Spontaneously active primary cultures obtained from dissociated embryonic medial medulla tissue were grown on microelectrode arrays for investigating burst patterns and pharmacological responses of respiratory-related neurons. Multichannel burst rates and spike production were used as primary variables for analysis. Pacemaker-like neurons were identified by continued spiking under low Ca++/high Mg++conditions. The number of pacemakers increased with time under synaptic blocking medium. Sensitivity to CO2 levels was found in some neurons. Acetylcholine changed activity in a complex fashion. Curare, atropine and gallamine modified ACh effects. Eserine alone was ineffective, but potentiated ACh-induced responses. Norepinephrine caused channel-specific increases or decreases, whereas dopamine and serotonin had little effect at 30 μM. GABA and glycine stopped most spiking at 70 μM. Developmental changes in glycine sensitivity (increasing with age) were also observed. It is concluded that pacemaker and chemosensitive neurons develop in medial medulla cultures, and that these cultures are pharmacologically histiotypic.

Repair of DNA damage is one of the most important processes undergone in a dividing cell. This is a two-part study undertaken to better understand some of the proteins involved in the sensing and repair of DNA damage in Drosophila melanogaster. The first portion of this experiment followed two Drosophila Rad51 paralogs, dmRad51D and dmXRRC2, and using constructs tagged with GFP, found that they entered the nucleus in response to drug induced DNA damage. Approximately one hour after the induction of DNA damage via bleomycin, dmRad51D and dmXRCC2 entered the nucleus of the Drosophila culture cells, where they remained for the next three to four hours. Following this period in the nucleus, the cells were visualized moving back into the cytosol. The second portion of this experiment was concerned with the four Drosophila Rad51 paralogs (dmRad51 D, dmXRCC2, Spn B, and Spn D) and two paralogs from Homo sapiens (hsRad51 D and dmRad51 D) and their interactions.

No description available.

No description available.

No description available.

Although the differential response of malignant cells to hyperthermia was observed at the end of the last century, few investigations into the phenomenon were made until fairly recently. At first the results obtained were inconsistent due mostly to the inefficient techniques in use at the time. The advent of cell and tissue culture, increased knowledge in tumor biology, advances in molecular biology, and the development of equipment which could raise and monitor the temperature of tumors during hyperthermic treatments, have made the study of the effects of supranormal temperature on malignant neoplasias substantially more reliable. The effectiveness of the combined treatment of hyperthermia and drugs has been established. A substantial amount of research to ascertain the most efficient uses of 2 combined treatments (or for finding or screening new drugs) remains to be done.

Despite rapid advances in clinical oncology, acquired drug resistance still poses a significant threat to the long-term efficacy of current treatment regimens. Because most chemotherapy drugs aim to activate apoptosis in cancer cells, expansion of the pharmacopeia to include treatments targeting novel tumor cell death mechanisms is a promising anti-cancer strategy. Induction of ferroptosis, an iron-dependent form of regulated cell death, shows particular therapeutic potential as aggressive metastatic and drug-resistant cancer cell states have been demonstrated to possess an exquisite dependency on glutathione peroxidase 4 (GPX4), a key suppressor of the ferroptotic cell death pathway. However, current GPX4 inhibitors are limited by poor pharmacokinetic properties that preclude their clinical use. The development of novel drug-like GPX4 inhibitors would benefit from the discovery of new chemical scaffolds to both enhance our understanding of the structural basis of small molecule binding and inhibition as well as facilitate the rational design of future GPX4-targeted therapeutics. In this dissertation, we employed three high-throughput screening strategies to identify novel scaffolds of interest for GPX4 inhibitor development. First, a Lead-Optimized Compound (LOC) library was screened and we conducted further characterization and structure-activity relationship (SAR) studies on hit compound LOC880. Compared to the original hit, analogs QW-095 and QW-105 showed improved binding affinity and GPX4 inhibitory activity in vitro and also induced lipid peroxidation in cells suggestive of ferroptotic death. Further enhancement of the potency and ferroptosis specificity of this scaffold is still needed, but the potentially noncovalent and allosteric mechanism of action presents a novel approach for targeting GPX4. Second, we conducted extensive SAR analyses on another promising hit from the LOC library screen, LOC1886, which led to the identification of the lead compound QW-314. This analog showed significantly improved potency and ferroptosis specificity in multiple cancer cell contexts, including a drug-tolerant persister cell model of minimal residual disease. Characteristic markers of GPX4 inhibition and ferroptosis are also observed in cells treated with QW-314, including GPX4 protein degradation and induction of lipid peroxidation, and QW-314 exhibited excellent selectivity for GPX4 over another glutathione peroxidase family selenoprotein GPX1 in an in vitro assay using cell lysates. Moreover, we determined a baseline of pharmacokinetic measures including aqueous solubility and metabolic stability in human and mouse liver microsomes for further medicinal chemistry optimization. Lastly, we screened a DNA-encoded library (DEL) and an Enamine Diversity library, identifying 10 additional chemical starting points for future investigation.

