The primary goal of this project was to design a more accessible human powered vehicle for lower-limb amputees. This was done using a variety of engineering concepts within the fields of both biomedical and aerospace engineering. This report will provide a background on why it is essential to have such vehicles and detail the overall design process to describe how specific design decisions were made. The final design will then be analyzed and followed up with a brief discussion and conclusion to elaborate on future steps and summarize the project as a whole.

A reliable method for real-time blood flow monitoring in vivo is critical for several medical applications, including monitoring cardiovascular diseases, evaluating interventional procedures and surgeries, and increasing the safety and efficacy of neuromodulation procedures. High-speed methods are particularly necessary for neural monitoring, due to the brain's heightened sensitivity to hypoxic and ischemic conditions. High-speed CBF monitoring methods may also provide a useful biomarker for the development of a closed-loop deep brain stimulation (DBS) system. Current methods such as laser Doppler, bold fMRI, and positron emission tomography (PET) often involve cumbersome instrumentation and are therefore not well- suited for chronic microvasculature monitoring. The purpose of this study is to develop a method for real-time measurement of blood flow changes using electrochemical impedance spectra (EIS). Utilizing EIS to measure CBF has the potential to be included in a chronic, closed-loop DBS system that is modulated by fluctuations in CBF, using minimal additional instrumentation. Five experiments in rodents were conducted, with the objective of 1) determining whether electrochemical impedance spectra showed impedance changes correlated with changes in blood flow, assessing the sensitivity, specificity, and limitations of detection of this method, and 2) determining whether cyclic voltammetry-based method could be used to produce EIS more rapidly than current methods. The experimental set-up included electrodes in the femoral artery with the administration of endothelin (ET-1) to induce blood flow changes (N=1), electrodes in the motor cortex using isoflurane variation to induce blood flow changes (N=3), and electrodes in the femoral artery with the administration of nitroglycerin (NTG) to induce blood flow changes (N=1). Preliminary results suggest that impedance changes in the higher frequencies (over 160 Hz) demonstrated higher sensitivity to blood flow changes in the femoral artery model compared to <100 Hz frequencies, with inconclusive results in the motor cortex model. Future in vivo experiments will be conducted using endothelin-1 to further establish the relationship between impedance and cerebral blood flow in the brain.

Traumatic brain injury (TBI) poses a significant global health concern with substantial health and economic consequences. Patients often face significant consequences after injury, notably persistent cognitive changes and an increased risk of developing neurodegenerative disease later in life. Apart from the immediate insult, the resulting inflammatory response can lead to neuroinflammation, oxidative stress, tissue death, and long-term neurodegeneration. Microglia and astrocytes play critical roles in these inflammatory processes, emphasizing the unmet need for targeted therapies. Vaccine formulations consisting of poly (a-ketoglutarate) (paKG) microparticles (MPs) encapsulating PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one) and myelin proteolipid protein (PLP) were developed for prior studies and have demonstrated the production of antigen-specific adaptive T-cell responses in the brain, spleen, and lymph nodes of mice, suggesting that these formulations may be able to prevent neuronal inflammation in mice after TBI. The vaccine efficacy was further evaluated through the image analysis of immunohistochemically stained brain tissue sections from naive, saline, and paKG(PFK15+PLP) MPs or paKG(PFK15) MPs treated mice. Though microglia (Iba1), astrocytes (GFAP) and CD86 were visualized in this method, only Iba1 was found to be significantly reduced in the contralateral hemisphere for paKG(PFK15+PLP) MPs and paKG(PFK15) MPs groups when compared to naive (p=0.0373 and p=0.0186, respectively). However, the naive group also showed an unexpectedly high level of CD86 after thresholding (compared to the TBI groups), indicating flaws were present in the analysis pipeline. Challenges of the image analysis process included thresholding setting optimization, folded tissues, bubbles, and saturated punctate signal. These issues may have impacted data accuracy, underscoring the need for rigorous optimization of experimental techniques and imaging methodologies when evaluating the therapeutic potential of the vaccines in mitigating TBI-induced neuroinflammation. Thus, future analyses should consider microglial morphology and employ more accurate thresholding in FIJI/ImageJ to better measure cellular activation and the overall positive signal.

Bone loss affects millions of people every year posing a major public health problem. Currently, autograft and allograft bones are the only options for treating bone loss. Although, they pose many limitations including donor availability, immunogenicity risks, and the potential to carry a risk of disease and/or infection transmission to name a few. Therefore, there is a pressing clinical need to create a novel treatment that will promote bone repair. Alpha-ketoglutarate (aKG) was investigated as it plays an important role in cellular energy metabolism as a key intermediate in the Krebs cycle. It has been shown to stimulate the production of collagen in the bone repair process. However, controlling the release of aKG is important in being able to control where and how much new bone growth is stimulated. To address this aKG was delivered via a hyaluronic acid hydrogel and its release was controlled via the degradation of poly(alpha-ketoglutarate) microparticles (paKG MPs). paKG MPs were synthesized and characterized based on size, shape, and uniformity. The release of aKG from paKG MPs was evaluated, as well as the addition of paKG MPs into norbornene functionalized hyaluronic acid and maleimide functionalized hyaluronic acid hydrogels. Initial cell work was also done to grow osteoblasts for future work. It was found that paKG MPs were of the desired size and shape. The release of aKG from the paKG MPs was found to be sustained. The addition of paKG MPs in norbornene functionalized hyaluronic acid (NorHA) was found to be ineffective due to the opaqueness of the MPs. Maleimide functionalized hyaluronic acid (MaHA) hydrogels were chosen as an alternative delivery system for this reason. Future tests will be done on the addition of paKG MPs into MaHA hydrogels. Osteoblasts were also successfully grown and will be used in future studies.

This paper is a summarization of a year of projects in Dr. Xiao Wang's Synthetic Biology lab, following from initial computational projects and moving into more experimental projects under the mentorship of Dr. Kylie Standage-Beier, dealing with molecular cloning and dose response curves produced by measuring fluorescence via flow cytometry. This is then integrated with a novel computational flow cytometry analysis software based on public MATLAB functions that convert flow cytometry files into MATLAB variables.

By 2050, feeding the world will require a 70% increase in food production with fewer water resources due to climate change. New strategies are needed to replace current approaches. C3 photosynthesis is inefficient due to photorespiration, but synthetic biology offers a way to increase photosynthetic efficiency and crop yields, such as the tartronyl-CoA (TaCo) pathway. This project assesses the TaCo pathway in the chloroplast of Chlamydomonas reinhardtii and represents a pivotal step toward its practical application in higher plants for use in agriculture and biotechnology.