Indiana University-Purdue University Indianapolis (IUPUI) / The single cell gel electrophoresis assay (comet assay) is a useful tool for monitoring individuals who may be at risk of DNA damage and the ensuing process of carcinogenesis or other disease states. Leukocytes in blood samples provide a means of obtaining cells for use in the comet assay. However instances may arise when samples must be stored for later analysis. The present study investigated the effects of storage conditions on DNA damage in the form of strand breaks and oxidized bases in rat and human leukocytes using the comet assay. Whole blood and buffy coat samples were stored at room temperature or 4ºC for 1, 2, 24, and 48 hours or cryopreserved at -80ºC for 1 day and 1, 2, 3, and 4 weeks. The results show that the time of storage is limited if the whole blood or buffy coat samples are stored at room temperature or 4ºC. However, if cryopreserved using glycerol or DMSO as the cryoprotectant, the samples may be stored for at least 4 weeks without DNA strand breaks or oxidative damage deviating significantly from the fresh samples.

Orientador: Rinaldo Poncio Mendes / Coorientador: James Venturini / Coorientador: Ana Pardini Vicentini / Coorientador: Daniela Vanessa Moris de Oliveira / Banca: Mário León Silva-Vergara / Banca: Anamaria Mello Miranda Peniago / Resumo: O reaparecimento de manifestações clínicas após tratamento eficaz, neste texto identificado como recaída, tem sido pouco avaliado. Assim, foram estudados os casos de recaída observados em 400 pacientes, 93 com a forma aguda/subaguda (FA) e 307 com a crônica (FC), que já tinham apresentado cura aparente, isto é, com cura clínica, normalização da velocidade de hemossedimentação e cura sorológica - caracterizada pela presença de teste negativo à imunodifusão dupla em gel de agar por dois anos. Vinte e um (5,2%) desses pacientes apresentaram recaídas da doença. Três (14,3%) eram do sexo feminino e 18 (85,7%) do masculino, com razão de masculinidade de 6:1. Dos 21 pacientes com recaída, 15 (4,8%) apresentavam a FC e 6 (6,4%) a FA (p>0,05). As reativações ocorreram de 46 a 296 meses após introdução do tratamento (Md=96) e de 4 a 267 meses (Md= 60) após sua suspensão. As formas clínicas não diferiram quanto aos tempos decorridos até a reativação. O diagnóstico sorológico de recaída pela IDD foi feito em apenas 45% dos casos, o que levou à avaliação de outros testes para esse fim. Assim, foi realizado o enzimaimunoensaio (ELISA) e o immunoblotting (IB). A sensibilidade da IDD e do ELISA / 0,710 foi 76,1% em amostras de soro obtidos no pré-tratamento (p=0,25). No diagnóstico de recaída, a sensibilidade da IDD foi menor que no pré-tratamento (80% vs 45%; p=0,017), enquanto o ELISA / 0,710 foi igual (80% vs 65%;p=0,125). A sensibilidade do IB para diagnóstico de recaída foi de 12,5% na identificação da gp70 e 43,8% na da gp43 (p<0,05). A avaliação da acurácia do teste ELISA revelou sensibilidade de 96%, especificidade de 95%, valor preditivo positivo de 95%, valor preditivo negativo de 96% e acurácia de 95,5% quando o cut-off utilizado foi a densidade óptica de 0,710, obtido em função da construção da receiver operator characteristc - ROC, para um intervalo de confiança ... / Abstract: The reappearance of clinical manifestations after efficacious treatment, here identified as relapse, has been rarely evaluated. Thus, the cases of relapse observed in a cohort of 400 patients, 93 with acute/subacute form (AF) and 307 with chronic form (CF) were studied. They had already reached apparent cure, characterized as clinical cure, normalization of the erythrocyte sedimentation rate and serological cure, with a negative double agar gel immunodiffusion test (DID) for two years after antifungal discontinuation. Twenty-one (5.2%) of these patients had relapses. Three (14.3%) were female and 18 (85.7%) were male, with a male:female ratio of 6:1. Of the 21 relapsed patients, 15 (4.8%) presented the CF and 6 (6.4%) the AF (p>0.05). The relapse occurred 46-296 months after introduction of the treatment (Md=8 yrs) and from 4 to 267 months (Md=5 yrs) after withdrawal. Clinical forms did not differ regarding to the time elapsed until relapse. DID was positive in only 45% of the relapsed cases, which led to the evaluation of other tests to diagnose this condition. Thus, the enzyme-linked immunosorbent assay (ELISA) was standardized and the cut off was determined using the