2016 SURP Participants

Anemia is the most common blood disorder. Anemia of chronic disease such as seen with end-stage renal disease often results from diminished production of the necessary growth factor, erythropoietin (EPO), or insensitivity to available EPO. A gap of knowledge exists in diagnostic methods to predict the response to exogenous erythropoiesis stimulating agents (ESAs) used for the treatment of anemia. We propose that this gap could be addressed using a proteomic approach to identify serum proteins or post-translationally modified protein states whose abundance is associated with ESA dosing. These differentially abundant proteins and modified protein isoforms could be used as surrogate biomarkers to guide ESA drug dosing. Preliminary data developed in our lab using combinatorial peptide aptamer arrays demonstrates abundant serum proteins are differentially enriched from patient serum samples as a function of ESA dosing. Our working hypothesis is that some of these differential protein enrichment results are associated with protein post-translations modifications derived from uremic toxins. Further, we hypothesize that specific protein post-translational modifications (PTMs) are viable biomarkers for response to drug dosing in end-stage renal disease patients. Our immediate goal in this study was to use a proteomic approach and hexapeptide combinatorial libraries to examine the effects of chemically induced protein post-translational modifications and/or the effects of uremic toxins on serum protein enrichment and purification. Our long term goal is the identification of proteins or protein post-translational modifications that can be used to develop individualized approaches to drug dosing.

Periodontitis is a microbial-induced chronic inflammatory disease that affects the gingival tissues supporting the tooth. Although some oral pathogens such as Porphyromonas gingivalis are considered keystone pathogens in the initiation and progression of periodontitis, the application of high-throughput sequencing technologies to the oral microbiota, has resulted in the recognition of several newly appreciated organisms which are associated with periodontal lesions. Among these is the Gram-positive anaerobic rod Filifactor alocis, which is present in high numbers in periodontal disease sites compared to healthy sites. Neutrophils are a major component of the innate host response and contribute to the maintenance of periodontal health by protecting the tissue against bacterial infection. The outcome of the interaction between periodontal bacteria and neutrophils is thus a key determinant of oral health status. The main objective of this project is to determine if human neutrophils challenged with opsonized or non-opsonized F. alocis, during the different stages of bacterial growth, will mount a different neutrophil bacterial killing response. One of neutrophils main antimicrobial mechanisms is the ability of the cell to mount a robust respiratory burst response which results in the generation of reactive oxygen species (ROS) within the bacteria-containing phagosome. Our preliminary in vitro data demonstrate that F. alocis can survive within human neutrophils up to 24 h post challenge, and that the bacterium, during its stationary growth phase, fails to induce neutrophil respiratory burst response. We predict that F. alocis inability to mount an appropriate respiratory burst response relies on the capacity of the bacterium to modulate the expression of the different components required to activate the antimicrobial oxygen-dependent response.

Severe spinal cord Injury (SCI) can cause limited mobility even if rehabilitative interventions lead to sufficient neuronal control. This is due to the deterioration of muscle mass over time when the muscle is inactive.Deleterious muscle adaptions can predispose individuals to metabolic disease and related health risk. Characterizing the effects of different interventions on lower limb skeletal muscle and intramuscular fat is of a great importance to optimize the rehabilitative protocols after SCI. The purpose of this project is to determine if exercise can change fat/muscle mass overtime. To test this we need to create and automatic system that will calculate the fat to muscle ratio from an MRI scan

Ovarian cancer ranks fifth in cancer-related deaths in women in the United States, and it is also the leading cause of death among gynecologic cancers. Since a majority of cases are diagnosed in the more progressive stages, treatment options are limited and have not been sufficient. They include cytoreductive surgery followed by chemotherapy with platinum derivatives, including carboplatin or paclitaxel. After the initial treatment however 70% of women will relapse and experience recurrent cancer. This is accredited to the resistance of the ovarian cancer stem cells. Therefore, our goal is to develop a combination therapy that will target both the cancer cells as well as the cancer stem cells. In our experiments, we have observed that Withaferin A (WFA) in combination with Cisplatin has a synergistic effect on the inhibition of cell proliferation. Withaferin A is a natural product isolated from Withania somnifera commonly used as an over-the-counter dietary supplement but more recently has been used to inhibit tumor growth. Cisplatin is a platinum-derivate chemotherapy drug, which causes severe side effects and cells eventually become resistant to it. The synergistic effect of the combination therapy would allow a reduction in dosage of Cisplatin minimizing the side effects and resistant cancer stem cells. ALDH1, CD44, CD133, PTTG, Notch1, E-Cadherin, Vimentin, Snail, and Wnt1 have been well defined cancer stem cell markers and their expression in the treatment groups will be studied to determine which are responsible for the self-renewal of cancer stem cells and also which serve as survival genes. This information will allow us to evaluate the expression of these genes and eventually control the pathways being affected. Cisplatin in combination with Withaferin A could be a novel yet effective, alternative chemotherapy treatment.

