This phase of the investigation was concerned with a study of the urinary excretion of estriol, estrone and estradiol -17B following the intramuscular injection of estradiol -17B in men with myocardial infarction. The results were compared with those of a control group of male subjects with no clinical evidence of this disease. The study was undertaken for the reasons set forth below. Myocardial infarction is rare in women before the menopause but postmenopausal women show a high incidence of this disease.

The study described in this thesis represents part of a larger programme of research on the preservation of blood. Though the benefits resulting from the transfusion of blood were recognized a very long time ago, it has been only within the last two decades that serious attempts have been made to preserve human blood. With the outbreak of the Second World War, the demand for blood both in civilian hospitals and on the battle field was increased tremendously. [...]

One of the most important phenomena in nature is the passage of material through semi-permeable membranes. In many cases, such as the passage of gases in the lungs, it is probably a physical process, but one can visualize the existence of special transfer processes in certain tissues. The transfer of material through the wall of the small intestine, or the reabsorption of material in the renal tubules, could conceivably be due to the existence of a special process. Van Slyke and Meyer (1), in 1912, were the first to suggest that such a process exists in all tissues.

The existence of y-aminobutyric acid (GABA), as a product of decarboxylation of glutamic acid, in living organism was first reported in 1910 by Ackerman and Kutscher (2, 3). This was soon afterwards confirmed by Abderhalden, Fromme and Hirsch (1). With the development of modern techniques for the separation of amino acids in the past decade, it has become apparent that GABA is widely distributed in animal and plant tissues as well as in micro-organisms (36).

Phenylketonuria is an inherited metabolic disorder which was discovered through the association of mental deficiency with the excretion of phenylpyruvic acid. Other names for this condition have ranged from the descriptive but unwieldy "phenylpyruvic oligophrenia" and "imbecillitas phenylpyruvica" to "Folling's disease", after its discoverer. [...]

Although 70 years have passed since Friedrich Miescher made this statement it is only during the last two decades that the full significance of the fundamental and central role of phosphorus in biological systems has been recognized. The presence of phosphorus as a key constituent in biological substances was first noted by Liebig (2) who isolated from marnma1ian muscle a crystalline material which was subsequently identified as inosinic acid. With the isolation during the present century of other naturally occurring phosphate esters, it has become apparent that phosphorylated compounds are invariably associated with the production of chemical energy.

Cyanocobalamin bound to normal human gastric juice (NHGJ) or to guinea pig stomach extract (GPSE) is taken up by guinea pig ileum to a greater extent than unbound cyanocobalamin. Almost all of the cyanocobalamin taken up in the presence of GPSE is removable by washing the intestinal tissue with ethylenediaminetetraacetate (EDTA), whereas the same washing procedure removes only one third of the cyanocobalamin taken up in the presence of NHGJ. The cyanocobalamin which is removed by washing with EDTA may be the same as that which is actively absorbed by guinea pig intestine.

