Vascular calcification greatly increases risk for heart disease, the leading cause of death in patients with diabetes and chronic kidney disease. Vascular calcification is an active process attributed to vascular smooth muscle cell (VSMC) mineralization. The sodium-dependent phosphate cotransporter, PiT-1, mediates VSMC calcification. The mechanisms by which PiT-1 causes calcification may include cellular matrix degradation, apoptosis, calcium phosphate vesicle release, and changes in gene expression. Of these mechanisms, my research focuses on the poorly understood signaling pathway by which PiT-1 induces VSMC calcification through up-regulating osteogenic (bone-forming) and chondrogenic (cartilage-forming) genes and down-regulating smooth muscle genes. These changes in gene expression make VSMCs more readily calcified in elevated phosphate. Recent literature suggests that VSMCs respond to elevated phosphate through PiT-1 with a novel cell signaling mechanism independent of phosphate uptake. My project concentrates on understanding the function of the PiT-1 protein's intracellular domain, which currently has no known function. I hypothesize that this section of PiT-1 is responsible for signaling in response to elevated phosphate, triggering a phosphorylation cascade that activates osteogenic transcription factors, such as Runx2, which initiate this osteogenic conversion. I am working toward integrating the gene for the intracellular domain of PiT-1 into the vascular smooth muscle cell genome to overexpress the truncated protein in cells. From my hypothesis that PiT-1 has a role in cell signaling, I predict that the overexpression of the intracellular domain of PiT-1 will reduce VSMC calcification by competing with the wild type PiT-1 protein for binding to proteins in the activation pathway, and inhibiting the signaling process. If my data support this hypothesis, I may engineer the intracellular domain of PiT-1 for therapeutic purposes.

The world’s future energy needs will almost certainly require extensive use of renewable sources of energy. Organic photovoltaics are a particularly lucrative source of energy due to their cheap materials and ease of production compared to inorganic solar cells. However, organic photovoltaics (OPVs) currently suffer several drawbacks: they have an efficiency of, at most, around 10% and they are unstable – they may begin to degrade within five to seven years. This project aims to alleviate these defects by gaining a greater understanding of the chemical interaction between interfaces within the solar cell. This project is a comprehensive study of the interactions between phenyl-C₆₁-butyric acid methyl ester (PCBM) and calcium. PCBM is an extensively researched molecule which acts as an electron acceptor in OPV devices. PCBM is typically paired with an electron donor such as poly-(3-hexylthiophene) in a working device, and calcium has energetic properties which allow for very good electron transfer at the cathode of such a device. We use adsorption micro-calorimetry under ultra-high vacuum conditions to study the reaction at the interface between calcium and PCBM, and surface analytical techniques to determine growth modes and chemical binding of calcium and PCBM. These experiments will elucidate the mechanisms of interface formation of this understudied but important OPV system.

Luminescent solar concentrators (LSCs) are solar energy harvesting devices that promise to revolutionize the next generation of solar energy by separating the task of sunlight absorption from power generation. These devices use phosphors that absorb sunlight and wave-guide reemitted light to photovoltaics (PVs) to generate electricity. One promising class of phosphors is that of doped semiconducting nanocrystals (known as quantum dots) due to their intense absorption and minimal reabsorption of dopant-emitted light. In these materials the semiconductor quantum dot absorbs sunlight and transfers the energy to an emissive dopant to prevent efficiency loss by reabsorption. CdSe quantum dots are well known for their intense absorption across the visible spectrum, and doping with Ytterbium would enable their emission to be used in LSCs. Moreover, the Yb³⁺ ions emit at an energy that is nearly perfectly matched with the bandgap of a silicon PV cell, boosting its efficiency. The focus of my research is the synthesis of Yb³⁺-doped CdSe. Ytterbium is incompatible with the tetrahedral CdSe lattice with respect to its larger oxidation state and coordination preference, and introducing it as a dopant is a challenge that has yet to be solved by the chemistry field. While employing air-free Schlenck techniques, I am modifying the doping methods that have proven successful in the synthesis of other Gamelin Group materials, including a process we recently developed called nanocrystal diffusion doping. Success can be measured by detecting quantum dot sensitized ytterbium luminescence. The goal is that this approach will lead to a repeatable procedure by which to synthesize stable Yb³⁺-doped CdSe for novel characterizations as well as for eventual application in a new generation of solar devices.