Retinitis Pigmentosa (RP) is title given to a number of hereditary conditions that lead to the degeneration of photoreceptors within the retina (1). RP can be inherited as an autosomal-recessive, autosomal-dominant, or X-linked trait (2). The P23H opsin mutation is the most common cause of autosomal dominant RP (3). P23H is a mutation that involves a Proline-to-Histidine substitution at the twenty-third codon within the Rhodopsin gene (4). Possession of a P23H mutant copy leads to deterioration of rod photoreceptors within the retina that eventually causes loss in vision. Initial symptoms of RP include night blindness and loss of peripheral vision because the rods are the photoreceptors that mediate signaling in low light conditions and are concentrated in peripheral vision. The retina samples used in my research project are the retina of both, wild type mice and mice possessing the P23H mutation. These retina samples come from mice at the ages of postnatal 30 days, 60 days, 90 days, 150 days, and 180 days (P30, P60, P90, P150, and P180). These retina samples have undergone gene therapy, involving the use of AAV vectors. Recombinant AAVs are used as vectors in gene therapy in the eye for a few reasons. These reasons include “their low immunogenicity, the ability to transduce a variety of non-dividing cells in the retina, and their ability to facilitate sustained expression of replacement genes.” (5) AAV8 Y733F (a tyrosine-to-phenylalanine mutant capsid vector) was the vector injected into the retinas of the mice used in my research project. AAV8 (Y733F) has been proven to be more effective in long-term gene therapy (6). The visual function of the mouse retinas used for my research project have previously been studied using electrophysiological recording from retinal ganglion cells, the output cells of the retina. My objective is to study these retina samples from an anatomical standpoint. I will cut tissue slices from the treated and untreated retina samples and perform immunohistochemistry to evaluate the infection by gene therapy. I will also determine the distribution of proteins important in the synapse between photoreceptors and their post synaptic partners. Cells which express good, poor, or no fluorescence will be identified. I will use confocal microscopy to examine the distribution of these proteins, based on their florescence.

Microcephaly is a component of several rare neurodevelopmental syndromes that are due to mutations of genes that are involved in such processes as chromatin structure, microtubule organization, RNA splicing, ribosomal biogenesis or lipid synthesis. In addition, microcephaly may be a consequence of viral infections during pregnancy. Mechanistically, microcephaly is caused by insufficient neurogenesis due to loss of neuroprogenitor cells and/or their impaired proliferation and/or loss of their neuronal progeny. In consequence, microcephalic individuals are often disabled including persistent cognitive deficits. Zika virus (ZV) is a small RNA virus that belongs to the Flaviviridae family of arthropod-borne viruses. During the recent Zika epidemic in South America, a disturbing association has been suggested between ZV infection during pregnancy and microcephaly. However, pro-microcephalic potential of ZV has never been examined in the laboratory. The nucleolus which is a center of ribosomal biogenesis is prominently present in many types of neurons as well as rapidly proliferating cells. Perturbation of any step of ribosomal biogenesis triggers ribosomal stress (RS) that suppresses cell growth and/or induces apoptosis in a p53-dependent or p53-independent manner. While in proliferating cells the RS is a major tumor suppressor mechanism, it is also proposed as a contributing factor to neurodevelopmental diseases that include microcephaly. Interestingly, many viral proteins that interact with RNA have an affinity towards the nucleolus. In such a location they may disturb activity of ribosomal biogenesis factors and activate RS. Indeed, a capsid protein of a close relative of ZV, West Nile Virus, has been shown to localize to the nucleolus, induce RS and the p53-mediated cell death. The hypothesis to be tested during this summer research project is that ZV capsid protein enters nucleolus of neuroprogenitors/immature neurons, activates RS, and, in consequence, triggers p53-mediated cell death.

The renal proximal tubule plays a crucial role in maintaining total body phosphate homeostasis. In the epithelium of the proximal tubule of the kidney is the type II sodium phosphate cotransporter (NPT2a), which is responsible for the transport of phosphate into the cell. NPT2a exists in the brush border membrane as part of a complex of membrane proteins that includes the Na-H exchanger regulatory factor (NHERF1), the parathyroid hormone receptor (PTHR1), protein kinase A (PKA), A kinase anchoring protein (AKAP 79/150), and ezrin. Previous reports suggest that PTH, a major physiological regulator of renal phosphate transport, causes dissociation of NPT2a from NHERF1, resulting in endocytosis of NPT2a, and decreased phosphate reabsorption in the proximal tubule. The molecular mechanisms by which this dissociation occurs have not been identified. We hypothesize that NHERF1 remains in the apical membrane through its association with ezrin, while NPT2a undergoes endocytosis in association with PTHR1 and AKAP 79/150. Using the well-established proximal tubule cell model, OK cells, we will test this hypothesis using Western blot, immunoprecipitation, and confocal microscopy to investigate the dissociative effect of PTH on the NPT2a complex.