Mounting evidence implicates persister cancer cells as the key element of minimal residual disease (MRD) from which cancer relapse occurs. The observation that persister cells are differentially and specifically sensitive to ferroptosis, a unique form of metabolically-linked cell death, presents a critical weak point through which identification and targeting of persister cells in MRD may become possible. To identify biomarkers for targetable cells, the drivers of ferroptosis sensitivity in persister cells must be identified. Using three chemotherapeutics and cell lines, we derived persister models across diverse tissues of origin and found that: 1) activating transcription factor 4 (ATF4), previously demonstrated as central to lung cancer persister state formation, is differentially expressed in prostate and fibrosarcoma persisters vs parentals, 2) proteins key to ferroptosis are underexpressed in persisters, and revert expression upon persister to parental reversion, 3) the lung persister lipidome is significantly rewired to drive ferroptosis and 4) upon persister to parental reversion and re-acquisition of ferroptosis resistance, the lipid signature also reverts back to a parental-like state, and 5) although ATF4 elimination in persisters does not revert ferroptosis sensitivity, mitochondrial elimination in persisters does abrogate ferroptotic sensitivity. Collectively, these findings reveal the mechanism of persister ferroptosis sensitivity across multiple cancer types, opening up the possibility of leveraging ferroptosis for elimination of minimal residual disease.

Evidence from various studies indicates that eccentric contractions produce more post-exercise changes in muscular function than do concentric contractions. Delayed muscular soreness, the pain and tenderness present 1 or 2 days after exercise, is negatively correlated with muscular performance and occurs particularly after eccentric work. The action of an analgesic, anti-inflammatory drug (Indocin) on muscular soreness indicates it may be effective in accelerating recovery of muscle function after eccentric work. In the study reported herein the effects of Indocin on muscular performance, as evaluated on the Cybex II isokinetic dynamometer, following prolonged concentric and eccentric work, were evaluated in 48 subjects who were randomly assigned to one of four drug groups. Subjects performed a 30 minute step test during which one limb led the stepping movement throughout (concentric contractions) and the contralateral limb trailed throughout (eccentric contractions). The muscular performance parameters of peak torque (PT), torque acceleration energy (TAE) and average power (AVP), evaluated at slow and high velocities, and the range of motion (ROM) at the knee joint were assessed prior to the step test and at five intervals thereafter. A non-significant decrease in PT and TAE at the contraction speed of 60 deg/sec were present in the eccentric limbs, greater reductions evidenced in the placebo group. Non-significant changes occurred in the concentric limbs, Non-significant changes in ROM and in muscular function parameters evaluated at 250 deg/sec were observed. / Master of Science

