Doctor of Philosophy

dissertationThe treatment of head and neck cancer is a complicated task which requires that many factors be considered before the best option can be chosen. The best treatment, providing the greatest efficacy with minimal side effects, is likely to be a localized nonsurgical treatment. Such a treatment would allow resolution of the primary tumor without the devastating systemic side effects associated with chemotherapy nor the social and psychological side effects associated with scarring and disfigurement resulting from surgical resection. Locally delivered gene therapy with viral vectors is a promising approach due to advances in delivery-enhancing materials. One such material is genetically engineered silk-elastinlike protein polymer (SELP), which due to its repeating structure and method of synthesis can be precisely modified to produce desired properties, such as pore size, swelling ratio, release rate, and mechanical strength. In this dissertation, it is shown that one particular analog of this polymer exhibits superior performance in the enhancement of adenoviral gene delivery, with enhancement of localization of gene expression of up to 55-fold, and reduction in acute immune response as measured by differential white blood cell count and hepatotoxicity due to viral administration. Further improvement of this material has been to include matrixmetalloproteinase (MMP)-sensitive sites in the polymer to provide it the capability to biodegrade more rapidly. Advantages to an MMP-responsive material include the ability to respond to changes in the tumor environment, as increased MMP activity can correlate with increased invasiveness and, in many cases, metastasis. The insertion of the MMPresponsive sequence caused sensitivity to both MMP-2 and MMP-9, with 63% and 44% increases in protein loss from sensitive hydrogels exposed to MMP-2 and MMP-9, respectively, compared to unexposed control. Further, MMP-2 and MMP-9 exposure caused 41% and 24% reduction in compressive modulus, and 95% and 66% increased release of 100nm polystyrene nanoparticles, respectively. These results show the potential of SELPs in increasing the safety and efficacy of adenoviral gene therapy

The development of a 14-day non-viral engineered CAR T-cell process

Immunotherapy utilizing chimeric antigen receptor (CAR) T cells is a promising strategy for the treatment of several types of cancer. Many preclinical and clinical studies engineer CAR T cells through a viral vector, presenting the potential for genotoxicity or insertional mutagenesis. We propose a 14-day non-viral process where we introduce the gene of interest via electroporation; integration can be achieved with the Sleeping Beauty transposon system. Minicircle (MC) DNA constructs containing the CAR, a surface marker (EGFRt), and a double mutant of dihydrofolate reductase (DHFRdm) are electroporated into previously frozen, unstimulated CD4/CD8 T cells with an RNA construct coding for the Sleeping Beauty transposase. After electroporation, cells are bead-stimulated with CD3/CD28 without the use of feeder cells throughout the process. CAR+ cells expressing DHFRdm are rendered insensitive to an FDA-approved small molecule drug, methotrexate (MTX), which allows for chemical selection of the cells of interest while avoiding a magnetic bead sort. The entire process is completed in 2 weeks with a media formulation that contains a serum-free replacement. Please click Additional Files below to see the full abstract

60 Year Old Male with Autonomic Dysreflexia in C7 AIS A secondary to a Suprapubic Catheter Inflation in the Membranous Urethra

Introduction: Autonomic dysreflexia is a potentially lifethreatening complication in patients with a spinal cord injury above the level of T6. A dangerous symptom is an increase in blood pressure induced by reflex sympathetic hyperactivity secondary to a noxious stimuli which can lead to cerebral hemorrhages, seizures, heart failure or pulmonary edema.1 The most common causes of autonomic dysreflexia are noxious stimuli to the 1) bladder such as urinary tract infection, distension, or catheterization; 2) bowel, such as constipation or 3) skin such as wounds, or tight clothing. Though Foley catheters and intermittent catheterizations are well known causes of dysreflexia there is little literature about suprapubic catheters inappropriately inflated causing autonomic dysreflexia.2 Case Description: The patient is a 60-year-old male with C7 AIS A SCI secondary to a motor vehicle crash in 2021 complicated by neurogenic bladder now with a suprapubic catheter (SPC) who presented to acute rehab for activities of daily living training. During his stay, he had an episode of hypertension (233/109) with associated diaphoresis, increased spasms and a “cold sensation behind [his] legs” occurring within one hour after an exchange of his SPC. At the time of exchange, the SPC was draining bright red blood and clots and flushing through the urethra. The patient was given topical nitroglycerin and sent to the Emergency Department for further evaluation. CT scan showed the SPC balloon inflated in the membranous/bulbous urethra. Urology deflated the balloon, retracted and replaced the SPC draining 1.2L of bloody urine leading to symptom improvement. Patient was evaluated by Urology who suspected a false passage in his bladder. Discussion: This case demonstrates how inappropriate urethral suprapubic catheter inflation may act as a noxious stimulus for autonomic dysreflexia. Though there are few cases documented describing suprapubic catheter placement in the urethra; this case clearly illustrates the common symptoms, such as hypertension, diaphoresis, and spasms which resolved after the reversal of the inciting stimulus. This case also provides CT imaging to visualize the aggravating stimulus inducing the patient’s autonomic dysreflexia. The patient’s symptoms improved almost immediately after removal of the suprapubic catheter exhibiting how important immediate diagnosis is to prevent complications. It is imperative for the patient to follow with Urology for workup for suspected false passage to decrease the chance of inappropriate placement in the future. Conclusion: Autonomic dysreflexia is a common emergency in the SCI population that can often go unrecognized and may lead to stroke, MI, renal failure, and pulmonary edema. A skilled physiatrist can promptly recognize autonomic dysreflexia, identify the source and treat the cause. Bladder complications such as UTI, bladder distension, catheter misplacement and clogging are the most common causes of autonomic dysreflexia and though a urethral SPC placement is rare, it must be a part of the differential in patients with SPCs, especially after a recent placement or exchange.https://jdc.jefferson.edu/rmposters/1015/thumbnail.jp

