Aptamers being used as sensors is an emerging field that has capabilities of being tomorrow’s diagnostic tools. As aptameric sensors have become more popular, their visualization systems have been limited. The majority of today’s aptameric sensors require expensive machinery such as a fluorometer in order to visualize results. We propose a system that will cut the need for instrumentation and be detected via the naked eye. With the selection of an aptamer to bind the pH indicating dye bromocresol purple (BCP) this may be achieved. When rendered active, the binding towards BCP will facilitate a color change from yellow to purple or vice versa. Previous studies have shown albumin contains the ability to facilitate this role and we now intend to use a DNA aptamer to achieve this as well. The BCP aptamer has the potential to serve as a signaling domain to any already selected aptamer thus making it a universal tool for both research and diagnostic measures. We have found that an alternative structure-switching systematic evolution of ligands by exponential enrichment (SELEX) method which left the dye unaltered was not sufficient for selecting an aptamer. We believe that a classical SELEX will enable us to select an aptamer that may be used to accomplish this role as a universal visual detector.

Polymerase chain reaction (PCR) is a method used in many research protocols to amplify a small amount of a short segment of DNA to millions of copies. PCR is used for many taxonomic studies, as well as for some medical diagnostic procedures. Through PCR, short DNA primers bind to the template DNA to allow the thermostable DNA polymerase to copy the DNA. Often, researchers create universal primers to target a conserved region of DNA in multiple species, for example, the 16S rRNA gene in bacteria. The problem with these universal primers is that they do not always perfectly match the target DNA. The mismatch primers can still bind to the template, but could affect the efficiency of the PCR amplification. The effect of mismatch primers on the efficiency of the amplification in PCR is the focus of this thesis. Four forward primers with various mismatch overhangs were generated and incorporated into a DNA template through an initial PCR. These primers contained the binding region complementary to the V3/V4 region of the 16S rRNA bacterial gene. Further quantitative PCR (qPCR) reactions were run on these newly-made templates using two sets of primers complementary to the 16S rRNA gene region – one with ambiguous base pairs, one with unambiguous base pairs. The qPCR amplification curves, the Cq values, and the initial concentrations of DNA products (seed values) were analyzed and compared. The results showed differences in the Cq values and seed values between the reactions containing mismatches and those not containing mismatches. Other variables including annealing temperature, addition of Illumina sequencing tails to the primers, and initial primer concentration were also tested to determine if these variables had an effect on the amplification. The results from these reactions using different variables were inconclusive.

Glycosaminoglycans are heterogeneous polysaccharides that mediate important biological functions. There has been considerable interest in deciphering the precise GAG sequences that are responsible for protein interactions. In fact, several GAG oligosaccharides have been discovered to date as targeting proteins with higher level of specificity. Yet, it has been difficult to develop GAG oligosaccharides as drugs. One of the key reasons for this state of art is that GAG synthesis is extremely challenging and is highly structure-specific. Thus, much of the biology and pharmacology of GAG remains unknown and unexploited to date. An alternative approach is to prepare GAG oligosaccharides using enzymatic depolymerization of polymeric GAGs. GAG lyases, including heparinases and chondritinases represent powerful tools that can theoretically generate multiple oligosaccharides in parallel. However, it is difficult to implement such procedures with high consistency. Moreover, GAG lyases can digest GAGs down to disaccharides. A priori, non-polymeric GAGs, or alternatively GAG oligosaccharides containing 4 to 10 residues, would be expected to function better as therapeutic agents because they would be more homogeneous and less non-specific than their polymeric precursors. Thus, we reasoned that immobilization of these enzymes may engineer altered biopolymer processing, which may afford longer oligosaccharides in higher proportions and greater consistency. Heparinase-I and chondroitinase ABC were immobilized on CNBr-activated Sepharose and compared with the free form of the enzyme. Immobilized GAG lyases retained high efficiency of depolymerization over a wide range of pH, temperature and reusability. Most importantly, the immobilized enzyme was found to produce larger proportions of oligosaccharides longer than di- and tetra-saccharides as compared to lyases in the free form. A two dimensional separation involves size exclusion chromatography followed by reversed phase ion-pairing ultra performance liquid chromatography coupled to electrospray ionization mass spectrometry was employed to separate and characterize oligosaccharide structures. We have identified 40 heparin oligosaccharides, including regular and rare structures ranging from dp4 to dp10 and 39 chondroitin sulfate oligosaccharides in high homogeneity and significant yields. Overall, this technology is likely to offer a simple and cost effective route to preparation of larger amounts of sequences that can be expected to bind and modulate protein function.

