This research work focused on studying the emission spectroscopy produced from the high energy ion-molecule collision processes in mass spectrometry. The collision experiments are described and divided into 4 chapters (Chapter 3, 4, 5, 6).N2O+● is an ion of atmospheric importance. In chapter 3 the investigation of the collision between high translational energy (4-8 keV range) N2O+● ions and Helium target gas in mass spectrometry using collision induced emission (CIE) spectroscopy is described.In chapter 4, the collision-induced emission (CIE) spectra from 4-8 keV collisions between projectile He+● ions and CO2 target gas (He+●/CO2) were obtained. In Chapter 5, to probe the validity of this hypothesis, CIE experiments were carried out to observe the photon emissions from keV collisions of a selection of projectile ions with O2 target gas. By studying the resulting CIE spectra, a second potential mechanism came to light, one that involves the nearly isoenergetic O2+. A → X state transition. In chapter 6, neutral hydroxymethylene and formaldehyde were generated by charge exchange neutralization of their respective ionic counterparts and then were reionized and detected as recovery signals in neutralization-reionization mass spectrometry in the modified VG-ZAB mass spectrometer.

The tumor suppressor p53 acts as a master transcription factor that controls hundreds of effecter genes in response to various cellular stresses. The flexibility of p53 to regulate its target genes with distinct functions (growth arrest, DNA repair, apoptosis etc.) is largely conferred by extensive and dynamic posttranslational modifications of the protein, including phosphorylation, acetylation, methylation, ubiquitination, sumoylation, neddylation and ADP-ribosylation. Recent evidence suggests that acetylation is indispensable for p53 activation. A major regulator of p53 acetylation, and hence p53 function, is a group of Class III histone deacetylases known as Sirtuins (SIRTs), that utilize nicotinamide adenine dinucleotide (NAD+) as substrate to catalyze the removal of acetyl groups from p53, resulting in the “silencing” of p53 activity. In an effort to determine whether a feedback loop exists whereby p53 is involved in the regulation of NAD+ metabolism, nicotinamide adenylyltransferase 2 (Nmnat2), a key NAD+ synthetase, was identified to be a novel target gene of p53, from which two transcript variants are expressed in human (TV1 and TV2). Two putative p53 response elements within the first intronic region of human Nmnat2 gene were also identified that can actively drive the expression of luciferase reporter gene in a p53-dependent manner. Most importantly, data suggests that Nmnat2, like SIRTs, is involved in the regulation of p53-mediated apoptosis and protein acetylation upon DNA damage. Furthermore, Nmnat2 isoforms exert opposite functions on SIRT-mediated deacetylation of p53. Specifically, ectopic expression of Nmnat2 TV2 isoform promotes p53 acetylation after DNA damage, whereas ectopic expression of Nmnat2-TV1 isoform suppresses it. Manipulation of SIRT activities by either RNA interference or specific inhibitors modifies p53 acetylation status the same way Nmnat2-TV2 isoform does. Collectively, the results suggest the existence of a p53-Nmnat2 feedback loop, whereby p53 can regulate its own activity positively or negatively, depending on the nature and extent of DNA damage.

No description available.

No description available.

This research work focused on studying the emission spectroscopy produced from the high energy ion-molecule collision processes in mass spectrometry. The collision experiments are described and divided into 4 chapters (Chapter 3, 4, 5, 6).N2O+● is an ion of atmospheric importance. In chapter 3 the investigation of the collision between high translational energy (4-8 keV range) N2O+● ions and Helium target gas in mass spectrometry using collision induced emission (CIE) spectroscopy is described.In chapter 4, the collision-induced emission (CIE) spectra from 4-8 keV collisions between projectile He+● ions and CO2 target gas (He+●/CO2) were obtained. In Chapter 5, to probe the validity of this hypothesis, CIE experiments were carried out to observe the photon emissions from keV collisions of a selection of projectile ions with O2 target gas. By studying the resulting CIE spectra, a second potential mechanism came to light, one that involves the nearly isoenergetic O2+. A → X state transition. In chapter 6, neutral hydroxymethylene and formaldehyde were generated by charge exchange neutralization of their respective ionic counterparts and then were reionized and detected as recovery signals in neutralization-reionization mass spectrometry in the modified VG-ZAB mass spectrometer.

