Current Research Projects

Josephine Chandler, Assistant Professor of Molecular Biosciences, University of Kansas

Mentor: Mario Rivera, Professor of Chemistry, University of Kansas

Project Title: Antibiotic-induced virulence in Burkholderia pseudomallei


Project Summary
The bacterium Burkholderia pseudomallei causes the human disease melioidosis and is currently the third leading cause of death in Northeast Thailand. Despite the increasing prevalence of melioidosis, mechanisms of B. pseudomallei pathogenesis remain poorly understood and treatment options are limited. This proposal is focused on a cytotoxic B. pseudomallei polyketide, malleilactone, which is important for B. pseudomallei pathogenesis in several infection models. Malleilactone is not produced in normal laboratory conditions, but its production can be elicited by certain antibiotics such as trimethoprim. Some of the antibiotics that induce malleilactone production are among the few available options to treat melioidosis. Our long-term goal is to define the underlying mechanisms of B. pseudomallei virulence, and use this information to identify novel therapeutic interventions to treat this challenging human disease. Here we propose to (i) synthesize and determine the mode of action of malleilactone, (ii) determine the spatial and temporal pattern of malleilactone expression and cytotoxicity in a murine model of melioidosis, and (iii) elucidate the regulatory pathway that triggers induction of the malleilactone biosynthetic genes using both directed and global approaches. These results are essential to gain a mechanistic understanding of how malleilactone increases B. pseudomallei pathogenesis and will provide insight into how this versatile pathogen has evolved the ability to adapt to different environments. In addition toxic polyketides are emerging as a broadly ubiquitous and poorly understood class of virulence factors in many pathogens, and we anticipate that B. pseudomallei and the robust animal models available for this species will provide insight into polyketide virulence mechanisms in a broader sense. Elucidating the underlying mechanisms that lead to malleilactone-dependent virulence will provide critical new information on B. pseudomallei pathogenesic mechanisms, and is an important step towards improving the currently limited treatment options for this devastating disease.

Jodi McGill, Assistant Professor of Immunology, Dept. of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University

Mentor: Roman Ganta, Professor and Director of Center for Excellence for Vector-Borne Diseases, College of Veterinary Medicine, Kansas State University

Project Title: Alternative functions for γδ T cells in the immune response to Mycobacterium


Project Summary
Mycobacterium bovis is a member of the M. tuberculosis complex and the causative agent of tuberculosis (TB) in cattle and zoonotic infections in humans. Bovine TB is an excellent model for understanding TB in humans, as the diseases are parallel in many aspects of pathogenesis and innate and adaptive immune responses. Further, the study of bovine and human TB exemplifies the One Health approach as discoveries in both species have been closely intertwined throughout history. γδ T cells are a set of non-conventional CD3+ T cells that share important characteristics of both the innate and adaptive arms of the immune system. γδ T cells are particularly recognized for their ability to respond robustly to Mycobacterium infection. Several characteristics of γδ T cells, predominantly those functions commonly shared with αβ T cells, such as IFNγ production have been identified and characterized. However, it is increasingly apparent that γδ T cells have the capacity for a diverse array of immune functions including chemokine production, antigen presentation and regulatory cytokine production. These alternative immune functions are not well described in the context of TB infection and their role in vivo, particularly at the site of infection, remains poorly defined. Aim 1 of the proposed project employs a novel γδ T cell-alveolar macrophage co-culture system to model interactions that occur between immune populations at the site of M. bovis infection in the lungs. Next-generation RNA-Sequencing and transcriptome analysis will be performed on samples from the co-culture system to identify novel and alternative functions for γδ T cells responding to Mycobacterium infection. γδ T cells are hypothesized to play a critical role in granuloma formation and immune cell recruitment; however, given the difficulty of studying the immune response to TB in vivo, little is known about their capacity to promote improved disease outcome during TB infection. Therefore, Aim 2 proposes to utilize RNAScope, a commercial technology similar to in situ hybridization to identify local γδ T cell responses that correlate with increased resistance to infection, or improved outcome during TB. If successful, the RNA-Seq analysis in Aim 1 will further inform studies of γδ T cell functions in infected tissues. The proposed studies will be amongst the first to correlate γδ T cell responses in the peripheral blood, with those that occur in the local tissues during M. bovis infection. Effectively engaging γδ T cells in vaccine-induced protection from TB is expected to enhance the efficacy of candidate TB vaccines. Aim 3 proposes to test two vaccine platforms that specifically target unique γδ T cell-specific pattern recognition receptors, to determine if these platforms effectively engage γδ T cells in vaccine-induced immunity to TB. The knowledge gained from these studies will contribute to our understanding of the basic biology of γδ T cells and are anticipated to significantly advance the fields of human and veterinary medicine alike.