curve receiver operator characteristic - ROC and confidence intervals of 95% and 99%, showing optical densities of 0.710 and 0.850, respectively. Then, serological evaluation was performed using ELISA/0.710 and ELISA/0.850, and immunoblotting identifying gp43 (IBgp43) and gp70 (IBgp70). ELISA 0.710 and DID showed the same sensitivity, 76.1%, in serum samples obtained before treatment (p=0.25). DID sensitivity was lower at relapse than before the initial treatment (45% vs 80%; p=0.017), whereas ELISA/0,710 was the same (65% vs 80%; p=0.125). IBgp70 showed a 12.5% and IBgp43 a 43.8% sensitivity for diagnosing relapse (p<0.05). ELISA/0.710 showed a 96% sensitivity, 95% specificity, 95% positive predictive value, 96% negative ... / Mestre

Submitted in partial fulfillment of the requirements for the degree of Doctor In Technology (Clinical Technology), Durban University of Technology, Durban, South Africa, 2016. / Erythropoietin (EPO) is widely used in patients with chronic renal failure and is a necessity. However, due to the cost implications and the medical complications in our population it is imperative to review the factors affecting the process of erythropoiesis and the analysis of cell proliferation and cell viability in the bioassay. Complications such as hypertension and risk of worsening a malignancy cannot be ignored. We had previously analysed variations of erythropoietin levels in haemodialysis patients over a six month period. This study aims to evaluate erythropoiesis in conjunction with various laboratory, demographic, clinical parameters and inflammatory markers, in the population of haemodialysis patients. EPO, antibody level and antibody activity were analysed in the population groups as EPO responsive and EPO sensitive patients. This is a prospective, experimental and controlled study. Fifty nine patients were randomly selected from haemodialysis units of Addington and King Edward VIII Hospitals following an informed consent and 15 healthy individuals were also selected as controls. Demographic parameters (age, sex), clinical parameters (weight, height, skin folding, EPO doses and blood pressures (BP) were recorded. Pre-dialysis serum was used to measure laboratory markers (haemoglobin, transferrin, ferritin, albumin, ESR, C reactive protein, creatinine and urea). EPO levels and antibody levels were measured by ELISA, the optical density of each well was determined within fifteen minutes using the microplate reader set at 450 nm. All results were statistically analysed using SPSS statistical package version 21 (IBMR). Patients requiring very high doses of EPO to reach Hb of 11g/dL, and they remained anaemic after at least three months of adequate EPO doses were considered to be EPO resistant. Those who responded to the usual EPO doses were labelled EPO sensitive. The bioassay was used to quantify cell proliferation and cell viability in the presence of EPO. The UT 7 cells were cultured in medium, in the presence of serum from the EPO resistant, EPO sensitive patients and the healthy, control subjects. Luminescence was read with the Glorunner Microplate Luminometer and was recorded in relative light units (RLU). The analysis revealed: a non-significant positive correlation between haemoglobin and erythropoietin levels. However, a strong negative correlation was found between CRP and albumin level (R= -0.591; (p=0.001), which was not significant. No correlation was found between haemoglobin or erythropoietin levels and CRP or albumin. There was a positive correlation with systolic and diastolic blood pressures and mean arterial pressures which was statistically significant (p <0.05). EPO dosages and Hb levels were correlated significantly (p < 0.05). No correlation of EPO levels and Hb; age and Hb was found to be significant (p = 0.08). The UT 7 cells cultured in serum in medium alone with RHuEPO containing cells were statistically significant (p <0.01)). Reduction of ATP stimulation between medium and serum was observed. However, mean arterial pressures had a significant association with EPO resistance (p = 0.041) odd ratio- 1.066. In conclusion, EPO level is not a useful tool for the monitoring of its use as it does not correlate with EPO goal of red blood production in our patients. The neutralizing antibodies did not correlate with any of our variables contributing to erythropoiesis, and are therefore not confirmed as playing a major role in erythropoiesis. From the analysis of our results the key contributing factors of EPO doses, malnutrition and age were more significant in erythropoiesis. However the higher doses of EPO significantly increased the blood pressures and the mean arterial pressures (MAP). The analysis of the bioassay showed lack of difference between EPO responsive and EPO sensitive patients. This observation warrants further studies to clarify the role of serum of haemodialysis patients in erythropoiesis. / D