Cell mass is an important indicator of cell health and status. A diverse set of techniques have been developed to precisely measure the masses of single cells, with varying degrees of technical complexity and throughput. Here, the development of a non-invasive, label-free optical technique, termed Live-Cell Interferometry (LCI), is described. Several applications are presented, including an evaluation of LCI’s utility for assessing drug response heterogeneity in patient-derived melanoma lines and the measurement of CD3+ T cell kinetics during hematopoietic stem cell transplantation. The characterization of mast cells during degranulation, the measurement of viral reactivation kinetics in Kaposi’s Sarcoma, and drug response studies in patient-derived xenograft models of triple-negative breast cancer are also discussed. Taken together, data from these studies highlight LCI’s versatility as a tool for clinical, translational, and basic research applications.

Chemo-radiation therapy is a principal modality for the treatment of head and neck cancers, and its efficacy depends on the interaction of tumor oxygen with free radicals. In this study, we adopted a novel hybrid diffuse optical instrument combining a commercial frequency-domain tissue oximeter (Imagent) and a custom-made diffuse correlation spectroscopy (DCS) flowmeter, which allowed for simultaneous measurements of tumor blood flow and blood oxygenation. Using this hybrid instrument we continually measured tumor hemodynamic responses to chemo-radiation therapy over the treatment period of 7 weeks. We also explored monitoring dynamic tumor hemodynamic changes during radiation delivery. Blood flow data analysis was improved by simultaneously extracting multiple parameters from one single autocorrelation function curve measured by DCS. Patients were classified into two groups based on clinical outcomes: a complete response (CR) group and an incomplete response (IR) group with remote metastasis and/or local recurrence within one year. Interestingly, we found human papilloma virus (HPV-16) status largely affected tumor homodynamic responses to therapy. Significant differences in tumor blood flow index (BFI) and reduced scattering coefficient (μs’) between the IR and CR groups were observed in HPV-16 negative patients at Week 3. Significant differences in oxygenated hemoglobin concentration ([HbO2]) and blood oxygen saturation (StO2) between the two groups were found in HPV-16 positive patients at Week 1 and Week 3, respectively. Receiver operating characteristic curves were constructed and results indicated high sensitivities and specificities of these hemodynamic parameters for early (within the first three weeks of the treatment) prediction of one-year treatment outcomes. Measurement of tumor hemodynamics may serve as a predictive tool allowing treatment selection based on biologic tumor characteristics. Ultimately, reduction of side effects in patients not benefiting from radiation treatment may be feasible.

Peripheral neuropathies are a set of common diseases that affect the peripheral nervous system, causing damage to vital connections between various parts of the body and the brain and spinal cord. Different clinical conditions are known to selectively impact various size nerve fibers, which often makes it difficult to diagnose which peripheral neuropathy a patient might have. The nerve conduction velocity diagnostic test provides clinically useful information in the diagnosis of some peripheral neuropathies. This method is advantageous because it tends to be minimally invasive yet it provides valuable diagnostic information. However, this test does not determine characteristics of peripheral nerve fiber size distributions, and therefore does not show any detailed information regarding the nerve fibers within the nerve trunk. Being able to determine which nerve fibers are contributing to the evoked potential within a nerve trunk could provide additional information to clinicians for the diagnosis of specific pathologies of the peripheral nervous system, such as chronic inflammatory demyelinating polyneuropathy or early diabetic peripheral neuropathy. In this study, three rat sciatic nerves are sectioned and stained with hematoxylin and eosin in order to measure the nerve fiber diameters within the nerve trunk. Stained samples are viewed using brightfield microscopy and images are analyzed using ImageJ. Histograms were created to show the frequency of various nerve fiber diameters. The nerve fiber diameters measured during this research are consistent with the range of previously published diameter values and will be used to support continuing research for a novel method to characterize peripheral nerve fiber size distributions using group delay.

Associated with health care reform, there has been a growing interest among healthcare professionals regarding use of energy-based therapies as a complementary therapeutic intervention. The purpose of this integrative literature review was to examine the impact of energy-based therapies (i.e. healing touch, reiki, therapeutic touch) on patients’ postoperative recovery. The methodology included identifying appropriate peer-reviewed, English-language research articles on the topic area that were published between 2006 to 2018. Following a search in select data bases, articles focusing on the topic were critiqued, analyzed and synthesized by the researcher. Consistent and inconsistent findings along with gaps in the literature are noted. In general, the research supported positive postoperative recovery outcomes when energy-based therapies were included along with standard postoperative nursing care. This theses highlights implication for nursing practice, education, and policy and identifies study limitations. Given that research is limited on this topic area, additional studies are needed to establish supporting evidence to ascertain the effects of energy-based therapies when used as a complementary postoperative intervention.

