Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The high-resolution 195Pt NMR signals (128.8 MHz) of most of the octahedral mixed-ligand Pt(IV) complexes in the series [PtCl6-n(OH)n]2-, n = 0-6, have been recorded in aqueous solutions at 293 K. These signals show characteristic 35/37Cl isotope-induced fine structure that results from the presence of several isotopologues in samples with a natural chlorine isotope distribution; each 37Cl isotope incorporated into the Pt coordination sphere of one of these complexes affords a fixed upfield (low frequency) isotope shift of between 0.17 and 0.22 ppm, depending on the particular complex. This assignment is confirmed by the excellent agreement between the natural abundances of the various isotopologues and the relative contributions of the corresponding signals to the overall area of the experimental spectrum of the particular isotoplogue set, obtained by a non-linear least-squares line fitting procedure. These results confirm that the 195Pt magnetic shielding in isotopomers differing only in the combination of the two chlorine isotopes coordinated in sites trans to hydroxido-ligands are indistinguishable under these experimental conditions, unlike those of similar isotopomers in the related series of aqua-complexes [PtCln(H2O)6-n]4-n, n = 3-5, as reported by Koch and co-workers. Moreover, the order of 195Pt shielding for the members of all stereoisomer pairs in the series of hydroxido-complexes is the reverse of that reported for the corresponding pairs in the aqua-series. These and other observations are interpreted qualitatively in terms of the relative strengths of the trans-influences of aqua-, hydroxido- and chlorido-ligands and the effect of these on bond displacements in these complexes. The 195Pt NMR spectra of especially the complexes cis-[PtCl2(OH)4]2- and [PtCl(OH)5]2- show remarkable fine structure in a ca. 45 % 18O-enriched aqueous solution; apart from additional signals resulting from 18O-containing isotopologues, the resonance signals of isotopomers differing in the combination of 16/18O isotopes in sites trans to chlorido-ligands are partially resolved. The effect of temperature on the 35/37Cl isotope-induced fine structure in the 195Pt signals of [PtCl6]2- and [PtCl(OH)5]2- was investigated in the range 283-308 K; some interesting differences are observed. 195Pt relaxation time measurements for [PtCl6]2- in this temperature range reveal that line-broadening is at least partially responsible for the loss of resolution between the signals of isotopologues of this complex as the temperature is increased, possibly due to the spin-rotation relaxation mechanism. The temperature coefficient of 195Pt shielding and the magnitude of isotope shifts in the spectra of the complexes in this series show interesting correlations with the 195Pt shielding itself; an interpretation of these observations is presented. / AFRIKAANSE OPSOMMING: Die hoëresolusie 195Pt NMR seine (128.8 MHz) van die oktaëdriese gemengde-ligand Pt(IV) komplekse in die reeks [PtCl6-n(OH)n]2- is waargeneem in waterige oplossing by ʼn temperatuur van 293 K. Hierdie seine toon ʼn karakteristieke 35/37Cl isotoop-geïnduseerde fynstruktuur as gevolg van die teenwoordigheid van verskeie isotopoloë in monsters met ʼn natuurlike chloor isotoopverspreiding. Die verplasing van ʼn 35Cl isotoop deur ʼn 37Cl isotoop in die Pt koördinasiesfeer van hierdie komplekse lei tot ʼn laefrekwensie isotoopverskuiwing van die 195Pt resonansiesein van tussen 0.17 en 0.22 ppm, afhangend van die spesifieke kompleks. Die toekenning van resonansieseine in hierdie spektra word ondersteun deur die goeie ooreenstemming tussen die berekende natuurlike verspreiding van isotopoloë en die persentasie area bydrae van die ooreenstemmende pieke tot die area van volledige stel seine van die chemiese spesie, soos bepaal deur ʼn nie-linieêre kleinste-kwadrate passingsmetode. Hierdie resultate bevestig dat vir isotopomere waarvan slegs die kombinasie van chloorisotope wat in posisies trans tot hidroksido-ligande gekoördineer is ʼn ononderskeibare 195Pt magnetiese skerming waargeneem word, m.a.w. ʼn enkele resonansiesein word vir hierdie isotopomere gemeet, anders as gerapporteer deur Koch en medewerkers vir die verwante aqua-komplekse [PtCln(H2O)6-n]4-n waar n = 3-5. Verder is die order van 195Pt magnetiese skerming vir stereoisomere in hierdie hidroksido-komplekse in elke stereoisomer paar die teenoorgestelde van dit waargeneem vir die ooreenstemmende aqua-komplekse. Hierdie waarnemings word kwalitatief geïnterpreteer in terme van die verskillende trans-invloede