Building biomedical research capacity in North Dakota
by serving public universities and tribal colleges within the state
NDINBRE Supported By
Institutional Development Award (IDeA)
National Institute of General Medical Sciences
National Institutes of Health
North Dakota IDeA Network of Biomedical Research Excellence
University of North Dakota - School of Medicine & Health Sciences
1301 N. Columbia Road, Stop 9037 - Grand Forks, ND 58202-9037
Telephone: (701) 777-6376 - Fax: (701) 777-6372
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Research

This American Society for Pharmacology & Experimental Therapeutics (ASPET) Institutional Summer Undergraduate Research Fellowship (SURF) Program provides hands-on summer research experiences to undergraduate students.† The scientific research focus of this SURF program is Neuropharmacology.† Research will be conducted under the mentorship of faculty in the Department of Biomedical Sciences at the University of North Dakota School of Medicine & Health Sciences in Grand Forks, ND.† Mentors, all of whom are members of ASPET will work closely with students to develop an independent research project.† Some possible research projects involve adrenergic regulation of neurogenesis & cognitive function; dopamine transporter (DAT) pharmacology & regulation, DAT & serotonin transporter structure function; epigenomic modification; neuroimmunology.  These research projects are relevant to a number of neurological disorders including Addiction, Alzheimerís disease, Anxiety, Depression, Epilepsy, Parkinsonís disease, and Schizophrenia.† Specific research activities being conducted by ASPET faculty mentors are:



Project Title: Novel Strategies for Attenuating the Inflammatory Contribution to Alzheimerís Disease
Mentor: Colin K. Combs, Ph.D.
Professor and Chair, Department of Biomedical Sciences
Research Aims:  This projectís goal is to determine the mechanisms by which immune cells in the periphery interact with brain resident immune cells to influence brain function during aging and Alzheimerís disease (AD).  Population and single cell transcriptomic analysis of brain resident immune cells, microglia, clearly demonstrate that these cells change their phenotype during AD.  Moreover, numerous epidemiologic and GWAS studies support a critical role for inflammatory changes during disease.  Although some of these changes are related to the microenvironment of the brain, the influence of peripheral immune cell behavior on the phenotype of microglia has become increasingly understood during disease.  Our prior work has shown that selectively manipulating peripheral immune cell behavior with immunomodulatory strategies is sufficient to provide protective changes in microglial phenotype.  Using a transgenic mouse model of Alzheimerís disease, this project seeks to understand the cross-talk between peripheral immune cells and microglia to better define molecular signals influencing the inflammatory changes that regulate disease progression.  Specific pharmacologic interventions using non brain-penetrant biologic and small molecule approaches will be tested for their ability to attenuate the peripheral to brain communication of inflammatory changes.

Techniques:  Students will learn rodent husbandry, genotyping and drug delivery via intravenous/intraperitoneal injection and oral gavage.  In addition, they will become familiar with PK/PD related to rodent therapeutic testing.  Finally, students will learn enzymatic assays, ELISAs, flow cytometry, immunohistochemistry, mass spectrometry, and microscopy techniques relevant to brain along with statistical analysis, hypothesis testing, and scientific writing skills.

Student Independence:  The SURF student will work with Dr. Combs to design a particular drug delivery paradigm to administer novel immunomodulatory therapeutics to control and AD mice. The student will also be responsible for literature review and suggestion of possible analyses of immune markers from the brain. Once the student has been trained by lab staff and passes an in-lab certification for the particular drug delivery paradigm and animal collection, he/she will be responsible for scheduling and delivering drugs and final brain and blood collections. The student will be expected to help plan the particular series of analyses from the collected tissue and present findings at weekly lab meetings. At all times, the student will have access to Dr. Combs, a lab manager, multiple post-docs, graduate students, and additional undergraduate students for any assistance that is needed.



Project Title: Noradrenergic Regulation of Neurogenesis and Cognitive Function
Mentor: Van A. Doze, Ph.D.
Associate Professor, Department of Biomedical Sciences
Research Project:  Norepinephrine (NE), an important neuromodulator in the brain, modulates cognitive function and synaptic plasticity. NE mediates its effects via activation of adrenergic receptors (ARs). We discovered that adult mice with chronically activated alpha1A-ARs exhibit significantly improved learning and memory, synaptic transmission, mood, and lifespan. In contrast, we found that mice lacking alpha1A-ARs have reduced cognitive function, mood, and lifespan. The mice with activated alpha1A-ARs also show increased neurogenesis in their hippocampi, an area of the brain critical for learning and memory. The molecular cues and genes regulating this process include a wide range of growth and survival factors, but a direct link between NE activity, gene regulation, and neurogenesis, has not been explored. This project will test the hypothesis that NE, through alpha1A-AR activation regulates differentiation and cell fate of neuronal and glial progenitors in the adult mouse brain, and subsequently enhances cognitive function. This project will characterize alpha1A-AR influences on adult mammalian neurogenesis and learning and memory.  These results may have important implications for the treatment of certain neurodegenerative disorders such as Alzheimerís disease and epilepsy.
Techniques: The SURF student will learn immunolabeling, stereology, behavioral studies, electrophysiology, transgenic methodology, and confocal imaging, as well as pharmacological principles, statistical analysis, and hypothesis testing.  The student will also be exposed to literature on adrenergic pharmacology, adult neurogenesis, transgenics, and Alzheimerís disease, as well as present research findings in posters and papers.
Student Independence: The SURF student will have independence to do experiments daily, with assistance as needed, and to analyze results for discussion with Dr. Doze.  The student will be able to suggest experimental directions as dictated by findings and formulate new ideas.