Increasing Legends Salon Revenue Per Square Foot

Katie MacNamara acquired Legends Hair Salon in Atascadero, California in January of 2021. After a few months of learning to run her own business she discovered that her retail revenue per square foot was not high enough. Her goal, and the project objective, was to raise her revenue while also increasing space for her stylists. The metrics used for these objectives were revenue per square foot and the square footage of the retail areas. The beginning state metrics were 48.75 square feet of retail and a retail revenue per square foot of $2.15 with a total of six stylist stations. The immediate solution that the team created was to make some facility layout changes such as moving the front desk to open up a retail area in the entryway and change the storage room to a pedicure room so that there was more space for another stylist booth. In addition to the facility changes, selling strategies were improved. Retail items were strategically placed along a customer’s “critical path” so they would be more likely to see them while going through the regular steps of a hair service. After making these immediate changes, the retail square footage increased to 78 square feet and the retail revenue per square foot was raised to$3.47. To help Katie to keep track of her sales data in a more user friendly way, a dashboard was created for her to input data and view the sales trends, total sales, revenue per square foot, and a quantity count of which brands are selling the most. A t-test was run to determine if the changes made were significant. The test data was from February-April of 2021 and February-April 2022 and the p-value was 0.036, further proving the assumption that the changes benefited Katie’s salon. From here, long term solutions were created with the assumption that, in five years, Katie would be able to purchase her salon and completely redesign her space including plumbing and moving walls. There were 3 different layouts created and a simulation model was run for each of them. According to the simulation, design 3 would bring in the most revenue. A Multi-Criteria Decision Analysis was created to further analyze the models. The model focused on the design\u27s ability to maximize revenue, increase retail space, increase retail visibility, increase customer comfortability, feasibility of implementation, increase in stylists stations, the A-efficiency score and sponsor approval. From this, solution 2 was the best design. Since Katie preferred solution 2 the most and it was only simulated to make $100 less than solution 3, it was chosen as the recommended 5-year plan

Coherent Cancellation of Photothermal Noise in GaAs/Al0.92_{0.92}Ga0.08_{0.08}As Bragg Mirrors

Thermal noise is a limiting factor in many high-precision optical experiments. A search is underway for novel optical materials with reduced thermal noise. One such pair of materials, gallium arsenide and aluminum-alloyed gallium arsenide (collectively referred to as AlGaAs), shows promise for its low Brownian noise when compared to conventional materials such as silica and tantala. However, AlGaAs has the potential to produce a high level of thermo-optic noise. We have fabricated a set of AlGaAs crystalline coatings, transferred to fused silica substrates, whose layer structure has been optimized to reduce thermo-optic noise by inducing coherent cancellation of the thermoelastic and thermorefractive effects. By measuring the photothermal transfer function of these mirrors, we find evidence that this optimization has been successful.Comment: 10 pages, 7 figure

Considerations towards a roadmap for collection, handling and storage of blood extracellular vesicles

There is an increasing interest in exploring clinically relevant information that is present in body fluids, and extracellular vesicles (EVs) are intrinsic components of body fluids (?liquid biopsies?). In this report, we will focus on blood. Blood contains not only EVs but also cells, and non-EV particles including lipoproteins. Due to the high concentration of soluble proteins and lipoproteins, blood, plasma and serum have a high viscosity and density, which hampers the concentration, isolation and detection of EVs. Because most if not all studies on EVs are single-centre studies, their clinical relevance remains limited. Therefore, there is an urgent need to improve standardization and reproducibility of EV research. As a first step, the International Society on Extracellular Vesicles organized a biomarker workshop in Birmingham (UK) in November 2017, and during that workshop several working groups were created to focus on a particular body fluid. This report is the first output of the blood EV work group and is based on responses by work group members to a questionnaire in order to discover the contours of a roadmap. From the answers it is clear that most respondents are in favour of evidence-based research, education, quality control procedures, and physical models to improve our understanding and comparison of concentration, isolation and detection methods. Since blood is such a complex body fluid, we assume that the outcome of the survey may also be valuable for exploring body fluids other than blood.Non peer reviewe

