Candida albicans is a fungus found in the normal biota of humans, but in immuno-compromised individuals, C. albicans forms complex biofilms on the surface of medical prosthetics, skin, oral cavities, the urinary tract, and other epithelial cell layers. Biofilms and the development of drug resistance has limited treatment options. Antimicrobial peptides (AMPs) are increasingly becoming attractive therapeutic agents for the treatment of these infections due to their multifunctional properties, multiple cellular targets, and the lower incidence of resistance development. Previous studies have shown that Os, an AMP derived from the tick defensin OsDef2, has antifungal activity against C. albicans. Preliminary antifungal mode of action studies indicated that Os induces the formation of reactive oxygen species although not a primary mode of killing. Os causes membrane permeabilization, which is inhibited by an excess of free laminarin and mannan. Furthermore, Os was shown to bind plasmid DNA but was inactive in high salt conditions. The aim of this study was to further explore the mode of action of Os in planktonic C. albicans (ATCC 90028) cells. A modified microbroth dilution assay was developed to allow rapid screening of salt sensitive AMPs such as Os. With this method the IC50 of the positive control, amphotericin B (AmpB), and Os were determined as 0.547 ± 0.056 μM and 1.163 ± 0.116 μM, respectively. The effects of AmpB and Os on cellular morphology were evaluated using scanning electron microscopy and transmission electron microscopy at their respective IC25, IC50 and IC75 values. When comparing the effects of Os with AmpB on the cell wall and membrane, Os had more severe and nonspecific effects. Os induced the formation of pits on the cell surface and pores in the cell membrane, as well as increased budding scars. Using isothermal titration calorimetry, no interaction between Os and the fungal cell wall components, mannan and laminarin, could be detected. Factors such as the lack of tryptophan and aspartate residues as well as β-sheet secondary structures may account for the lack of interaction. However, with the modified microbroth dilution assay in the presence of excess of mannan or laminarin (20 mg/mL), reduced activity from Os was observed. The formation of soluble macro-complexes between Os and the cell wall components at high concentrations may account for reduced activity. The ability of Os to cause membrane depolarization was evaluated with bis-(1,3-dibutylbarbituric acid) trimethine oxonol. The control, melittin, caused a linear increase in depolarization with a significant increase at 0.63 μM, while Os caused a sigmoidal increase in depolarization with a significant increase at 2.5 μM. Therefore, membrane depolarization occurs following membrane permeabilization which occurs at 2 μM. Finally, the localisation of 0.5 μM and 6.4 μM (IC25, IC75) 5-FAM-Os, and concurrently the effect on vacuoles loaded with CellTracker Blue-CMAC, was determined with flow cytometry and confocal laser scanning microscopy (CLSM). Findings were that Os, at a concentration below its IC50, binds to the cell membrane, then translocates and binds DNA. At a concentration above its IC50, Os accumulates in the cytoplasm and causes destruction of membranes, including that of vacuoles, leading to cell death. In conclusion, this study shows that Os is a membrane acting AMP that can be further developed for clinical application as an antifungal drug. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2020 / NRF / Biochemistry / MSc (Biochemistry) / Unrestricted

Chronic Obstructive Pulmonary Disease (COPD), which is characterized by limitation of pulmonary air flow, is now the third major cause of death worldwide. Barrero et al. have reported that the elevation of extracellular hyperacetylated histone H3.3 in the lungs of COPD patients is associated with cytotoxicity and disease progression. They found that extracellular hyperacetylated H3.3 was cytotoxic to lung structural cells and resistant to proteasomal degradation, and that mouse antibodies to either the C- or N- termini of H3.3 could partially reverse H3.3 toxicity in vitro. Thus, we hypothesize that human antibodies directed against H3.3 may be effective biotherapeutics useful to control progression of COPD in vivo. The discovery and development of human monoclonal antibodies (mAbs) is a fast growing field of biotherapeutics. In addition to full length mAbs, antibody fragments also have been used in antibody discovery research. We have used phage display technology in this project to discover human anti-H3.3 antibody Fab fragments. This technology utilizes genetically engineered phage particles containing genes encoding diverse Fab fragments displayed on the particles. The “Ylanthia” library from MorphoSys AG, a synthetic fully human Fab antibody phage display library with 1.3 x 1011 independent clones, was panned against purified recombinant human H3.3 immobilized on 96-well plates. Seven H3.3-binding Fab fragments with unique DNA sequences were isolated after four rounds of panning. Following their expression in E.coli and purification, Fab purities and electrophoretic mobilities were evaluated on SDS-PAGE. The concentration-dependent binding activities of all seven Fabs to human H3.3 were tested by ELISA. All seven Fabs were shown by ELISA to bind H3.3 but not histones 2A, 2B or 4. Since H3.3 is localized to the nucleus, western blotting was used to demonstrate that seven Fabs recognize purified, recombinant H3.3 and denatured natural histone(s) from nuclear extracts of human 293T cells. In order to characterize these molecules further, biological activity assays will be done to test their potential to reverse the toxic effects of H3.3 in cell culture. If these Fabs prove active in cell culture, they will be converted to IgGs and tested in animal models as potential biotherapeutics for COPD. / Pharmaceutical Sciences

