Understanding the mechanisms behind unexplained abnormal heart rhythms is important for diagnosis and prevention of arrhythmias. Many studies have investigated the mechanisms at organ, tissue, cellular and molecular levels. There is considerable information available from tissue level experiments that investigate local action potential properties and from optical imaging to observe activity propagation properties at an organ level. By combining those electrophysiological properties together, in the present study we developed a simulation model that can help in estimation of the resulting body surface potentials from a specific electrical activity pattern within the myocardium. Some of the potential uses of our model include: 1) providing visualization of an entire electrophysiological event, i.e. surface potentials and associated source which would be optical imaging data, 2) estimation of QT intervals resulting from local action potential property changes, 3) aiding in improving defibrillation therapy by determining optimal timing and location of shocks.

Orthostatic intolerance (OI), i.e., the inability to maintain stable arterial pressure during upright posture, is a major problem for astronauts after spaceflight. Therefore, one important goal of spaceflight-related research is the development of countermeasures to prevent post flight OI. Given the rarity and expense of spaceflight, countermeasure development requires ground-based simulations of partial gravity to induce appropriate orthostatic effects on the human body, and to test the efficacy of potential countermeasures. To test the efficacy of upright lower body positive pressure (LBPP) as a model for simulating cardiovascular responses to lunar and Martian gravities on Earth, cardiovascular responses to upright LBPP were compared with those of head-up tilt (HUT), a well-accepted simulation of partial gravity, in both ambulatory and cardiovascularly deconditioned subjects. Results indicate that upright LBPP and HUT induced similar changes in cardiovascular regulation, supporting the use of upright LBPP as a potential model for simulating cardiovascular responses to standing and moving in lunar and Martian gravities. To test the efficacy of a short exposure to artificial gravity (AG) as a countermeasure to spaceflight-induced OI, orthostatic tolerance limits (OTL) and cardiovascular responses to orthostatic stress were tested in cardiovascularly deconditioned subjects, using combined 70º head-up tilt and progressively increased lower body negative pressure, once following 90 minutes AG exposure and once following 90 minutes of -6º head-down bed rest (HDBR). Results indicate that a short AG exposure increased OTL of cardiovascularly deconditioned subjects, with increased baroreflex and sympathetic responsiveness, compared to those measured after HDBR exposure. To gain more insight into mechanisms of causal connectivity in cardiovascular and cardiorespiratory oscillations during orthostatic challenge in both ambulatory and cardiovascularly deconditioned subjects, couplings among R-R intervals (RRI), systolic blood pressure (SBP) and respiratory oscillations in response to graded HUT and dehydration were studied using a phase synchronization approach. Results indicate that increasing orthostatic stress disassociated interactions among RRI, SBP and respiration, and that dehydration exacerbated the disconnection. The loss of causality from SBP to RRI following dehydration suggests that dehydration also reduced involvement of baroreflex regulation, which may contribute to the increased occurrence of OI.

Recent pharmaceutical developments have investigated using supramolecular nanoparticles in order to increase the bioavailability and solubility of drugs delivered in various methods. Modification of the carbohydrate cyclodextrin increases the ability to encapsulate hydrophobic pharmaceutical molecules by forming a carrier with a hydrophobic core and hydrophilic exterior. Guest molecules are commonly added to these inclusion complexes in order to add stability and further increase targeting abilities of the carriers. One such guest molecule is adamantine combined with a poly(ethylene glycol) chain. Vesicles are formed by hydrating a thin film of amphiphilic cyclodextrin and guest molecules in buffer solution that mimics physiological conditions. The solution is subject to freeze-thaw cycles and extrusion, and the complexes are separated out via size exclusion chromatography. Dynamic Light Scattering instrumentation is used to observe the particle size distribution. Cargo release can be observed in fluorescent dye-loaded vesicles by addition of a membrane-cleaving agent under a fluorimeter instrument. Future work involving this drug delivery technology includes synthesizing a chemically sensitive guest that will cleave in the presence of an intra-cellular anti-oxidant, and finally observing the uptake of these vesicles into live cells and testing the delivery of cargo in vitro under physiological conditions.

