Summer Projects

Examples of previous research projects

2014 Student Projects Examples

MRI Brain Tissue Segmentation: A Case Study for Diagnosis of Alzheimer

Mentor: Ayman El-Baz, PhD, Associate Professor, Department of Bioengineering, J. B.
Speed School of Engineering, University of Louisville, Louisville, KY 40292;
KBRIN Summer Student: Austin Black is a junior student at the Bellarmi ne
University, Louisville, KY 40205.

Description of the Proposed Project
A novel framework for developing a Computer-Aided Diagnosis (CAD) of Alzheimer based on structural MRI data. Classification methods for dementia have demonstrated high performances, but are not yet used in clinical practice. A major reason for this is that these methods are not well enough validated for clinical use. we aim to take a step forward to the clinical use of computer-aided diagnosis for Alzheimer by participating a challenge to compare the performance of the proposed CAD system to the current state of the art image-based diagnosis methods. The proposed CAD system is divided in t.wo parts: (i) brain segmentation and (ii) features extraction and diagnosis. The first step is done based on integrating three models: geometrical model to preserve the brain geometry, stochastic model to account for the brain inhomegeinty and shape model to enhance the classification accuracy. The second step is done based on analyzing the features of the segmented brain tissues such as Corpus Callosum (CC) length, shape analysis of the cerebral cortex, and the cerebral white matter (CWM) thickness.
Experiments & Activities for KBRIN Summer Intern: Preliminary Training for Austin:

  • Learning the existing imaging software for preprocessing medical imaging
    (week 1+).
  • Learning the MA TLAB programming language to design the software for
    automatic analysis of magnetic resonance (MR) images. (week 1&2).
  • Learning the existing tool for performing the statistical analysis (week 3+)
  • Validation the results obtained by the designed software for automatic
    analysis of MR images (weeks 1-4).

Experiments to be performed (weeks 5-9):

  • Segmentation of Brain from MR images.
  • Building the 3D model for the segmented brain.
  • Classification/Diagnosis of the segmented brain.

Lipids that accumulate in the vitreous cause collagen and hyaluronan to liquefy.

KBRIN Summer Student: Emily Dennis

Emily Dennis will study human vitreous liquefaction using Raman spectroscopy. Her hypothesis to test is: lipids that accumulate in the vitreous cause collagen and hyaluronan to liquefy. Liquefaction leads to retinal tears and blindness.

Gas sensors for analysis of acetone in exhaled breath

Mentor: Xiaoan (Sean) Fu
Department of Chemical Engineering, University of Louisville

KBRIN Summer Student: Allison Durr

Gas sensors have found wide applications in monitoring environmental air quality, detecting explosive chemicals for homeland security and analyzing exhaled breath for diagnosis of diseases. However, because of interference of trace volatile organic compounds (VOO;) in air and exhaled breath, it is extremely difficult to use one gas sensor to analyze target VOCs in air and exhaled breath. In recent years, microfabricated gas sensor array or so called electronic noses have demonstrated great promises for analysis of complex VOC mixtures in air and exhaled breath. However, the challenges of low sensitivity, poor selectivity and narrow operation ranges hinder gas sensor an ays for applications in breath analysis. The objective of this Summer KBRIN project is to study gas sensors based on gold nanoparticles with different surface molecular modification for analyzing target acetone in breath. It has been known that high acetone concentration in breath is directly related to glucose level in blood of diabetic patients. So far, there is no gas sensor can be used to analyze acetone in breath. The hypothesis of this project is that the sensitivity and selectivity of gold nanoparticle-based gas sensors are controlled by the surface molecular modification (or surface antenna). The approach is to investigate gas sensors made from gold nanoparticles with different surface molecular modification. The gas sensor arrays will be fabricated using microelectromechanical systems (MEMS) technologies. An undergraduate student Allison Durr from the department of Chemistry at the Eastern Kentucky University will conduct this research. The student will study the effects of gold nanoparticle surface functionalization on the sensitivity and selectivity and detection range of gas sensors for sensing acetone. This project will provide the student with research experience in Chemistry, MEMS, gas sensor, and breath analysis for biomedical application.

