Category Archives: Week 6

Abstract

Limitations to dopamine D2 ligands for antipsychotic efficacy.

The D2 receptor has often been examined as the target for antipsychotic drugs due to its distribution in critical areas of the brain involved in movement and reward. Beta-arrestin biased drugs such as UNC9994 function as antagonists through G-protein signaling and partial agonists through beta-arrestin signaling, which has been shown to decrease psychotic properties while minimizing unwanted side effects. Of the five dopamine receptors, D2 closely mirrors D3 and D4. Therefore, it has been proposed that these novel therapeutics may be having an effect on the dopamine D3 and D4 receptors. Using BRET assays with HEK293 cell lines, aripiprazole and its congener UNC9994 have been tested on the dopamine D2-D4 receptors, and their effects were recorded as dose-response curves. By comparing the effect of the drugs to the effect of the natural ligand dopamine, it has been discovered that these agents do have an effect at the D3 and D4 receptors. Due to the role of non-D2 dopamine receptors in contributing to many of the symptoms of schizophrenia, novel therapeutic agents remain to be developed that could have the receptor specificity needed to attenuate psychotic phenotypes while minimizing unwanted side effects.

Investigating the Role of PTPRZ1 in Astrocytes

Astrocytes are a non-neuronal cell population in the brain which perform many important functions, including regulating synapse formation and function and helping to form the blood brain barrier. Despite the importance of these cells in vivo, little is known about how they carry out these critical functions. Protein Tyrosine Phosphatase Receptor Z1 (PTPRZ1) is a transmembrane protein which is heavily expressed in astrocytes and oligodendrocytes, another non-neuronal cell in the brain. While some function has been found for PTPRZ1 in controlling oligodendrocyte maturation, little is known about the function of the protein in astrocytes. The present study investigates the role of this protein by determining its location of action, its expression across development, and finally what it is doing in astrocytes. To determine the location in which PTPRZ1 is acting, immunohistochemistry (IHC) was used on mouse brain sections. These tests showed expression of PTPRZ1 along the branches of astrocytes, with a co-localization between the PTPRZ1 and our astrocyte marker GFP. We also found the protein to be present on western blot in the brains of mature animals. We plan to continue this work by investigating PTPRZ1 levels over different developmental time-points using western blot and quantitative PCR analysis.

Abstract Draft (199 words)

Identifying the endoreplication pathway in Cryptococcus neoformans

Aaliyah Davy                                                                                                                     Mentors: Ci Fu, PhD., Joseph Heitman, MD., PhD.                                             Department of Molecular Genetics and Microbiology

Cryptococcus neoformans is an opportunistic fungal pathogen that affects the immunocompromised and rarely, the immunocompetent. The fungus has two different sexual cycles- that is, bisexual and unisexual. Central to bisexual reproduction is cell and nuclear fusions that are indicative of ploidy duplication and the yeast-hyphal morphology transition. In this study, we investigated the currently unidentified endoreplication cycles that these cells go through to attain ploidy replication in the unisexual cycle. We hypothesized that one of six cell cycle genes we have chosen, that have differential expression throughout unisexual reproduction, will have some impact on the pathway. To test this, we generated gene deletion constructs with dominant selectable marker neomycin (G418), and conducted biolistic transformation on the unisexual strain XL280α. Transformants were streaked on additional G418 plates and verified by PCR. We tested whether deletion of these genes impact unisexual reproduction. This endoreplication pathway has also been linked to the formation of titan cells (greatly enlarged versions of the pathogen) in host infection, so we tested the significance of these genes in titan cell formation as well.

Abstract Draft

Randomly self-assembling copolymers have repeatedly demonstrated great promise toward eliciting therapeutic immune responses, and specifically for autoimmune diseases. However, their mechanisms of action are not yet fully understood. Many factors such as pH and temperature can affect the nature of the self-assembly of copolymers thus influencing immunogenicity. We tested the hypothesis that factors such as pH and temperature can be manipulated to optimize the self-assembly of copolymers thus eliciting a more desirable immune response through shifting the nature of the T-cell response. This was implemented by analyzing the effect of pH, temperature, and sonication on the stability of the copolymers dissolved in solution. According to our results, increasing heat and pH were the most effective methods to optimize the formation of fibers. We next plan to use these copolymer solutions to vaccinate mice and use methods like ELISA to analyze the nature of their immune responses. This will help us gain a better understanding of the ideal steps to be taken to increase the solubility of these copolymers while simultaneously optimizing their immunomodulatory actions. Through exploring different methods to increase the solubility of copolymers in solution, we can work to better understand how to optimize the immunogenicity of copolymers in vaccinations.