van die chlorido-, aqua- en hidroksido-ligande en die effekte daarvan op bindingslengtes in die komplekse. In ʼn ongeveer 45 % 18O-verrykte monster toon die 195Pt seine van veral die komplekse cis-[PtCl2(OH)4]2- en [PtCl(OH)5]2- uitsonderlike fynstruktuur vanweë die addisionele seine van 18O-bevattende isotopoloë en die parsiële resolusie van die seine van isotopomere wat verskil in die kombinasie van 16/18O isotope wat trans tot chlorido-ligande gekoördineer is. ʼn Studie is gemaak van die uitwerking van temperatuur op die 35/37Cl isotoop-geïnduseerde fynstruktuur in die 195Pt seine van die komplekse [PtCl6]2- en [PtCl(OH)5]2- in die gebied 283-308 K; interessante verskille is waargeneem. 195Pt magnetiese relaksasietyd metings vir die kompleks [PtCl6]2- in waterige oplossing in hierdie temperatuurgebied toon dat verbreeding van resonansieseine ten minste gedeeltelik verantwoordelik is vir die waargenome verlies aan resolusie tussen die seine van isotopoloë namate die temperatuur styg; die verbreeding van seine kan waarskynlik aan die spin-rotasie relaksasiemeganisme toegeskryf word. Die temperatuurkoëffisiënt van 195Pt magnetiese skerming en die grootheid van isotoopverskuiwings in die spektra van die hidroksido-komplekse in hierdie reeks toon interessante korrelasies tot die 195Pt magnetiese skerming; ʼn interpretasie van hierdie waarnemings word voorgestel.

This thesis describes the development and the use of quantitative deuterium Nuclear Magnetic Resonance spectroscopy (NMR) as a tool in two areas of environmental research: the study of long term climate-plant interactions and the source tracking of persistent organic pollutant. Long-term interactions between plants and climate will influence climate change during this century and beyond, but cannot be studied in manipulative experiments. We propose that long tree rings series can serve as records for tracking such interactions during past centuries. The abundance of the stable hydrogen isotope deuterium (D) is influenced by physical and biochemical isotope fractionations. Because the overlapping effects of these fractionations are not understood, studies of the D abundance of tree rings led to conflicting results. We hypothesized that both types of fractionations can be separated if the D abundance of individual C-H groups of metabolites can be measured, that is if individual D isotopomers are quantified. The first paper describes a technique for quantification of D isotopomers in tree-ring cellulose by NMR. The technique showed that the D isotopomers distribution (DID) was non-random. Therefore, the abundance of each isotopomer potentially contains individual information which suggests an explanation for the conflicting results obtained by measuring the overall D abundance (dD). In the second paper, this technique was used to study hydrogen isotope exchange during cellulose synthesis in tree rings. This revealed that some C-H positions exchange strongly with xylem water, while others do not. This means that the exchanging C-H positions should acquire the D abundance of source water, which is determined by physical fractionations, while non-exchanging C-H positions of tree-ring cellulose should retain biochemical fractionations from the leaf level. Therefore, the abundance of the corresponding D isotopomers should contain information about climate and physiology. When analysing tree-ring series, the DIDs should reflect information about temperature, transpiration and regulation of photosynthesis. In the third paper, we showed that CO2 concentration during photosynthesis determines a specific abundance ratio of D isotopomers. This dependence was found in metabolites of annual plants, and in tree-ring cellulose. This result shows that D isotopomers of tree-ring series may be used to detect long-term CO2 fertilisation effects. This information is essential to forecast adaptations of plants to increasing CO2 concentrations on time scales of centuries. In the fourth paper, the source of persistent organic pollutants in the environment was tracked using DID measurements. The dD values of two compounds of related structures were not enough to show indisputably that they did not originate from the same source. However, the DIDs of the common part between the two compounds proved that they did not originate from the same source. These results underline the superior discriminatory power of DIDs, compared to dD measurements. The versatility of DID measurements makes them a precious tool in addressing questions that can not be answered by dD measurements. / Betson, Tatiana R

No description available.