There are 5 tasks for this 10-weeks KBRIN research project:
1. Learn to search literature and prepare a brief review of gas sensor
2. Learn to synthesize gold nanoparticles and surface functionalization
3. Prepare gold nanoparticles-based gas sensor
4. Characterize the gas sensor array for sensing acetone in air and exhaled breath
5. Study sensitivity and interference the gas sensor
6. Learn to do data analysis and write a scientific paper

During the 10-weeks research, My PhD student Zhenzhen Xie supervises a 40-hour work per week for the student. I have one-to-one weekly meeting with her to provide advice and check research progress. The student learns literature search; synthesis of gold nanoparticles and surface functionalization, preparation of gold nanoparticle-based gas sensor, characterization of the gas sensor for sensing acetone in air and exhaled breath and data analysis. She will also learn to write a scientific report. She will have opportunities to interact with my post-doctoral researchers and collaborators in breath analysis for diagnosis of lung cancer. The student will attend the mandatory weekly meeting during the entire summer session and make a presentation at one Wednesday group meeting. In addition, the student will present at the final poster session to be held on August 1, 2014.

Ribosomal biogenesis in the developing nervous system.

Mentor: Dr. Michal Hetman at the Kentucky Spinal
Cord Injury Research Center and Department of Neurological Surgery, University of Louisville

KBRIN Summer Student: Shane Elliott

A sophisticated ribonucleoprotein complex called ribosome is the nexus for protein synthesis.
Although protein synthesis is well recognized as a driver of brain development and a critical
component of brain maintenance, little is known about significance of ribosomal biogenesis in
the brain. Hence, there is a need for more studies in th is area especially as ribosomal
biogenesis appears impaired in several neurodevelopmental and neurodegenerative diseases.
For instance, reduced ribosomal biogenesis has been suggested to contribute to Alzht3imer's
disease as well as several developmental syndromes including Rett's, Prader-Willi's or BowenConradi's
(Hetman and Pietrzak, 2012). In the laboratory of dr. Michal Hetman an
interdisciplinary approach is applied to investigate involvement of ribosomal biogenesis in
neuronal growth and maintenance (Gomes et al., 2011 ; Kalita et al. , 2008; Pietrzak et al. , 2011 ).
The current research opportunity is directly related to these studies. Specifically, a summer
student will join a team effort to identify how making new ribosomes in a specialized nuclear
structure that is called nucleolus affects neurite outgrowth as well as distribution of ribosomes
and mRNAs throughout a neuron including neurites and synapses. In summer 2014 a special
emphasis will be placed on descriptive analyses of ribosomal biogenesis and its regulation
throughout development of the rat brain. Students are expected to become fully involved in this
research performing experiments, analyzing data and contributing to the resulting publications.
The laboratory uses a variety of molecular, cellular and genetic techniques in cultured cedi- and
whole animal systems. To learn more about this opportunity please refer to the cited references
or visit the laboratory webpage (http://louisville.edu/kscirc/faculty-laboratory-websites/laboratoryof-
neurosignalling/laboratory-of-neurosignalling.html).

Students will benefit from participating in this research by
1. learning molecular-cellular neurobiology techniques
2. developing critical thinking skills and learning experimental design and analysis
3. developing presentation skills of scientific data
4. learning about mechanisms of neurodevelopment and neurodegeneration.

Does Withaferin A (WFA) act synergistically with ca rboplatin and paclitaxel and will a combination of carboplatin, paclitaxel, and WFA kill the cancer stem cells as well as the differentiated cancer stem cells.

KBRIN Summer Student: Kara Garcia

Epithelial ovarian cancer (EOC) remains the leading cause of death in women among gynecologic cancers and is the 5th highest cause of cancer-related deaths in women in the United States. The majority of ovarian cancers are diagnosed at advanced stage due to the mainly non-specific symptoms. Currently, the treatment for ovarian cancer entails cytoreductive surgery followed by chemotherapy, employing mainly platinum/taxane combination. Although this regimen is initially effective in a high percentage of cases (70 to 80%), unfortunately 70% of women develop recurrent cancer within few months of initial treatment as a result of platinum-resistance. In addition, cisplatin (CIS) is associated with multiple severe side effects such as nausea, vomiting, myelosuppression, hepatotoxicity, neurotoxicity, nephrotoxicity and ototoxicity. Therefore, need for new treatment options that target cancer cells and in particular putative cancer stem cells is mandatory either at first-line setting or even more at the first- and second-line management of recurrent ovarian cancer.