Protein aggregation is a frequently cited problem during the development of liquid protein formulations, which is especially problematic since each protein exhibits different aggregation behaviour. Aggregation can be controlled by judicious choice of solution conditions, such as salt and buffer type and concentration, pH, and small molecule additives. However, finding conditions is still a trial and error process. In order to improve formulation development, a fundamental understanding of how excipients impact upon protein aggregation would significantly contribute to the development of stable protein therapeutics. The underlying mechanisms that control effects of excipients on protein behaviour are poorly understood. This dissertation is directed at understanding how excipients alter the conformational and colloidal stability of proteins and the link to aggregation. This knowledge can be used for finding novel ways of either predicting or preventing/inhibiting protein aggregation. Experiments using static and dynamic light scattering, intrinsic fluorescence, turbidity and electrophoretic light scattering were conducted to study the effect of solution conditions such as pH, salt type and concentration on protein aggregation behaviour for three model systems: lysozyme, insulin and a monoclonal antibody. Emphasis is placed on understanding the effects of solution additives on protein-protein interactions and the link to aggregation. This understanding has allowed the rational development of stable formulations with novel additives, such as arginine containing dipeptides and polycations.

<p>Despite improvements in detection and treatment, cancer is the leading cause of death worldwide. Current treatment modalities have not been able to improve the mortality rates and significant toxicities limit efficacy. Therefore there is a need for development of novel therapeutics.</p> <p>Oncolytic viruses have the ability to efficiently replicate in and destroy tumours while leaving normal tissues unharmed. These treatment platforms have been gaining momentum in recent years due to pre-clinical and clinical successes. Oncolytic viruses are extremely safe with limited toxicity observed in phase I/II clinical trials, and objective responses have been observed in some patients treated with oncolytic viruses. However, there is still room to improve on these therapeutic platforms.</p> <p>Recently, the importance of the induction of anti-tumour immunity during oncolytic virotherapy has been realized and harnessing this immune response can be used to improve current oncolytic virus platforms. To this end we have conducted numerous studies assessing our ability to improve oncolytic VSV through the addition of transgenes to enhance the immunostimulatory properties of oncolytic VSV treatment. These studies showed that only the addition of a highly secreted form of human IL-15 was able to improve VSV therapy through enhanced anti-tumour immunity. However, expressing cell-autonomous transgenes from oncolytic VSV was unable to modify the therapeutic efficacy of VSV due to limited replication, both temporally and geographically within the tumour, and the indirect vascular shutdown induced by VSV infection of tumours. We believe that the drastic vascular shutdown observed following VSV therapy is an important component to the success of VSV and we have investigated which steps in this process are critical for induction of anti-tumour immunity.</p> <p>The research presented in this thesis further enforces the requirement for induction of anti-tumour immune responses in the success of OV therapy. Our findings also indicate that manipulating the tumour as a whole, rather than the virus, will lead to improved oncolytic therapeutics.</p> / Doctor of Philosophy (PhD)

Next-Generation protein sequencing (NGPS) creates newfound ways of fully identifying every protein species in a single biological organism. It is an effort to use technology to determine proteomic data. The purpose of this research project is to use the current technology to sequence proteins and potentially find treatments for some diseases that are common today. Through NGPS, scientists can identify low abundant proteins including those that go through post-translational modifications (PTM) [1]. NGPS will allow us to fully determine protein sequences from protein samples using mass spectrometry with the ultimate goal of being able to determine the primary sequence of the protein in the given sample [1]. Antibodies are a specific class of proteins that aid our bodies in the immune response. Due to their variability in the complementary-determining region (CDR), NGPS will be used to determine the heavy and light chain sequences [2]. The goal of this technology is to fully determine the primary sequence of a protein in a given sample. The randomness of an antibody’s variable (V), diversity (D), and joining (J) genes (VDJ recombination) makes each protein unique. VDJ recombination refers to the process of T cells and B cells randomly assembling different gene segments. This process allows the antibody to make specific receptors that can recognize different molecules presented on the surface of antigens. Proteases are enzymes that break down proteins and peptides. By using different proteases with varying cutting rules, we can digest the antibody and run it through high mass accuracy determining instrument [1]. NGPS allows us to utilize mass spectrometry technology to measure proteins or polypeptides. Because of these measurements, post-translation