Summaries in Afrikaans and English / Hierdie studie spreek die emosionele skoolgereedheid van voorskoolse kinders aan. Die voorskoolse kind met sy basiese emosies vanuit sielkundig opvoedkundige perspektief word in die studie van naderby beskou. Tydens die studie is die basiese emosies volgens Plutchik (1980) wat bepalend vir die emosionele skoolgereedheid van die skooltoetreder is, geidentifiseer. Na aanleiding van nuwe insigte waartoe gekom is, het die navorser 'n eie emosionele skoolgereedheidsevalueringslys volgens Plutchik (1980) se basiese emosies antwerp, wat as 'n maatstaf vir die evaluering van emosionele skoolgereedheid kan dien. 'n Empiriese ondersoek is onderneem om te bepaal of 'n aantal proefpersone volgens n emosionele skoolgereedheidsevalueringslys emosioneel skoolgereed is al dan nie. Emosionele tekorte is vanuit die emosionele skoolgereedheidsevalueringslys geidentifiseer, waarna diagnostiese en terapeutiese tegnieke vir emosionele skoolgereedmaking kortliks bespreek is. Opvoedkundige sielkundiges kan die diagnostiese en terapeutiese tegnieke as hulpmiddel gebruik om emosionele tekorte, wat moontlik 'n blokkasie vir emosionele skoolgereedheid inhou, aan te spreek. / This stidu addresses the emotional school preparedness (readiness) of pre-primary children. The pre-primary child with his basic emotions from a psychological educational perspective is examined more closely in this study. During this study the basic emotions according to Plutchik {1980) that determine the emotional school readiness of the school entrant, were identified. On account of new insights gained, the researcher designed a school readiness evaluation list of her own according to Plutchik's (1980) basic emotions. This list can serve as a guideline for the evaluation of emotional school readiness. An empirical study was undertaken to determine whether a number of experimental subjects {children) were ready for school or not. Emotional deficiencies were identified by means of the emotional school readiness evaluation list, whereafter diagnostic and therapeutic techniques for the emotional preparation of children were briefly discussed. Educational psychologists can use these diagnostic and therapeutic techniques as an aid to address emotional deficiencies, which may impede the emotional school readiness of children. / Educational Studies / M. Ed. (met spesialisering in Voorligting)

The collaboration between the School of Engineering and the Department of Physical Therapy at the University of North Florida has introduced the possibility of creating a new environment for pediatric physical therapy assessments. There are currently no methods for remotely monitoring children with impairments. However, with embedded sensor technology in the form of power mobility and accepted therapy assessment tools, remote monitoring can become a possibility. As a part of this work, a ride-on toy car was developed as a remote monitoring device and a case study with a child with a mobility impairment was used as a proof of concept. In this thesis, the background information on the project, the case study diagnosis and history, and the model used to develop this project are detailed.

Summaries in Afrikaans and English / Hierdie studie spreek die emosionele skoolgereedheid van voorskoolse kinders aan. Die voorskoolse kind met sy basiese emosies vanuit sielkundig opvoedkundige perspektief word in die studie van naderby beskou. Tydens die studie is die basiese emosies volgens Plutchik (1980) wat bepalend vir die emosionele skoolgereedheid van die skooltoetreder is, geidentifiseer. Na aanleiding van nuwe insigte waartoe gekom is, het die navorser 'n eie emosionele skoolgereedheidsevalueringslys volgens Plutchik (1980) se basiese emosies antwerp, wat as 'n maatstaf vir die evaluering van emosionele skoolgereedheid kan dien. 'n Empiriese ondersoek is onderneem om te bepaal of 'n aantal proefpersone volgens n emosionele skoolgereedheidsevalueringslys emosioneel skoolgereed is al dan nie. Emosionele tekorte is vanuit die emosionele skoolgereedheidsevalueringslys geidentifiseer, waarna diagnostiese en terapeutiese tegnieke vir emosionele skoolgereedmaking kortliks bespreek is. Opvoedkundige sielkundiges kan die diagnostiese en terapeutiese tegnieke as hulpmiddel gebruik om emosionele tekorte, wat moontlik 'n blokkasie vir emosionele skoolgereedheid inhou, aan te spreek. / This stidu addresses the emotional school preparedness (readiness) of pre-primary children. The pre-primary child with his basic emotions from a psychological educational perspective is examined more closely in this study. During this study the basic emotions according to Plutchik {1980) that determine the emotional school readiness of the school entrant, were identified. On account of new insights gained, the researcher designed a school readiness evaluation list of her own according to Plutchik's (1980) basic emotions. This list can serve as a guideline for the evaluation of emotional school readiness. An empirical study was undertaken to determine whether a number of experimental subjects {children) were ready for school or not. Emotional deficiencies were identified by means of the emotional school readiness evaluation list, whereafter diagnostic and therapeutic techniques for the emotional preparation of children were briefly discussed. Educational psychologists can use these diagnostic and therapeutic techniques as an aid to address emotional deficiencies, which may impede the emotional school readiness of children. / Educational Studies / M. Ed. (met spesialisering in Voorligting)