The most frequent clinical presentation of peripheral arterial occlusive disease (PAOD) is intermittent claudication, which may be caused by impaired vasodilation. Patients demonstrate both local and systemic impairments in vasodilation, but as the collateral circulation is the primary site of resistance to the ischemic zone, impaired collateral vasodilation would have the greatest potential to induce claudication. Collateral function following arterial occlusion is not well defined, but immature collaterals may demonstrate impaired vasodilation in animal models, although this is potentially improved with exercise training. Furthermore, as females exhibit poorer physical function with ischemia and less improvement with therapeutic exercise, there appears to be a sexually dimorphic response to PAOD, warranting a comparison in collateral vasodilation between sexes. In this study, the femoral artery was ligated to induce chronic ischemia in sedentary and exercise-trained mice, and at 7 or 28 days post-surgery, the diameter of the gracilis collateral arteriole was measured at rest and after gracilis muscle contraction using intravital microscopy. No major sex differences were observed in any group. At day 7, both the resting and dilated diameters were increased, while vascular reactivity was minimal. By day 28, resting diameter decreased while maximal diameter was unchanged, causing an increase in functional vasodilation. Exercise training also improved vasodilation by decreasing collateral resting diameter. These results are consistent with reported trends in endothelium-dependent and smooth muscle-dependent vasodilation, which are impaired in immature vessels and improved with maturation and exercise, but the significance of the observed variations in resting diameter remains unclear. Large resting diameters at day 7 could be due to a loss of sympathetic tone or the proliferative and non-contractile state of smooth muscle cells, while decreased resting diameters at day 28 could indicate that a smooth muscle contractile phenotype has been restored, or that the gracilis collateral is no longer the primary collateral. However, the further research is required to determine the functional relevance of collateral resting diameter and its importance in the ischemic limb circulation.

Often caused by trauma or tumor removal, large bone defects frequently result in delayed or non-union. The current gold standard for treatment is autograft. However, due to limitations, such as the size and location of the defect, these cannot always be utilized. A common alternative to autograft is the use of BMP-2 with a collagen scaffold, however, this treatment is limited by numerous side effects. In recent years, genetic materials such as microRNAs (miRNAs) have offered possible alternative therapies. MiRNAs are small non-coding RNA molecules that generally range from 20-24 nucleotides, serve as repressors of gene expression, and are involved in a wide range of biological activities. Their functions can be inhibited or upregulated by delivering antagomiRs or miRNA mimics, respectively. Two miRNAs involved in bone regeneration are of particular interest in this study, miR-26a and miR-133a. Previous studies demonstrated miR-26a is involved in osteoblastic differentiation and miR-133 is a negative regulator of Runx2, the key transcription factor of osteogenesis. Therefore, we hypothesized the delivery of miR-26a and antagomiR-133a will increase bone formation in critical-sized bone defects. The research outlined in this thesis investigates the healing efficacy of these genetic cargos delivered by novel peptide nanoparticles, RALA, soak loaded into a collagen-hydroxyapatite scaffold. vi To test this hypothesis, scaffolds soak-loaded with RALA/microRNA were implanted into calvarial defects in Wistar Rats. The defects were then left to heal for 8 weeks and were longitudinally monitored using micro-computed tomography (μCT). At 8 weeks, rats were euthanized and calvaria tissue was harvested for histological analysis. The μCT data demonstrates that the scaffolds with microRNAs show promise as a novel therapy for bone defects. The histological analysis showed the treatments promote healing by normal bone formation activity. While there was no statistical difference (p ≥ 0.11276) between groups for the healing variables, this is believed to be due to the small sample size and low power (60%). All of the miRNA treatment groups had samples with considerably higher healing responses than the gene-free group. In conclusion, the findings of this study support the use of this cell-free implant system as a potential novel clinical therapy, as an alternative to bone grafting, for treating large bone defects.