modifications, including glycosylation, can be detected, unlike in DNA sequencing technology. Protein sequencing has the opportunity to play a major role in the fight against the COVID-19 outbreak and serve as curative measures for the treatment and Type 2 Diabetes [3]. Proteomics can serve as the basis of vaccine development as well as monitoring treatment. Utilizing techniques such as mass spectrometry could reveal the structure of the virus and ultimately allow for engineered tissues to produce the protein in large amounts in a lab setting. Currently, many companies are utilizing highly sensitized technology to carry out the goals of NGPS. The Oxford Nanopore is a company that uses technology to develop and explore more ways to undergo protein analysis. The methods used by this company involve using protein nanopores to mutate residues in pores to determine the overall sequence. The company utilizes modified aptamers that are drawn to the nanopore current. These aptamers can bind with some, but not all pores, allowing for the differentiation between target and non-target proteins. Nicoya Life Sciences is another company that uses Open Surface Plasmon Resonance (SPR) to detect molecular interactions. SPR uses an analyte (a mobile molecule) to bind to a ligand and observe changes in the refractive index. SPR allows researchers the ability to characterize the binding kinetics and affinities of monoclonal antibodies. SPR is an extremely promising technique to sequence proteins due to its flexibility in being able to work with a variety of molecules including lipids, nucleic acids, cells, viruses, nanoparticles, proteins, antibodies, carbohydrates, and more. The original goal behind NGPS was to establish a method to sequence proteins to aid in the early detection of common diseases such as Type 2 Diabetes. After significant research, it is now known that NGPS can be done in a variety of ways to accomplish a common goal—sequencing proteins and understanding how amino acids affect the human body. In the case of diseased states, NGPS can help researchers find ways to diagnose, treat, and cure diseases early on. Focusing on antibodies allows us to manipulate the body’s immune response to rid the host of pathogens. NGPS, however, is advancing at a much slower rate than anticipated by companies due to its many limitations including not being able to sequence large peptides, difficulties in material and composition of the sample, and needing to label small peptides to begin degradation. Ideally, finding a way to combine the high accuracy and specificity of certain techniques, the ability to detect low abundant proteins in others, and the flexibility of Open SPR would allow researchers and companies to create the standard for NGPS. Creating effective antibodies is precisely why NGPS has such great potential today. Ultimately, I found that as a standalone, Open SPR is the most effective method. However, as the research shows, there are limitations with each method, including Open SPR. The conclusion shows that it is necessary to find a combination of these techniques and create an accurate method, potentially using different technologies, to establish the most effective way to sequence proteins.

Cell-free protein synthesis is an emerging technology that has many applications. The open nature of this system makes it a compelling technology that can be manipulated to answer many needs that are unavailable in other systems. This dissertation reports on engineering this technology for: 1) sense codon emancipation for incorporation of multiple unnatural amino acids; 2) expressing a hard-to-express anticancer biotherapeutic and introducing a just-add-water system; 3) a biosensing ligand that interacts with nuclear hormone receptors. Emancipating sense codons toward a minimized genetic code is of significant interest to science and engineering. A promising approach to sense codon emancipation is the targeted in vitro removal of native tRNA. Here we introduce a new in-vitro or "cell-free" approach to emancipate sense codons via efficient and affordable degradation of endogenous tRNA using RNase-coated superparamagnetic beads. The presented method removes greater than 99% of tRNA in cell lysates, while preserving cell-free protein synthesis activity. The resulting tRNA-depleted lysate is compatible with in vitro-transcribed synthetic tRNA for the production of peptides and proteins. Biotherapeutics have many promising applications, such as anti-cancer treatments, immune suppression, and vaccines. However, due to their biological nature, some biotherapeutics can be challenging to rapidly express and screen for activity through traditional recombinant methods. In this work, we demonstrate the use of cell-free systems for the expression and direct screening of the difficult-to-express cytotoxic protein onconase. Using cell-free systems, onconase can be rapidly expressed in soluble, active form. Furthermore, the open nature of the reaction environment allows for direct and immediate downstream characterization without the need of purification. Also, we report the ability of a "just-add-water" lyophilized cell-fee system to produce onconase. Here we introduce a Rapid Adaptable Portable In-vitro Detection biosensor platform (RAPID) for detecting ligands that interact with nuclear hormone receptors (NHRs). The biosensor is based on an engineered, allosterically-activated fusion protein, which contains the ligand binding domain from a target NHR. The presented RAPID biosensor platform is significantly faster and less labor intensive than commonly available technologies, making it a promising tool for detecting environmental EDC contamination and screening potential NHR-targeted pharmaceuticals.