A doença renal crônica (DRC) gera manifestações orais e alterações salivares nos pacientes, entretanto poucos estudos avaliaram os efeitos desta doença nas glândulas salivares. O objetivo deste trabalho foi avaliar os efeitos da nefropatia crônica experimental na morfologia, metabolismo, estresse oxidativo e concentrações iônicas nas glândulas parótida (PA) e submandibulares (SM) de ratos. A nefropatia crônica experimental foi induzida pelos 5/6 de nefrectomia (grupo DRC) e os resultados foram comparados com os grupos Sham e Controle no tempo experimental de 12 semanas. Foram avaliados os pesos corpóreos iniciais e finais, níveis séricos e urinários de ureia e creatinina, a depuração de creatinina e excreção proteica urinária. A morfologia do córtex renal foi avaliada pela contagem de glomérulos em 30 campos x200 de aumento por grupo, além disso, foram avaliadas a presença de glomeruloesclerose e fibrose intersticial no grupo DRC. Nas glândulas PA e SM, foi realizada contagem de ductos em 30 campos x400 de aumento por grupo e avaliação de fibrose e de glicogênio no parênquima glandular. A análise metabólica avaliou os efeitos da DRC e estímulos simpáticos e parassimpáticos da salivação nos fluxos metabólicos glicolítico, oxidativo e anaplerótico utilizando infusões com [U-13C]Glicose ou [1,6-13C2]Glicose, [2-13C]Glicose e [U-13C]Ácidos graxos, e espectroscopia de ressonância magnética nuclear (RMN) de próton (1H) e carbono 13 (13C) em alta resolução de extratos glandulares como técnica de detecção de incorporação de 13C em metabólitos intermediários. Atividades enzimáticas avaliadas foram: hexoquinase [HK], fosfofrutoquinase-1(PFK-1), piruvato quinase (PK), lactato desidrogenase (LDH), glicose-6-fosfato desidrogenase (G6PD), amilase, peroxidase e catalase. Além disso, foram quantificados os conteúdos de malondialdeído [MDA], glicogênio e concentrações iônicas de cálcio, fósforo e sódio. Foram observadas diminuições significativas dos pesos corpóreos finais, aumentos significativos das concentrações séricas e diminuições das concentrações urinárias de ureia e creatinina, diminuição da depuração de creatinina e aumento da excreção proteica no grupo DRC comparado aos grupos Controle e Sham. Na avaliação do córtex renal encontramos diminuição significativa da contagem glomerular, infiltrado inflamatório, atrofia tubular, glomeruloesclerose e fibrose intersticial. Na avaliação histológica glandular foi observado aumento significativo da contagem de ductos e diminuição de glicogênio na glândula SM. Na análise metabólica observamos a prevalência do fluxo glicolítico nas glândulas salivares. O estímulo salivar promoveu aumentos significativos na razão 12C-Lactato/12C-Alanina nos grupos Controle e Sham, com exceção do grupo DRC PA, onde em repouso se observou um aumento, comparando com os grupos Sham e Controle. No grupo DRC PA foram observadas diminuições significativas dos níveis de 12C-Alanina, 12C-Creatina e 12C-Glicose sem estímulo e aumento de 12C-Acetato com estimulo. No DRC SM detectaram-se aumento dos níveis de 12C-acetato e diminuição significativa da relação 13C3-Lactato/13C3-Acetato pós-estímulo indicativos de inibição do metabolismo oxidativo. Estas alterações nos níveis de metabólitos e suas razões, bem como nos níveis de enriquecimento 13C, denotam alterações profundas a nível metabólico nas glândulas salivares devido a DRC. Nas análises enzimáticas foram encontradas na glândula PA diminuições significativas das atividades da PFK-1 e LDH em relação ao grupo Controle e aumentos significativos da peroxidase e catalase comparado aos grupos Controle e Sham. Na glândula SM foi observada diminuição significativa na atividade enzimática da PFK-1 comparado ao grupo Controle. Além disso, foram observados aumentos significativos do conteúdo de MDA nas glândulas PA e SM e aumento significativo da concentração de sódio na glândula SM comparado aos grupos Controle e Sham. As modificações estruturais, metabólicas, oxidativas e iônica evidenciam a disfunção glandular na DRC e podem estar associadas às alterações