<p>Transcription is the process by which cells translate genetic information stored in DNA into RNA. Transcription is a highly regulated and discontinuous process, and elongation is frequently blocked by DNA damage, pause sites, or intrinsic or external inhibitors. Due to the essential nature of transcription, the cell has numerous ways of dealing with these blockages to transcription, only some of which are understood. We examined the fate of RNA polymerase stalled by DNA-protein crosslinks as well as elongation inhibitors Streptolydigin and Actinomycin D.</p><p> We use 5-azacytidine, a cytosine analog that covalently traps cytosine methyltransferases, as a model system for DNA-protein crosslinks (DPCs) in<italic>Escherichia coli</italic>. Our lab previously showed the importance of the tmRNA system for survival during DPC-formation, implying that transcription and translation are blocked by DPCs. For tmRNA to function, the A-site must be cleared, requiring either RNA polymerase to be released first or the nascent RNA chain to be cleaved. Using cell growth assays, we tested mutants related to A-site cleavage factors known to affect transcription initiation, elongation, and termination. Of these mutants, only DksA seemed to have a mild effect, and only at late stages of growth phase. However, western blots for tmRNA tagging showed that <italic>dksA</italic> mutants have increased rather than decreased tmRNA tagging, indicating that another unknown factor is responsible for enabling tmRNA activity. </p><p> Since the issue of repair of DPCs remains unresolved, and the repair of DPCs could affect the blocked elongation complex, we used the same cell growth assay to look for potential repair pathways. We found that <italic>dnaK</italic> knockouts were slightly resistant to 5-azacytidine treatment which, coupled with our previous finding that <italic>dnaJ</italic> mutants are hypersensitive to DPCs, implies a potential DnaK-independent role for DnaJ in DPC repair.</p><p> Previous <italic>in vitro</italic> studies have shown that Stl-stalled RNAP is stable, while <italic>in vivo</italic> studies argued that Stl-inhibited polymerases are released from the DNA transcript, implying that there is a release factor responsible for removing RNAP from DNA <italic>in vivo</italic>. Using cell growth assays, Western blots for tmRNA tagging, and <italic>in vitro</italic> studies, we showed the transcription-coupled repair factor Mfd is responsible for releasing Stl-stalled RNAP, and that treatment with an elongation inhibitor such as Stl is an effective treatment against cells overexpressing the transcription-coupled repair pathway. </p><p> The tmRNA western blots also implied that Mfd has termination abilities in wildtype cells, leading us to perform RNAseq analysis on <italic>mfd</italic> knockout and overexpressing cells. We found that global transcription patterns are changed by altering Mfd levels, thus allowing us to propose a novel transcription regulatory role for Mfd.</p><p> We extended our elongation inhibitor studies to the eukaryotic inhibitor Actinomycin D and found that transcription-coupled repair pathway is again involved in responding to stalled RNAP. We also screened rifampicin-resistant RNAP mutants for Actinomycin D resistance and found several with the desired phenotype. We thus propose that Actinomycin D inhibition is more complicated than just steric hindrance due to DNA intercalation.</p> / Dissertation

Cytochrome P450s are versatile biocatalysts that play crucial roles in many important biological processes. Functional annotation of orphan P450 enzymes is the focus of this work. Catalytic activities of orphan human P450 2S1, 2W1, and 4X1 were investigated with untargeted metabolomic approaches. A series of lysophospholipids and free fatty acids were identified as novel substrates for P450 2W1. The isomer- and enantiomer-selectivity of P450 2W1-catalyzed lysophospholipid oxidations were characterized. The identities of the oxidation products were defined, and steady-state kinetics of the P450 reactions were determined. Epoxidation and hydroxylation of 18:1 lysophospholipid are considerably more efficient than that of the 18:1 free fatty acid. No endogenous substrate was identified for human P450 2S1 or 4X1. Heterologous expression systems for human P450 2U1, 4V2, 4Z1, and 46A1 were established for functional studies. A new software system was established to search for isotopic patterns in LC-MS data. It was used to identify monooxygenated enzymatic products after incubation with 16O2/18O2 gas.

Genomic instability and deregulated proliferation are hallmarks of human tumors. Cytotoxic chemotherapeutics, including cisplatin, have been used for over forty years to combat cancers that have deregulated proliferation and alterations in DNA damage response pathways. During initial clinical investigation in breast cancer, cisplatin did not prove to be more efficacious when compared to other treatments. However, in the past ten years oncologists identified a patient sub-group that appeared to be exceptional responders to neoadjuvant cisplatin treatment. These new insights may be beneficial to patients with triple negative breast cancer (TNBC). TNBC is defined by the lack of expression of the estrogen receptor, progesterone receptor, and a lack of amplification of the HER2 gene and thus standard of care targeted therapies are ineffective. Historically, cisplatin has proven to be effective against several tumor types but resistance to cisplatin-based therapy is common. With the increase in the use of cisplatin against TNBC in the clinic, we sought to develop cell-line models to identify mechanisms of cisplatin resistance in the basal-like 1 (MDA-MB-468) and basal-like 2 (HCC-1806) subgroups of TNBC. The cisplatin resistant cell line models do not undergo apoptosis in response to cisplatin-induced DNA damage. Caspase-14 was identified as a potential anti-apoptotic mediator in the cisplatin-resistant MDA-MB-468 while an abrogation of the MEK/ERK signaling pathway conferred apoptotic resistance in the cisplatin-resistant HCC-1806 cells. These findings provide insight into identifying signaling pathways involved in drug resistance and eventually potential future biomarkers that may predict clinical response to cisplatin in patients with TNBC.