Summer Undergraduate Research Program 2021

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KY INBRE at UK group photo

 

Hunter Akers

Hunter Akers

Home Institution:
University of Pikeville

PI:
Dr. Douglas Harrison 

Zachary Farrell

Zachary Farrell

Home Institution:
Northern Kentucky University

PI:
Dr. Elizabeth Duncan 

Genetic analysis of soma-germline communication during spermatid differentiation
Sperm are perhaps the most highly specialized and modified cells in the animal world. They are specifically designed to be efficient couriers of the male’s genetic information. Animal sperm vary in shapes and sizes to carry out this task. Nonetheless, the general process of sperm production is largely similar from insects to mammals. Construction of a sperm is accomplished with the assistance of other cells in the testis. While many studies have uncovered genes that are active in sperm during their development, little is known about the genes and functions of the support cells in the testis. In the fruit fly, Drosophila melanogaster, each 64-spermatid cluster develops as an interconnected cyst and is encapsulated by a pair of somatic support cells, called cyst cells. Recent work in the lab has uncovered that communication between these somatic cyst cells and the spermatids is necessary to direct the separation of spermatids within a cyst during the late stages of spermatogenesis. To better understand the roles of the somatic support cells, this project will identify factors required in cyst cells to complete sperm development. For this project, we will specifically impair the functions of particular candidate genes only in the support cells of the testis to examine the impact on sperm formation. Because the process of spermatogenesis is similar across species, this work will not only aid in understanding cellular collaboration in Drosophila spermatogenesis, but it will contribute to our understanding of spermatogenesis in general.

Tumor suppressor genes are essential for cellular function in multicellular organisms, but many Tumor Suppressor Genes (TSGs) and tumor suppressing mechanisms remain unknown or their mechanisms poorly understood, particularly as they function in vivo. In the Duncan lab, we study planarian flatworms, which are best known for their remarkable regenerative abilities. In addition, they share many striking similarities to cancer cells, including genomic patterns that are known to mark TSGs in human cells, e.g. wide peaks of histone H3 K4 trimethylation (H3K4me3). We have analyzed the patterns of H3K4me3 signal across the planarian genome and identified many loci with this pattern. Moreover, we have identified the enzyme, Set1, that is largely responsible for creating this signature in planarian stem cells. Noteably, depletion of set1 in planarians induces stem cell phenotypes that suggest loss of TSG function, including hyperproliferation and an abnormal response to DNA damage. In his KBRIN project this summer, Zach is cloning genes we have identified as Set1 targets, i.e. potential TSGs. He is then creating dsRNA from these cloned constructs, delivering it to planarian animals, and assessing the effects. He is using both live animal imaging and immunofluorescence to screen for genes that cause phenotypes relevant to tumor suppression. This work will help discover new roles for known genes and uncover the functions of unknown planarian genes.

Jacob Lewis

Jacob Lewis

Home Institution: Morehead State University

PI: Dr. Eve Schneider

Ruby Mason

Ruby Mason

Home Institution: Western Kentucky University

PI: Dr. Jessica Blackburn

Description to come

Development of Research Tools to Study the Role of PRL-3 in Cancer. PRL-3 or PTP4A3 is an oncogenic phosphatase upregulated in a multitude of cancers with roles in tumorigenesis and metastasis. Despite the remarkable number of studies deciphering great insights into the physiological roles of PRL-3, structural challenges and high degree of homology between its family members remain challenging to therapeutically target PRL-3. Therefore, development of new tools to bypass these challenges are needed. Towards this end Nanobodies have emerged as an important research tool and has potential to be implemented in therapeutics as well. In the current project a bacterial immunotoxin PE38 will be fused to the nanobody that is highly specific to PRL-3 by the traditional insertional cloning into a bacterial expression construct pSKB3. The construct is then purified as a Immunotoxin fused to nanobody that has the ability to specifically bind to PRL-3 and potentially be toxic to the cancer cells. This strategy if successful can be used as a translational approach to therapeutically target PRL-3.