<p>Subcutaneous administration has become a common approach for drug delivery of biotherapeutics, such as monoclonal antibodies, which is achieved mainly by absorption through the lymphatic system. This dissertation focuses on the computational modeling of the fluid flow and solute transport in the skin tissue and the quantitative investigation of lymphatic uptake. First, the various mechanisms governing drug transport and lymphatic uptake of biotherapeutics through subcutaneous injection are investigated quantitatively through high-fidelity numerical simulations, including lymphatic drainage, blood perfusion, binding, and metabolism. The tissue is modeled as a homogeneous porous medium using both a single-layered domain and a multi-layered domain, which includes the epidermis, dermis, hypodermis (subcutaneous tissue), and muscle layers. A systematic parameter study is conducted to understand the roles of different properties of the tissue in terms of permeability, porosity, and vascular permeability. The role of binding and metabolism on drug absorption is studied by varying the binding parameters for different macromolecules after coupling the transport equation with a pharmacokinetic equation. The interstitial pressure plays an essential role in regulating the absorption of unbound drug proteins during the injection, while the binding and metabolism of drug molecules reduce the total free drugs. </p> <p>  </p> <p>The lymphatic vessel network is essential to achieve the functions of the lymphatic system. Thus, the drug transport and lymphatic uptake through a three-dimensional hybrid discrete-continuum vessel network in the skin tissue are investigated through high-fidelity numerical simulations. The explicit heterogeneous vessel network is embedded into the continuum model to investigate the role of explicit heterogeneous vessel network in drug transport and absorption. The solute transport across the vessel wall is investigated under various transport conditions. The diffusion of the drug solutes through the explicit vessel wall affects the drug absorption after the injection, while the convection under large interstitial pressure dominates the drug absorption during the injection. The effect of diffusion cannot be captured by the previously developed continuum model. Furthermore, the effects of injection volume and depth on the lymphatic uptake are investigated in a multi-layered domain. The injection volume significantly affects lymphatic uptake through the heterogeneous vessel network, while the injection depth has little influence. At last, the binding and metabolism of drug molecules are studied to bridge the simulation to the experimentally measured drug clearance. </p> <p><br></p> <p>Convective transport of drug solutes in biological tissues is regulated by the interstitial fluid pressure, which plays a crucial role in drug absorption into the lymphatic system through the subcutaneous (SC) injection.  An approximate continuum poroelasticity model is developed to simulate the pressure evolution in the soft porous tissue during an SC injection. This poroelastic model mimics the deformation of the tissue by introducing the time variation of the interstitial fluid pressure. The advantage of this method lies in its computational time efficiency and simplicity, and it can accurately model the relaxation of pressure. The interstitial fluid pressure obtained using the proposed model is validated against both the analytical and the numerical solution of the poroelastic tissue model. The decreasing elasticity elongates the relaxation time of pressure, and the sensitivity of pressure relaxation to elasticity decreases with the hydraulic permeability, while the increasing porosity and permeability due to deformation alleviate the high pressure. </p> <p><br></p> <p>At last, an improved Kedem-Katchalsky model is developed to study solute transport across the lymphatic vessel network, including convection and diffusion in the multi-layered poroelastic tissue with a hybrid discrete-continuum vessel network embedded inside. The effect of different drug solutes with different Stokes radii and different structures of the lymphatic vessel network, such as fractal trees and Voronoi structure, on the lymphatic uptake is investigated. The drug solute with a small size has a larger partition coefficient and diffusivity across the openings of the lymphatic vessel wall, which favors drug absorption. The Voronoi structure is found to be more efficient in lymphatic uptake. </p> <p><br></p> <p>The systematic and quantitative investigation of subcutaneous absorption based on high-fidelity numerical simulations can provide guidance on the optimization of drug delivery systems and is valuable for the translation of bioavailability from the pre-clinical species to humans. We provide a novel approach to studying the diffusion and convection of drug molecules into the lymphatic system by developing the hybrid discrete-continuum vessel network. The study of the solute transport across the discrete lymphatic vessel walls further improves our understanding of lymphatic uptake. The novel and time-efficient computational model for solute transport across the lymphatic vasculature connects the microscopic properties of the lymphatic vessel membrane to macroscopic drug absorption. The comprehensive hybrid vessel network model developed here can be further used to improve our understanding of the diseases caused by the disturbed lymphatic system, such as lymphedema, and provide insights into the treatment of diseases caused by the malfunction of lymphatics.</p>