Arghya Paul, Assistant Professor of Chemical & Petroleum Engineering, University of Kansas

Mentor: Stevin Gehrke, Fred Kurata Memorial Professor of Chemical Engineering, University of Kansas

Project Title: Role of biomechanical cues for stem cell based myocardial infarction therapy


Project Summary
Myocardial infarction and hypertrophic cardiomyopathy followed by heart failure is a major cause of death worldwide. As the terminally differentiated adult cardiomyocytes possess a very limited innate ability to regenerate, much research has focussed on exploring the potential of adult stem cells and induced pluripotent stem cells to repair the damaged myocardium. However with regards to benefits till date, experimental and clinical trials have shown sub-optimal to modest results. The main drawback for this is that the mechanisms involved for the in vivo therapy is not well understood. Suggested pathways include permanent or partial cell fusion between stem cells and resident cardiac cells, transdifferentiation of stem cells into cardiac and vascular cells and secretion of proangiogenic paracrine factors. However, none of them have considered the fact that the dynamic cardiac microenvironment can also induce significant biological effects on the transplanted stem cells that can influence their overall fate and functionality. In this project we will study, for the first time, the fundamental microenvironmental interactions between mesenchymal stem cells and contractile cardiomyocytes in a continuously beating 3D microenvironment that can influence the clinical outcomes when transplanted in patients with cardiomyopathy. Furthermore, we will also study the potential of the mechano-biologically activated stem cells, pre-conditioned in this 3D cardiac microenvironment, for myocardial regeneration therapy in animal model. Established collaborations with members (physicians and scientists) from the Midwest Stem Cell Therapy Center (MSCTC) at KU Medical Center, The KU Center for Epigenetics and Stem Cell Biology (CESCB), KU Center for Molecular Analysis of Disease Pathways (CMADP) and University of Cincinnati Cardiovascular Disease Center will provide further support and guidance to successfully pursue the project.

 

Past Research Projects

Yong Zeng, Assistant Professor of Chemistry, University of Kansas (2015-2017)

Mentor: Susan Lunte, Ralph N. Adams Distinguished Professor of Chemistry and Pharmaceutical Chemistry, Director of Adams Institute for Bioanalytical Chemistry, University of Kansas

Project Title: Microfluidic single-cell analysis of cancer exosomes


Project Summary
Most eukaryotic cells secrete numerous membrane-derived vesicles of 30-150 nm in size termed exosomes. As an emerging mechanism for cell-to-cell communication, exosomes have been recently found to play important roles in a wide range of biological processes, including cancer development and metastasis. For instance, increasing evidences support the cancer-derived exosomes can reprogram the behavior of recipient cells to promote tumor growth and metastasis. Despite the significance of exosomes, our understanding of their biogenesis, molecular classification, and biological functions remain very limited. One of the challenges is to analyze exosomes released from single cells. Because cells in a tumor are known to be remarkably heterogeneous, single-cell analysis of exosomes is crucial to understanding their pathological roles in cancer. However, current “gold standard” methods can only perform ensemble measurements of exosomes released from a large cell population because of their poor isolation yield, insufficient analysis sensitivity and low throughput. In this proposal, the PI aims to develop for the first time a high-throughput single cell exosome analysis system (SCEAS) capable of probing the secretion and molecular composition of exosomes at the single cell level. The goal will be achieved via two specific aims: 1) develop a microfluidic exosome barcode chip for multiplexed, ultrasensitive immunofluorescence detection of exosomes; and 2) develop and validate a Single Cell Exosome Analysis System (SCEAS) for quantitative phenotyping of exosomes derived from single cancer cells. Success of the work will yield a key tool to enable the studies of heterogeneous exosome release by tumor cells at the single cell level, which would facilitate better understanding of intercellular signaling pathways underlying cancer development, metastasis, and drug resistance.