Withaferin A (WFA) has been a part of Indian traditional medicine for centuries. It is available in US over-the-counter as a dietary supplement and is known to treat various disorders due to its anti-inflammatory, anti-bacterial, and cardio protective properties. In recent years, WFA has been suggested as a potential anti-cancer compound shown to prevent tumor growth, angiogenesis, and metastasis in various types of cancer. Mechanisms by which WFA attains its anticancer activity include inactivation of Akt and NF-KB to achieve apoptosis, decrease in prosurvival protein Bcl-2, G2/M cell cycle arrest, generation of reactive oxygen species (ROS), induction of Par-4, activation of caspase 3 and 9 activities, DNA damage, inhibition of HSP90, regulation of FOX03a and Bim inhibition of Notch-1 and down regulation of expression of HPV E6 and E7 oncoproteins.

There are two main questions my research is directed to answer. The first is whether or not Withaferin A (WFA) acts synergistically with ca rboplatin and paclitaxel. The second question is if combination of carboplatin, paclitaxel, and WFA is able to kill the cancer stem cells as well as the differentiated cancer stem cells. For my research I have been treating cells seeded in 96 well plates with various concentrations of drug as a signal agent or drug combinations. Effects of signal agent or in combination are assayed by performing cell proliferation assays using MlT assays. I input this data into the CalcuSyn software .which generates an isobologram that will determine whether the effect is synergistic, antagonistic, or additive. Cell apoptosis is measured by using Annexin V assays followed by FACS analysis. Western Blots are performed for the cell extracts treated with each drug alone or in combination. These include cells t reated with paclitaxel (SnM), carboplatin (25uM), WFA (O.SuM), WFA (l.SuM), Paclitaxei+Carboplatin, Paclitaxel + Carboplatin + WFA (0.5), and Paclitaxel + Carboplatin + WFA (l.SuM) , and untreated cells as control. Cancer stem cells regulation is analyzed by using the specific antibody for each surface marker for CD34, CD44, CD24, Cdl17, CD133 and EpCAM. The data is analyzed by quantitating levels of each surface maker protein and its comparison with control (untreated cells). I will also look for the mechanisms that regulate the cancer stem cells populations such as Notchl signaling mechanisms that regulate the self-renewal and maintenance of cancer stem cells populations. In addition, I will also analyze generation of ROS in response to each drug alone as well as in combination. The data will be published in the form of an abstract or a short communication.

Extracellular sodium and potassium concentration, Na+/K+ ATPase expression: Role in cancer cell size, metastasis, and cell proliferation.

Our laboratory has previously shown that the highly metastatic breast cancer cell line MDA-MB453 cells lack mRNA and protein expression of Na+/K+ ATPase. However, these cells have three times higher potassium uptake as compared to the normal cells. Structurally, these cells are very small in size and lack cell-cell junctions. Based on these studies we hypothesized that extracellular sodium and potassium concentrations and Na+IK+ ATPase expression regulate cell size and metastasis. Erika Hardin as a KBRIN student will work on the project which will define whether the extracellular sodium and potassium concentrations and Na+/K+ ATPase expression will affect cell size, proliferation, and metastasis using a control non-cancerous MCF-lOA cell line and 4 breast cancer cell lines. She will flow cytometry to determine cell size. She will then use BrDU incorporation to determine cell proliferation and movement of cells grown on a porous membrane (200 j..lm pore size) to the lower compartment to determine metastasis. As a KBRIN student Ericka will learn several molecular and microscopic techniques including but not limited to transfection, western blot, confocal microscopy, and flow cytometry.

Specifically Ericka learned new innovative techniques in microscopy along with the regular molecular biology techniques.