salivares que caracterizam a doença. / Chronic kidney disease (CKD) promotes oral manifestations and salivary changes in patients, but few studies have evaluated the effects of this disease in the salivary glands. The aim of this study was to evaluate the effects of experimental chronic nephropathy in morphology, metabolism, oxidative stress and ionic concentrations in parotid (PA) and submandibular glands (SM) of rats. Experimental chronic nephropathy was induced by 5/6 nephrectomy (CKD group) and the results were compared with the Sham and Control groups at 12 weeks experimental period. The initial and final body weights, serum and urinary levels of urea and creatinine, creatinine clearance and protein excretion were evaluated. The morphology of the renal cortex was assessed by counting glomeruli in 30 fields x200 magnification per group, moreover, the presence of glomerulosclerosis and interstitial fibrosis in CKD group were also evaluated. The PA and SM histological evaluation was made by ducts count in 30 fields x400 magnification per group and assessment of fibrosis and glycogen in the glandular parenchyma. Metabolic analysis measured the effects of CKD and sympathetic and parasympathetic stimulation of salivation in glycolytic, oxidative and anaplerotic metabolic fluxes using infusions with [U-13C]Glucose or [1,6-13C2]Glucose, [2-13C]Glucose and [U-13C]Fatty acids, using high resolution proton (1H) and carbon 13 (13C) nuclear magnetic resonance (NMR) spectroscopy of glandular extracts as detection technique of 13C incorporation in intermediary metabolites. Enzyme activities were measured: hexokinase [HK], 1-phosphofructokinase (PFK-1), pyruvate kinase (PK), lactate dehydrogenase (LDH), glucose 6-phosphate dehydrogenase (G6PD), amylase, peroxidase and catalase. Furthermore, the malondialdehyde [MDA], glycogen content and calcium, phosphorus and sodium ionic concentrations were quantified. Significant decreases of final body weights, significant increases in serum concentrations and decreases in urinary concentrations of urea and creatinine, decreased creatinine clearance and increased protein excretion were observed in CKD group comparing with the Control and Sham groups. In the evaluation of the renal cortex, a significant decrease in glomerulus count, inflammatory infiltration, tubular atrophy, glomerulosclerosis and interstitial fibrosis were found. In glandular histological evaluation was observed a significant increase in the ducts count and reduced glycogen content in SM gland. Metabolic analysis revealed the prevalence of glycolytic flux in the salivary glands. The salivary stimulus promoted significant increases of 12C-Lactate/12C-Alanine ratio in the Control and Sham groups, except the DRC PA group, which at rest had an increase of the ratio compared to the sham and control groups. In DRC PA group significant decreases in the levels of 12C-Alanine, 12C-Creatine and 12C-Glucose without stimulation and increase of 12C-Acetate with stimulus were observed. In CKD SM were detected increased levels of 12C-acetate and significant decrease of the 13C-Lactate/13C-Acetate indicative of post-stimulus inhibition of oxidative metabolism. These changes in levels of metabolites and their ratios, as well as the enrichment levels 13C denote major changes at the metabolic level in salivary glands due to DRC. In the enzymatic analyzes, at DRC PA gland were found significant decreases of PFK-1 and LDH activities compared to the Control group and significant increases of peroxidase and catalase compared to control and Sham groups. In DRC SM gland was observed a significant decrease in the enzymatic activity of PFK-1 compared to Control group. In addition, significant increases in MDA content in PA and SM and significant increase in sodium concentration were observed in the SM compared to the control and Sham groups. Structural, metabolic, oxidative stress and ionic changes demonstrate the occurrence of glandular dysfunction in CKD, which might be associated with the salivary changes that characterize the disease.

