Mass spectrometry is being increasingly used in biomarker research mainly due to its ability to achieve high selectivity coupled with high sensitivity. This dissertation focuses on quantitative mass spectrometric investigations of two protein biomarkers i.e. serum thymidine kinase 1 (TK1) and human osteopontin (OPN). First part of this research was focused on developing a liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for measuring the activity of TK1 in serum by monitoring the conversion of a TK1 specific exogenous substrate, 3’-deoxy-3’-fluorothymidine (FLT), to its mono-phosphorylated form 3’-deoxy-3’-fluorothymidine monophosphate (FLT-MP). A method to quantify FLT-MP on LC-MS/MS was developed and validated. The method was linear over the range of 2.5-2000 ng/mL with a mean correlation coefficient of 0.9935. Using the developed method, serum TK1 activity was measured in serum from hepatocellular carcinoma (HCC) patients and age-matched controls under standardized conditions. A sub-population of the HCC patient samples showed an almost 20-fold enhanced TK1 activity compared to the controls. A method was developed and validated for quantifying human osteopontin from plasma using immunoaffinity isolations coupled with microflow liquid chromatography and tandem mass spectrometry (MFLC-MS/MS). A biologically relevant tryptic peptide ‘GDSVVYGLR’ which is unique to hOPN was identified and used as a signature peptide for this method. The method was validated over a range of 25-600 ng/mL. The performance of the method was compliant with USFDA validation guidance. In addition, a stable isotope labeled (SIL) peptide GDSVVYGLR* and an extended SIL peptide TYDGRGDSVV*YGLRSKSKKF’ were evaluated as internal standards (IS) to account for signature peptide digestion instability and variability. In the digestion variability studies, the use of extended SIL peptide as internal standard limited the total variability within ±30%. Alternatively, when SIL peptide was used as internal standard the variability ranged from -67.4% to +50.6 %. The applicability of the validated method was demonstrated by analyzing plasma samples obtained from 10 healthy individuals and 10 breast cancer patients. More than 9-fold increase in the mean plasma hOPN concentration was seen in 30% of the breast cancer patient samples (n=10) in comparison to the healthy volunteer samples. In a proof of concept investigation, a stable isotope labeled signature peptide was evaluated as an internal standard to compensate for immunocapture variability during quantification of human osteopontin (hOPN) by immunoaffinity coupled LC-MS/MS. Immunocapture variability was induced by varying the antibody amount per well. The immunocapture variability ranged from -80.9 % to +77.0 % when the IS was added after immunocapture and from -37.5% to +20.3% when the IS was added before immunocapture. The lower variability demonstrates the ability of SIL-IS peptide to compensate for variation during immunocapture.

Parkinson’s disease and Essential tremor are the two most prevalent movement disorders in the world, but due to overlapping clinical symptoms, accurate differential diagnosis is difficult. As a result, approximately 60% of patients with movement disorders symptoms will have their diagnosis changed at least once before death. By their subjective nature, clinical exams are inherently imprecise, leading to the desire to create an objective, quantifiable test for movement disorders; a test that currently is elusive. Eye movements have been studied for a century, and are widely appreciated to be quantifiably affected in those with neurological disease. Through a collaborative effort between the VA hospital and VCU, over 1,000 movement disorder subjects had their eye movements recorded, utilizing an SR Research Eyelink 2. Patients with Parkinson’s disease exhibited an ocular gaze tremor during fixation, normal reflexive saccades, and reduced blink rate. Subjects with Essential tremor exhibited slowed saccadic dynamics, with increased latencies, in addition to a larger number of square wave jerk interruptions of otherwise stable fixation. After diagnostic features of each disorder were identified, prospective data collection could occur in a blinded fashion, and oculomotor features used to predict clinical diagnoses. It was determined that measures of fixation stability were capable of almost perfectly differentiating subjects with PD, and a novel, combined parameter was capable of similar results in ET. As a group, it appears as if these symptoms do not progress as the disease does, but subanalyses show that individual patients on constant pharmaceutical doses tracked over time do slightly change and progress. The near perfect separation of disease states suggest the ability of oculomotor recording to be a powerful biomarker to be used for the differential diagnosis of movement disorders. This tool could potentially impact and improve the lives of millions of people the world over.

Past studies evaluating the esthetics of orthodontic treatment have been done using 2-D images. New 3-D imaging offers an improved, real-life representation of a subject. The purpose of this study was to determine how laypeople perceived differences in lip position (flat versus ideal lip fullness) in 2-D compared to 3-D. 3dMD images of 8 Caucasian subjects were adjusted to an ideal and flat lip position in 3-D and then in 2-D from the profile view. 2 surveys were created with paired ideal and flat images on the screen, either in 2-D or 3-D, and evaluators were asked to choose which image they preferred and by how much. Evaluators were more likely to be neutral, and were less decisive of their preference in 3-D compared to 2-D. People might be less sensitive to small differences in facial soft tissue and esthetics than previous research in 2-D has led orthodontists to believe.