Clustered, regularly interspaced, short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) constitute a bacterial and archaeal adaptive immune system. The ongoing arms race between prokaryotic hosts and their invaders such as phages led to the emergence of anti-CRISPR proteins as countermeasures against the potent antiviral defense. Since the first examples of anti-CRISPRs were shown in a subset of CRISPR-Cas systems, we endeavored to uncover these naturally-occurring inhibitors that inactivate different types of CRISPR-Cas systems. In the first part of my thesis, we have identified and characterized Type II anti-CRISPR proteins that inactivate several Cas9 orthologs. We share mechanistic insights into anti-CRISPR inhibition and show evidence of its potential utility as an off-switch for Cas9-mediated mammalian genome editing. Although the RNA programmability of Cas9 enables facile genetic manipulation with great potential for biotechnology and therapeutics, limitations and safety issues remain. The advent of anti-CRISPR proteins presents opportunities to exploit the inhibitors to exert temporal, conditional, or spatial control over CRISPR. In the second part of my thesis, we demonstrate that anti-CRISPR proteins can serve as useful tools for Cas9 genome editing. In particular, we have demonstrated that anti-CRISPRs are effective as genome editing off-switches in the tissues of adult mammals, and we further engineered anti-CRISPR proteins to achieve tissue-specific editing in vivo. Taken together, my thesis research aimed to mine for natural anti-CRISPR protein inhibitors and repurpose these proteins to complement current Cas9 technologies in basic and clinical research.

Atrial fibrillation (AF) is the disordered activation of the atrial myocardium, which is a major cause of stroke. Currently, the most effective, minimally traumatic treatment for AF is percutaneous catheter ablation to isolate arrhythmogenic areas from the rest of the atrium. The standard in vitro evaluation of ablation catheters through lesion studies is a resource intensive effort due to tissue variability and visual measurement methods, necessitating large sample sizes and multiple prototype builds. A computational test bed for ablation catheter evaluation was built in SolidWorks® using the morphology and dimensions of the left atrium adjacent structures. From this geometry, the physical model was built in COMSOL Multiphysics®, where a combination of the laminar fluid flow, electrical currents, and bioheat transfer was used to simulate radiofrequency (RF) tissue ablation. Simulations in simplified 3D geometries led to lesions sizes within the reported ranges from an in-vivo ablation study. However, though the ellipsoid lesion morphologies in the full atrial model were consistent with past lesion studies, perpendicularly oriented catheter tips were associated with decreases of -91.3% and -70.0% in lesion depth and maximum diameter. On the other hand, tangentially oriented catheter tips produced lesions that were only off by -28.4% and +7.9% for max depth and max diameter. Preliminary investigation into the causes of the discrepancy were performed for fluid velocities, contact area, and other factors. Finally, suggestions for further investigation are provided to aid in determining the root cause of the discrepancy, such that the test bed may be used for other ablation catheter evaluations.

Sig sigma is a methodology where companies strive to reproduce results ending up having a 99.9996% chance their product will be void of defects. In order for companies to reach six sigma, statistical process control (SPC) needs to be introduced. SPC has many different tools associated with it, control charts being one of them. Control charts play a vital role in managing how a process is behaving. Control charts allow users to identify special causes, or shifts, and can therefore change the process to keep producing good products, free of defects. There are many factories and manufacturing facilities having implemented some sort of statistical process control. St. Jude Medical implemented control charts to monitor different tools on the manufacturing line. How the data is entered and stored poses a difficult situation for the person monitoring the processes. The program used to keep the control charts is not user friendly and difficult to use. Another program can be produced to provide a greater level of efficiency. The goals of this project are to stress how important control charts are in the manufacturing world, what problems are currently seen for operators and supervisors, and how a new and improved program can help fix the current situation. This paper goes into the reasons for the change as well has what has been improved.

