The p53 tumor suppressor protein has been well-characterized for its role in inducing growth arrest, senescence, and apoptosis upon various types of stresses. Recently, however, roles of p53 have expanded beyond the canonical functions, and now include cellular processes such as metabolism, oxidative balance, and ferroptosis. Through RNA-seq screening, we first identified phosphoglycerate dehydrogenase (PHGDH), a rate-limiting enzyme in the serine biosynthesis pathway, as a novel metabolic target of p53. p53 suppresses PHGDH expression and inhibits de novo serine biosynthesis. Notably, upon serine starvation, p53-mediated cell death is significantly enhanced in response to Nutlin-3 treatment. Moreover, PHGDH has been demonstrated to be frequently amplified in human melanomas. We found that PHGDH overexpression significantly suppresses the apoptotic response, whereas RNAi-mediated knock-down of endogenous PHGDH promotes apoptosis under the same treatment. Together, our findings demonstrate an important role of p53 in regulating serine biosynthesis through suppressing PHGDH expression, and reveal serine deprivation as a novel approach to sensitize p53-mediated apoptotic responses in human melanoma cells. In addition, we also identified spermidine/spermine N1-acetyltransferase 1 (SAT1) as a novel metabolic target of p53. SAT1 is a rate-limiting enzyme in polyamine catabolism critically involved in the conversion of spermidine and spermine back to putrescine. Surprisingly, we found that activation of SAT1 expression induces lipid peroxidation and sensitizes cells to undergo ferroptosis upon reactive oxygen species (ROS)-induced stress, which also leads to suppression of tumor growth in xenograft tumor models. Notably, SAT1 expression is down-regulated in human tumors, and CRISPR-cas9-mediated knockout of SAT1 partially abrogates p53-mediated ferroptosis. Moreover, SAT1 induction is correlated with the expression levels of arachidonate 15-lipoxygenase (ALOX15), and SAT1-induced ferroptosis is significantly abrogated in the presence of PD146176, a specific inhibitor of ALOX15. Together, these data indicate a novel regulatory role of p53 in polyamine metabolism and provide insight into the regulation of p53-mediated ferroptotic responses. Our studies on PHGDH and SAT1 led us to the question of whether these unconventional functions of p53 contribute to its role as a tumor suppressor. In fact, previous view regarding the mechanism of p53-mediated tumor suppression, which was long thought to be growth arrest, apoptosis, and senescence, has recently been challenged by several knockout and knock-in mouse studies. Previously, we established mice (p533KR/3KR) in which p53 acetylation at lysine residues K117, K161, and K162 were abolished by replacing lysine with arginine. p533KR/3KR mice completely lost p53-mediated cell cycle arrest, apoptosis, and senescence functions in response to stresses. However, unlike p53-null mice which rapidly develop spontaneous thymic lymphomas, all of the p533KR/3KR mice remain tumor-free, indicating that other aspects of p53 functions are sufficient to prevent tumor formation. Notably, p533KR retains the ability to regulate metabolic targets including TIGAR and SAT1, as well as ferroptosis regulator SLC7A11. In this study, we have identified two novel acetylation sites- K98 and K136, in the mouse p53 DNA-binding domain. Whereas loss of K98 or K136 acetylation (p53K98R, p53K136R) alone has modest effect on p53 transcriptional activity, simultaneous mutations at all of these acetylation sites (p534KR98: K98R+3KR, p534KR136: K136R+3KR, p535KR: K98R+K136R+3KR) completely abolish the ability of p53 to regulate TIGAR, SAT1, and SLC7A11. In addition, p534KR98, p534KR136, and p535KR are defective in Erastin-induced ferroptosis. Notably, p534KR98/4KR98, p534KR136/4KR136, and p535KR/5KR knock-in mice lost intact tumor suppression and developed spontaneous tumors. This suggests that p53-mediated ferroptosis may function as a critical barrier to prevent tumor formation independently from growth arrest, apoptosis, and senescence. Interestingly, both p534KR98/4KR98 and p534KR136/4KR136 mice displayed significantly delayed tumorigenesis comparing with p53-null and p535KR/5KR mice. We found that unlike p535KR, p534KR98 retains the capacity to inhibit mammalian target of rapamycin (mTOR) signaling pathway through activating the expression of two mTOR negative regulators, Sestrin2 and DDIT4. Altogether, our findings underscore the extensive scope of p53 functions in metabolic regulation, oxidative stress response, and ferroptosis, and provide novel insights into the tumor suppression mechanism of p53.