Sickle Cell Disease (SCD) is a hereditary monogenic disorder that affects millions of people worldwide and is associated with symptoms such as stroke, lethargy, chronic anemia, and increased mortality. SCD can be quickly detected and diagnosed using a simple blood test as an infant, but as of now, there is currently limited treatment to cure an individual of sickle cell disease. Recently, there have been several promising developments in CRISPR-Cas-associated gene-editing therapeutics; however, there have been limitations in gene-editing efficiency monitoring, which if improved, could be beneficial to advancing CRISPR-based therapy, especially in SCD. The CRISPR-Chip, a three-terminal graphene-based field effect transistor (gFET), was used to detect genomic samples of individuals with SCD, with and without amplification. With the dRNP-HTY3’ complex, CRISPR-Chip was able to specifically detect its target sequence with and without pre-amplification. With the dRNP-MUT3’ complex, CRISPR-Chip was only able to specifically detect one of its two target sequences. Facile detection, analysis, and editing of sickle cell disease using CRISPR-based editing and monitoring would be beneficial for simple diagnostic and gene-editing therapeutic treatment of other single nucleotide polymorphisms as well, such as beta-thalassemia and cystic fibrosis.

Reoccurrence of deformity can affect upwards of 64% of children with clubfoot. The ability to use foot function as a measure of reoccurrence has not been previously assessed. The purpose of this investigation was to utilize foot pressure analysis to predict the probability of reoccurrence in children with unilateral clubfoot. Retrospective foot pressure data revealed predictive algorithms detecting the probability of experiencing any type of reoccurrence (overall reoccurrence) and for experiencing a tibialis anterior tendon transfer (TATT). The equation for overall reoccurrence reported sensitivity and specificity of 0.82 and 0.81 and the equation for TATT reported values of 0.81 and 0.84. These algorithms were then applied prospectively to a cohort of children with unilateral clubfoot. Interim sensitivity and specificity results at a 1.5-year follow-up demonstrate that the equations for overall reoccurrence and TATT were highly specific but not sensitive (0.84, 0.73 specificity; 0.11, 0 sensitivity). This is an indication that these algorithms were more accurate when identifying the absence of reoccurrence. However, these results may change as the prospective subjects continue to age. Overall, the results of this investigation show that foot pressure analysis can predict the presence/absence of reoccurrence. The algorithms developed herein have the potential to improve long and short-term outcomes for children with clubfoot. Providing clinicians with the probability of reoccurrence will improve their ability to be proactive during the treatment decision making process.

Normal movement execution requires proper coupling of motor and sensory activation. An increasing body of literature supports the idea that incorporation of sensory stimulation into motor rehabilitation practices increases its effectiveness. Paired associative stimulation (PAS) studies, in which afferent and efferent pathways are activated in tandem, have brought attention to the importance of well-timed stimulation rather than non-associative (i.e., open-loop) activation. In patients with tetraplegia resulting from spinal cord injury (SCI), varying degrees of upper limb function may remain and could be harnessed for rehabilitation. Incorporating associative sensory stimulation coupled with self-paced motor training would be a means for supplementing sensory deficits and improving functional outcomes. In a motor rehabilitation setting, it seems plausible that sensory feedback stimulation in response to volitional movement execution (to the extent possible), which is not utilized in most PAS protocols, would produce greater benefits. This capability is developed and tested in the present study by implementing a brain-computer interface (BCI) to apply sensory stimulation synchronized with movement execution through the detection of movement intent in real time from electroencephalography (EEG). The results demonstrate that accurate sensory stimulation application in response to movement intent is feasible in SCI patients with chronic motor deficit and often precedes the onset of movement, which is deemed optimal by PAS investigations that do not involve a volitional movement task.

Four dimensional imaging has become part of the standard of care for diagnosing and treating non-small cell lung cancer. In radiotherapy applications 4D fan-beam computed tomography (4D-CT) and 4D cone-beam computed tomography (4D-CBCT) are two advanced imaging modalities that afford clinical practitioners knowledge of the underlying kinematics and structural dynamics of diseased tissues and provide insight into the effects of regular organ motion and the nature of tissue deformation over time. While these imaging techniques can facilitate the use of more targeted radiotherapies, issues surrounding image quality and accuracy currently limit the utility of these images clinically. The purpose of this project is to develop methods that retrospectively compensate for anatomical motion in 4D-CBCT and correct motion artifacts present in 4D-CT to improve the image quality of reconstructed volume and assist in localizing respiration-influenced, diseased tissue and mobile structures of interest. In the first half of the project, a series of motion compensation (MoCo) workflow methods incorporating groupwise deformable image registration and projection-warped reconstruction were developed for use with 4D-CBCT imaging. In the latter half of the project, novel motion artifact observation and artifact- weighted groupwise registration-based image correction algorithms were designed and tested. Both deliverable components of this project were evaluated for their ability to enhance image quality when applied to clinical patient datasets and demonstrated qualitative and quantitative improvements over current state-of-the-art.