Damaging variants in FOXI3 cause microtia and craniofacial microsomia

Q1Q1Pacientes con Microtia y Microsomía craneofacialPurpose: Craniofacial microsomia (CFM) represents a spectrum of craniofacial malformations, ranging from isolated microtia with or without aural atresia to underdevelopment of the mandible, maxilla, orbit, facial soft tissue, and/or facial nerve. The genetic causes of CFM remain largely unknown. Methods: We performed genome sequencing and linkage analysis in patients and families with microtia and CFM of unknown genetic etiology. The functional consequences of damaging missense variants were evaluated through expression of wild-type and mutant proteins in vitro. Results: We studied a 5-generation kindred with microtia, identifying a missense variant in FOXI3 (p.Arg236Trp) as the cause of disease (logarithm of the odds = 3.33). We subsequently identified 6 individuals from 3 additional kindreds with microtia-CFM spectrum phenotypes harboring damaging variants in FOXI3, a regulator of ectodermal and neural crest development. Missense variants in the nuclear localization sequence were identified in cases with isolated microtia with aural atresia and found to affect subcellular localization of FOXI3. Loss of function variants were found in patients with microtia and mandibular hypoplasia (CFM), suggesting dosage sensitivity of FOXI3. Conclusion: Damaging variants in FOXI3 are the second most frequent genetic cause of CFM, causing 1% of all cases, including 13% of familial cases in our cohort.https://orcid.org/0000-0003-3822-7780https://orcid.org/0000-0002-0729-6866Revista Internacional - IndexadaA1N

Summary of the ISEV workshop on extracellular vesicles as disease biomarkers, held in Birmingham, UK, during December 2017

This report summarises the presentations and activities of the ISEV Workshop on extracellular vesicle biomarkers held in Birmingham, UK during December 2017. Among the key messages was broad agreement about the importance of biospecimen science. Much greater attention needs to be paid towards the provenance of collected samples. The workshop also highlighted clear gaps in our knowledge about pre-analytical factors that alter extracellular vesicles (EVs). The future utility of certified standards for credentialing of instruments and software, to analyse EV and for tracking the influence of isolation steps on the structure and content of EVs were also discussed. Several example studies were presented, demonstrating the potential utility for EVs in disease diagnosis, prognosis, longitudinal serial testing and stratification of patients. The conclusion of the workshop was that more effort focused on pre-analytical issues and benchmarking of isolation methods is needed to strengthen collaborations and advance more effective biomarkers

Differential, Phosphorylation Dependent Trafficking of AQP2 in LLC-PK1 Cells

The kidney maintains water homeostasis by modulating aquaporin 2 (AQP2) on the plasma membrane of collecting duct principal cells in response to vasopressin (VP). VP mediated phosphorylation of AQP2 at serine 256 is critical for this effect. However, the role of phosphorylation of other serine residues in the AQP2 C-terminus is less well understood. Here, we examined the effect of phosphorylation of S256, S261 and S269 on AQP2 trafficking and association with recycling pathway markers. We used LLC-PK1 cells expressing AQP2(S-D) or (S-A) phospho mutants and a 20°C cold block, which allows endocytosis to continue, but prevents protein exit from the trans Golgi network (TGN), inducing formation of a perinuclear AQP2 patch. AQP2-S256D persists on the plasma membrane during cold block, while wild type AQP2, AQP2-S256A, S261A, S269A and S269D are internalized and accumulate in the patch. Development of this patch, a measure of AQP2 internalization, was most rapid with AQP2-S256A, and slowest with S261A and S269D. AQP2-S269D exhibited a biphasic internalization profile with a significant amount not internalized until 150 minutes of cold block. After rewarming to 37°C, wt AQP2, AQP2-S261A and AQP2-S269D rapidly redistributed throughout the cytoplasm within 20 minutes, whereas AQP2-S256A dissipated more slowly. Colocalization of AQP2 mutants with several key vesicular markers including clathrin, HSP70/HSC70, EEA, GM130 and Rab11 revealed no major differences. Overall, our data provide evidence supporting the role of S256 and S269 in the maintenance of AQP2 at the cell surface and reveal the dynamics of internalization and recycling of differentially phosphorylated AQP2 in cell culture