published_or_final_version / Molecular Biology / Master / Master of Philosophy

The commencement of in vitro differentiation is mediated by genetic changes that result in selective expression of genes and a shift in metabolism. The role of nitrate reductase, a key enzyme of nitrate assimilation, during differentiation was examined in this study using an in vitro Nicotiana tabacum (tobacco) callus culture system. In particular, the effects of nitrogen and light/dark regimes on callus differentiation and nitrate reductase were investigated. Methodology required for the analysis of nitrate reductase regulation during in vitro tobacco callus differentiation was established. Optimised in vivo, in situ and in vitro nitrate reductase assays yielded similar values and patterns during tobacco callus culture development, and the in vivo assay was selected for nitrate reductase activity measurement during subsequent experiments. Western blot analysis of tobacco callus acetone-extracted protein after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a spinach polyclonal nitrate reductase antibody yielded major bands at 71 and 48 kD, with numerous minor bands. Extraction of callus protein in the presence of various protectants did not prevent cleavage of putative nitrate reductase polypeptide. Slot blot detection of nitrate reductase mRNA using a [32p]- labelled nitrate reductase cDNA probe isolated from the plasmid pBMC102010 was not possible due to non-specific binding to nitrocellulose filters. Northern blotting of RNA fractionated by agarose gel electrophoresis using a [32p]-labelled nitrate reductase cDNA probe identified a single mRNA species at 3.5 kb, the expected size of tobacco nitrate reductase mRNA. In vitro tobacco callus differentiation on 60 or 120 mM nitrogen regimes and under light/dark (16/8 h), continuous dark or continuous light treatments were comparable in terms of fresh weight, protein and nitrate uptake. Higher levels of in vivo nitrate reductase activity were observed prior to visible shoot primordia in all treatments, suggesting that the developmental status of callus mediated the regulation of nitrate reductase. Putative nitrate reductase protein levels were not correlated with in vivo nitrate reductase activity during initial stages of tobacco callus differentiation under various light treatments; nitrate reductase mRNA levels could not be ascertained. These results suggested that post-translational control mechanisms were involved in nitrate reductase regulation during in vitro tobacco callus differentiation. / Thesis-(M.Sc.)-University of Natal, Durban, 1993.

Copies of author's previously published articles inserted. / Bibliography: leaves 89-126. / vii, 126, [88] leaves, [1] leaf of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Finds that the metabolism of substance P (SP) and the subsequent production of SP-(7-11) is important in regulating the biological activity of SP on chondrycytes in the synoviol joint. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1996?

Additional appendix (5 p.) is pasted onto back end-paper. / Bibliography: leaves 219-244. / v, 244, [14] p., 67 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Investigates the complexity of the vagal reflexes arising from the upper gastrointestinal tract by recording single unit vagal afferents and efferents in the ferret. The potential involvement of various neurotransmitters in mediating and modulating gastrointestinal tract inputs is also explored. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1999

Erratum pasted onto front end-paper. / Copies of author's previously published article inserted. / Bibliography: leaves 167-180. / 180 leaves, [51] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Reanalyses the effects of the absence of CreA in the cell, raises antibodies for the detection of CreA and identifies new loci involved in carbon catabolite repression by using different genetic selection methods. / Thesis (Ph.D.)--University of Adelaide, Dept. of Genetics, 1999?

Additional appendix (5 p.) is pasted onto back end-paper. Bibliography: leaves 219-244. Investigates the complexity of the vagal reflexes arising from the upper gastrointestinal tract by recording single unit vagal afferents and efferents in the ferret. The potential involvement of various neurotransmitters in mediating and modulating gastrointestinal tract inputs is also explored.