Michael Veeman, Assistant Professor of Biology, Kansas State University (2015)

Mentor: Michael Herman, Professor of Biology, Co-Director of Ecological Genomics Institute, Associate Dean of Academic Affairs and Research, K-State Graduate School, Kansas State University

Project Title: Morphogenetic effector networks in the Ciona notochord


Project Summary
The gene regulatory networks active in early development set up tissue-specific patterns of gene expression, but little is understood about how the restricted expression of large sets of tissue-specific effector genes leads to the unique aspects of morphogenesis and differentiation in different tissues. These effector gene sets have complex regulatory and functional interactions that have proven challenging to dissect. The proposed research integrates new methods for transcriptional profiling, targeted gene disruption, and quantitative multidimensional imaging into a systems biology approach to dissecting morphogenetic effector networks in a carefully chosen model organ, the Ciona notochord. Ascidians such as Ciona are close chordate relatives of the vertebrates with conserved embryonic anatomy, but with a particularly small, simple embryo, a very compact genome and unusually straightforward transgenesis. The Ciona notochord undergoes a broad range of complex, conserved, medically relevant morphogenetic behaviors but consists of only 40 cells that can easily be imaged in their entirety with fine subcellular detail. Aim 1 is to identify all the genes expressed and upregulated in the notochord using a direct RNA-seq approach on purified notochord and not-notochord cells from timepoints spanning key steps in morphogenesis. Aim 2 is to systematically disrupt a large and carefully chosen subset of these genes and quantify the resulting phenotypes across many dimensions of cell size, shape and tissue architecture. This will enable the identification of both major and minor players in notochord morphogenesis and will allow these genes to be linked into networks of phenotypic similarity with important functional implications. Aim 3 is to use RNA-seq on embryos in which each of the transcription factors upregulated in the notochord has been disrupted so as to identify transcriptional regulatory relationships genome-wide. ChIP-seq will be used to test if these relationships are direct or indirect and thus to build a comprehensive effector gene regulatory network for the notochord. Together these aims will allow a systematic dissection of the transcriptional regulatory architecture of morphogenesis and differentiation in this tractable model organ with unprecedented scope and detail. This new ability to relate gene regulatory network structure to effector gene function will be a major step towards an integrative understanding of how genome sequences encode the dynamic cell properties of differentiating tissues.

Brian Ackley, Associate Professor of Molecular Biosciences, University of Kansas (2012-2015)

Mentor: Erik Lundquist, Professor and Research Coordinator, Dept. of Molecular Biosciences, University of Kansas

Project Title: Identifying mRNAs associated with a synaptogenic calcium-mediated pathway


Project Summary
Voltage-gated calcium channels (VGCCs) are the engines that drive the synapse. They are required for vesicle exocytosis, and it is now clear that these molecules are critically important to the dynamics of formation, maintenance, adaption and elimination that underlie changes in neural networks. Therefore, as we study these molecules and their mode of action, we will gain a much clearer understanding of the basic assembly of the nervous system. VGCCs have been linked to human diseases and disorders, most closely to epilepsy. In epileptic conditions excessive neural activity could result in structural changes in the brain. Also, treatment with anti-epilepsy drugs (AEDs) that inhibit VGCCs and/or activity could have detrimental effects on memory formation. In fact, epilepsy is commonly co-morbid with cognitive and psychiatric disorders, and AED treatment has been linked to poor cognitive function. We have identified genetic pathways that separate neural transmission and the facilitation of synapse formation downstream of VGCC activity. Here our goal is to further the understanding of how these proteins contribute to neuronal development. Using animals that have mutations that inactivate or hyperactive synaptic VGCCs we will obtain transcriptome profiles to identify genes that are transcriptionally regulated by VGCC functional status. We will then target those genes for knockdown by RNAi to find molecules that contribute to VGCC-dependent synapse addition. Finally we will seek to visualize how calcium may be dynamic during times when synapses are being modified during development to correlate intracellular levels of calcium with specific changes in synapses. The organization of the C. elegans neuromuscular system provides a powerful genetic and cell biological model to study development. The primary motorneurons have many similarities to vertebrate CNS neurons, which are more difficult to study in vivo. C elegans may provide important insights into the mechanisms that underlie the formation and spacing of these types of synapses in vivo.