Project Title:  Regulation of Membrane Transporters by Palmitoylation
Mentor: James D. Foster, Ph.D.
Assistant Professor, Department of Biomedical Sciences
Research Project:  The dopamine, norepinephrine and serotonin transporters (DAT, NET and SERT, respectively) are membrane proteins responsible for clearance of their corresponding substrates dopamine (DA), norepinephrine (NE) and serotonin (5-HT) from the extraneuronal space during neurotransmission.  Each monoamine controls distinct behavioral and physiological functions in the nervous system.  Extraneuronal monoamine levels are controlled spatially and temporally by transporter mediated reuptake of released transmitter into presynaptic neurons.  Abnormalities in transmitter levels and subsequent neurotransmission are linked to neurological disorders including major depression, schizophrenia, bipolar disorder, attention deficit hyperactivity disorder, Tourette syndrome, and Parkinson disease, through mechanisms that are incompletely understood.  In addition, the transporters are sites of action for therapeutic drugs such as methylphenidate, bupropion, selective serotonin and serotonin-norepinephrine reuptake inhibitors, used to treat these disorders, and are also targets for addictive drugs including cocaine, amphetamine (AMPH), and methylenedioxy methamphetamine (MDMA) that elevate transmitter levels.
We previously discovered that rat, mouse and human DATs are modified by S-palmitoylation, a post-translational modification in which C16 saturated palmitic acid is added via a thioester linkage to cysteine. Our studies indicate that DAT palmitoylation has the capacity to impact dopaminergic signaling acutely by regulating DA transport kinetics independent of surface losses and chronically by opposing DAT degradation. We have also identified NET, SERT and the sodium hydrogen ion exchanger isoform 1 (NHE1) as palmitoylated transporters but the sites of modification and their influence on transporter function and membrane microdomain localization are unknown. We have begun to identify the signaling molecules and pathways involved in the regulation of transporter palmitoylation and resulting functional outcomes and our proposed project for the SURF student will be to characterize transporter palmitoylation in response to one of the following: drugs of abuse (AMPH, MDMA, etc), growth factors (insulin, BDNF, etc.) or specific signaling pathway inhibitors, and resulting effects on transport capacity and cell surface transporter expression as time permits.
This project is suitable for execution by an undergraduate student in a 10-week time period since the assays are currently successfully employed in the Foster Lab and conditions used have been worked out for studying DAT palmitoylation.  In addition, the studies have scientific merit and relevance to human disease.  The study will introduce a student to many pharmacological, biochemical and cell signaling principles related to transporters, drug abuse, and neurologic disorders as well as to basic scientific principles in experimental methodology.
Techniques:  The SURF student will learn cell culture with pharmacologic treatments, SDS-PAGE, immunoblotting, acyl-biotinyl exchange (palmitoylation assay), substrate uptake assays with pharmacological principles, cell surface biotinylation assay in addition to statistical analysis, and hypothesis testing. The student will also be given exposure to transporter literature, and the opportunity to write up research findings for posters or papers.
Student Independence:  The SURF student will perform daily experiments, with guidance as needed, and will analyze results for discussions with Dr. Foster.  Initially, it is expected that more assistance will be needed while the student gains more independence. In the latter weeks of the experience, the student will have significant input into the interpretation of results and planning of further experiments.



Project Title:  Substrate and Antagonist Molecular Determinants in the Dopamine and Serotonin Transporters.
Mentor: L. Keith Henry, Ph.D.
Associate Professor, Department of Biomedical Sciences
Research Project: The project will focus on identification and characterization of molecular determinants that discriminate substrates from antagonists in the serotonin and dopamine transporters. We have identified that substrate selectivity for neurotransmitters and drugs of abuse such as MDMA (ecstasy) occur through divergent interactions. Understanding these key differences could lead to unique pharmacological approaches to selectively block the actions of drugs of abuse. This project is ideal for an undergraduate and will focus on generating site-directed mutants in amino acids attributed to outer gate function as well and the phosphorylation sites located on the N-terminus. The functional readouts will include expression and transport levels using radiolabeled and fluorescent transport assays along with Western Blot techniques.

Techniques: The SURF student will learn basic and advanced principles of pharmacology and small molecule interactions with targets using biochemical and computational approaches. These will include (but not limited to) transport assays, binding assays, Wester Blots, chemical crosslinking, molecular mutagenesis and cloning, and affinity purification.

Student Independence: The SURF student will be given a project that is independent of other students but will be provided guidance and mentorship appropriate for their experience and background with the understanding they are expected to move toward independence on their project. Dr. Henry will oversee this process and assess regularly via questions and monitoring.