<p>More than 50% of wastewater biosolids are applied to agricultural fields as fertilizer in the U.S. This technique has been used for decades as a widely accepted beneficial reclamation method for biosolids, which meet the established regulatory levels for nutrients, metals, and pathogens. A major drawback to land application is the potential environmental release of non-regulated organic contaminants, which accumulate in biosolids during the wastewater treatment process. Recent studies have been performed to identify and quantify the presence of emerging contaminants in biosolids, and others have investigated the effects of compounds already identified as `priority pollutants' and whose use is waning. However, there is limited research on the effect of emerging organic contaminants on soil microbial ecology and nutrient cycling. Because many of the compounds found in biosolids are specifically designed to elicit biological modifications (e.g., antimicrobials), there is a risk that these compounds will disrupt microbial soil functions, decrease soil productivity, and ultimately affect the long term viability of these ecosystems, resulting in unforeseen economic and social costs. Therefore, there is a clear need to characterize the effects of novel contaminants on soil health.</p><p>This dissertation was divided into three distinct parts examining the impacts of emerging organic contaminants on soil microbial ecology with increasing complexity to better reflect environmental conditions. To assess the ecological impacts, the functional endpoint of denitrification was selected because it provides a vital indication of soil health. Denitrifying bacteria play a critical role in this process, and thus, were used as indicator organisms for determining contaminant ecotoxicological potential. Furthermore, antimicrobial agents (a.k.a., bactericides or biocides) were selected as model contaminants because they are designed specifically to deactivate microorganisms, are heavily used in the U.S with over $1 billion in yearly sales, and have been measured in biosolids.</p><p>Overall, the objectives of this dissertation were to: 1) develop a rapid, high-throughput functional assay that measured denitrification inhibition for screening potential ecological impacts of biosolids-derived antimicrobial agents, 2) determine the potential effects of common and emerging biosolids-derived antimicrobial agents on denitrification by a model soil denitrifier, Paracoccus denitrificans PD1222, 3) examine the impacts of the most commonly used antimicrobial, triclosan (TCS), on wastewater treatment efficiency in bench scale sequencing batch reactors (SBRs) coupled with anaerobic digesters, 4) examine the impacts of biosolids aged and spiked with TCS on denitrification under simulated agricultural soil conditions, and 5) evaluate potential impacts of TCS in `traditional' biosolids on denitrification in agricultural soil under field conditions.</p><p>The first phase of research pertaining to Objectives 1 and 2 examined the baseline interactions between biosolids-derived antimicrobial agents and soil microbial ecology. However, to isolate the effect of an individual contaminant from the myriad of contaminants found in biosolids, there was a need for developing a rapid, high-throughput method to evaluate general ecotoxicity. In the first part of this dissertation, we developed a novel assay that measured denitrification inhibition in a model soil denitrifier, Paracoccus denitrificans Pd1222. Two common (TCS and triclocarban) and four emerging (2,4,5 trichlorophenol, 2-benzyl-4-chlorophenol, 2-chloro-4-phenylphenol, and bis(5-chloro-2-hydroxyphenyl)methane) antimicrobial agents found in biosolids were analyzed as model contaminants. Overall, the assay was reproducible and measured impacts on denitrification over three orders of magnitude exposure. The lowest observable adverse effect concentrations (LOAECs) were 1.04 &mu;M for TCS, 3.17 &mu;M for triclocarban, 0.372 &mu;M for bis-(5-chloro-2-hydroxyphenyl)methane, 4.89 &mu;M for 2-chloro-4-phenyl phenol, 45.7 &mu;M for 2-benzyl-4-chorophenol, and 50.6 &mu;M for 2,4,5-trichlorophenol. Compared with gene expression and cell viability based methods, the denitrification assay was more sensitive and resulted in lower LOAECs. Of the six compounds examined, four resulted in LOAECs that were below or within an order of magnitude of concentrations that were measured in the environment, indicating potential ecological impacts.