A doença renal crônica (DRC) gera manifestações orais e alterações salivares nos pacientes, entretanto poucos estudos avaliaram os efeitos desta doença nas glândulas salivares. O objetivo deste trabalho foi avaliar os efeitos da nefropatia crônica experimental na morfologia, metabolismo, estresse oxidativo e concentrações iônicas nas glândulas parótida (PA) e submandibulares (SM) de ratos. A nefropatia crônica experimental foi induzida pelos 5/6 de nefrectomia (grupo DRC) e os resultados foram comparados com os grupos Sham e Controle no tempo experimental de 12 semanas. Foram avaliados os pesos corpóreos iniciais e finais, níveis séricos e urinários de ureia e creatinina, a depuração de creatinina e excreção proteica urinária. A morfologia do córtex renal foi avaliada pela contagem de glomérulos em 30 campos x200 de aumento por grupo, além disso, foram avaliadas a presença de glomeruloesclerose e fibrose intersticial no grupo DRC. Nas glândulas PA e SM, foi realizada contagem de ductos em 30 campos x400 de aumento por grupo e avaliação de fibrose e de glicogênio no parênquima glandular. A análise metabólica avaliou os efeitos da DRC e estímulos simpáticos e parassimpáticos da salivação nos fluxos metabólicos glicolítico, oxidativo e anaplerótico utilizando infusões com [U-13C]Glicose ou [1,6-13C2]Glicose, [2-13C]Glicose e [U-13C]Ácidos graxos, e espectroscopia de ressonância magnética nuclear (RMN) de próton (1H) e carbono 13 (13C) em alta resolução de extratos glandulares como técnica de detecção de incorporação de 13C em metabólitos intermediários. Atividades enzimáticas avaliadas foram: hexoquinase [HK], fosfofrutoquinase-1(PFK-1), piruvato quinase (PK), lactato desidrogenase (LDH), glicose-6-fosfato desidrogenase (G6PD), amilase, peroxidase e catalase. Além disso, foram quantificados os conteúdos de malondialdeído [MDA], glicogênio e concentrações iônicas de cálcio, fósforo e sódio. Foram observadas diminuições significativas dos pesos corpóreos finais, aumentos significativos das concentrações séricas e diminuições das concentrações urinárias de ureia e creatinina, diminuição da depuração de creatinina e aumento da excreção proteica no grupo DRC comparado aos grupos Controle e Sham. Na avaliação do córtex renal encontramos diminuição significativa da contagem glomerular, infiltrado inflamatório, atrofia tubular, glomeruloesclerose e fibrose intersticial. Na avaliação histológica glandular foi observado aumento significativo da contagem de ductos e diminuição de glicogênio na glândula SM. Na análise metabólica observamos a prevalência do fluxo glicolítico nas glândulas salivares. O estímulo salivar promoveu aumentos significativos na razão 12C-Lactato/12C-Alanina nos grupos Controle e Sham, com exceção do grupo DRC PA, onde em repouso se observou um aumento, comparando com os grupos Sham e Controle. No grupo DRC PA foram observadas diminuições significativas dos níveis de 12C-Alanina, 12C-Creatina e 12C-Glicose sem estímulo e aumento de 12C-Acetato com estimulo. No DRC SM detectaram-se aumento dos níveis de 12C-acetato e diminuição significativa da relação 13C3-Lactato/13C3-Acetato pós-estímulo indicativos de inibição do metabolismo oxidativo. Estas alterações nos níveis de metabólitos e suas razões, bem como nos níveis de enriquecimento 13C, denotam alterações profundas a nível metabólico nas glândulas salivares devido a DRC. Nas análises enzimáticas foram encontradas na glândula PA diminuições significativas das atividades da PFK-1 e LDH em relação ao grupo Controle e aumentos significativos da peroxidase e catalase comparado aos grupos Controle e Sham. Na glândula SM foi observada diminuição significativa na atividade enzimática da PFK-1 comparado ao grupo Controle. Além disso, foram observados aumentos significativos do conteúdo de MDA nas glândulas PA e SM e aumento significativo da concentração de sódio na glândula SM comparado aos grupos Controle e Sham. As modificações estruturais, metabólicas, oxidativas e iônica evidenciam a disfunção glandular na DRC e podem estar associadas às alterações