Substance abuse leads to alterations in cognition that affects processes such as impulse control and valuation. Decreased impulse control and aberrant valuation are responsible for continued drug seeking and are thought to be escalated by external stress stimuli. Stress leads to release of an endogenous opioid neuropeptide called dynorphin which binds to the Kappa Opioid Receptor (KOR). Upon KOR binding, dynorphin induces a protein signaling cascade that also promotes drug seeking behavior. In this study, we investigated the dynorphin/KOR system in the medial prefrontal cortex (mPFC) due to its critical role in cognition. We examined the properties of dynorphin release in the mPFC of C57BL/6 mice in response to different external stressors to determine if this nucleus is a potential therapeutic target for stress-induced drug seeking behaviors. Using a pharmacological approach, we first showed that systemic administration of U50,488, a KOR agonist, leads to KOR activation in the mPFC. U50,488 administration also disrupted cognition by impairing performance in a working memory behavioral task. We next tested whether different stress modalities stimulated mPFC dynorphin release and disrupted cognitive performance. Surprisingly, repeated forced swim stress did not cause dynorphin release in the mPFC and did not disrupt cognitive performance although it did activate dynorphin release in the Dorsal Raphe nucleus, as expected. In contrast, different stressors, including repeated foot shock and precipitated morphine withdrawal did effectively lead to KOR activation in the mPFC. This indicates that dynorphin release in the mPFC is dependent on the type of behavioral stress. Future experiments will utilize an in-vivo dynorphin sensor, kLight, to detect dynorphin release in real-time in response to these stressors. Exploration of the differences in dynorphin/KOR system functioning in response to different stress modalities is important for establishing how this system may be targeted to alleviate stress-induced drug seeking behaviors.

Chronic respiratory infections and diseases are the third leading cause of death globally. The Frevert lab studies the protein versican (Vcan), an extracellular matrix proteoglycan, whose expression is highly upregulated during lung injury and inflammation. However, it is unclear whether this upregulation is an anti-inflammatory or a pro-inflammatory response. We are testing the hypothesis that the cellular source of Vcan determines its inflammatory actions; versican from myeloid cells is anti-inflammatory but versican from stromal cells is pro-inflammatory. Vcan deletion in our cells of interest can test this hypothesis. To do this we have generated Vcan^fl/fl genetically engineered mice, which allow for conditional removal of functional versican with Cre Recombinase. Cre excises versican’s exon 4, recombining exons 3-5 and creating a premature stop codon. This produces a truncated non-functional form of versican. The goal of this research project is the development of cell-specific protocols for in vitro deletion of versican in both myeloid cells (bone marrow-derived macrophages) and stromal cells (lung explant fibroblasts). These protocols will allow for further evaluation of versican’s role in the inflammatory response when different cell types are confronted by a bacterial or viral agonist. So far, I’ve been investigating the dose response and time course for exposure of macrophages to Cre to optimize its efficiency and have been able to demonstrate by qPCR that intact versican decreases and non-functional versican increases. The inflammatory response is quantified through qPCR analysis of the fold increase of Ifn-b compared to the house-keeping gene MRPL32. Ifn-b is a cytokine released by the innate immune system in response to viral pathogens. Next, I will investigate the conditions necessary for efficient Cre deletion of Vcan in fibroblasts. These experiments allow us to investigate the effects of Vcan made by different cell types furthering our understanding Vcan’s function during injury and inflammation.

Capacitation involves biochemical and physiological processes in sperm with motility changes (including hyperactivation) that allow sperm to bind to the zona pellucida, undergo acrosome reaction and penetrate the ovum. Bovine serum albumin (BSA), has been thought to facilitate capacitation in mouse sperm through depleting the sperm membrane of cholesterol. Other albumin preparations including human serum albumin (HSA), fatty acid free (FAFHSA), and recombinant albumin (RHSA) may affect capacitation differently. Moreover, cholesteryl ester transfer protein (CeTP) may facilitate capacitation but it should not be present in yeast-derived RHSA as opposed to human blood-derived HSA; RHSA and HSA may support capacitation at different extents. These preparations were tested in motility experiments after 3-5.75 hr (mean 4 hr) capacitating incubation. Sperm were purified by 40/80% PureSperm gradients and washed in HTF-BSA, then diluted in final albumin to 10-20M/mL. FAFHSA, BSA, HSA, and RHSA had hyperactivation percentages of 2.83%, 3%, 11.3%, and 15.75% respectively. RHSA had a motility of 71.3% on average while the others ranged from 83.6% to 88%, affecting the hyperactivation results. RHSA also caused incubated sperm to agglutinate or aggregate, but its elevated hyperactivation rates relative to HSA are insignificant. These results fit previous data suggesting that BSA and FAFHSA do not support hyperactivation (therefore, capacitation) as both are abnormally below 10% hyperactivation. Since BSA contains low CeTP, this suggests CeTP may help facilitate capacitation. Previous studies demonstrated the amount of CeTP in albumin correlated well with albumin’s ability to support acrosome reaction. The present results suggest that capacitating motility patterns may not necessarily correlate with the ability to acrosome react; this hypothesis could be tested. Since fertilization is largely misunderstood; understanding the roles of Albumin or CeTP may aid in developing infertility treatments, contraceptives and family planning for a growing human populace.