Thesis (MAppSc in Applied Physics)--University of South Australia, 1994

Tissue factor (TF) is the essential cofactor for FVIIa. Binding to transmembrane tissue factor increases the catalytic efficiency of FVIIa allowing activation of FX and FIX which initiates coagulation and propagates stable clot. Transmembrane TF resides in a cryptic configuration on the cell surface and in the circulation with low procoagulant activity. However TF can be rapidly switched to an active configuration in order to contribute to thrombus propagation. The precise nature of this switch is unknown, however it is known to be an extracellular event. The extracellular part of TF consists of 2 fibronectin type III domains. The disulfidebond in the membrane proximal domain (Cys186-Cys209) is a cross-strand bond which links adjacent strands in the same ?? sheet. It has the configuration, characteristic dihedral strain energy and bond length of an allosteric disulfide bond. This indicates that it has the potential to undergo thiol/disulfide exchange to change the function of the TF protein. We confirm that the integrity of the Cys186-Cys209 disulfide is required for coagulant function and that tissue factor contains free thiols in the cryptic state which are lost when TF becomes de-encrypted. Membrane based tissue factor procoagulant activity is blocked by the mono-thiol alkylators N-ethylmaleimide and methyl methanethiosulfonate; but increased by ECl/formation of the disulfide via the thiol oxidiser, HgCb or thiol cross-linkers, eimidohexane and bismalemidoethane. The increase in activity correlates with a conformation change in the TF protein adjacent to the disulfide. We show that redox active protein disulfide isomerase is associated with cryptic tissue factor and propose that the cryptic conformation of tissue factor is maintained through formation of an Snitrosylated complex with protein disulfide isomerase. Our results indicate that the activation of TF involves a change of conformation of the domain 2 of TF caused by formation of the cross-strand Cys186-Cys209 disulfide bond. We suggest that this is likely to be the physiological change that facilitates productive binding of FIX and FX in coagulation.

The complement system is one of the most ancient immune defense mechanisms, providing rapid protection against invading micro-organisms. It is essential for the complement cascade to be under tight regulation in order to prevent excessive production of complement proteins. C5a is the most potent anaphylotoxin produced by the complement system, it binds C5a receptor (C5aR, CD88) and C5L2 (GPR77). C5a binding to C5aR induces leukocyte chemotaxis and release of inflammatory mediators. Over-production of C5a is known to be involved in many inflammatory and pathological conditions such as RA, I1R injury and sepsis, making it an attractive therapeutic target. Human and mouse C5aR share low homology and blocking C5a/C5aR signaling with small molecules has been challenging. We generated human C5aR knockout/knockin (hC5aR KI) mice in which the mouse C5aR coding region was replaced with that of human C5aR to utilize them for the development of human therapeutics targeting C5aR. hC5aR KI mice showed normal development, and leukocytes from hC5aR KI mice responded well to mouse C5a. We used two approaches to generate monoclonal antibodies (mAbs) against hC5aR. We used a mouse cell line transfected with hC5aR or neutrophils from hC5aR KI mice to immunize wild-type mice and generated high-affinity antagonistic mAbs which are specific to human C5aR. Anti-hC5aR mAb 7F3 blocked C5a-induced signaling completely without agonistic activity in vitro. In the animal model of K/BxN inflammatory arthritis, 7F3 both prevented and reversed inflammation. Currently, the function of the second C5a receptor, C5L2, remains controversial. There are contradicting reports from C5L2 KO mice that were generated by independent groups. We assessed the function of human C5L2 using an antagonistic mAb that specifically blocks C5L2 function and not C5aR. In vitro analysis using the C5L2-blocking mAb showed that C5a does not signal via C5L2 to affect chemotaxis or phagocytosis by neutrophils, indicating that C5L2 is not a signaling receptor for C5a, at least in these cellular functions.

