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.

No description available.

No description available.

Chlamydia is a clinically significant organism that exhibits a unique stage-specific developmental cycle, involving the interconversion between two metabolically distinct forms. The completion of eight chlamydial genome sequences identified three different RNA polymerase sigma factor (ó) genes. Temporal gene expression analysis has predicted that each ó may play an integral role in controlling the development cycle. This thesis examines the role of the chlamydial alternate sigma factor, ó54 (rpoN) and the potential mechanism for the control the developmental cycle and disease pathogenesis. To achieve this, we searched the genome for putative ó54 promoters, validated the findings by DNA-binding assays, and examined the roles of the genes predicted to be regulated by ó54. This study applied a bioinformatics approach to search for additional ó54 regulated genes in C. trachomatis L2. A reduced consensus sequence (TGGCACnnnnnTTGC) identified two previously published ó54 promoter sequences upstream of CT652.1 and CT683. A modified consensus sequence (TGG-N9-TGC) was applied to the C. trachomatis D genome in Findpatterns yielded 512 potential targets of which 20 by virtue of sequence orientation and distance upstream of the predicted ORF start codon Primer extension analysis of total RNA isolated at 24 hours post-infection mapped the 5' RNA end upstream for acpS (CT100), yhf0_1 (CT258), SAM (CT404), lpxA (CT531), hypothetical proteins CT652.1 and CT683, and htrA (CT823) to the predicted ó54 promoters. Three candidates (CT291, CT404, and CT847) were mapped to putative ó70-like ó66 promoters. No transcript start sites were detected for the remaining ó54 promoter candidates. Two transcripts were detected from predicted ó66 and ó54 tandem promoters upstream of CT404. Primer extension analysis of the CT404 transcripts from RNA isolated at 4, 8, 12, 24 and 32 hours post-infection showed a decrease between 12 hours and 24 hours post-infection in transcripts thought to be generated from the predicted ó66 promoter. Transcripts from the predicted ó54 promoter were identified throughout development. Temporal gene expression profiles of the candidate genes with predicted ó54 promoters (CT652.1, CT683, CT100, CT258, CT531 and CT823) were resolved throughout the C. trachomatis L2 developmental cycle using real-time PCR. Transcripts for CT608 and CT609 were detected early in the cycle, while strong transcript levels were detected for CT258, CT531 and CT823 after the appearance of CT609 (rpoN). Low levels of CT652.1 and CT683 were measured, in the mid to late phase of the cycle, and transcripts for CT100 appeared at lower levels during the middle phase of the cycle. The functional assay of the predicted ó54 promoters required the generation of recombinant C. trachomatis L2 ó54 (rRpoN). The C. trachomatis rpoN was cloned into a bacterial expression system (pQE70) and the recombinant proteins purified for subsequent DNA mobility shift assays. Expression of rRpoN was hampered by low copy numbers, and unusual physical characteristics. DNA binding and mobility shift assays using rRpoN extracts against the chlamydial CT652.1 ó54 promoter, plus two characterised E. coli ó54 promoters (hypA and hycA), were successful if E. coli core RNA polymerase was added to the assay. All 20 candidates with predicted ó54 promoters were analysed with EMSA using rRpoN extract. The promoters upstream of CT100, CT223, CT258, CT322, CT652.1 and CT683 showed affinity towards the recombinant rRpoN-E. coli core RNA polymerase holoenzyme complex. Searches for potential chlamydial ó54 transcription initiation activators were made using the Multiple Em for Motif Elucidation (MEME) software, looking to identify the DNA binding motifs. The upstream promoter regions of CT100, CT223, CT258, CT322, CT531, CT652.1, CT683 and CT823 in C. trachomatis L2 and orthologs found in other species of Chlamydia were analysed. The software identified a near palindromic sequence upstream of CT100 orthologs in C. trachomatis D and C. trachomatis MoPn (CAACCCAAC and CACCACAAC) where as a CT531- and CT823-specific motif was also discovered (CCGTTGTAGAATCTC). It is beginning to emerge that ó54 may regulate the expression of proteins required for the formation of the cell wall. Since the expression of the ó54 transcript, rpoN, coincides with the morphological change from the non-infectious RB to the infectious EB, predictions could be made concerning which genes are potentially regulated by ó54.

Bibliography: leaves 242-298. / xxxiii, 298 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis investigates the effects of polyunsaturated fatty acids (PUFAs) on macrophage oxygen radical production. The role of fatty acid structure in the ability to stimulate the fMLP response is also examined. The mechanisms by which fatty acids induce their effects on mononuclear phagocytes are partially elucidated. The mechanisms of the biological effects of the PUFAs in terms of intracellular signalling pathway are partly defined. / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 1997

Cardiac hypertrophy is clinically defined as a relative increase in heart size associated with a thickening of the ventricular wall. It is a common feature of individuals suffering from different cardio-vascular or metabolic conditions and leads to heart failure. The structural, functional and molecular mechanisms which induce hypertrophy independent of hemodynamic alterations are poorly characterized. In this study, questions about whether cardiac-specific neuro-endocrine activation or metabolic imbalance are sufficient to induce hypertrophic structural and functional remodeling are addressed using genetically manipulated mouse models of primary cardiac hypertrophy. (For complete abstract open document)

Bibliography: leaves 242-298. / xxxiii, 298 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis investigates the effects of polyunsaturated fatty acids (PUFAs) on macrophage oxygen radical production. The role of fatty acid structure in the ability to stimulate the fMLP response is also examined. The mechanisms by which fatty acids induce their effects on mononuclear phagocytes are partially elucidated. The mechanisms of the biological effects of the PUFAs in terms of intracellular signalling pathway are partly defined. / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 1997

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.

Zugl.: Berlin, Freie Univ., Diss., 2005 / Dateiformat: zip, Dateien im PDF-Format. - Erscheinungsjahr an der Haupttitelstelle: 2005

Charité, Universiẗat-Med., Diss., 2006--Berlin. / Dateiformat: zip, Dateien im PDF-Format.