Annually approximately 1.5 million Americans suffer from a traumatic brain injury (TBI) increasing the risk of developing a further neurological complication later in life [1-3]. The molecular drivers of the subsequent ensuing pathologies after the initial injury event are vast and include signaling processes that may contribute to neurodegenerative diseases such as Alzheimer’s Disease (AD). One such molecular signaling pathway that may link TBI to AD is necroptosis. Necroptosis is an atypical mode of cell death compared with traditional apoptosis, both of which have been demonstrated to be present post-TBI [4-6]. Necroptosis is initiated by tissue necrosis factor (TNF) signaling through the RIPK1/RIPK3/MLKL pathway, leading to cell failure and subsequent death. Prior studies in rodent TBI models report necroptotic activity acutely after injury, within 48 hours. Here, the study objective was to recapitulate prior data and characterize MLKL and RIPK1 cortical expression post-TBI with our lab’s controlled cortical impact mouse model. Using standard immunohistochemistry approaches, it was determined that the tissue sections acquired by prior lab members were of poor quality to conduct robust MLKL and RIPK1 immunostaining assessment. Therefore, the thesis focused on presenting the staining method completed. The discussion also expanded on expected results from these studies regarding the spatial distribution necroptotic signaling in this TBI model.

The development of novel aqueous cross-coupling strategies has emerged as a rapidly expanding area of research within organic synthesis. However, many of these cross-coupling reactions require the pre-formation of an organohalide substrate, which often involves toxic halogenating reagents and harsh reaction conditions. This work details the development of a tandem halogenation/cross-coupling procedure in which an electron-rich arene or heteroarene is brominated through an enzymatic halogenation reaction catalyzed by a vanadium dependent haloperoxidase (VHPO) and then used without workup in a subsequent aqueous Suzuki cross-coupling reaction. This sequential process allows the arylated product to be accessed in a single pot from the unfunctionalized substrate via the brominated intermediate. Optimization of the enzymatic halogenation step was performed for three different substrates, resulting in the discovery of conditions for the bromination of 2,3-dihydrobenzofuran, chromane, and anisole in high yield (>95%). The scope of the reaction was then investigated for a range of electron-rich arene and heteroarene substrates. Next, Suzuki cross-coupling conditions were developed in a reaction mixture of pH 5 citrate buffer and acetonitrile and applied to the arylation of 2,3-dihydrobenzofuran utilizing an array of arylboronic acid coupling partners. Finally, the two procedures were combined to perform a tandem enzymatic halogenation/aqueous Suzuki cross-coupling of 2,3-dihydrobenzofuran to give the arylated product in 74% yield.

In intracranial aneurysms, multiple factors and biochemical pathways are believed to be involved in the event of a rupture. The epidermal growth factor receptor (EGFR) activation pathway is of particular interest as a way to understand and target the mechanism of rupture due to its established role in cellular proliferation and inflammation. Furthermore, unfolded protein responses in vascular cells’ endoplasmic reticulum (ER), known as ER stress, have emerged as a potential downstream mechanism by which inflammatory EGFR activation may lead to aneurysm rupture. The purpose of this project was to investigate the role of EGFR inhibition on the aneurysm rupture rate in a preclinical model, investigate the role of ER stress induction on the aneurysm rupture rate, and confirm which cellular phenomenon lies upstream in this mechanistic cascade. Based on analyses of aneurysm rupture rate and gene expression in the Circle of Willis, ER stress and inflammatory unfolded protein responses were found to be downstream of initial EGFR activation, which may be an effective therapeutic target for preventing aneurysm rupture in a clinical setting.

In cold chain tracking systems, accuracy and flexibility across different temperatures ranges plays an integral role in monitoring biospecimen integrity. However, while two common cold chain tracking systems are currently available (electronic and physics/chemical), there is not an affordable cold chain tracking mechanism that can be applied to a variety of temperatures while maintaining accuracy for individual vials. Hence, our lab implemented our understanding of biochemical reaction kinetics to develop a new cold chain tracking mechanism using the permanganate/oxalic acid reaction. The permanganate/oxalic acid reaction is characterized by the reduction of permanganate (MnVII) to Mn(II) with Mn(II)-autocatalyzed oxidation of oxalate to CO2, resulting in a pink to colorless visual indicator change when the reaction system is not in the solid state (i.e., frozen or vitrified). Throughout our research, we demonstrate, (i) Improved reaction consistency and accuracy along with extended run times with the implementation of a nitric acid-based labware washing protocol, (ii) Simulated reaction kinetics for the maximum length reaction and 60-minute reaction based on previously developed MATLAB scripts (iii) Experimental reaction kinetics to verify the simulated MATLAB maximum and 60-minute reactions times (iv) Long-term stability of the permanganate/oxalic acid reaction with water or eutectic solutions of sodium perchlorate and magnesium perchlorate at -80°C (v) Reaction kinetics with eutectic solvents, sodium perchlorate and magnesium perchlorate, at 25°C, 4°C, and -8°C (vi) Accelerated reaction kinetics after the addition of varying concentrations of manganese perchlorate (vii) Reaction kinetics of higher concentration reaction systems (5x and 10x; for darker colors), at 25°C (viii) Long-term stability of the 10x higher concentration reaction at -80°C.

An effort to experiment on the novel Usutu virus in pure in silico methods was made to determine conformational changes with non polar point mutations in the amino acid sequence. The first method consisted of creating a Python program to exhaustively identify codons, amino acids, and dinucleotide bridges & nonbridges, including viral characteristics defined by Mollentze in 2021. The second method consisted of creating point mutations to non polar amino acids in deemed key sites of the Usutu virus envelope protein and finding the RMSD from the original structure. This resulted in one of two outcomes - either the experiment showed that the Usutu virus envelope protein is highly resistant to point mutations or in silico methods are inconsistent and biased, leading to inaccuracy.