Lens organ cultures have been used to test the direct effect of moieties on lens metabolism. As one of the major functions of the lens is to stay clear, regardless of the metabolic changes, the aim of our study was to measure the ultimate endpoint, the optical properties of the lens, in relationship organ culture time, and the incubation levels of dexamethasone, glucose, and glucose with camel’s milk. Human, porcine and rat lenses were studied. Lenses were incubated in minimum essential medium (MEM) Eagle with Earle’s BSS without L-glutamine or phenol red at 37 °C, 5% CO2 atmosphere for 13 days. We tested a range of levels of dexamethasone, glucose, and glucose with camel’s milk. Lens absorbance and light scattering was measured using a spectrometer. Lens opacity was graded from lens photographs. Dexamethasone and glucose caused cataract formation in human and rat lenses. Camel’s milk ameliorated the rate of cataract formation. Glutathione levels had no significant difference when camel’s milk was added to glucose. In conclusion, the optical properties of organ cultured human, porcine and rat lenses could be a valuable tool to determine the direct efficacy of drugs and moieties in causing or protecting the lens from opacification.

We are interested in the underlying mechanisms that cause a human disease, Retinitis Pigmentosa (RP), by using transgenic zebrafish as a model. The human Rhodopsin (hRHO) gene is known to play a role in causing RP. Mutations within the gene may be responsible for the phenotype of vision loss in these patients. I will create constructs of the wildtype and P23H mutation within the hRHO and insert them into plasmid DNA. A tag with fluorescent properties will allow me to see the gene expression if it is adhered to the hRHO constructs; I will use the fluorescent protein mCherry to see photoreceptors. Once the embryos are injected with the final construct DNA, I will be able to compare wildtype to the P23H mutation fish. I expect to see a degeneration of rod photoreceptors in the P23H transgenic zebrafish line compared to the wildtype zebrafish.

In between the blastula and gastrula stages of embryogenesis the sea lamprey undergoes a process known as programmed genome rearrangement (PGR), where this organism deletes about 20% of their DNA from all somatic cells. This process is unique among sea lamprey in the fact that they are the only vertebrate known to delete such a large portion of their DNA. The material which sea lamprey deletes contains repetitive sequences, as well as protein coding sequences for numerous genes. One possible reason for this genetic rearrangement could be because the genes which are deleted are crucial for proper germline development, yet unnecessary for somatic cells to function properly. Therefore these sequences could be deleted as a safeguard, to reduce the risk developing cancer later on in their life cycles, from the unintentional misexpression of these genes. The overall goal of this project is to determine if these genes can act synergistically with MYC in zebrafish T-ALL models to drive cancer progression. In this experiment genes that are deleted during PGR are injected into Zebrafish with T-cell acute lymphoblastic leukemia, to determine how the presence of this gene influences the latency time of tumor development, the rate of progression through the Zebrafish’s body, and the ability of self-renewal among the cancer cells.

Abstract #1

The proposed research plan is to investigate neuronal plasticity within a neural circuit with altered sensory input. Synaptic connections are dependent on neural activity and this regulates how a brain can develop within an animal. We are addressing how enhancing or depressing sensory input alters the development of a sensory-motor circuit while larval Drosophila develop. The reason to choose Drosophila as a model organism for this study is that development is regulated in part by environmental temperature and the ease in genetically altering the organism to allow one to optically stimulate or depress defined sensory circuits. Larval Drosophila melanogaster offer many advantages to investigate the development of neural circuits based on altered input. Modern optogenetics (Channel rhodopsin for excitation or halohrhodopsin for inhibition) are feasible approaches we will use to alter sensory neuronal activity of subclasses of “md neurons” which provides the larvae with the sense of touch and gravitation force. This work has a relationship to developmental questions in animals within a microgravity environment. We are curious if this research with depressed sensory input on earth could simulate what might be observed for larval development in space (microgravity).

