<p>Background and Objectives: There are some methodological and clinical challenges in conducting HIV related research. A subset of such challenges include: non-availability of a universally accepted method to quantify subclinical atherosclerosis in HIV patients; ultrasound imaging techniques aimed at quantifying atheroma burden and endothelial dysfunction have been proposed, however there is no universally accepted ultrasound protocol; conflicting inferences on the nature ofthe relationship between anti-retroviral therapy (ART) and cardiovascular disease (CVD) due to small sample sizes; and missing data from longitudinal studies and ultrasound data. The objective of this thesis is to investigate selected aspects of the afore-mentioned issues, and to provide recommendations for future research.</p> <p>Methods:</p> <p>Project 1: We compared the construct validity of carotid artery intima media thickness (IMT) and brachial artery flow mediated vasodilation (FMD); two non-invasive ultrasound techniques used in measuring the extent of sub-clinical atherosclerosis. Baseline and one-year follow-up data were obtained for a sample of 257 subjects aged 35 years or older, recruited into an ongoing study of cardiovascular risk in HIV. An ultrasound technique having statistically significantly strong association with known CVD risk factors was adjudged to have good construct validity. The relationship between baseline IMT or FMD and known CVD risk factors was studied using multiple regression analysis. We modelled the relationship between progression of IMT or FMD and risk factors using fixed-effects models.</p> <p>Project 2: To more precisely investigate the relationship between ARTs and IMT (as a surrogate for CVD), we pooled cross-sectional baseline, record-level data for 1,032 patients recruited across three cohort studies in Canada, France and USA in a metaanalysis. We investigated the association between exposure to ARTs and CVD using hierarchical linear models.</p> <p>Project 3: On missing data, we studied the impact ofan inclusive strategy for conducting multiple imputation (MI) on the efficiency ofregression parameter estimates using Monte-Carlo simulation. In an inclusive strategy, all final analysis variables are included in a multivariate normal model to impute plausible values for missing data. This issue is not well studied for longitudinal HIV data.</p> <p>Results and Conclusions:</p> <p>Project 1: Baseline IMT was significantly associated with age (p < 0.001), male gender (p = 0.034), current smoking status (p < 0.001), systolic blood pressure (p < 0.001) and total:HDL cholesterol ratio (p = 0.004). IMT progression was significantly associated with age (p < 0.001), male gender (p = 0.0051) and current smoking status (p = 0.011). Neither extent nor progression ofFMD was significantly associated with any of the examined vascular risk factors. IMT was adjudged to have better construct validity than FMD.</p> <p>Project 2: Similar to some (but not all) previous studies, AR Ts do not appear to lead to CVD independent of traditional risk factors. However, exploratory analysis of two-way interactions suggests statistically significant moderating effects between ARTs and traditional risk factors. These results warrant further investigation into potential moderating effects between ARTs and known CVD risk factors.</p> <p>Project 3: In conducting MI, simulation results show that a strategy that includes all final analysis model variables in the imputation model provides the least combined variability and bias for final regression estimates. This is important to note because final regression estimates are used in making clinically relevant inferences in practice.</p> / Thesis / Doctor of Philosophy (PhD)