Dalton McCown

Dalton McCown

Home Institution: Alice Lloyd College

PI: Dr. Chintan Kakani

Ana Mort

Ana Mort

Home Institution: Bellarmine University

PI: Dr. Jakub Famulski

Description to come

Neural crest cells are responsible for forming multiple tissues in the body. The periocular mesenchyme is a subgroup of neural crest cells that forms the anterior segment of the eye, which includes the lens, cornea, and the iris. In the Famulski lab, zebrafish were used as the model organism to study these periocular mesenchyme cells on the molecular level. The periocular mesenchyme cells were isolated and a single cell transcriptome analysis identified the genes highly expressed by the cells. Si:ch211-251b21.1 and hgd were two of these upregulated genes. Si:ch211-251b21.1 is suspected to be a glutamate receptor of the kainate family, while hgd is involved in pigment synthesis. Knocking out both genes with the CRISPR Cas9 system resulted in a particular phenotype that included maldevelopment of the anterior segment. My project will focus on the role of the potential glutamate receptor si:ch211-251b21.1 for eye development. The subgroup of glutamate receptors in zebrafish embryos will be inhibited with drugs known from previous studies. These drugs include CNQX, UBP 302, Kainate acid, and sym2081. CNQX and UBP 302 are Kainate receptor antagonists, that will block the glutamate receptor function. Kainate acid and sym2081 are Kainate receptor agonist which could produce an enhanced effect of the kainate receptor on the cell. After drug exposure, in situ hybridization and antibody stainings will be performed to examine if the drugs had an impact on the expression and function of si:ch211-251b21.1. The expected result includes a similar phenotype from the CRISPR Cas9 knockout and changes in expression of the si:ch211-251b21.1 gene.

Abigail Secen

Abigail Secen

Home Institution: Asbury College

PI: Dr. Douglas Harrison

Hannah Tanner

Hannah Tanner

Home Institution: Eastern Kentucky University

PI: Dr. Robin Cooper

Genetic analysis of soma-germline communication during spermatid differentiation
Sperm are perhaps the most highly specialized and modified cells in the animal world. They are specifically designed to be efficient couriers of the male’s genetic information. Animal sperm vary in shapes and sizes to carry out this task. Nonetheless, the general process of sperm production is largely similar from insects to mammals. Construction of a sperm is accomplished with the assistance of other cells in the testis. While many studies have uncovered genes that are active in sperm during their development, little is known about the genes and functions of the support cells in the testis. In the fruit fly, Drosophila melanogaster, each 64-spermatid cluster develops as an interconnected cyst and is encapsulated by a pair of somatic support cells, called cyst cells. Recent work in the lab has uncovered that communication between these somatic cyst cells and the spermatids is necessary to direct the separation of spermatids within a cyst during the late stages of spermatogenesis. To better understand the roles of the somatic support cells, this project will identify factors required in cyst cells to complete sperm development. For this project, we will specifically impair the functions of particular candidate genes only in the support cells of the testis to examine the impact on sperm formation. Because the process of spermatogenesis is similar across species, this work will not only aid in understanding cellular collaboration in Drosophila spermatogenesis, but it will contribute to our understanding of spermatogenesis in general.

Description to come

 


KY INBRE at UK group photoAbove are three UofL KY INBRE SURP students (L to R) Jessica Stein, Bo Stoll, and Noah Saltsman (and guest) on the Throwback Thursday Belle of Louisville Cruise


 

 

 

 

  

Skyllar Gayhart

Skyllar Gayhart

Home Institution: Berea College

PI: Dr. Ayman El-Baz

Elise Major

Elise Major

Home Institution: Bellarmine University

PI: Dr. Hermann Frieboes

Renal cancer is the sixth most common cancer in men and the eighth most common cancer in women. Renal cell carcinoma (RCC) is the most common and a highly aggressive type of malignant renal tumor, representing around 70% of all renal cancers. The World Health Organization (WHO) states that the most common sub-types of RCCs are clear cell RCCs (ccRCCs) and non-clear cell RCCs (nccRCC) including papillary RCCs (paRCCs) and chromophobe RCCs (chrRCCs), accounting for approximately 70%, 15%, and 5% of all RCCs, respectively. This taxonomy of RCCs is of immense importance as each sub-type has its own prognosis. Biopsy procedure, the gold standard, is the only technique that can provide a definite diagnosis for renal cancer. However, it is used as the last resort due to its high invasiveness, high cost, and turnaround and recovery times (approximately a week). Therefore, investigation of non-invasive imaging modalities to provide a reliable, accurate, less-expensive, and rapid diagnosis of renal tumors at an early stage is underway. In this research, we aim to identify and integrate the optimal discriminating morphological, textural, and functional markers that best describe the malignancy status of a given renal tumor. The integrated discriminating markers may lead to the development of a novel comprehensive renal cancer computer-aided diagnostic (RC-CAD) for accurate, fast, early, and inexpensive identification of renal cancer. The obtained preliminary results demonstrated an accuracy > 95% for differentiating between benign and malignant RCC renal tumors and identify the RCC sub-types for optimal medical management.