Submitted in complete fulfillment for the Degree of Doctor of Philosophy in Biotechnology, Durban University of Technology, Durban, South Africa, 2016. / Colon cancer is the third most common cancer worldwide with a high morbidity and mortality rate. Therapies are less effective during metastasis, therefore prevention and earlier detection is key to reducing the risk of colon cancer. Increased dietary fibre and probiotic intake is known to lower the risk of colon cancer. Probiotics are defined as “live microorganisms which when administered orally in an adequate amount confer a health benefit on the host”. The International Dairy Federation recommends a viable minimum level of 6–7 log10cfu/g in a probiotic product being consumed. Different biopolymer matrices have been used for encapsulation of probiotics; however, loss of viability is still a major challenge. Citrus pectin is a dietary fibre polysaccharide broken down into smaller fragments to form modified citrus pectin (MCP). The unique bioactivity of MCP against carcinogenesisis is linked to its sugar β-galactose inhibiting the cell signalling protein marker, galectin-3 (gal-3), which is intimately involved in endothelial cell morphogenesis. The vascular endothelial growth factor (VEGF) signalling, which invariably drives angiogenesis can be activated when gal-3 binds to integrins. The bioactivity and uptake of MCP may be improved through a novel approach if conjoined with a supplement for example probiotic. Therefore, the synergistic inhibitory effect of modified citrus pectin alginate (MCPA) probiotic microbeads on gal-3 and VEGF in an azoxymethane (AOM) induced colon carcinogenesis Balb/c mouse model was investigated. A microencapsulation process was used to produce a MCPA microbead containing probiotic, Lactobacillus acidophilus ATCC 4356. Efficiency of the microbead was evaluated in vitro (simulated conditions) and in vivo (Balb/c mouse model). Genomic identification of faecal lactobacilli samples from the treated mice was analyzed. Optimization of AOM dose-time with 10 and 15 mg/kg AOM intraperitoneal (ip) administered to Balb/c mice for 2 and 4 weeks were performed. The optimal AOM dose was initiated prior to intake of MCPA, AP (alginate calcium) probiotic microbeads and MCP in Balb/c mice for 16 weeks; samples were analyzed for colon histopathology and immunohistochemistry. The MCPA probiotic microbeads significantly enhanced the viability of L. acidophilus ATCC 4356 compared to the AP microbeads in vitro (p< 0.05). Exposure of the MCPA probiotic microbeads to 3 h of simulated gastric juice (SGJ) resulted in 82.7% survival of L. acidophilus ATCC 4356. Also, the faecal lactobacilli count in the MCPA probiotic treated mice significantly increased after 28 days by 10.2% compared to the AP probiotic, MCP treated and control mice (p< 0.0001). A total of 4DNA encoding 16S rRNA gene closest to the genera namely Lactobacillus, Bacillus, Enterococcus and Bifidobacterium were identified from faecal samples of the colon cancer-induced Balb/c mice. Azoxymethane at 15 mg/kg for 4 weeks induced optimal gal-3 and VEGF immunoexpression. Furthermore, MCPA probiotic treatment significantly reduced gal-3 immunoexpression in the colon of AOM induced cancer Balb/c mice compared to the control mice (p< 0.0001). The immunoexpresion of VEGF in the MCPA and AP probiotic treated groups was weakly positive and significantly reduced when compared to the control group (p<0.05), while the MCP treated group was barely positive (p< 0.001). Modified citrus pectin alginate is a novel effective means of oral delivery of bacterial cells and bioactive compounds. It has a good biodegradability, inexpensive, non-toxic, proven efficiency, and stability at low temperatures warranting its use as a drug carrier by pharmaceuticals. Modified citrus pectin alginate probiotic microbeads increase bioactivity and chemoprevention against colon pre-cancerous lesions and adenocarcinoma through inhibition of gal-3 and VEGF in the mouse model. Modified citrus pectin alginate can be used in probiotic therapy, which may improve the prevention of colon cancer. / D