Gas filled microbubbles (MBs) stabilized by a shell (e.g. lipids) are commonly used as ultrasound (US) contrast agents. Attaching biomolecules to the surface of MBs allows for molecular US imaging of various diseases. With the increased interest in targeted US imaging, new platforms to prepare disease-targeted MBs are necessary. Furthermore, attaching signaling agents to MBs creates multi-modal imaging opportunities, enhancing visualization and quantification of disease biomarkers. In this thesis, MBs labeled with 99mTc and/or rhodamine dye by taking advantage of the strong interaction between biotin and streptavidin are reported. Radiolabeling of MBs was achieved in good radiochemical yield (~ 30%). 99mTc-labeled MBs were targeted to vascular endothelial growth factor receptor 2 (VEGFR2) using an anti-VEGFR2 antibody and to prostate specific membrane antigen (PSMA) using small-molecule based PSMA inhibitors. In vitro evaluations showed successful binding of MBs to the target while in vivo targeting assessments were unsuccessful. New strategies to target MBs to the site of interest were then developed through the use of the bioorthogonal reaction between tetrazine (Tz) and trans-cyclooctene (TCO). A biotinylated derivative of Tz was loaded on streptavidin coated MBs to create a Tz-derivatized MB (MBTz). Targeting MBTz to extracellular markers of cancer such as VEGFR2, PSMA and urokinase plasminogen activator receptor (uPAR) in vitro was achieved using TCO-conjugated antibodies. In vivo targeting was successful for VEGFR2 and PSMA, but not uPAR. Translating the new strategy to other US contrast agents was then investigated. Gas vesicles (GVs) produced in halobacteria were conjugated with TCO using amide-coupling chemistry. A 99mTc-labeled derivative of Tz was loaded on TCO-GVs (RCY= 59%) and their distribution assessed by SPECT/CT imaging and ex vivo tissue counting. Having established a convenient platform to conjugate molecules to GVs and MBs, future work focuses on developing a new generation of human compatible molecular US imaging probes. / Dissertation / Doctor of Philosophy (PhD)

Performing non-destructive testing (NDT) on materials is a helpful tool for maintenance and quality control because the materials are not destroyed or disturbed; ultrasound imaging is one type of NDT. Ultrasound imaging of heterogeneous materials contains many echoes from the material itself. These echoes come from changes in the acoustic impedance, i.e. changes in the relation between the density and the sound speed of the material. However, these echoes will show speckle characteristics in images, making it hard to detect any defects in the imaged material. In this work, a method of suppressing this speckle noise is proposed. The proposed method is a 2D Wiener filter, which with the help of an image of the healthy material models changes in the material when a new image is taken later. The filter models the changes of the speckle noise between images of a defected- and healty material and then supresses the speckle from the image with defects. The filter works well on the artificial images used in this work but have yet to be tested on actual data. A version of a weighted moving average filter was also looked into, but this filter did not produce usable results.

