The removal of milk fouling deposits often requires the diffusion of electrolyte solutions such as sodium hydroxide through a gel. Very often more than one single anion and one single cation are involved and thus the modelling of such diffusion requires a multicomponent description. Diffusion of electrolyte solutions through gels can be modelled using the Maxwell-Stefan equation. The driving forces for diffusion are the chemical potential gradients of ionic species and the diffusion potential, i.e., the electrostatic potential induced by diffusion of the ions. A model based on the Maxwell-Stefan equation was applied to electrolyte solutions and electrolyte solutions with a gel. When modelling the diffusion of electrolyte solutions, the resulting equations were found to be a partial differential algebraic equation system with a differentiation index of two. The identification of this characteristic of the system enabled a solution method using the method of lines to be developed. When modelling the diffusion of electrolyte solutions through a gel an explicit expression for diffusion potential was developed and hence the diffusion equations were solved. Numerical solutions were presented for a number of case studies and comparisons were made with solutions from literature and between different electrolyte systems. It was found that the results of diffusion of electrolytes were in good agreement with those of experiments and literature. In the case of diffusion of electrolytes through a gel, swelling of the gel was predicted. The model can be improved by adding thermodynamic factors and can be easily extended to multiple ion systems.

Surface modification is an extensively researched approach in order to overcomethe limitations, and improve the performance of orthopedic and dental implants. It is atthe surface of the implant materials that the initial interactions of tissues or body fluidstake place. Therefore, surface properties of biomaterials are the important factors that cancontrol these biological responses. Molecular imprinting is a surface modificationtechnique that creates specific recognition sites on the surface of biomaterials. Todevelop the recognition sites, a functional monomer is assembled with templatebiomolecule and then crosslinked. After removal of the template, the surface can rebindthe molecules. Therefore, desired reactions can be initiated at the interface between tissueand implants by modifying surfaces to selectively bind certain types of biomolecules,such as proteins. The objective of this project was to observe the potential of molecularimprinting technique for creating biomaterials that can recognize specific biomolecules.Fluorescently labeled lysozyme or RNase A was used as a template biomolecule and theprotein-imprinted scaffolds were fabricated by sol-gel processing. To interpret the densityof binding sites created, the quantity of surface-accessible protein was determined. Theamount of protein available on the surface was proportional to the amount loaded.Protein-imprinted scaffolds were evaluated for their ability to selectively recognize thetemplate biomolecule. Further, for these selectivity studies, a combination of theimprinted protein and a competitor protein were rebound to the polysiloxane scaffolds.The template protein rebound to the surface was measured more than twice as much ascompetitor. These scaffolds were then tested to understand their interaction with cells.The results of DNA and alkaline phosphatase activities indicate that the scaffolds thusdeveloped support growth and adhesion of osteoblastic cells. These initial selectivity andcytocompatibility studies show the potential of molecular-imprinted polysiloxanescaffolds to be used as tissue engineered materials for stable and controlled interactions atthe tissue-implant interface.

Molecular imprinting, a common method used in separations and chromatography to isolate specific molecules via surface binding, has been adapted for applications in biomaterials and related sciences. The objective of this study was to determine the effectiveness of different approaches to molecular imprinting by testing for preferential binding of protein on polysiloxane scaffold surfaces. To test preferential rebinding, the scaffolds were exposed to a mixture of the template protein and a competitor protein with similar size but different chemistry. Lysozyme-imprinted polymers rebound 8.13 0.99% of lysozyme without any competition and 5.1 0.3% of the protein during competition. Lysozyme C peptide was imprinted into polysiloxane scaffolds to investigate the epitope approach to molecular imprinting. Without competition, 8.95 11.53% of the lysozyme preferentially bound to the scaffolds, while under competition 1.85 9.47% bound to the scaffolds. Lastly, bone morphogenetic protein 2 (BMP-2) was imprinted into the polymer scaffolds. Results revealed that BMP-2 imprinted scaffolds bound 10.09 6.625% under noncompetitive conditions and a very small 0.65 4.55% during competition. Trends of preferential binding via peptide imprinting and BMP-2 imprinting can be seen, and show promise in future tissue engineering material applications and biomaterial compatibility.