</p><p>In the second part of the dissertation, the impacts of emerging contaminants were examined first under laboratory conditions mimicking wastewater treatment processes (Objective 3) and then agricultural fields (Objective 4). For this phase, TCS, which is the most widely used antimicrobial agent and identified in the first phase for potential ecological impacts, was used as the model contaminant. To mimic wastewater treatment processes, bench scale SBRs coupled with anaerobic digesters were set up and operated. The SBRS and digesters were seeded with activated and anaerobically digested sludge from the North Durham Water Reclamation Facility (NDWRF, Durham, NC). Reactors were fed synthetic wastewater with or without 0.73 &muM of TCS. Samples were taken periodically to monitor chemical oxygen demand (COD), ammonium (NH₄<super>+</super>), nitrate (NO₃<super>-</super>), nitrite (NO<sub>2</super>-</super>), total suspended solids (TSS), volatile suspended solids (VSS), dissolved oxygen (DO), and phosphate (PO₄<super>3-</super>) and pH. In addition, biomass samples were collected for DNA extraction and microbial community analysis using terminal restriction fragment length polymorphism (T-RFLP) of 16S SSU rDNA. Methane production was also monitored for the anaerobic digesters. In addition, the final digested biosolids that were generated from the SBRs fed with and without TCS were analyzed for TCS concentration, TSS, VSS, TKN, phosphorus (as P₂O₅), potassium (as K₂O), and pH. Overall, biological processes associated with nitrogen removal (nitrification and denitrification), were impacted by TCS entering the SBRs regardless of the starting microbial community. Both of the SBRs that were not receiving TCS reached steady-state at greater than 92% NH₄<super>+</super>, removal within the first week of operation, whereas the SBRs receiving TCS took 42 and 63 days to reach steady-state removal at that level. However, while NH₄<super>+</super> removal was temporarily inhibited, elevated levels of NO₃<super>-</super> and NO₂<super>-</super> in the effluent of the TCS fed SBRs, suggested longer-term impacts on nitrite oxidizing bacteria (NOB) and denitrifiers. After Day 58, the NO₃<super>-</super> effluent concentration for the SBRs receiving TCS was 3.9 ± 0.16 mg/L, which was 2.4 times greater than the NO₃<super>-</super> effluent of the SBRs not receiving TCS (1.7 ± 0.08 mg/L). Similarly, after Day 58, the NO₂<super>-</super> effluent of the SBRs receiving TCS reached a steady-state concentration of 8.7 ± 0.75 mg/L. The mean NO₂<super>-</super> concentration in the controls after Day 58 was 7.7 times lower at 1.1 ± 0.78 mg/L, but was still trending towards 0 when the reactors were stopped. No inhibition was observed for COD and PO₄<super>3-</super> removal. In addition, non-metric multidimensional scaling (NMS) ordination analysis showed that the microbial communities between SBRS fed with and without TCS were similar on Day 0, but increased in difference to Day 41, around when the major changes in nitrification were observed. After a slight increase in similarity between the control and TCS SBR microbial communities on Day 41, the communities increased in difference to Day 63.</p><p>To mimic agricultural field conditions, containers of soil were amended with the biosolids generated from the SBRs. The containers were maintained in a growth-chamber to simulate field lighting and watering conditions. Three biosolids treatments were examined: 1) biosolids generated from the SBRs not fed TCS, but that still had low backgrounds of TCS (a.k.a., Control Biosolids); 2) biosolids generated from the SBRs fed with TCS (a.k.a., Aged TCS Biosolids); and 3) biosolids that were generated by the SBRs not fed TCS, but spiked with TCS 24 h before application (a.k.a., Spiked TCS Biosolids). Alfalfa was planted in half of the containers receiving the Control