4.
1. AndrŽs, C., Lurin, C., Small, I.: The multifarious roles of PPR proteins in plant mitochondrial gene expression. Physiologia Plantarum 129(1): 14-22 (2007).
2. Barkan, A., Walker, M., Nolasco, M., Johnson, D.: A nuclear mutation in maize blocks the processing and translation of several chloroplast mRNAs and provides evidence for the differential translation of alternative mRNA forms. EMBO J. 13: 3170-3181 (1994).
3. Schmitz-Linneweber, C., Williams-Carrier, R., Barkan, A.: RNA immunoprecipitation and microarray analysis show a chloroplast pentatricopeptide repeat protein to be associated with the 5' region of mRNAs whose translation it activates. Plant Cell 17: 2791-2804 (2005).
Peeters, N., Chapron A., Giritch, A., Grandjean, O., Lancelin, D., Lhomme, T., Vivrel, A., Small, I.: Duplication and quadruplication of Arabidopsis thaliana cysteinyl- and asparaginyl-tRNA synthetase genes of organellar origin. J. Mol. Evol. 50: 413-423 (2000).
|