A Polycyclic Aromatic Hydrocarbon Environmental Chemical Mixture Increases Growth of Inflammatory Breast Cancer Tumor Emboli

Exposure to polycyclic aromatic hydrocarbon (PAH) chemicals is widespread due to their presence in emissions from tobacco smoke, wood stoves, and organic fuel burning throughout the world. Many PAHs are classified as carcinogens, and prior studies have shown an increase proliferation in an estrogen-receptor positive human breast adenocarcinoma cell line due to exposure to low doses of a complex PAH mixture. The objective of this study was to observe the effects of the same PAH chemical mixture on an aggressive human inflammatory breast cancer cell line using a 3D tumor emboli assay. We hypothesized that greater concentrations of PAH mixture would lead to greater tumor emboli growth. Cells were seeded in ultra-low attachment well plates and once emboli were formed, they were treated with different concentrations of PAH mixture, called Elizabeth River Sediment Extract (ERSE). Microscopic and statistical analysis revealed that low nanomolar doses of ERSE result in greater tumor emboli size compared to untreated emboli, in a dose-dependent manner. There was a positive correlation between emboli size and increased ERSE concentration, although a high micromolar ERSE dose was cytotoxic to the cells. Collectively, these results suggest that low-dose exposure to this PAH mixture can enhance growth of aggressive breast cancer cells, and may have a wider and more substantial impact on cancer progression and outcome.

Abstract Draft

Self-assembling peptide nanofibers have been shown to have self-adjuvanting properties—stimulating targeted immune responses in vivo and raising long lasting antibody responses without an accompanying inflammatory response. Despite these novel properties, the mechanisms behind nanofiber induced immune responses are still not known. To investigate these mechanisms, the self assembling Q11 peptide sequence was used. Dendritic cell (DC) activation was measured in vitro using the DC2.4 dendritic cell line, which immunohistochemistry identified nanofiber localization within the lymph node in a murine model of immunization. DC activation in vitro was shown to be dependent on the presence and concentration of the Pan-DR-epitope (PADRE) within the nanofiber. Measurements made by flow cytometry showed a dose dependent upregulation of CD83 and MHCII when PADRE containing nanofibers were added to DC cell culture media. Additionally, immunohistochemical analysis of lymph nodes containing nanofibers conjugated with the fluorescent molecule Tetramethyl-6-Carboxyrhodamine (TAMRA) showed a colocalization of injected nanofibers with both macrophages and DCs in vivo. These results indicate that nanofiber induced immune responses function in both canonical pathways, involving DCs, and non-standard pathways, involving macrophages. This provides a framework to begin to understand the complex immune responses generated by self-assembled peptide nanofiber vaccines.

Caution: First Draft

This first draft of my abstract is the result of careful and meticulous revisions to quite a few sentences to reduce the word count to fewer than 200 words. Who knew how tough it would be to fit so many of the ideas swirling around my brain into less than 200 words? Words fly when you’re having fun.

ABSTRACT – Both ABL and DDR kinases appear particularly hyperactive in the context of chemoresistance, in which cancer cells become resistant to chemotherapy. Furthermore, preliminary data suggest that receptor tyrosine kinases, DDR1 and DDR2, are ABL kinase targets. Thus, the hypothesis that DDR and ABL kinases form a feedback loop and that hyperactivation of the ABL-DDR network occurs during therapy resistance was tested. Due to the suggestion that DDR kinases are implicated in subsets of breast and lung cancers, DDR1/2 expression in these cancers was evaluated. It was demonstrated that increased ABL2 activity using the constitutively active form of ABL2 (ABL2-PP) in lung cancer cells induces phosphorylation of DDR1, whereas treatment with the ABL kinase allosteric inhibitor, GNF5, and the ABL and DDR kinase inhibitor, Nilotinib, decreases DDR1 phosphorylation without decreasing total DDR1 amounts. These data suggest that DDR kinases are downstream targets of ABL kinases, which supports the idea that DDR and ABL kinases constitute a novel signaling axis. If hyperactivation of the ABL-DDR network is indeed important during therapy resistance, there is potential that inhibition of the multifunctional ABL kinases through targeted therapy will resensitize cancer cells to the treatment to which they had been resistant before.