This project explores the application of modeling and simulation to understand disease progression and patient response to therapy. The goal is to provide clinicians with insight into tailoring treatment to individual patients. The student will be trained in machine learning techniques, as well as laboratory benchwork.

Noah Saltsman

Noah Saltsman

Home Institution: Spalding University

PI: Dr. Nicholas Mellen

Sarah Stasel

Sarah Stasel

Home Institution: Western Kentucky University

PI: Dr. Xiao-An Fu

Central circuits that control swallow are located in dorsal medulla, and project to (pre-)motoneurons in ventral medulla. These circuits have not been characterized. The goal of this summer project is to cross index optical recordings and immunohistochemistry to identify the anatomical networks that mediate swallow, and to taxonomize their constituents based on their immunohistochemical profiles.

We will use a transgenic mouse expressing the genetically-encoded Ca2+ indicator GCaMP6F in the germline to carry out optical recordings in vitro from these networks. To elicit orofacial behaviors, a stimulating electrode will be displaced along the dorsal half of the brainstem, and the effect of stimuli will be recorded optically from the sagittal face of the sectioned brainstem, while motor output will be recorded via suction electrodes sampling activity from ventral root C4 and the hypoglossal nerve (XIIn). Thereafter, brainstems will be fixed, and a 400 um thick section will be cut from the face from which optical recordings were made, and processed immunohistochemically, and then imaged using the confocal microscope in the microscopy core of the spinal cord injury center. We will screen for choline acetyltransferase (ChAT), somatostatin receptor (SST), and phox2b. The first is expressed in motoneurons, the second in constituents of respiratory rhythm-generating networks, and the third in visceral afferent pathways.

Noah will shadow on the electrophysiology experiments, keeping notes on stimulus location and amplitude. He will then analyze the data using machine vision software developed in-house. He will be trained in the sectioning and immunoprocessing of tissue samples, and he will shadow on the confocal microscope.

From Dr. Fu:
Development of a new analytical method for analysis carbonyls in exhaled breath. The analysis of human breath has a great potential to be developed as a powerful non-invasive tool for evaluating inhaled toxicants and diagnosis of diseases including cancers, chronic obstructive pulmonary disease (COPD), bacteria and virus infections. However, there are some critical challenges hindering breath analysis for clinical applications. These challenges include identifying trace volatile organic compounds (VOCs) related to disease biochemical processes and reducing interference of other VOCs in exhaled breath. We have developed a novel microreactor approach for chemoselective capture of carbonyl VOCs in exhaled breath. The innovations of this approach enable quantitative analysis of both aldehydes and ketones by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) and gas chromatography-mass spectrometry (GC-MS). The goal of this KY_INBRE summer project is to compare a new reagent 4-(2-aminooxyethyl)-morpholin-4-ium chloride (AMAH) coated micrchips with our current reagent 2-(aminooxy)ethyl-N,N,Ntrimethylammonium iodide (ATM) loaded microchips for capture of carbonyl compounds in exhaled breath. The obtained results will enable analysis of carbonyl compounds in exhaled breath by gas chromatography-mass spectrometry (GC-MS). The undergraduate research student Sarah Stasel from Western Kentucky University will focus on characterization of the new reagent (AMAH) for capture efficiencies of a few deuterated compounds including deuterated propanal, 2-butanone, pentanal using our current microchips. These compounds are found in exhaled breath. She will develop analytical methods for separation and detection of AMAH adducts of these compound by UHPLS-MS and GC-MS. The capture efficiencies will be compared with current reagent ATM loaded microchips. Finally, the AMAH loaded microchips will be used for analysis of exhaled breath samples.