and Aged TCS Biosolids to assess differences due to vegetation. To assess the overall ecotoxicity of biosolids aged and spiked with TCS, the function, abundance, and diversity of the soil denitrifying communities were examined. The impacts on total bacteria abundance and diversity were also examined for comparison. Specifically, the denitrifying enzyme activity (DEA) assay was used to measure functional impacts, quantitative polymerase chain reaction (qPCR) was used to measure impacts on abundance, and T-RFLP was used to measure impacts on diversity. Correlations between these methods were also examined for possible interactions between denitrifier function and community structure and to provide insight into targets of inhibition. Lastly, a denitrification inhibition score was developed to quantify global impacts of TCS on denitrification. The containers with plants that received biosolids aged with and spiked with TCS showed potential long-term inhibition based on measurement of soil denitrification at 26.9 ± 4.6 &mu;g/kg and 68.6 ± 26.9 &mu;g/kg of TCS, respectively. Denitrifier abundance and diversity, however, were more sensitive to TCS in biosolids and inhibition was observed throughout the experiment, with maximum inhibition on Days 7 and 28. Inhibition of denitrifier abundance and diversity was observed at TCS concentrations as low as 17.9 ± 1.93 &mu;g/L, which was about 10 to 3000 times lower than concentrations reported by other studies that showed impacts on other functional endpoints (i.e., respiration, phosphatase activity, NO₃<super>-</super> and NO₂<super>-</super> production, and Cy17 stress biomarker abundance), even after taking pH into account. Five significant correlations were developed, three of which related qPCR and the DEA assay, or abundance and activity. However, the analyses that were correlated did not yield the same results as far as significant inhibition in the presence of TCS. Thus, while the results suggested some relatedness between activity, abundance, and diversity, the results generally support the use of multiple methods to determine the ecotoxicity of biosolids-derived organic contaminants. As a result, a denitrification inhibition score was developed that took into account all three methods to determine the overall ecotoxicity of TCS in biosolids. Overall, the denitrification inhibition score showed that denitrification was inhibited by both biosolids that were aged and spiked with TCS over the extent of the 84 day experiment, but maximum inhibition occurred after a week to about a month. While the denitrification inhibition score indicated that the TCS in the biosolids aged with TCS was less bioavailable than in the spiked biosolids, the impacts of the aged and spiked biosolids could have also been due to differences in TCS concentrations.</p><p>Objective 5 consisted of a long-term soil sampling campaign on four agricultural fields receiving Class B municipal biosolids. Soil samples were taken before and after biosolids application and were analyzed to elucidate potential impacts of TCS in the biosolids on denitrification. Again, to assess the overall impacts of TCS on the soil denitrifying community, the DEA assay, qPCR, and T-RFLP were used to measure impacts on function, abundance, and diversity, respectively. Similar to Objective 4, the analysis included an examination of potential correlations between denitrifying community structure and function, and quantification of global impacts using the denitrification inhibition score. As expected, the results in this pilot-study reflected the complexity of the system that was analyzed and many more samples, which account for variables including, but not limited to soil characteristics, biosolids characteristics, biosolids application rates, and chemical composition and quantities, would be needed to show any statistically significant differences. Nevertheless, several key results were obtained. Again potential long-term inhibition of denitrification was observed using the DEA assay, however the effects of exhaustion of resources, such as NO₃<super>-</super>, or significant changes in the local environment were suspected, but could not be verified. Inhibition was also observed for denitrifier abundance, but little to no inhibition was observed when examining the relative number of denitrifying species. Thus, while the abundance of denitrifiers was reduced, and denitrification was