To be continued… as I add the results of the experiments I plan to perform next week, fresh from the oven!

Note: “Fresh from the oven” is solely meant to convey the idea that new and not yet acquired data are still to be incorporated into my abstract. I do not actually carry out my experiments in an oven. 😉

Update: For those of you who are interested, here is the final draft of my abstract, in all of its glory:

Novel signaling axis between ABL and DDR kinases in chemoresistance and metastasis: promise for targeted therapy in breast and lung cancers?

In the context of metastasis and chemoresistance in lung and breast cancer cells, the ABL family of tyrosine kinases and the receptor tyrosine kinases, DDR1 and DDR2, have been revealed to be hyperactive. My preliminary data suggest that the DDR kinases are ABL kinase targets. We hypothesize that DDR and ABL kinases form a feedback loop and that hyperactivation of the ABL-DDR network occurs during therapy resistance in subsets of breast and lung cancers. My data demonstrate that increased ABL2 activity using the constitutively active form of ABL2 (ABL2-PP) in lung cancer cells induces phosphorylation of DDR1; however, treatment with the ABL kinase allosteric inhibitor, GNF5, and the ABL and DDR kinase inhibitor, Nilotinib, decreases DDR1 phosphorylation without decreasing total DDR1 expression. These results suggest a novel ABL-DDR signaling axis, and preliminary data suggest that hyperactivation of this pathway may be important during metastasis and therapy resistance. Importantly, we demonstrated that pharmacological inhibition of the ABL kinases resensitizes therapy-refractory tumor cells to standard-of-care chemotherapy.

Abstract Draft

The Role of Glucagon in Insulin Secretion

Glucagon and Insulin are products of the alpha and beta cell respectively, that are essential to maintaining glucose homeostasis. It’s also known that incretins, signaling hormones secreted in our small intestine are essential insulin secretagogues. In understanding ways to mediate insulin production in the beta cell, more must be known specifically about the relationship between glucagon and the glucagon like peptide (GLP-1).  In hypoglycemia conditions glucagon is secreted to increase blood glucose concentrations, whereas in hyperglycemia conditions GLP-1 stimulates insulin secretion to reduce blood glucose concentrations.  In this study, we examined the relationship between glucagon and GLP-1 because we hypothesize that glucagon and GLP-1 work together to increase insulin secretion.  We examined this relationship by performing glucose tolerance tests on our beta cell glucagon receptor knockout mice model(GCGR), to understand the response between glucagon and the beta cell. Then we inhibited the GLP-1 receptor to further understand the relationship between glucagon and GLP-1. We used a Perifusion machine to examine the relationship between the mice’s insulin response specifically in the beta cell to different buffers. If glucagon and the GLP-1 receptor support our hypothesis, future work can be dedicated to creating new agonists that could improve treatment for type 2 diabetic individuals.

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Abstract Draft

This is a first draft of my abstract

Ecologic Factors Influencing Hormone Concentrations in Female Baboons

Although studies have shown how ecologic factors influence human female hormone concentrations and male baboon hormone concentrations, little is known about how these same factors effect female baboon hormone concentrations. This study measures correlations between estrogen, progesterone, and glucocorticoid levels and rank, weather, parity, age in the female yellow baboon population in Kenya’s Amboseli National Park. To determine the hormone concentrations, each fecal sample collected in Kenya was purified into a serum. Radioimmunoassays were then run on each sample, and hormone concentrations were correlated with field data on the individual the sample was from, time of sample, rank, and temperature. Although we do not yet have results, our hypothesis is that female baboon hormone concentrations and female human hormone concentrations will be similarly affected by parity and age, and that weather will effect both male and female baboon hormone concentrations similarly. Rank in female baboons is matrilineal, that is, a daughter baboon will be lower in rank than her mother suggesting that rank and hormone concentrations would not be correlated.