Heart disease is a major health pandemic, and the myocardial infarction (MI), also known as a heart attack, is the leading cause of death. This is because adult cardiomyocytes (CMs) present in the heart cannot divide and proliferate, preventing the heart from regenerating itself and repairing tissue damage. From developmental studies in rodents, we know the Notch signaling pathway is crucial in mediating expansion and proliferation of CMs during development, as impairments in Notch lead to cardiac defects. Notch is inactive in adult CMs, which suggests it plays a role in CM renewal; however, this mechanism is still unknown. Thus, our goal is to investigate the role of Notch in CM proliferation and cell cycle regulation. For the purpose of this study, we are using CMs differentiated from a human embryonic stem cell line, RUES2. Differentiation was completed over a 17-day period by culturing the cells as a monolayer. On Day 0, Chiron 99021 is added to activate Wnt signaling, promoting mesoderm formation. Wnt-C59 is added at Day 2 to inhibit Wnt signaling and differentiate the cells into progenitors, and B27 supplement at Day 6 promotes full differentiation into CMs. This adherent protocol recapitulates every step of natural heart development in vitro. Purity of the cell populations, as assessed by flow cytometry staining for cardiac troponin T (cTnT), was 97.1 ± 0.9% cTnT+. We then determined the proliferative capabilities of CMs in the presence of a Notch inhibitor, DAPT. DAPT inhibits gamma-secretase, a transmembrane protein that normally proteolytically cleaves Notch during signaling, thus inactivating the Notch pathway. Treatment with DAPT significantly decreased cell proliferation by about 50%, confirming that Notch directly affects CM proliferation. The results of this study increase our knowledge of CM physiology and the mechanisms behind cell cycle withdrawal, and provide new insights into improving CM renewal.

In recent years, gravitational waves have yielded insights across many fields, from the expansion of the universe and black hole populations to the origin of heavy elements and nuclear physics. The Laser Interferometer Gravitational-Wave Observatories (LIGO) are michelson-type interferometers formed by two, 4-kilometer-long arms with suspended test masses (mirrors) at the ends. The test masses reflect high-power lasers to be combined at the intersection of the two arms and create an interference pattern, which is sensitive to gravitational waves passing through the interferometer. For a stable interference pattern, the test masses must be oriented precisely; the low frequency orienting of the test masses is done using optical levers (OpLevs). They consist of optics that launch light from a diode laser which reflects off the test mass and hit a quadrant photodiode, which measures the tip and twist of the test mass as a function of beam spot displacement. However, this measurement has elevated noise at low frequencies, limiting its accuracy. The reduction of this noise by a factor of 10-100, would allow for a more accurate orienting of the test mass. The source of noise is likely to be found in the fiber optics connected to the launching telescope. To isolate this noise I recreated parts of the LIGO OpLev setup with a level of positional noise well below LIGO's current OpLev noise at 0.1 Hz (below a nanometer per square root hertz). To achieve noise at this level I have designed a low noise pre-amplifier, improved the physical stability of the setup, and analyzed data to identify the noise sources. With this reduced noise setup we plan to search for the source of LIGO's OPLEV low frequency noise. Reduced noise would increase the uptime of the observatories thus increasing the number of gravitational wave detections.

Can parenting styles offset the effects of neighborhood disadvantage on child life-course trajectories? Much research indicates that children raised in an economically disadvantaged neighborhood face a greater risk of high school dropout or expulsion, low-wage employment, and incarceration. Yet not all youths raised in disadvantaged neighborhoods follow this trajectory, and success stories abound. This study examines how low-income parents’ childrearing strategies positively or negatively affect the life-course trajectories of at-risk youth. Using the Denver Youth Survey (DYS), a nationally representative, longitudinal sample of high risk youth, I intend to test whether parents’ childrearing strategies affect young adults’ academic accomplishment, income, job type, and criminal record. I hypothesize that positive parenting, defined as monitoring, control, and warmth towards the child, is associated with a reduced likelihood of youths experiencing impediments to socio-economic mobility. As an alternative hypothesis, I test whether impulsivity of the child mediates the association between parenting style and positive life-course outcomes. In doing so, I aim to contribute to the intersecting literature on life-course theory, child developmental psychology, and criminology, as well as provide evidence to aid the development of policy assisting at-risk youths overcome their disadvantaged circumstances.