eventually depressed, the number of species in the soil remained constant. When looking at the denitrification inhibition score, which took all three measurements into account, increased inhibition over time was observed with the exception of the measurements on Days 30 and 103, which indicated overall, but weak inhibition of denitrification by the application of biosolids. NMS ordinations showed no correlation between the shift in denitrifying microbial community and TCS. Because of the complexity of the soil and biosolids and because of the myriad of contaminants likely in the biosolids, the results may not be significant and a more in-depth study was recommended.</p><p>Overall, the results presented in this dissertation provide a systematic evaluation of the effects of biosolids-derived TCS on agricultural soil microbial ecology. First, it was demonstrated that statistically significant inhibition of denitrification could be used as a potential indicator of biosolids-derived emerging organic contaminant ecotoxicity. The denitrification assay that was developed was then used to analyze ecotoxicological potential of six emerging biosolids-derived antimicrobial agents, and found inhibition of denitrification at environmentally relevant concentrations. The most widely used antimicrobial agent, TCS, was further shown to inhibit wastewater treatment processes, as well as, denitrification in simulated agricultural conditions after being aged with and spiked into biosolids. In addition, evidence showing potential inhibition of denitrification by TCS in `traditional' biosolids under field conditions was also obtained. Based on these results, this dissertation asserts that biosolids-derived emerging organic contaminants pose a potential risk to agricultural soil microbial ecology and overall soil health. Future studies, however, are needed to examine the impacts of other contaminants that might be flagged with the assay developed in this dissertation under more complex conditions mimicking the environment. Furthermore, other research is needed to examine the role microbial communities play in the bioavailability of emerging contaminants, especially TCS, and a more extensive, in-depth study is needed to characterize the individual impacts of emerging contaminants on soil microbial communities under field conditions.</p> / Dissertation

A genetically determined high level of intrinsic normal tissue radiosensitivity may account for the 5% of patients who experience unexpectedly severe normal tissue side effects following radiotherapy. The pre-treatment identification of these individuals by a diagnostic test or “predictive assay “ may allow appropriate modification of treatment plans and improve the therapeutic index of radiotherapy. Results from studies of cell-based assays measuring the response of a single cell type taken from patients to in vitro irradiation have been inconsistent, leading to the opinion of many that they are of no value in the prediction of normal tissue radiosensitivity. A systematic review of the literature presented here, however, suggests that poor methodology of study design often with inadequate control for those factors other than normal tissue radiosensitivity which influence radiotherapy toxicity and lack of reporting of assay precision means that it is difficult to form any conclusions, positive or negative about the diagnostic accuracy of the cell-based assays studied so far. Analysis of individual patient data extracted from these studies suggests that at least some of these assays may possess some discriminatory value. This finding justified an attempt to develop a novel cell-based assay based on the kinetics of radiation-induced .H2AX in peripheral blood lymphocytes. Assay failure rate was high and intra- and inter-sample assay reproducibility was poor for quantification by microscopy but were better for flow cytometric analysis. A study of 8 volunteers, however, demonstrated that intra-individual variation was higher than inter-individual variation in assay results, strongly suggesting that poor assay reproducibility due to technical or biological factors may limit the assay’s potential to identify radiosensitive individuals. This suspicion needs to be confirmed in a clinical study of patients of known radiosensitivity. As blood sample storage conditions affect assay results these will need to be standardized to prevent confounding of results.