Mizuki Azuma, Associate Professor of Molecular Biosciences, University of Kansas (2012-2015)

Mentor: Berl Oakley, Irving S. Johnson Distinguished Professor of Molecular Biosciences, University of Kansas

Project Title: Role of Ewing sarcoma proteins EWS/FLI1 and EWS in mitosis


Project Summary
Ewing sarcoma remains the second most common form of bone cancer in children. A genetic hallmark of this cancer is a chimeric fusion protein EWS/FLI1, containing EWS-derived sequences at the amino-terminal region fused to the carboxyl-terminal regions of the ETS transcription factor FLI1. Despite that the chimeric EWS/FLI1 is proposed to function as a transcription factor that regulates aberrant transcription of its target genes, the pathogenesis of Ewing sarcoma is poorly resolved. We previously reported that both the knockdown of endogenous EWS, and expression of the EWS/FLI1 fusion protein in zebrafish embryos and HeLa cells produce mitotic defects, and biochemical interaction between EWS/FLI1 and wildtype EWS leads to inhibition of EWS activity by EWS/FLI1. We hypothesize that the interaction of EWS/FLI1 with endogenous EWS induces mitotic defects leading to chromosome instability and to malignant transformation.

Aim 1: To determine the role of EWS in midzone formation. The midzone, a structure formed between segregating chromosomes during metaphase-anaphase, is essential for cytokinesis. Our preliminary studies demonstrated that the function of EWS is required for midzone formation and for preventing the formation of multinucleated cells. We will identify the downstream target molecules of EWS/FLI1 and EWS during midzone using Ewing sarcoma cells.

Aim 2: To determine how knockdown of EWS leads to aneuploidy in zebrafish. The zebrafish provides an excellent in vivo model to study the function of EWS because it contains the microenvironment necessary for tumor formation, it has high fecundity (200 eggs/female/week) to ensure statistical reliability for genetics, it is suitable for live imaging analysis, and it is an established model for chemical screens. Our preliminary data suggests that the EWS mutant cells exhibit high incidence of aneuploidy. We will elucidate how EWS maintains genomic stability.

Aim 3: To determine whether expression of EWS/FLI1 leads to aneuploidy and Ewing sarcoma in zebrafish. We already generated transgenic EWS/FLI1 zebrafish and in a process of establishing the lines and analyzing it. Utilizing this line, we will address whether/how EWS/FLI1 leads to aneuploidy and Ewing sarcoma.

Prajnaparamita Dhar, Associate Professor of Chemical & Petroleum Engineering, University of Kansas (2012-2015)

Mentor: Stevin Gehrke, Fred Kurata Memorial Professor of Chemical Engineering, University of Kansas

Project Title: Probing Lipid-Protein Interactions in Biological Self-Assembly


Project Summary
We hypothesize that there exists an optimal range of surface viscosities in an effective lung surfactant that provides both rapid adsorption and spreading to the air-water interface during inspiration while maintaining a stable film at ultra-low surface tensions during expiration. The primary goal of this research is to determine, for the first time, this optimal surface viscosity of lung surfactant using an active microrheology technique unique to our lab. Our goal is to determine how best to achieve this optimum by controlling the cholesterol fraction of a synthetic replacement lung surfactant. Three orders of magnitude increased sensitivity of our microrheology technique as compared to macroscopic rheometers allows precise monitoring of changes in the molecular organization of the lung surfactant film in the presence of cholesterol, enabling accurate measurements of surface viscosity of surfactant films. Ultimately, determining the optimal cholesterol concentration will enable a better design of synthetic surfactants to treat Neonatal Respiratory Distress Syndrome (NRDS) and may give insights into the causes of surfactant inactivation in Adult Respiratory Distress Syndrome (ARDS).

We hypothesize that small fractions (1-5 wt. %) of cholesterol reduce the crystalline ordering of saturated lipids in lung surfactant monolayers, leading to a reduction in the shear viscosity, which enhances the surfactant's ability to flow and cover the alveolar interface. We also hypothesize that excess cholesterol ( >10 wt %) decreases the effectiveness of lung surfactants in ARDS by increasing the minimum surface tension of the interfacial film. This inability to reach ultra-low surface tensions is hypothesized to be a consequence of significantly reduced interfacial energy of the film (line tension). Low interfacial film energy can influence the mechanical cohesion in the surfactant film and lead to the failure of the film on compression, which ultimately causes the film to become unstable at lower surface tensions. Furthermore, lipid(cholesterol)- protein interactions can also alter these mechanical and structural properties by changing their molecular organization at the interface. By determining the mechanical properties of both model and clinically relevant surfactant film in the presence of physiological and elevated amounts of cholesterol, we can understand how increased cholesterol might lead to surfactant inactivation in ARDS and determine better replacement surfactants for treatment. The mechanical properties thus determined by the active microrheology technique will be correlated with isotherms, fluorescence microscopy, and grazing incidence synchrotron X-ray diffraction to determine how cholesterol alters the molecular packing of lung surfactant lipids, which determines the mechanical properties of monolayers.