A Work in Progress

The Dzirasa lab uses chronic social defeat stress to create an ecologically relevant model for depression in mouse models. The model results in the expression of two distinct phenotypes, resilient and susceptible, similar to the phenotypes seen in human patients who have different responses to stressful events.The aim of my project was to correlate the behavioral changes associated with chronic anti-depressant treatment of socially defeated mice with biochemical changes of the level of phosphorylation of the protein MeCP2 in the lateral habenula. Previous work by the West lab studying the methyl DNA-binding protein MeCP2 has found that MeCP2 is phosphorylated by monoamine neurotransmitters. Increased levels of MeCP2 phosphorylation in the lateral habenula were found with chronic treatment of imipramine, indicating that MeCP2 may have a role in the brain’s biochemical response to chronic anti-depressant treatment. In order to correlate MeCP2 phosphorylation during anti-depressant treatment with behavioral recovery of a prosocial phenotype in socially defeated mice, mice will undergo two rounds of behavioral tests before and after imipramine treatment and then will be tested for levels of MeCP2 phosphorylation. If phosphorylation is correlated with behavioral recovery, susceptible mice that display resilient behavior after imipramine treatment will also have increased levels of MeCP2 phosphorylation in the lateral habenula.

The beginning of the poster struggle

My abstract draft:

Will they learn to court? 
Studying the role of Or47b in drosophila courtship learning behavior

Behaviors are made up of both innate and plastic components and courtship is no exception. It has been discovered that courtship can be learned in drosophila melanogaster (fruit flies), given that the innate component is absent. However, we do not know the exact mechanism that enables this learning. In this study, we are focusing on the olfactory system because we hypothesize that olfaction is important in learning; more specifically that Or47b is a key olfactory receptor neuron that regulates the learning of courtship behavior. To do this, we are using fru mutant fruit flies and fru and Or47b double mutant fruit flies and group housing them with other males or females for a few days and then observing their courtship behavior. We are currently waiting for results, however if our hypothesis is correct, we expect the double mutant fruit flies to not court even after group housing them. These results could better our understanding of the mechanisms driving behavior changes in general.

Abstract Draft

The effect of serration angle on spine puncture mechanics

Spines, defined as rigid biological structures that come to a point, are physical features found across a wide range of organisms; however general relationships between spine structure and function remain unclear. This study explores a specific aspect of the form-function relationship of spines, by investigating the influence of serration angle on spine puncture mechanics. Autodesk Fusion 360 was used to 3D-model spines with differing serration angles, which were then printed out with hard resin. The models will undergo materials testing as they are punctured into and retracted from ballistics gel. The maximum force needed for both puncture and retraction will be recorded and analyzed to see whether a change in serration angle leads to a change in puncture mechanics. Maximum puncture force is expected to increase with serration angle, as serrations angled more towards the front of the spine will increase the surface area on which the ballistics gel can resist the spine’s entry. An inverse trend with maximum retraction force is also expected, as similar gel-spine interactions will occur but in the reverse direction.

A look into the writing process…

*This abstract is the first draft of what will ultimately be used for the poster presentation at the end of this program*

The gut microbiome & depression: what is the best method for DNA extraction?

Our gut microbiomes play an important role in immune functioning, nutrient processing, and regulating many aspects of brain functioning. This gut-brain axis has been implicated in major depressive disorder, and inducing a depressive phenotype in mice using social defeat (SD) is one commonly used model to study the shifts in microbial diversity that occur. To do so, an effective protocol to extract and sequence the bacterial DNA from the fecal matter of subject mice is required. As it stands, methods and results in the field vary widely. This study compared two DNA extraction kits—the Maxwell RSC PureFood GMO & Authentication Kit and the MoBio PowerSoil DNA Isolation Kit. The Promega kit was hypothesized to be superior due to its high degree of automation. In order to assess the quality of the extracted DNA, spectrophotometer, fluorometer and electrophoresis were used. Results from 16S rRNA gene sequencing are awaited for phylogenetic classification of the samples. Analyses indicate that the Promega kit is more efficient, requiring less sample, but may have co-purified contaminants. Sequencing results will indicate effects of this, if any. Further work must explore microbial changes after SD and treatment, and the roles of specific bacterial species on the CNS.