Hypertrophic cardiomyopathy (HCM) is the most common monogenic disorder and the leading cause of sudden arrhythmic death in children and young adults. It is typically asymptomatic and first manifests itself during cardiac arrest, making it a challenge to diagnose in advance. Computational models can explore and reveal underlying molecular mechanisms in cardiac electrophysiology by allowing researchers to alter various parameters such as tissue size or ionic current amplitudes. The goal of this thesis is to develop a computational model in MATLAB and to determine if this model can accurately indicate cases of hypertrophic cardiomyopathy. This goal is achieved by combining a three-dimensional network of the bidomain model with the Beeler-Reuter model and then by manually varying the thickness of that tissue and recording the resulting membrane potential with respect to time. The results of this analysis demonstrated that the developed model is able to depict variations in tissue thickness through the difference in membrane potential recordings. A one-way ANOVA analysis confirmed that the membrane potential recordings of the different thicknesses were significantly different from one another. This study assumed continuum behavior, which may not be indicative of diseased tissue. In the future, such a model might be validated through in vitro experiments that measure electrical activity in hypertrophied cardiac tissue. This model may be useful in future applications to study the ionic mechanisms related to hypertrophic cardiomyopathy or other related cardiac diseases.

The prevalence and devastating nature of cardiovascular diseases has led to many advancements in the therapies used to treat the millions of patients that suffer as a result of these conditions. As coronary artery disease (CAD) is the most common of these cardiovascular conditions, it is a major focus of research among the medical industry. Although lifestyle changes and drug therapies can treat early CAD, more advanced cases often require more definitive interventions. In conjunction with angioplasty, stenting of an occluded vessel has shown significant success in preventing restenosis. However, as with nearly every therapeutic process in the medical field, several complications have arisen in stented patients that pose a need for further improvement of the devices. As a result, the stent industry is constantly striving towards improving the characteristics and outcome of their product and with these efforts comes the need for extensive testing and research. Continuous improvement and innovation in the field of tissue engineering has brought about the possibility of creating laboratory grown tissue engineered vascular grafts (TEVGs) for the purpose of replacing and/or bypassing damaged or occluded regions of the vasculature. By employing the techniques used to produce TEVGs, a blood vessel mimic (BVM) bioreactor system has been developed with the intent of using the resulting construct as a model for testing the cellular response of a human blood vessel to an intravascular device such as a stent. This would allow gathering of more significant data in the early stages of device development and may reduce the overall costs and time required to refine a design. Although the BVM system has previously been used to cultivate viable constructs that were subsequently used to observe the response to a deployed stent, the flow conditions within the original design are not representative of the physiologic conditions in a native vessel. This aspect of the original system presented a need for development in order to be considered by researchers as an accurate in vitro representation of the target vessels in which the stents are used. One of the primary concerns of this environment is creating and maintaining physiologic flow conditions that will represent those present in native vessels in order to facilitate cells sodded on the construct to grow as they would under native conditions. The two key aspects of flow are pulsatility and wall shear stress. Studies in this thesis were carried out to determine the best and most feasible methods for implementing appropriate levels of pulsation and wall shear stress in the previously established BVM bioreactor system with the intention of maintaining the original system’s simplicity and high throughput potential. Pulsatile flow was created by elevating backpressure in the BVM chamber while using a different pump head and pump tubing. Wall shear stress was adjusted by altering the viscosity of the perfusate and flow rate through the system. Both pulsatile flow and shear stress were established without any major changes to the overall configuration of the system. Pulsatile pressures of ~80 mmHg and wall shear stress forces of ~6.4 dyn/cm2 were established with minimal alteration to the original system. Pulsatility was created by using a 3-roller peristaltic pump head in place of the originally specified 8-roller head to create pulses that were then regulated with backpressure created by restricting down stream flow. Increasing the viscosity and corresponding flow rate allowed for instigation and control of wall shear stress at the inner wall of the BVM graft. Although the resulting protocols presented here require refinement for ultimately successful implementation, they are important underpinnings that will facilitate the eventual development of an ideal BVM system that is highly suitable for use as a high-throughput intravascular device testing model.