Application of de novo peptide sequencing methods to discern effects of ethanol on histone posttranslational modifications

KBRIN Summer Student: W. Blake Kusiak
Mentor: Michael L. Merchant, PhD

Liquid chromatography coupled with mass spectrometry (LCMS} enables the analyses of biological molecules for goals such as the (a) the discovery of proteins for markers of disease and to identify posttranslational modifications (PTM} to proteins. Histones are positively charged nuclear proteins that combine with eukaryotic DNA to form chromatin and their modification helps regulate gene transcription. We hypothesized high resolution LCMS methods and de novo peptide sequencing methods could be used to reveal novel histone PTMs. To initiate this work we compared four informatics algorithm software programs (Sequest, Mascot, X!tandem, and PEAKS} for their ability to identify proteins from a tryptic digest of human urine. PEAKS identified 272 proteins, X!tandem identified 239, Sequest found 166, and Mastcot found 125 proteins. Of these, 60 proteins were identified in common by all four programs. Proteins unique to each program were 115 by PEAKS; 42 by X!tandem, 8 by Sequest, and 13 by Mascot. Using PEAKS, we studied the histone PTMs extracted from . alcohol-treated stimulated or unstimulated CD4+ T cells. Cell nuclear extracts were separated using HPLC and SDS-PAGE. Gel bands were excised, proteins digested with trypsin and then analyzed using LCMS. Bioinformatic software was used for identification and relative quantification of proteins and protein post-translational modifications. Our HPLC data showed that histones from ethanol-treated CD4+ T cells (stimulated or unstimulated) suggested an increase in hydrophobicity compared to untreated CD4+ T cells; most likely due to changes in histone acetylation and/or methylation.

Glutamate Induced Apoptosis in Olfactory Neuroepithelial Progenitor Cells Obtained from Bipolar Patients and Non-bipolar Controls

KBRIN Summer Student: David Thompson
Mentor: Rif S. El -Mallakh, MD
Mood Disorders Research Program
Department of Psychiatry and Behavioral Sciences
University of Louisville

Background: Bipolar disorder is a severe psychiatric condition in which afflicted individuals may display loss of brain tissue and loss of cognitive and functional ability over the long-term course of the illness. It has been proposed that apoptosis is an important factor in this clinical decline. Previous work in our laboratory utilizing a novel cellular model has found that neuronal cells obtained from people with bipolar illness may be more sensitive to glutamate-induced excitotoxicity than cells obtained from non-bipolar controls. Clinical and translational work by others has suggested that lithium, a common treatment of this condition, may protect cells from apoptosis. Hypothesis: The hypothesis that will be examined this summer is that lithium treatment of cells in our model will protect cells obtained from bipolar individuals so that the response to glutamate toxicity will be normalized.' Methods: We will utilize Olfactory Neuroepithelial Neural Progenitor Cells (ONPs) obtained from individuals with bipolar disorder (n=3), and matched, non-bipolar controls (n=6). We will examine multiple markers of apoptosis in cells treated with apoptotic doses of glutamate, with or without lithium treatment. Student's Role: We will involve the student in all aspects of this investigation. In the growing and treatment of cells, the ELISA assays for the measurement of antiapoptotic and pro-apoptotic proteins, the analysis of the data, and ultimately, the presentation of the results at the Summer Program poster presentation and Research! Louisville.

2012 Student Projects Examples

Isolation of new mutations in genes of the JAK signaling pathway
Nimoh Boateng (Kentucky State University), Leslie Denhard (Berea College) & Claudia Rodriguez (Bellarmine University)
Mentor: Douglas Harrison
The Janus kinase (JAK) signaling pathway has many essential roles in development and homeostasis in animals from flies to humans. In Drosophila melanogaster, there are homologues of all the primary components for both transduction and regulation of the pathway. There is a family of three proteins, Unpaired and two Unpaired-likes, that are believed to be the secreted ligands that activate the JAK pathway. Although mutations in the unpaired gene have been isolated and characterized, there are no known mutations in the unpaired-like genes. This summer, the, participating students conducted a chemical mutagenesis screen to generate and recover potential mutations in the unpaired-like genes. Approximately 35 candidate mutations were recovered and are now being genetically tested for allelism with the unpaired-like genes. In addition, there is a suppressor of cytokine signaling (SaCS) homologue in flies that is very similar to a class of mammalian regulators of the pathway. No mutations previously existed for this gene. To generate mutant alleles, a Ptransposable element inserted near the fly sacs gene was mobilized and assayed for imprecise excision, a process that deletes flanking genomic DNA. More than 40 imprecise excisions were identified in this screen and are being analyzed molecularly to determine the extent of deleted DNA. These genetic reagents will be invaluable in the continuing characterization of the JAK signaling pathway in Drosophila.