In this thesis we look for potential chronic inflammation biomarkers because studies have shown that farmers with musculoskeletal disorders might be affected by the environment to develop musculoskeletal disorders. Animal farmers are highly exposed to dust, aerosols, molds and other toxins in the air and environment leading to musculoskeletal disorders, respiratory disorders, airway symptoms and febrile reactions. There is reason to believe that the farmers have a constant or chronic inflammation that develops into musculoskeletal disorders. By using a proteomic approach with Two-dimensional Gel Electrophoresis and silver staining our goal was to find biomarkers by quantifying protein spots that differ significantly from farmers with musculoskeletal disorders compared to rural controls. In our study we found 8 significant proteins, two from Alpha-2-HS-glycoprotein, one from Apolipoprotein A1, three from Haptoglobin, one from Hemopexin and 1 from Antithrombin. All 5 proteins are involved in inflammation response in some way and some proteins are linked to chronic inflammation. Out of the 5 proteins Alpha-2-HS-glycoprotein, Apolipoprotein A1 and Hemopexin seem like the most likely proteins to investigate further as potential inflammation biomarkers.

Gaucher’s disease (GD) is an autosomal recessive lysosomal storage disorder which is caused by a mutation in the gene encoding acid β-glucocerebrosidase (GBA, EC 3.2.1.45). Deficient activity in GBA leads to a wide variety of clinical phenotypes, including visceral symptoms such as hepatospenomegaly as well as neurological symptoms. Current enzyme replacement therapy is effective in treating visceral symptoms but cannot cross the blood-brain barrier to target neurological manifestations. Another drawback to current therapy is the high cost to patients due to present protein expression strategies. Recently, protein transduction domains, such as the synthetic PTD4 domain, have been proposed as a therapeutic strategy for drug delivery to the central nervous system. In the present study, we use an economical yeast expression system, Pichia pastoris, to produce a recombinant fusion protein GBA-PTD4, and semi-preparative hydrophobic interaction chromatography and gel filtration chromatography for purification. Results show that final preparations are near homogenous, with GBA-PTD4 accounting for approximately 76% of total protein and only one major contaminant. A cell line expressing GBA without a transduction domain was also created in anticipation of further cellular uptake studies. Future research will focus on large scale enzyme expression in fermentation systems and more direct purification methods such as immunoaffinity chromatography for better protein recovery. / Graduate

X-ray computed tomography (CT) polymer gel dosimetry (PGD) is an attractive tool for three-dimensional (3D) radiation therapy (RT) treatment verification due to the availability of CT scanners in RT clinics. Nevertheless, wide-spread use of the technique has been hindered by low signal-to-noise CT images largely resulting from gel formulations with low radiation sensitivity. However, a new gel recipe with enhanced dose sensitivity was recently introduced that shows great promise for use with CT readout. This dissertation describes development of an CT PGD system for 3D verification of RT treatments using the new gel formulation. The work is divided into three studies: gel characterization, commissioning of a multislice CT scanner and investigation of a dose rate dependence observed during gel characterization. The first component of this work examines the dosimetric properties of the new gel formulation. The response of the gel is found to be stable between 15 - 36 hours post-irradiation and excellent batch reproducibility is seen for doses between 0 - 28 Gy. A dose rate dependence is found for gels irradiated between 100 - 600 MU/min, indicating machine dose rate must be consistent for calibration and test irradiations to avoid dosimetric error. An example clinical application is also presented using an IMRT treatment verification that demonstrates the potential of the system for use in modern RT. The second component of this work focuses on commissioning a multislice CT scanner for CT PGD. A new slice-by-slice background subtraction technique is introduced to account for the anode heel effect. Additional investigations show recommendations for optimizing image quality in CT PGD using a single slice machine also apply to multislice scanners. In addition, the consistency of CT numbers across the multislice detector array is found to be excellent for all slice thicknesses. Further work is performed to assess the tube load characteristics of the scanner and develop a scanning protocol for imaging large gel volumes. Finally, images acquired throughout the volume of an unirradiated active gel show variations in CT data across each image on the order of 7 HU. However, these variations are not expected to greatly influence gel measurements as they are consistent throughout the gel volume. The third component of this work examines the dose rate dependence found during gel characterization. Studies using gel vials and 1 L cylinders indicate the response of the gel does not depend on changes in mean dose rate on the order of seconds to minutes. However, the machine dose rate remains, indicating variations in dose rate on the order of milliseconds influence the response of the gel. An attempt is made to mitigate the effect by increasing the concentration of antioxidant in the gel system but results in reduced overall response. Further work is performed to determine if self-crosslinking of one of the gel components contributes to the observed machine dose rate dependence. In summary, this dissertation has significantly advanced the field of gel dosimetry by providing a prototype CT PGD system with enhanced dose resolution for complex RT treatment verification. / Graduate / 0992 / 0495 / 0756 / holly.johnston@utsouthwestern.edu