Thesis (MNutr (Interdisciplinary Health Sciences. Human Nutrition))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Introduction Complications of severe infection or acute trauma include a cascade of immunological dysfunctions known as SIRS (Systemic Inflammatory Response Syndrome), that affect response to treatment, prolonging and complicating the course of illness and jeopardizing clinical outcome. Timing and the nature of nutritional support in the Intensive Care Unit (ICU) setting may influence this process. Against this background, and despite some trials demonstrating beneficial clinical outcomes for the use of immune-modulating diets (IMD), the findings of the US summit on immune-enhancing enteral therapy concluded that the currently available enteral immune-enhancing formulas are “first-generation products” which may not be appropriate in patients with SIRS or severe sepsis. This highlights a need for alternative nutritional products that target the specific needs of this patient population. As such, Intestamin® is designed for use in severely stressed patients as an immune-modulating enteral feed supplement which aims to improve maintenance of gut barrier integrity and immune response. Aim The aim of this pilot study was to investigate the effect of Intestamin® administration to critically ill patients, and in particular, to determine if administration would impact on nosocomial infections, ventilation days and the length of stay in the ICU. Methods The study design was an open label, retrospective case control, analytical study, of patients admitted to the ICU in The Bay Hospital, Richards Bay, between January 2002 and November 2003, who received Intestamin®. Patients were selected for the study from post-surgery and post-trauma patients at high risk of sepsis and SIRS, and critically ill patients with manifested SIRS or severe sepsis. Development of respiratory and urinary sepsis was used as surrogate markers for progression to severe sepsis and SIRS. Additionally, duration of ventilation and ICU stay were considered representative of the response to treatment and degree of clinical complications. Results The findings of the study demonstrated a significant difference in the rates of respiratory infection(p=0.05), positive sputum and tracheal aspirate cultures(p=0.03) and urinary catheter tip cultures(p=0.04). with statistically lower rates in the intervention group compared to the control group. There were no significant differences in the rates of urinary tract infection, septicaemia or in combined sepsis rates between the two groups. There were statistically significant higher rates of positive pus cell counts in the sputum(p=0.003) and urine(p=0.01) in the intervention group, compared to the control group. No corresponding reduction in ventilation days or ICU stay was observed. Conclusion In this patient population, early enteral nutrition with specially formulated IMD, (Intestamin®), did result in a significant reduction in respiratory infections, but not in other types of sepsis, ICU or ventilator days in critically ill ICU patients. This positive finding in some, but not all endpoints collected, may reflect confounding factors in the small patient population or the choice of clinical endpoints, rather than a genuine limitation in the benefit. IMD remains a tantalizing and scientifically plausible intervention in this patient population, with larger clinical trials necessary to confirm outcomes. The study supports the safe use of Intestamin by the nasojejenal route in this patient population. / AFRIKAANSE OPSOMMING:Inleiding Komplikasies van erge infeksie of akute trauma sluit ‘n kaskade van immunologiese disfunsie in, bekend as SIRS (Sistemiese Inflammatoriese Respons Sindroom), wat die respons op behandeling affekteer, die verloop van siekte verleng en kompliseer asook die kliniese uitkoms beïnvloed. Tydsberekening en die aard van die voedingsondersteuning in die Intensiewe Sorg Eenheid (ISE) mag hierdie proses beinvloed. Teen hierdie agtergrond, en ten spyte van sommige studies wat die voordelige kliniese uitkoms vir die gebruik van immuun-modulerende diete (IMD) toon, het die “US summit” oor immuunverbeterde enterale terapie tot die gevolgtrekking gekom dat die huidige beskikbare enterale immuun-verbeterde formules, “eerste-generasie” produkte is, wat moontlik nie toepaslik is vir pasiente met SIRS of erge sepsis nie. Dit beklemtoon ’n behoefte aan alternatiewe voedingsprodukte wat die spesifieke behoeftes van die genoemde pasient populasie teiken. Intestamin® is ontwerp vir gebruik in erge gestresde pasiente as ‘n immuun-modulerende enterale voedingssupplement doelgerig om spysverteringskanaal integriteit te onderhou en immuniteit te verbeter. Doel Hierdie loodsstudie se doel was om die effek van Intestamin® toediening aan kritiek siek pasiente te ondersoek, spesifiek om vas te stel of die toediening impakteer op nosokomiale infeksies, ventilasie dae en dae in ISE. .Metode Die studie ontwerp was ‘n oop, retrospektiewe, geval kontrole, analitiese studie van pasiente opgeneem in die ISE van The Bay Hospital, Richardsbaai, tussen Januarie 2002 en November 2003, wat Intestamin® ontvang het. Pasiënte is geselekteer vir die studie uit post-chirurgies en post-trauma pasiente wat hoë risiko was vir sepsis en SIRS, en kritiek siek pasiente wat reeds manifisteer het met SIRS of erge sepsis. Ontwikkeling van respiratoriese en urinêre sepsis is gebruik as surrogaat merkers vir die progressie na erge sepsis en SIRS. Addisioneel is duur van ventilasie en ISE verblyf beskou as verteenwoordigend vir die respons op behandeling en die graad van kliniese komplikasies. Resultate Die bevindinge van die studie het betekenisvolle verskille aangedui in die voorkoms van respiratoriese infeksies(p=0.05), positiewe sputum en trachiale aspiraatkulture(p=0.03) en urine kateterpunt-kulture(p=0.04) met statistiese laer voorkoms in die intervensie groep in vergelyking met kontroles. Geen statistiese verskille in die voorkoms van urineweg-infeksies, septisemia of in gekombineerde sepsis voorkoms tussen die twee groepe is gevind nie. Daar was statistiese betekenisvolle hoër voorkoms van etterselle hoeveelhede in die sputum(p=0.030 en uriene(p=0.01) van die intervensie groep in vergelyking met die kontrole groep. Geen ooreenkomstige vermindering in ventilasie dae of ISE verblyf is opgemerk nie. Gevolgtrekking In hierdie pasiënt populasie, het vroeë enterale voeding met spesifieke geformuleerde IMD (Intestamin®), ‘n beduidende vermindering in respiratoriese infeksies getoon, maar nie in ander tipes sepsis, ISE of ventilasie dae by kritiek siek pasiente nie. Hierdie positiewe bevindinge in sommige. maar nie al die versamelde eindpunte nie, reflekteer moontlike bydraende faktore in die klein pasiënt populasie of die keuse van kliniese eindpunte, eerder as a ware beperking in die voordele. IMD bly steeds ‘n uitdagende en wetenskapilik uitsonderlike intervensie in hierdie pasiënt populasie, wat groter kliniese studies benodig om die uitkoms te bevestig. Die studie ondersteun die veilige gebruik van Intestamin® via die nasojejenale roete in kritiek siek pasiënte.