Genetic Screen for Cell Cycle Mutants in Aspergillus nidulans.
Rui Chen (Berea College), Becky Ford (Transylvania University) & Stephanie Jenkins (Bellarmine University)
Mentor: Peter Mirabito
Control of the cell division cycle is required for the growth, development, and health of all organisms. In order to understand the mechanisms of cell cycle control, the genes/proteins involved in cell cycle control must be identified and their actions and interactions studied. Fungi have long been used in genetic analysis of cell cycle control and the information gleaned from fungi has proven fundamental to our understanding of the cell cycle in all organisms, including man. A genetic screen was carried out for cell cycle genes in Aspergillus nidu/ans, a filamentous fungus which has long been used to study cell cycle control. The goal was to make new cell cycle mutants and to use those mutants to identify new cell cycle genes. Each student isolated several mutants and cloned at least one gene. The DNA sequence of those genes have not yet been determined. This project provided a collection of new cell cycle mutants which will be the subject of future, more intensive studies of cell cycle control in Aspergillus nidulans.

Sequencing the Ambystoma mitochondrial genome
Katie France (Bellarmine University)
Mentor: Randall Voss
Salamanders of the genus Ambystoma are models in biomedical and life history research. A mitochondrial genome project was initiated for two ambystomatid salamanders that exhibit considerable morphological variation: A. andersoniand A. dumerilii. Long PCR, DNA-cloning, and DNA sequence analysis was used to obtain approximately 30% of the A. andersoni genome and 15% of the A. dumeriliigenome. Comparative DNA sequence analyses indicated low (1-4%) nucleotide sequence variation between these and two other species with nearly complete mitochondrial DNA sequence. Overall, these data support the idea that ambystomatid salamanders are recently derived yet exhibit considerable morphological and life history variation.

Mutations of pyrimidine degradation in Drosophila.
Lindsay Poling (Transylvania University), Emily Steinmetz (Bellarmine University) & Alice Wright (Western Kentucky University)
Mentor: John Rawls
Degradation of pyrimidines produces ~-alanine, a compound implicated in a wide range of biochemical processes (cuticle pigmentation in insects, CoA biosynthesis, neurotransmitter agents); thus, this pathway performs both degradative and biosynthetic roles. A variety of loss-of-function mutations of the first pathway enzyme have been discovered recently among patients exhibiting toxic reactions to pyrimidine analog chemotherapy. To examine the role of this pathway genetically, a variety of screens were carried out to create mutations of the first two enzymes in Drosophila melanogaster. Mobilization of a transposon near the pydl gene generated 31 strains exhibit suppression of the phenotypes exhibited by mutants of pyrimidine biosynthesis. DNA sequence analysis of a subset of these mutations show that they are partial deletions of the pydl gene. Making use of this suppression phenotype, mobilization of a transposon near the pyd2 gene produced 6 mutant strains, at least two of which are deletions of the 5' end of the pyd2 gene. EMS mutagenesis screens for pyd2 mutants recovered 7 viable, suppressor alleles. Another EMSscreen for recessive lethals inthe pyd2 region was also performed, yielding 18 lethaIs,two ofwhich arealleles of pyd2. DNA sequence analysis of these mutations are underway. Data so far indicate that, in flies as in humans, pydl is required for pyrimidine degradation, but is not an essential gene (null mutations develop essentially normally). On the other hand, pyd2 is required in flies for pyrimidine degradation but is also required for normal development. Thus, pyd2 is probably involved in more than pyrimidine degradation.