There are six objectives for the 10-weeks KY INBRE training and research project:

  1. Learn to do literature search and prepare a brief review of analysis of volatile organic compounds in exhaled breath
  2. Learn to use UHPLC-MS and GC-MS for analysis of VOCs
  3. Learn to operate microchips and preparing samples for quantitative analysis
  4. Characterize capture efficiencies of ATM and AMAH loaded microchips
  5. Learn to analyze exhaled breath samples using the microchips
  6. Learn to do data analysis and write a scientific report and manuscript for publication

During the 10-weeks research, my PhD student Zhenzhen Xie supervises and trains the KY INBRE student. I will have one-to-one weekly meeting with the student to provide advice for the research project.

Jessica Stein

Jessica Stein

Home Institution: Murray State University

PI: Dr. Tamer Mohamed

Bo Stoll

Bo Stoll

Home Institution: Thomas More University

PI: Dr. Michal Hetman

Induction of Cardiomyocyte Proliferation for Heart Failure Treatment
Background: Heart failure is often caused by loss of cardiomyocytes. To identify a combination of cell cycle regulators that can induce stable cytokinesis in adult post-mitotic cardiomyocytes, we screened a combination of cell-cycle regulators expressed in proliferating fetal cardiomyocytes. Overexpression of cyclin-dependent kinase 1 (CDK1), CDK4, cyclin B, and cyclin D efficiently induced cell division in post-mitotic mouse and human cardiomyocytes. In vivo, lineage tracing revealed that the four cell cycle regulators induced cardiomyocyte cell division of adult cardiomyocytes, resulting in significant improvement in cardiac function following acute or subacute myocardial infarction. These findings reveal a discrete combination of genes that can unlock the proliferative potential in cells that had exited the cell cycle (Mohamed et al., Cell, 2018).

Goal: Achieve a cell cycle cocktail of genes/shRNA that fits in one cardiomyocyte-specific viral vector and generate >20% proliferating cardiomyocytes.

Spinal cord injury (SCI) is a devastating condition in which loss of neurons, their axons and axon myelinating oligodendrocytes (OLs) is a major driver of a long lasting functional deficits including disrupted sensation and locomotion. SCI causes immediate loss of neural tissue at the site of impact. In addition, delayed secondary damage broadens tissue loss enhancing deficits. Reducing the secondary injury is a clinically translatable neuroprotective strategy that could attenuate functional deficits and improve patients outcome after SCI. To identify new targets for neuroprotective therapies that could be relevant in SCI, we determined gene expression changes specifically in OLs at various times after SCI. We have identified several candidate genes that may regulate secondary injury-associated death of OLs.

The summer research rotation student could help with further validation of the relevant targets. The following questions could be addressed:

  1. Which of the candidate genes are upregulated after experimental SCI at the protein level?
  2. Upon overexpression in rat OLs in cell culture, which of them are cytotoxic?

Techniques that could be learned include: primary OL cultures, plasmid DNA preparation, transfection of primary OLs, immunofluorescence staining, epifluorescence and confocal microscopy, image analysis, tissue processing for histology.

Suggested readings:
Experimental approaches:

  • Kilanczyk E, S Saraswat Ohri, SR Whittemore, M. Hetman. (2016) Anti-oxidant protection of NADPH-depleted oligodendrocyte precursor cells is dependent on supply of reduced glutathione. ASN Neuro, 8(4). pii: 1759091416660404. doi:10.1177/1759091416660404. PubMed PMID: 27449129.
  • Slomnicki LP, SA Myers, S Saraswat Ohri, MV Parsch, KR Andres, JH Chariker, EC Rouchka, SR Whittemore, M Hetman. (2020) Improved locomotor recovery after contusive spinal cord injury in Bmal1-/- mice is associated with protection of the blood spinal cord barrier. Sci Rep. 10(1):14212. doi:10.1038/s41598-020-71131-6. PMID: 32848194.

Review on pathogenesis of SCI:
https://www.nature.com/articles/nrdp201718

Makayla Wright

Makayla Wright

Home Institution: Northern Kentucky University


PI: Dr. Dae-Sung Hwangbo

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Description to come