Mild Traumatic Brain Injury

Mild Traumatic Brain Injury (mTBI): Role of rotational kinematics through the mechanism of shear shock

ABSTRACT— Despite decades of research, the exact mechanism in mild Traumatic Brain Injury (mTBI) remains unknown. The purpose of this study was to determine whether rotational velocity or acceleration is the primary contributor to mTBI and explore the effect of shear shock waves generated from rotational acceleration. It was hypothesized that rotational acceleration, not rotational velocity, has a primary role in mTBI, and shear shock waves due to the nonlinear pressure waves are a mechanism for mTBI. This study was conducted with a dual modeling and experimental approach. First, strain and deformation response was observed and recorded through the finite element analysis Simulated Injury Monitor (SIMon model). These results were then compared to deformations and strains recorded during pig brain rotational impacts using three dimensional imaging analysis. So far, the simulations reflect the relatively lower strain and stresses created through rotational velocity. Results indicate that rotational acceleration through the mechanism of shear shock waves is a prevalent mechanism in mTBI and could largely alter how future head injury criterions are created and how protective gears for sports players and the military are crafted.

 

Does sunlight affect the toxicity of organic contaminants?

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants produced from human activity, such as combustion of biofuels, often found in aquatic ecosystems. Embryonic fish have been shown to be significantly sensitive to PAH exposure. Further, PAH toxicity can be modulated by environmental factors such as hypoxia and temperature. This study investigated how sunlight exposure modifies the toxicity of carbazole, a hetereocyclic PAH. Zebrafish embryos were exposed at 6 hours post fertilization (hpf) until 96 hpf using photoactivated and parent carbazole. Photoactivation was induced using a sunlight simulator. Survival rates, deformities, CYP1A activity, and oxidative stress were examined. Additionally, chemical analysis of photoactivated carbazole is under development to identify photoproducts. Exposure to parent carbazole did not cause mortality, but did cause minor embryonic developmental effects. Contrarily, photoactivated carbazole caused significantly higher rates of mortality and deformities at 72 hpf at concentrations of 1000 µg/L and higher. Photoactivated carbazole induced higher CYP1A activity, indicating that photoproducts acted as aryl hydrocarbon receptor (AHR) agonists despite carbazole being a known AHR inhibitor. Such induction may be related to the increased toxicity, which remains to be tested. These results show that sunlight can play a role in modifying the toxicity of certain organic contaminants.

 

Draft of Abstract

Below you will find the first draft of the abstract that summarizes my summer research project in the Kuhn lab. Please keep in mind that this abstract is very preliminary especially regarding result interpretation and conclusions. Enjoy with a grain of salt.

 

The neurobiology of depression remains largely unknown and has not been studied in respect to sex-differences. Rapid tryptophan depletion (RTD) is a method used to lower levels of endogenous tryptophan (TRP) and serotonin (5-HT) and was used in this study to explore possible sex differences in the way rats react to tryptophan depletion. This study administered one of three treatments to 36 rats: 1) RTD, 2) a balanced amino acid treatment (BAL), or 3) None. Plasma and brain samples were collected and later analyzed using high performance liquid chromatography (HPLC) to determine concentrations of TRP and 5-HT. While RTD had similar depletion effects in the plasma of both females and males, there were significant differences in how TRP and 5-HT levels shifted in the hippocampal brain region of males and females. Compared to female controls, RTD females experienced significantly lowered levels of 5-HT and TRP, more so than RTD males compared to male controls. This suggest that sex-mediated differences in the serotonergic systems of rats is responsible for the different responses to tryptophan depletion. Further work must explore this neurobiological distinction between male and female rats and clinical research should focus on determining whether a similar difference exists in humans.

WC: 200