salivares que caracterizam a doença. / Chronic kidney disease (CKD) promotes oral manifestations and salivary changes in patients, but few studies have evaluated the effects of this disease in the salivary glands. The aim of this study was to evaluate the effects of experimental chronic nephropathy in morphology, metabolism, oxidative stress and ionic concentrations in parotid (PA) and submandibular glands (SM) of rats. Experimental chronic nephropathy was induced by 5/6 nephrectomy (CKD group) and the results were compared with the Sham and Control groups at 12 weeks experimental period. The initial and final body weights, serum and urinary levels of urea and creatinine, creatinine clearance and protein excretion were evaluated. The morphology of the renal cortex was assessed by counting glomeruli in 30 fields x200 magnification per group, moreover, the presence of glomerulosclerosis and interstitial fibrosis in CKD group were also evaluated. The PA and SM histological evaluation was made by ducts count in 30 fields x400 magnification per group and assessment of fibrosis and glycogen in the glandular parenchyma. Metabolic analysis measured the effects of CKD and sympathetic and parasympathetic stimulation of salivation in glycolytic, oxidative and anaplerotic metabolic fluxes using infusions with [U-13C]Glucose or [1,6-13C2]Glucose, [2-13C]Glucose and [U-13C]Fatty acids, using high resolution proton (1H) and carbon 13 (13C) nuclear magnetic resonance (NMR) spectroscopy of glandular extracts as detection technique of 13C incorporation in intermediary metabolites. Enzyme activities were measured: hexokinase [HK], 1-phosphofructokinase (PFK-1), pyruvate kinase (PK), lactate dehydrogenase (LDH), glucose 6-phosphate dehydrogenase (G6PD), amylase, peroxidase and catalase. Furthermore, the malondialdehyde [MDA], glycogen content and calcium, phosphorus and sodium ionic concentrations were quantified. Significant decreases of final body weights, significant increases in serum concentrations and decreases in urinary concentrations of urea and creatinine, decreased creatinine clearance and increased protein excretion were observed in CKD group comparing with the Control and Sham groups. In the evaluation of the renal cortex, a significant decrease in glomerulus count, inflammatory infiltration, tubular atrophy, glomerulosclerosis and interstitial fibrosis were found. In glandular histological evaluation was observed a significant increase in the ducts count and reduced glycogen content in SM gland. Metabolic analysis revealed the prevalence of glycolytic flux in the salivary glands. The salivary stimulus promoted significant increases of 12C-Lactate/12C-Alanine ratio in the Control and Sham groups, except the DRC PA group, which at rest had an increase of the ratio compared to the sham and control groups. In DRC PA group significant decreases in the levels of 12C-Alanine, 12C-Creatine and 12C-Glucose without stimulation and increase of 12C-Acetate with stimulus were observed. In CKD SM were detected increased levels of 12C-acetate and significant decrease of the 13C-Lactate/13C-Acetate indicative of post-stimulus inhibition of oxidative metabolism. These changes in levels of metabolites and their ratios, as well as the enrichment levels 13C denote major changes at the metabolic level in salivary glands due to DRC. In the enzymatic analyzes, at DRC PA gland were found significant decreases of PFK-1 and LDH activities compared to the Control group and significant increases of peroxidase and catalase compared to control and Sham groups. In DRC SM gland was observed a significant decrease in the enzymatic activity of PFK-1 compared to Control group. In addition, significant increases in MDA content in PA and SM and significant increase in sodium concentration were observed in the SM compared to the control and Sham groups. Structural, metabolic, oxidative stress and ionic changes demonstrate the occurrence of glandular dysfunction in CKD, which might be associated with the salivary changes that characterize the disease.