Background: Histotripsy is a non-thermal, non-invasive high-intensity focused ultrasound (HIFU) ablative technique that causes mechanical fragmentation of tissue, resulting in liquefied acellular debris with histologically clear demarcated boundaries between treated and non-treated tissues. The acellular debris may include tumor antigens with preserved immunogenicity and the potential to generate systemic immune response against tumor cells. Soft tissue sarcomas (STS) are a common form of cancer in dogs with biological behavior similar to STS in humans. Long-term tumor control requires complete removal with extensive surgical resection, which in many cases is not feasible. As a result, there is need for alternative therapies. Objectives: The primary objective of this study was to demonstrate safety and feasibility of histotripsy in a small animal model of spontaneous STS. The secondary objective was to characterize the impact of histotripsy on the immunologic response. Materials and methods: Pet dogs diagnosed with spontaneous STS were recruited. CT scan of the chest, abdomen, and the tumor was performed for staging and treatment planning. Pretreatment biopsies were obtained. Safety was monitored with physical examinations, owner reports, and CBC/serum biochemistry. Partial tumor ablation was performed using a 500 kHz prototype histotripsy system. A spherical treatment zone of up to 3 cm diameter in each tumor was treated with histotripsy according to the patient-specific treatment plan using 1-2 cycle pulses applied at a pulse repetition frequency (PRF) of 500 Hz. Anatomical ablation zones were evaluated with contrast CT at 1- and 4-days post-treatment, with tumor resection at 4-6 days post-treatment. Tumor microenvironment (TME) gene expression was evaluated with the Nanostring Canine IO panel, and the systemic immune response was evaluated using multiplex serum cytokine levels. Results: Ten dogs were recruited and treated. Tumor histologies included 3 grade III STS, 4 grade II STS, 2 grade I STS, and 1 malignant mesenchymoma. Six dogs were alive, three dogs were euthanized due to disease progression, and one dog was lost to follow up. Histotripsy-related complications were generally self-limiting, with only one patient having increased cutaneous injury score from 1 to 2 (scale 1-5) post-treatment, likely due to prefocal cavitation at the skin. No significant adverse events impacting patient outcome were noted in any of the patients. Visible histotripsy cavitation bubble clouds were seen on real-time ultrasound imaging in nine of ten treatments. Post-treatment histopathology indicated sharply defined regions of ablation that were clearly identifiable grossly and histologically in all samples. Treatment zones were characterized by loss of cell viability, hyalinization, and acute hemorrhage. Post-treatment contrast-enhanced CT images revealed clear, demarcated regions of histotripsy ablated tissue in seven of ten patients. Differential gene expression analysis identified 79 genes with at least 2-fold change following treatment. Genes associated with inflammation, immune cell migration, and immune cell interactions were the highest upregulated. Amongst the gene set analyses, the myeloid compartment gene sets obtained the highest significance score. There were no statistically significant differences between pre- and post-treatment cytokine concentrations for any of the analytes. Conclusions: Histotripsy can achieve safe and effective tumor ablation in dogs diagnosed with STS. Histotripsy induced pro-inflammatory changes within the tumor microenvironment. Histotripsy as an immunotherapeutic treatment option needs to be further investigated. Histotripsy has a potential to be a precise, non-invasive treatment for canine STS. / Master of Science / Histotripsy is a non-thermal high-intensity focused ultrasound (HIFU) ablative technique that uses controlled acoustic cavitation to cause mechanical fragmentation of tissue. To date, there are no reports investigating histotripsy for the treatment of soft tissue sarcoma (STS). This study aimed to investigate the in vivo feasibility of ablating STS with histotripsy and to characterize the impact of partial histotripsy ablation on the acute immunologic response in canine patients with spontaneous STS. CT of the chest, abdomen, and the tumor was performed for staging and treatment-planning. Pretreatment biopsies were obtained. Safety was monitored with physical examinations, through owner reports, and CBC/serum biochemistry. A custom 500 kHz histotripsy system was used to treat ten dogs with naturally occurring STS. Anatomical ablation zones were evaluated with contrast CT at 1- and 4-days post-treatment, with tumor resection at 4-days post-treatment. Safety was determined by monitoring vital signs during treatment and post-treatment physical examinations, routine lab work, and owners' reports. Ablation was characterized using radiologic and histopathologic analyses. Systemic immunological impact was evaluated by measuring changes in cytokine concentrations, and tumor microenvironment changes were evaluated by characterizing changes in infiltration with tumor-associated macrophages (TAMs) and tumor-infiltrating lymphocytes (TILs) using multiplex immunohistochemistry and differential gene expression. Results showed histotripsy ablation can achieve safe and effective tumor ablation in all ten dogs. Immunological results showed histotripsy induced pro-inflammatory changes in the tumor microenvironment. Histotripsy as an immunotherapeutic treatment option needs to be further investigated. Overall, this study demonstrates histotripsy's potential as a precise, non-invasive treatment for STS.

Current ACL reconstruction techniques are performed with little regard to cutaneous nerves despite the evidence that iatrogenic nerve injury often leads to painful neuromas, reflex sympathetic dystrophy, or anterior knee/kneeling pain which directly impacts patients whose profession requires kneeling (Cerulli et al. 2013; Ruffilli et al. 2016; Hurley et al. 2018). In this study, the femoral cutaneous and saphenous nerves from two embalmed lower extremities were dissected in exquisite detail to create topographic maps of the anterior thigh and knee, detailing the nerves' anatomical course and relationship to relevant landmarks. To further characterize the cutaneous innervation at the knee joint, ultrasound techniques to locate sensory nerves were also explored. In both specimens, all branches of the femoral cutaneous and saphenous nerves were located, dissected, and mapped. In addition, the ability to locate terminal branches of the anterior femoral cutaneous nerve in a cadaveric specimen on ultrasound was successfully demonstrated. However, due to the small sample size, further research implementing similar methods is necessary to ensure the results are representative of the population and can be applied to surgical innervation. These findings highlight the importance of considering cutaneous nerves in ACL reconstruction techniques to minimize the risk of iatrogenic nerve injury and associated complications.

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