It has been shown that the chemical nor-binaltorphimine (norBNI) inactivates the k-opioid receptor (KOR) for days or weeks in vivo, and also results in downstream activation of c-Jun N-terminal kinase (JNK). If JNK is blocked in vivo by SP610025 or by knocking out the JNK1 gene, norBNI inactivates the KOR for just hours. We hypothesize that there is a JNK mediated inactivation of the KOR. To further investigate this, I will create constructs that will allow us to study the interactions of JNK and arrestin, two proteins that are involved in the transduction of KOR signaling. I aim to express a version of each JNK1 splice variant tagged with luciferase and arrestin3 tagged with Venus in human embryonic kidney (HEK293) cells that have been transfected with rat KOR. Venus and luciferase are two bioluminescent molecules that emit different wavelengths of light depending on their distance from one another. After treating these cells with norBNI, I can dtermine the level of their interaction by using a bioluminescence resonance energy transfer (BRET) assay. These experiments will provide useful data in determining the extent of the interactions between JNK1 and arrestin3. This data will help us understand the protein cascade that results in the deactivation of the KOR, and if certain drugs activate certain splice variants. Understanding this pathway has implications in creating analgesics with smaller potential for abuse and addiction, and creating pharmaceuticals that could help with stress related depression and addiction relapse.

Affecting millions of people across the globe, the WHO deems myocardial infarctions (heart attacks) as the largest cause of death worldwide. The heart’s weak regenerative capacity leads to scarring of the dead tissue and a reduction in cardiac output that presents numerous health consequences. Tissue engineering is a promising outlook in restoring the structural integrity and contractile force of the damaged area. Recent findings have demonstrated that engineered heart tissues grown from stem cells can display significantly improved characteristics when subjected to mechanical stimuli. Inspired by these results, this project seeks to improve tissue performance even further by investigating a novel concept where stem cell-derived cardiac tissues are treated with the same mechanical profile experienced by native heart tissue. In order to simulate and implement these forces, tissues are grown and then anchored onto two posts that can vary in distance and stiffness. Upon contracting, post stiffness is increased providing a resistance to each beat. This resistance simulates the afterload stress that the ventricular wall contracts against to eject blood into the aorta. Next, the tissues are stretched by increasing the distance between each post. The stress produced by elongating the tissues simulates the preload stress exhibited by the ventricular wall as blood is pumped into the chamber prior to the next contraction. Finally, combining the stiffness (afterload) and stretching (preload) schemes allows the tissues to experience the entire force profile exhibited by the native heart. Simulating the cardiac mechanical environment gives the engineered tissues the stimulus to yield contractile forces and a cellular structure at par with their native counterparts. This investigation thus aims to advance regenerative medicine by bringing stem-cell derived cardiac tissues one step closer towards curing myocardial infarctions.

The aim of this study was to further the understanding of mechanisms that underlie the pruritic and antipruritic effects of kappa opioid receptor drugs. The kappa opioid receptor (KOR) has been studied for its potential therapeutic benefits. It has been previously shown that the kappa opioid receptor antagonist, 5’-guanidinonaltrindole (GNTI), causes compulsive, hind-leg scratching in mice, while the kappa opioid receptor agonist, nalfurafine, inhibits this pruritic effect. There are applications for this antipruritic effect in medicine, to counteract symptoms in kidney-failure patients. By learning more about the mechanisms of this anti-pruritic effect, better drugs can be developed in the future with increased specificity and decreased side effects. We pre-treated male C57/BL6 mice with either saline or nalfurafine (0.05mg/kg, s.c. flank), and challenged them 20 minutes later with 5’-GNTI (0.03mg/kg, s.c. behind the neck) or saline. The number of hind-leg scratches in the 30 minutes following GNTI injection were counted as the measure of itch. We compared the results between kappa opioid receptor (KOR) knock-out mice, mu opioid receptor (MOR) knock-out mice, and wild-type mice, to determine the role of KOR and MOR in GNTI-induced itch and nalfurafine inhibition of itch. We concluded that while the pruritic effects of 5’-GNTI are not mediated by KOR or MOR, the antipruritic effects of nalfurafine was KOR dependent.