published_or_final_version / abstract / Microbiology / Doctoral / Doctor of Philosophy

Polyadenylation is an essential post-transcriptional modification resulting in a mature mRNA in eukaryotes. Three cis-elements the Far Upstream Element (FUE), Near Upstream Element (NUE), and Cleavage Site (CS) - guide the process of cleavage and polyadenylation with the help of multi-subunit protein complexes cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF) along with cleavage factors and poly(A) polymerase. Protein-protein interactions play an important role in the cleavage and polyadenylation process. WD repeat proteins play an important role in protein-protein interactions and have diverse functions in plant system. In the present study WD repeat proteins AtCstF50 and AtFY were studied for their role in polyadenylation process. Mammalian CstF50 is a WD repeat protein that is one of the subunit of CstF that aids in the cleavage step by associating with CPSF and cleavage factors. AtCstF50 was functionally characterized using T-DNA knock-out lines and by identifying the proteins that interacts with it in the process. Results shows that AtCstF50 is essential and was identified as part of CPSF complex, which is different from its mammalian counter part. CPSF was known to interact with Fip (factor interacting with PAP), Poly(A) polymerase and Poly(A) binding protein and AtCstF50 also interacts with these complexes. AtFY is a 3’ end processing factor which contains WD repeats is one of the subunits of the CPSF complex in Arabidopsis polyadenylation machinery. The AtFY interacts with FCA and promotes the alternative polyadenylation and also plays a role in polyadenylation site choice of FCA mRNA. We characterized the FY expression and localization of FY in the cell by fusing with RFP reporter. Results show that FY accumulates in the nucleus while FY with deleted calmodulin binding domain localizes both to the nucleus and outside the nucleus. The individual N-terminal and C-terminal domains also localized in the nucleus suggesting that they are multiple nuclear localization signals in FY and calmodulin might play a direct or indirect role in FY localization. Using a tethering assay we proved that AtFY is able to recruit the 3’ end processing complex in the proximal polyadenylation site choice of the reporter mRNA.

The shikimate pathway is responsible for the biosynthesis of essential aromatic metabolites and, as such, its enzymes are targets for the design of potential antimicrobial and herbicidal agents. The enzyme 3-dehydroquinate dehydratase (dehydroquinase, DHQase) catalyses the conversion of 3-dehydroquinate to 3-dehydroshikimate, the third step of the shikimate pathway. There are two types of DHQase, unrelated structurally and mechanistically. Type I DHQase catalyses the rection by via a covalently attached imine intermediate. Type II DHQase catalyses the reaction by way of an enolate intermediate. This thesis describes the synthesis of a series of potential inhibitors of type II DHQase. Inhibitors with C and N at C-3 and with both sp2 and sp3 character at this position were prepared. A potential type I DHQase inhibitor was also prepared. The biological evaluation of these inhibitors against type II DHQases from Mycobacterium tuberculosis and Streptomyces coelicolor and type I DHQase from Salmonella typhi is described. Inhibitors were evaluated by spectrophotometric assay. However, this proved inappropriate for some inhibitors with the S. coelicolor enzyme. The development of an alternative 1H NMR assay and its application to the evaluation of S. coelicolor DHQase inhibitors is therefore also described. Some insights into the structure activity relationships of type II DHQases, obtained from the results of these studies, are discussed.

No description available.

The colony formation assay (CFA) is the gold standard for measuring cytotoxic effects on cells. To increase efficiency, the CFA was converted to a 96-well format using an automated colony counting algorithm. The 96-well CFA was validated using ionizing radiation (IR) on the FaDu and A549 cancer cell lines. Its ability to evaluate combination therapies was investigated using cisplatin and IR. The 96-well CFA was transferred to a robotic platform for evaluation as a high-throughput screen (HTS) readout for the discovery of novel anti-cancer compounds, and radiosensitizers. Screening yielded eight putative anti-cancer hits, and five putative radiosensitizing hits. Secondary screening confirmed 6/8 anti-cancer compounds, and 0/5 radiosensitizing compounds. Thus, the 96-well CFA can be adopted as an alternative assay to the 6-well CFA in the evaluation of cytotoxicity in vitro, providing a possible readout to be utilized in HTS for discovering anti-cancer compounds, but with limited applicability in discovering radiosensitizers.