Microgels are lightly cross-linked hydrogel particles in the sub-micrometer to micrometer size range with a capacity to drastically change their volume in response to changes in the external environment. Microgels have an ability to bind and store substances such as biomacromolecular drugs, notably proteins and peptides, and release them upon stimuli, making them potential candidates as drug delivery vehicles and functional biomaterials. This thesis aims at clarifying important factors affecting peptide-microgel interactions. These interactions were studied by micromanipulator-assisted light and fluorescence microscopy focusing on microgel deswelling in response to peptide binding, as well as re-swelling in response to peptide release or enzymatic degradation. To evaluate peptide uptake in microgels, solution depletion measurements were used whereas the peptide secondary structure was investigated by circular dichroism. In addition, the peptide and enzyme distribution within microgels was analyzed with confocal microscopy. Results presented in this thesis demonstrate that peptide incorporation into microgels, as well as peptide-induced microgel deswelling, increases with peptide length and charge density. In addition, results demonstrate that the peptide charge (length) rather than peptide charge density determines microgels deswelling. End-to-end cyclization is shown to not noticeably influence peptide-microgel interactions, suggesting that peptide cyclization can be used in combination with oppositely charged microgel carriers to improve the proteolytic and chemical stability of the peptide compared to the corresponding linear variant. Peptide secondary structure is found to drastically affect peptide incorporation into, and release from, oppositely charged microgels. Furthermore, it is shown that microgel charge density, peptide molecular weight, and enzyme concentration all greatly influence microgel bound peptide degradation. Of importance for applications, protective effects of microgels against proteolytic peptide degradation are observed only at sufficiently high microgel charge densities. Enzyme-mediated microgel degradation is shown to increase with increasing enzyme concentration, while an increased peptide loading in microgels causes a concentration-dependent decrease in microgel degradation. Taken together, results obtained in this work have provided some insight into factors of importance for rational use of microgels as delivery systems for protein or peptide drugs, but also in a host of other biomedical applications using weakly cross-linked polymer systems.

Palyginus gelių gamybos metodus nustatyta, kad gamybos laikas naudojant magnetinę maišyklę yra 7,5 karto ilgesnis nei naudojant neutralizatorių. Pastebėta, kad gelio gamybos metodas reikšmingai įtakoja klampą. Nustatyta, kad gelio formavimosi laikui hidroksietiloceliuliozės koncentracija įtakos neturi ir visų tirtų koncentracijų geliai susiformuoja per tą patį laiką. Išmatavus 1,5, 2 ir 3 proc. koncentracijų hidroksietilceliuliozės gelių klampas pastebėta, kad didėjant polimero koncentracijai, reikšmingai didėja gelių klampa. Nustatyta, kad maišymo trukmė neturi įtakos gelių klampai ir fiziniam stabilumui. Tiriant temperatūros įtaką gelių klampai ir stabilumui nustatyta, kad didžiausia klampa pasižymi geliai, kurie gaminami aukščiausioje 100 °C temperatūroje. Tyrimo rezultatai parodė, kad temperatūrinis rėžimas neturi įtakos gelių stabilumui. Tiriant gelinių pagrindų sąveiką su hidrofilinėmis/hidrofobinėmis medžiagomis pastebėta, kad geliai išlaiko savo pradinį fizinį stabilumą sąveikaujant su natrio chloridu, etanoliu, pieno rūgštimi, cinko oksidu, mentoliu ir kamparu. Nustatyta, kad 1 % pieno rūgšties užtikrina visišką gelio stabilumą. Ištyrus hidroksietilceliuliozės gelinio pagrindo sąveika su cinko oksidu nustatyta, kad norint užtikrinti geresnį cinko oksido dalelių pasiskirstymą ir lėtesnę sedimentaciją, gelis turėtų būti maišomas 5 min. 400 aps/min greičiu. / The study showed that gel production method using neutralizer is simplier than using magnetic stirrer. It was found that the gel production method significantly influences the viscosity. However, In order to reduce time, it is recommended to produce gel using the neutralizer. The study showed that the gel formation time is not affected by different hydroxyethylcellulose concentrations. By increasing concentration of hydroxyethylcellulose, viscosity increased. 1,5% gel viscosity without neutralizer is 49,70% higher compared with the gel, which is neutralized by 1 percent 10% trolamine solution. It was found that gel mixing time does not affect gel viscosity and physical stability. Furthemore, the results showed that gels, which were produced at 100° C temperature, are more viscous, than gels, which were produced at 40 °C temperature. Thus, it is recommended to produce gel by using higher temperature. The further study showed that the interaction between hydroxyethylcellulose and hydrophilic / hydrophobic materials does not affect the stability of gels. It was found that 1 % 90 percent lactic acid ensures complete stability of gel. The study showed that in order to ensure slower sedimentation and better distribution of zinc oxide particles, gel should be mixed 5 minutes 400 r/min.