D.Litt et Phil. (Psychology) / Please refer to full text to view abstract

There is considerable evidence to suggest that dopamine (DA), in addition to its role as a precursor of norepinephrine (NE) and ephinephrine, has important physiological actions in its own right. One physiological action of DA seems to be that of a neurotransmitter in the mammalian brain (Hornykiewicz, 1966). In addition, there is evidence that abnormalities of dopaminergic transmission in the central nervous system (CNS) may be of clinical importance. For example, dopaminergic over activity in the mesolimbic forebrain may be a primary feature in the etiology of schizophrenia (Meltzer and Stahl, 1976). The drugs used to treat schizophrenia act as DA antagonists in the brain (Snyder et al., 1974; Robinson et al., 1979). Drugs such as phenothiazines and butyrophenones have been shown in clinical studies to be effective in treating the fundamental symptoms of psychosis (Snyder et al., 1974). The results of animal experiments indicate that their principal mode of action is blockade of DA receptor sites in the CNS (VanRossum, 1966). However, these neuroleptics are generally nonspecific in their effects upon DA neurons and thus, cause major undesireable side effects. If new drugs could be discovered that were more structurally selective for different DA systems, then, perhaps these undesireable side effects could be eliminated. In order to develop such drugs, a closer look would have to be made at different DA systems in an attempt to demonstrate DA receptors which are topographically distinct and can thus be selectively regulated by both agonistic and antagonistic agents. The demonstration of more than one DA receptor in mammalian CNS is the subject of the research presented in this thesis.