According to the World Health Organization, cardiovascular diseases are the leading cause of mortality worldwide. The low regenerative capacity of the heart renders it ineffective in healing itself after heart failure. Our lab has developed efficient methods to derive cardiomyocytes (cardiac muscle cells) from human embryonic stem cells (hESCs). However, it has become apparent that the cardiomyocytes derived using current differentiation methods exhibit immature morphological and functional properties. An important characteristic of mature cardiomyocytes is the presence of T-tubules, which are invaginations of the plasma membrane into the cell interior. These subcellular structures are critical for synchronized calcium release and contraction of mature cardiomyocytes. The protein caveolin-3 is a critical component of T-tubules, and thus its visualization can be used to assess the development of these important subcellular structures. I am currently evaluating the expression of caveolin-3 in hESC-derived cardiomyocytes using immunostaining techniques. For these studies, I expose the sample to anti-caveolin-3 as the primary antibody followed by a complementary secondary antibody conjugated to a fluorescent marker for visualizing the protein. Titration of several parameters, such as time of incubation and dilution of the antibodies allows for staining optimization. I have obtained detailed images of T-tubules aligning with striations in control heart tissue, but the staining is diffuse for the hESC-derived cardiomyocytes grown in standard 2D cultures. I am currently exploring whether this is a technical issue or the result of developmental immaturity. To assess the latter, I will stain hESC-derived cardiomyocytes that are more mature due to mechanical conditioning in 3D constructs. Optimization of this technique will facilitate our efforts to find novel agents that can accelerate the maturation of hESC cardiomyocytes. Finding ways to mature these cells will then provide a better platform for therapeutic applications such as drug screening, disease modeling and cardiac repair.

Myocardial infarction and resulting heart failure continues to be the leading cause of death worldwide. The heart does not respond effectively to injury through tissue regeneration because cardiomyocyte proliferation after differentiation occurs at a greatly diminished rate. The goal of this research is to study stem cell-derived cardiomyocytes to better understand the mechanisms and possible controls of cardiomyocyte proliferation. Achieving this goal would allow for the intitiation of cardiomyocyte proliferation, which would better direct new technologies aimed at regenerating cardiac tissue after injury. RUES2 human embryonic stem cells are differentiated into cardiomyocytes using a directed differentiation protocol that manipulates the TGFβ and Wnt signaling pathways, by the controlled addition of Activin A, BMP4, chiron, and Xav signaling factors. After twelve to fifteen days, cells are harvested and stained for cardiac troponin T (cTnT), which is a protein present in the sarcomere of cardiomyocytes. Flow cytometery is used to identify cardiomyocytes expressing cTnT and indicate the purity of the differentiated culture. Cardiomyocyte proliferation is measured by exposing cells to Bromodeoxyuridine (BrdU), which is a thymidine analog that is incorporated into synthesized DNA. A double immunohistochemistry stain for Beta Myosin Heavy Chain (β-MHC) and BrdU is used to indicate proliferating cardiomyocytes. RNA is isolated from cardiomyocyte cultures and analyzed using quantitative real time PCR. Specific sequences of RNA present in cells can indicate relative gene expression, and allow for genes of interest to be monitored through stages of differentiation to determine peak expression. Ongoing experiments are investigating the activity of the Notch signaling pathway during directed differentiation of cardiomyocytes and its relation to cardiomyocyte proliferation. Results from these experiments will better indicate time points for enhancing cardiomyocyte proliferation using Notch signaling and help further characterize methods for induced proliferation as a potential therapeutic after cardiac injury.