Michael Johnson, Associate Professor of Chemistry, University of Kansas (2012-2015)

Mentor: Craig Lunte, Professor of Chemistry, University of Kansas

Project Title: Neurotransmitter Interactions on Sub-Second Timescales


Project Summary
The specific mechanisms of interaction between neurotransmitter and neuromodulator systems in the CNS, which make up a complex, three-dimensional signaling network that controls critical brain functions, are not clearly understood largely because the analytical methodology required to study these sub-second interactions is underdeveloped. This deficiency represents a significant roadblock toward understanding neurological function because signaling events in the brain that influence outward physical responses and cognitive events occur within this sub-second time regime. Therefore, the central aim of this proposed research is to develop and apply tools that will allow for the quantitative study of these neurotransmitter interactions in tissues and in vivo. Fast-scan cyclic voltammetry (FSCV) at 7 μm-diameter carbon-fiber microelectrodes, used here to measure the sub-second release of dopamine as well as other electroactive neurotransmitters and neuromodulators, will be combined and temporally-aligned with the μs-timescale photoactivated release of photoactivatable compounds. Methodology for photoactivating p-hydroxyphenacyl (pHP) forms of neurotransmitters will be developed and applied. Precise concentrations of photoactivated compound will be calculated by electrochemical measurement of the 4-hydroxyphenylacetic acid (4HPAA) by-product, a procedure we have previously developed. This central aim will be accomplished by completion of the following Specific Aims:

1. Employ caged compounds to Identify aberrant mechanisms of sub-second intracellular communication in mice that model Huntington’s disease. Dopamine release will be electrically evoked in brain tissue slices harvested from YAC128 Huntington’s disease model mice while caged compounds are activated by irradiation with light pulses delivered through a 100 μm diameter fiber optic cable. The light pulses will be time-synchronized with the electrical pulses. Caged glutamate will be photoactivated at selected times prior to and during electrical stimulation. Photoreleased glutamate will be quantified by the electrochemical measurement of the 4HPAA by-product in order to identify precise glutamate concentrations required to influence dopamine release. Using this approach, we will test our working hypothesis that transient, sub-second increases in extracellular glutamate result in greater decreases of dopamine release, per μmole of glutamate photoreleased, in rodents that model Huntington’s disease compared to wild-type control animals.

2. Develop microfluidic platforms for the application of photoactivatable compounds and measurement of their effects. Microfluidic chips that allow for the rapid application and photoactivation of chemical components in a chemical well containing to brain tissue samples will be designed, constructed, and implemented. Two general designs will be pursued initially: (1) a microfluidic device/perfusion chamber that allows for the measurement of dopamine release by FSCV in conjunction with the spot application of caged compounds at selected points on a brain slice and (2) a microfluidic device for capillary electrophoresis with on-line derivatization and laser-induced fluorescence detection of amino acid neurotransmitters released from small brain tissue samples.

3. Develop, evaluate, and apply a line of synthetic agonists and antagonists for studying dopamine system function. We will synthesize selected photoactivatable agonists and antagonists of dopamine D2-family receptors using the coumarin photoprotecting group. These compounds will be evaluated for their ability to increase or decrease electrically-evoked dopamine release immediately following photoactivation in brain tissue slices.

4. Identify sub-second neurotransmitter release characteristics in zebra fish. The purpose of this aim is (1) to develop the methodology that will allow the electrochemical measurement of sub-second fluctuations of neurotransmitters, such as dopamine and serotonin, and (2) characterize sub-second neurotransmitter release properties. Techniques that will be developed include: acute removal of the brain, slicing of brain tissue, stimulation of brain tissue, and measurement of neurotransmitter release. Fast-scan cyclic voltammetry at carbon-fiber microelectrodes will be used to measure the sub-second release of dopamine and serotonin. The development of these techniques will allow detailed study of how neurotransmitter levels are regulated by release, uptake, and feedback inhibition in zebrafish. Furthermore, we expect that zebrafish brains, due to their small size, will be amenable to use on microfluidic chips for analysis.

 


Recent News

May 2017
CMADP Research Project Investigator co-recipient of NCI R33 grant

April 2017
CMADP Graduate, one of ACS's 'Must See Presenters', speaks on latest "chemobrain" findings at Spring 2017 ACS National Meeting

February 2017
CMADP Project Investigators co-author Top Downloaded article in Lab on a Chip

CMADP Graduate's research featured on cover of Genetics and in other journals

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