Abstract #2

In the Cooper lab, I have been working on a project that focuses on manipulating the cholernigic system and how it modulates neural circuits and understanding receptor subtypes by assessing behavior and development in Drosophila. The main part in the project I have been here to focus on is a genetic approach using optogenetics. This approach is used along with thermogenetics, pharmacology, electrophysiology, and touch assays to help reinforce the important uses of using protein channel activated systems by using genes that transcribe specific proteins in neural circuits. There are two ways we have been testing optogenetics on fruit flies which is by shining light on 3rd instar larvae and assessing the change in their locomotion when light is shined onto their central nervous system. Another way to manipulate behavior and development is by using light pulses that are set up to shine on embryos at certain times to see if the light when shined onto these light sensitive channels, will either be inhibiting or activating their growth and behavior within the CNS. So far I have been caught up with the other techniques by using mutated flies where sensory neurons have inserted genes to make different proteins that may be activated or inhibited by temperature, or different neurotransmitters. Along with the project, I have been learning many other general techniques used in the lab which are useful in physiology research. In the lab I am always aware as to stay on track to reach our goals and their importance which enriches my experience of what research is really like.

A key problem in development and regeneration is understanding how cells position themselves in coordinate space and undergo patterning. Although the genetic control of patterning during regeneration remains poorly understood, a rich literature on vertebrate limb development provides candidate genes to explore. This project aimed to visualize spatiotemporal gene expression of Homeobox d9 (Hoxd9) and Sonic hedgehog (Shh) in regenerating axolotl limbs, two genes that are required for limb patterning. Using available gene information for Ambystoma mexicanum we designed primers to these genes and developed RNA probes for in situ hybridization. We present images of gene expression during early and late blastema phases and cone stages of regeneration. Using these patterns we analyzed expression to detect restriction in regional positioning (ventral, dorsal, anterior and/or posterior). We discuss our findings in the context of how these genes are expressed in discrete compartments and function to pattern vertebrate limbs.

Circadian rhythms are endogenous, yet entrainable, physiological processes that can be found in all living organisms. This project focuses on detecting rhythms located in the gut microbiome of the house sparrow, studying if Enterobacter aerogenes (MntH and coaE) found there display circadian rhythms using techniques such as PCR, cryosectioning, and histology. By detecting these rhythms, enough evidence may be provided to support that the house sparrow is a superior model species to study circadian rhythms.

We are examining the influence of the Lhx5 transcription factor in ocular development using a zebrafish model. The aim of this project is to accurately genotype fish with a Lhx5 mutation using a genotyping protocol, which consists of a genomic DNA isolation followed by amplification using the Polymerase Chain Reaction and concluded with an RFLP analysis to determine the heterozygous individuals. Upon determining these individuals, we perform an in cross and are left with 25% double mutant negative embryos and these embryos can be used for analysis of ocular development. Ultimately, we are looking to see if the expression of the Lhx5 gene and its corresponding mutation has an adverse effect on ocular development and how this may correlate to ocular disorders, specifically Coloboma.

Spinal Muscular Atrophy (SMA) is a genetic disease that causes degeneration of motor neurons and muscle wasting. SMA is the number one genetic cause of infant mortality and results from recessive autosomal mutations in the Survival Motor Neuron 1 (SMN1) gene that reduce the amount or activity of the SMN protein. SERF1/H4F5 was discovered as a potential genetic modifier of SMA as patients with the most severe forms of SMA frequently have a deletion in the SERF1 gene in addition to mutations within SMN1. This hypothesis has not been tested in any animal model. We are using a Drosophila melanogaster SMA model to test if the SERF gene in flies modifies the SMA phenotype. To address, we created both SERF overexpression and RNAi knockdown lines by using the standard UAS-GAL4 system. Previous work in our lab and others has suggested that the SERF encoded protein plays a role in protein homeostasis, in particular in modulating the assembly or stability of selected protein complexes. Our lab has established that overexpression of SERF increases Smn protein abundance with a correlated restoration of larval body size and locomotion in various SMN mutants. Enhanced SERF expression is not, however, sufficient to rescue the lethality of SMN null alleles. The data collected during the KBRIN program shows that the knockdown of SERF mRNA significantly impairs larval growth in multiple SMA mutant flies. Additionally, the SMA mutants show reduced pupal and adult viability after knockdown of SERF mRNA. While larval growth and viability were reduced in the SMN mutants with RNAi knockdown, larval locomotion per se appears not to be affected although adult animals lacking SERF show much reduced mobility. Our study provides the first test in any experimental organism of the longstanding hypothesis that SERF1 is a true genetic modifier of spinal muscular atrophy. In addition our results provide preliminary evidence for the mechanism involved – specifically, SERF1 stabilize the SMN protein, the limiting factor in patients with spinal muscular atrophy. These data offer the potential for the development of diagnostic or therapeutic tools relevant to this devastating human disorder.