A method for the selective concentrating of DNA targets using capillary affinity gel electrophoresis is presented. Complementary ssDNA targets are retained through hybridization with oligonucleotide probes immobilized within polyacrylamide gels while non-complementary targets are removed. The captured DNA targets were concentrated by step elution, where a localized thermal zone was applied in small steps along the capillary. Evaluation of the selective capture of a 150 nt DNA target in a complicated mixture was carried out by factorial analysis. Gels with a smaller average pore size were found to retain a higher amount of complementary targets. This was thought to be due to the ssDNA target migrating through the gel by reptation, eliminating hairpin structures, making the complementary region of the target available for hybridization. This method was applied to a series of DNA targets of different lengths, 19 nt, 150 nt, 250 nt and 400 nt. The recovery of the method ranged from 0.5 to 4% for the PCR targets, and 13 to 18% for the 19 nt oligonucleotide target. The purity was calculated to be up to 44% for the PCR targets and up to 86% for the 19 nt target. This was an improvement in purity of up to 15 times and 1100 times in comparison to the original samples for the PCR targets and 19 nt oligonucleotide, respectively. The 19 nt targets were selective concentrated and delivered into a microfluidic based DNA biosensing platform. The purity of the sample improved from 0.01% to 50% while recovery decreased from 100% to 20% for a sample with 0.5 nM complementary and 1 μM non-complementary targets. An improvement in the response of the sensing platform was demonstrated on 19 nt oligonucleotide targets delivered by selective concentration versus concentration alone into the microfluidic biosensing system.

A method for the selective concentrating of DNA targets using capillary affinity gel electrophoresis is presented. Complementary ssDNA targets are retained through hybridization with oligonucleotide probes immobilized within polyacrylamide gels while non-complementary targets are removed. The captured DNA targets were concentrated by step elution, where a localized thermal zone was applied in small steps along the capillary. Evaluation of the selective capture of a 150 nt DNA target in a complicated mixture was carried out by factorial analysis. Gels with a smaller average pore size were found to retain a higher amount of complementary targets. This was thought to be due to the ssDNA target migrating through the gel by reptation, eliminating hairpin structures, making the complementary region of the target available for hybridization. This method was applied to a series of DNA targets of different lengths, 19 nt, 150 nt, 250 nt and 400 nt. The recovery of the method ranged from 0.5 to 4% for the PCR targets, and 13 to 18% for the 19 nt oligonucleotide target. The purity was calculated to be up to 44% for the PCR targets and up to 86% for the 19 nt target. This was an improvement in purity of up to 15 times and 1100 times in comparison to the original samples for the PCR targets and 19 nt oligonucleotide, respectively. The 19 nt targets were selective concentrated and delivered into a microfluidic based DNA biosensing platform. The purity of the sample improved from 0.01% to 50% while recovery decreased from 100% to 20% for a sample with 0.5 nM complementary and 1 μM non-complementary targets. An improvement in the response of the sensing platform was demonstrated on 19 nt oligonucleotide targets delivered by selective concentration versus concentration alone into the microfluidic biosensing system.