Project Title: Epigenomic Modification of Microglia from Early to Advanced Stages of Parkinson's Disease
Mentor: James E. Porter, Ph.D. (Not hosting SURF participants Summer 2021)
Associate Professor, Department of Biomedical Sciences
Research Project:  Parkinsonís Disease (PD) is a neurodegenerative illness characterized by the loss of dopaminergic neurons in the substantia nigra and the production of alpha-synuclein aggregations called Lewy bodies.  Development of appropriate neuroprotective therapies for PD is hindered by limited understanding of critical molecular events that constitute the underlying cause of PD.  Recent studies have suggested alpha-synuclein may be epigenetically regulated and influence surrounding cells by initiating the inflammatory processes observed in PD.  We hypothesize that secreted alpha-synuclein induces DNA methylation changes in microglia that result in temporal activation of inflammatory phenotypes associated with PD progression.  Using a previously characterized transgenic animal model, we will test the hypothesis by co-extracting and purifying mRNA and DNA isolated from microglia at time points representing early and advanced stages of disease progression.  Purified microglial nucleic acid will then be used to characterize temporal gene expression and DNA methylation changes when compared to age-matched wild-type animals, using mRNA-sequencing and double-digest reduced representation bisulfite sequencing, respectively.  Differences in genome-wide methylation will be linked to gene expression changes and analyzed for variations identified between each stage of PD progression.  Network-based bioinformatics analyses will be employed to identify the most influential elements of these observed changes.  Collectively, the proposed work will identify and validate crucial epigenetically-regulated genes and signaling networks contributing to the progression of PD, which will be consequential as well as translational to the scientific community at large.
Techniques: The SURF student will learn animal dissection, primary cell isolation, cell sorting flow cytometry, and nucleic acid purification techniques as well as statistical analysis, hypothesis testing, and scientific writing skills.
Student Independence:  The SURF student, with support of a Postdoctoral Fellow, will be expected to master two standard protocols routinely performed in the laboratory (cell sorting flow cytometry and nucleic acid purification), which are essential for success of this research project.  The SURF student will also learn steps required for the analysis, interpretation and troubleshooting of raw data generated from these experiments.



Project Title:  Dopamine Transporter Regulation
Mentor: Roxanne A. Vaughan, Ph.D.
Professor, Department of Biomedical Sciences
Research Project:  The dopamine transporter, DAT, is a synaptic protein that drives reuptake of dopamine from the synapse into the presynaptic neuron, and is the major mechanism for regulation of DA neurotransmission.  DAT activity is regulated by phosphorylation of multiple serine and threonine residues, driven by multiple protein kinases, but many major questions remain as to the mechanisms underlying the responses. Regulatory mechanisms linked to phosphorylation are induced not only by exogenous activators of Protein Kinase C (PKC), but also by amphetamine and methamphetamine, indicating the involvement of these abused drugs in endogenous regulatory mechanisms. More recently we have extended our studies to regulatory processes of the synthetic cathinones methcathinone (MCAT) and methylenedioxypyrovalerone (MDPV).  These psychoactive and addictive compounds are of major concern in the drug abuse community, but their mechanisms of action are currently poorly understood.
Preliminary work in our lab has shown that pretreatment of DAT-expressing cells with MCAT and MDPV induce DAT regulatory responses and affect phosphorylation of DAT residue Threonine (T)53.  We have generated a phospho-specific antibody against T53 for analysis of this site, and our proposed project for the SURF student will be to characterize this phosphorylation process for dose and time courses of responses to MCAT and MDPV, using previously characterized responses to amphetamine or PKC as controls.  As time permits, the student will determine levels DAT transport activity in parallel conditions and assess DAT regulatory responses in a phosphorylation-null T53A mutant.  The results will indicate if MCAT and MDPV exert physiological regulatory dysfunction in DAT as part of their overall actions.
This project is suitable for execution by an undergraduate student in that the constructs and assay conditions have been worked out, and the questions to be answered are scientifically important.  The study will introduce a student to many pharmacological principles related to DAT and drug abuse, as well as to basic scientific principles in experimental methodology.
Techniques:  The SURF student will learn DAT SDS-PAGE and pan/phosphospecific immunoblotting, DAT uptake assays and pharmacological principles, cell culture and transfection, statistical analysis, and hypothesis testing.  The student will also be given exposure to DAT literature, and the chance to write up research findings for posters or papers.
Student Independence:  The SURF student will have independence to execute daily experiments, with assistance as needed, and to analyze results for discussion with Dr. Vaughan.  The student will be able to suggest experimental directions as findings dictate and formulate plans for execution of ideas.
  Colin K. Combs, Ph.D.
  Van A. Doze, Ph.D.
  James D. Foster, Ph.D.
  L. Keith Henry, Ph.D.
  James E. Porter, Ph.D.
  Roxanne A. Vaughan, Ph.D.

Colin K. Combs, Ph.D.

Van A. Doze, Ph.D.

James D. Foster, Ph.D.

L. Keith Henry, Ph.D.

James E. Porter, Ph.D.

Roxanne A. Vaughan, Ph.D.