This thesis describes a broad range of coordination and organometallic chemistry on a series of ruthenium and rhodium complexes towards the aim of activating unsaturated N Ξ N, C=O, and C=C bonds. The dinitrogen complex [RuTp(pzP)(N2)]BPh4 (5) (where Tp= tris(pyrazolyl)borate, and PzP= 1-(2-diphenylphosphinoethyl)pyrazole) was synthesized via displacement of the chloride ligand from the complex [RuTp(pzP)Cl] (1). It was found that N2 coordination proceeded through an unusual oxidation/reduction cycle with the Ru(III) intermediate, [RuTp(pzP)CI]BF4 (6), isolated upon reaction of 1 with AgBF4 in THF. Investigations of the coordination chemistry of the related Tp complex [RuTp(Bp)PPh3] (4) (where Bp= bis(pyrazolyl)borate), resulted in several unusual reactions occurring on the Bp chelate. Reaction of 4 with AgBF4 gave the unusual product [RuTp(BpF')PPh3] (6), which had the Bp B-hydride substituents replaced by fluoride substituents from the BF4 anion, (i.e. BpF,). Alternatively, reaction of 4 with AgOTf, or SOCb, led to the synthesis of the products [RuTp(BpoH)PPh3]OTf (34), and [RuTp(BpoH)PPh3]Cl (35), respectively, which have a single hydroxyl substituent substituted in place of the two B-hydrides to yield a highly unusual neutral borane chelate (BpOH). A series of ruthenium tris(pyrazolyl)methane (Tpm) complexes [RuTpm(PPh3)2Cl]BPh4 (44.BPh4) RuTpm(PPh3CI2] (46), [RuTpm(PPh3)2CI]BPh4 (44.BPh4) [RuTpm(PPh3)(MeCN)Cl]BPh4 (50), [RuTpm(PPh3HMeCN)](BF4)2 (51), [RuTpm(PPh3)(MeCNh](BF4)2 (52), and [RuTpm(MeCNhCI]BPh4 (54.BPh4) were synthesized. These complexes varied in the number of labile acetonitrile ligands they contained, the net charge of the complex, and the presence or absence of strongly coordinating phosphine coligands on the complex. The influence of these properties on the catalytic activity of the complexes for the transfer hydrogenation of acetophenone was investigated. It was shown that the net charge and number of labile MeCN donors on the complex had little influence on the activity of the catalyst. It was also observed that the catalyst [RuTpm(MeCN)2CI]BPh4 (54.BPh4), which does not contain a strongly coordinating PPh3 ligand, would rapidly decompose during catalysis. A series of bis(tert-butylthiomethyl)pyridyl (SNS tBU ) pincer complexes [Ru(SNStBU)(PPh3)Cb] (65), [Ru(SN StBU)(PPh3)(MeCN)CI]BPh4 (66), [Ru(SNStBU)(PPh3)(MeCN)2](BF4h (67), and [Ru(SNStBU)(MeCN)Cb] (68) were synthesized and their as catalysts for the transfer hydrogenation of acetophenone was investigated. The activity of these complexes for as transfer hydrogenation catalysts was shown to increase as the number of labile coligands on the complex increased, with an extremely high transfer hydrogenation activity obtained using 68. The catalytic activity of 68 is one of the fastest to be reported in the literature, achieving a superlative TOF (turnover frequency) of 87360 h- I. The coordination of SNStBu in 66 and the related complexes [Ru(SNS (MeCN)2CI]BPh4 (70), and [Ru(SNS)(MeCN)2CI]2[??-Ag(MeCN)2]2(BF4)4(71) was observed to yield a range of different conformational isomers. These isomers were studied in detail using low temperature NMR and 20 NOESY and COSY IH_1H correlation experiments. The complex 71 was also characterized crystallographically and was shown to have an unusual tetrametallic macrocyclic structure with two [Ru(SNS)(MeCN)2C1r moieties bridged by two [Ag(MeCN)2r ions through a chloro and thioether donor group. The hydrogenation of unsaturated olefinic bonds was achieved using a series of Rh N-heterocyclic carbene (NHC) complexes of the type [Rh(L)(COD)]BPh4 (where L= and NHC-pyrazolyl chelate). A series of NHC-pyrazoly ligands (L) were synthesized that contained varying degrees of steric bulk on the pyrazolyl and NHC donor group. The influence of these steric parameters on the rhodium complex structure and activity of the complexes as catalysts for the hydrogenation of styrene was investigated. It was found that increasing the steric bulk around Rh decreased the activity of the catalyst.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 4 uppsatser.