In humans and other organisms, monoaminergic systems are crucial in neuronal function and behavior. The monoamine transporters (MATs), which can be found on the presynaptic plasma membrane of neurons in the central nervous system (CNS), are crucial in the regulation of neurotransmitter concentration in the synaptic cleft. As the duration and concentration of neurotransmitters in the cleft affect further downstream signaling responses, these proteins are important targets for both understanding neuronal physiology and compounds of interest. Multiple theories exist proponing the contribution of MATs to a variety of mental and neurological disorders, including depression. This theory establishes that depression is caused by imbalances in monoamine neurotransmitters. Compounds such as Fluoxetine (FLX) are classified as selective serotonin reuptake inhibitors (SSRIs), these drugs selectively block the reuptake of neurotransmitters at the serotonin transporter (SERT). Since differences in MAT selectivity of inhibitory compounds are influential to selecting efficacious antidepressant treatments, we utilized a unique fluorescent analysis technique to explore three therapeutic compounds of interest (in-vitro) which contain structural similarity to FLX. Our results confirm the selectivity of FLX at SERT, and classify the novel compounds studied into different potential categories of reuptake inhibitors. We hope these compounds will be studied further to elucidate their potentially therapeutic roles and mitigation of undesired side effects seen in other medications.

Liver cancer ranks the third most common cause of cancer related mortality worldwide. Radiomicrosphere therapy (RMT), a form of radiation therapy, involves administration of Yttrium-90 (90Y) microspheres to the liver via the hepatic artery. 90Y microspheres bremsstrahlung SPECT/CT or PET/CT imaging could potentially identify an extrahepatic uptake. An early detection of such an uptake, thus, could initiate preventative measures early on. However, the quantitative accuracy of bremsstrahlung SPECT/CT images is limited by the wide and continuous energy spectrum of 90Y bremsstrahlung photons. 90Y PET/CT imaging is also possible but limited by the extremely small internal pair production decay. These limitation lead to inaccurate quantitation of microsphere biodistribution especially in small tumors. SPECT/CT and PET/CT acquisition of a Jasczak phantom with eight spherical inserts filled with 90Y3Cl solution were performed to measure the quantitative accuracy of the two imaging modalities. 90Y microsphere SPECT/CT data of 17 patients who underwent RMT for primary or metastatic liver cancer were acquired. Technetium-99m macroaggregated albumin (99mTc-MAA) SPECT/CT scans were also collected, but available for only twelve of the patients. SPECT/CT images from phantoms were used to determine the optimal iteration number for the iterative spatial resolution recovery algorithm. Methods for image based calculation of calibration factors for activity estimation from the patient and phantom 90Y bremsstrahlung SPECT/CT images were developed. Tumor areas were segmented using an active contour method. The 99mTc-MAA and 90Y microsphere SPECT/CT images were co-registered a priori for correlation analysis. Comparison of uptake on 99mTc-MAA and 90Y microsphere SPECT/CT images was assessed using tumor to healthy liver ratios. Furthermore, a three dimensional absorbed dose estimation algorithm was developed using the voxel S-value method. Absorbed doses within the tumor and healthy part of the liver were investigated for correlation with administered activity. Improvement in contrast to noise ratio and contrast recovery coefficients (QH) on patient and phantom 90Y bremsstrahlung SPECT/CT images as well as PET/CT images were achieved. Total activity estimations in liver and phantom gave mean percent errors of -4 ± 12% and -23 ± 41% for patient and phantom SPECT/CT studies. The pre and post-treatment images showed significant correlation (r = 0.9, p < 0.05) with mean TLR of 9.2 ± 9.4 and 5.0 ± 2.2 on 99mTc-MAA and 90Y microspheres SPECT/CT respectively. The correlation between the administered activity and tumor absorbed dose was weak (r = 0.5, p > 0.05), however, healthy liver absorbed dose increased with administered activity (r = 0.8, p < 0.05).

Approximately 2 to 3 million people in the United States suffer from chronic wounds, which are defined as wounds that do not heal in 30 days time; an estimated $25 billion per year is spent on their treatment in the United States. In our work, we focused on treating chronic wounds with bacterial infections using hyperbaric and topical oxygen therapies. We used a mathematical model describing the interaction between bacteria, neutrophils and oxygen. Optimal control theory was then employed to study oxygen treatment strategies with the mathematical model. Existence of a solution was shown for both therapies. Uniqueness was also shown for hyperbaric therapy. We then used a forward-backward sweep method to find numerical solutions for the therapies. We concluded by putting forth ideas for how this problem could progress toward finding applicable treatment strategies.