Diss.--Technische Hochschule, Brunswick.

Hyperprolactinemia (hyperPRL) is a highly prevalent adverse side effect in antipsychotic therapy as most antipsychotic drugs are dopamine D2 receptor antagonists. Peony-Glycyrrhiza Decoction (PGD, 芍藥甘草湯) is a classic Chinese herbal formula initially used to treat muscle pain and spasm. Our pilot clinical studies have confirmed the effectiveness of PGD in alleviating antipsychotic-induced hyperPRL in patients with schizophrenia. In the present study, we further examined the effects of PGD, its individual herbal preparations and major compounds in suppressing prolactin (PRL) hyperactivity in in vitro and in vivo models and underlying mechanisms. PGD treatment significantly suppressed PRL secretion in MMQ cells, an exemplary model of hyperPRL that is derived from pituitary adenoma cells. PGD also suppressed PRL synthesis of MMQ cells in a dose-dependent manner; however, these suppressive effects were completely abolished by pretreatment with 10 μM haloperidol, a dopamine D2 receptor antagonist. PGD did not affect hyperactive PRL in GH3 cells that lack the D2 receptor expression, but PGD significantly increased the expressions of the D2 receptor and dopamine transporters (DAT) in PC12 cells. In the rat model of hyperPRL produced by repeated injection with the dopamine blocker metoclopramide (MCP), PGD (5 - 10 g/kg daily) treatment for 14 days significantly reduced elevated serum PRL and the reduced magnitude was similar to that of 0.6 mg/kg bromocriptine (BMT), a dopamine D2 receptor agonist used for treating hyperPRL. Both PGD and BMT did not alter serum estradiol, but PGD reversed MCP-induced decreased serum progesterone to control level, whereas BMT did not. Similar to BMT, PGD treatment displayed a great effect in reversing the MCP-induced reduction of the expressions of D2 receptor, DAT and tyrosine hydroxylase in both the pituitary and the hypothalamus, in particular the arcuate nucleus, but both had least effects on the expressions of PRL in the pituitary and hypothalamus. The anti-hyperPRL effects of individual herbal preparation and major compounds of PGD were further examined in cultured cells. The three herbal preparations, Paeoniae Radix (PR) alone, Glycyrrhiza Radix (GR) alone and the pooled PR and GR individual preparation (PR+GR), and the three major constituents 18β-glycyrrhetinic acid (GA), liquiritigenin (LQ) and paeoniflorin (PF), were tested. All these preparations and constituents displayed significant effects in suppressing PRL hyperactivity and enhancing the expressions of dopamine mediators. However, PR had the most robust anti-hyperPRL effects compared to PGD and other preparations and constituents. The present study provides experimental evidence confirming the clinical effects of PGD in suppressing antipsychotic-associated hyperPRL. Not only D2 receptor is involved in the anti-hyperPRL effect of PGD, it is also associated with the modulation of other dopamine mediators and sex hormones. The finding that the magnitudes of the anti-hyperPRL effects of PGD and of combined PR and GR are similar suggests that boiling the herbs together or or separately shows the same effects. Additionally, PR preparation appears to be more efficacious in reducing hyperPRL compared to GR preparation which deserves to be further investigated. / published_or_final_version / Chinese Medicine / Doctoral / Doctor of Philosophy

La información fácilmente obtenible para los suelos agrícolas son textura, contenido de materia orgánica y densidad aparente. Otras variables como la conductividad hidráulica saturada y la cantidad de agua almacenada en relación con el potencial agua del suelo son, en muchas ocasiones, difíciles de medir en el campo. Las funciones de transferencia edafológica (FTE) transforman datos asequibles en aquellos que necesitamos. Los objetivos de este trabajo fueron evaluar la aplicabilidad de FTE disponibles en la literatura a suelos de la zona de La Plata (Argentina) y desarrollar nuevas FTE para estos suelos. Se utilizaron datos obtenidos experimentalmente de retención hídrica, textura y materia orgánica. Las FTE seleccionadas para evaluar su eficacia estimativa en estos suelos fueron dos: una paramétrica (FTE de Saxton et al., 1986) y la otra de estimación puntual (FTE de Rawls et al., 1982). Para la FTE de Saxton et al. (7), en dos de las cuatro tensiones analizadas se encontraron diferencias significativas entre los valores medidos y los estimados. La FTE de Rawls et al. (6) para todas las tensiones estimó valores significativamente diferentes a los medidos. Se generó una FTE a partir de los datos generados de estimación puntual de retención hídrica a las tensiones estudiadas. La misma fue efectiva para las tensiones de 33, 100 y 1500 kPa. / Available data for agricultural soils are texture, organic matter content and bulk density. Determining other variables such as saturated hydraulic conductivity and the relationship between water content and soil-water potential can be expensive and time consuming. Pedotransfer functions (PTF) translates available data into results we need. The objectives of this work were to evaluate the applicability of PTF available in the literature to soils of the area of La Plata (Argentina) and generate new PTF for these soils. Results obtained experimentally from the water characteristic function of soils of La Plata surroundings were used. The PTF selected to evaluate were two: a parametric PTF (PTF of Saxton et al., 1986), and a point estimation PTF ( PTF of Rawls et al., 1982). For the PTF of Saxton et al. (7), there were significant differences between measured and estimated values for two water potentials. The PTF of Rawls et al. (6) estimated values significantly different from those measured for all water potentials tested. A point estimation PTF was generated for La Plata soils which was effective for 33, 100 and 1500 kPa.

Diss. Uppsala : Uppsala universitet, 2007.

Thesis, Basel.

Inaugural-Dissertation--Universität Basel, 1930. / Cover title. Vita. "Biographisches Register": p. 225-236. Bibliography: p. 237-247.

Osteoarthritis (OA) is a complex, multifactorial, and slowly progressive disease where there is currently no effective medical treatment. Research in understanding the mechanisms of OA has been advanced by preclinical studies in rodent models of OA. Recent evidence highlights the role of different classes of lipids in OA pathogenesis. Therefore, the main aim of this thesis was to apply both targeted and untargeted (global) lipidomics mass spectrometry based an alytical methods, in conjunction with univariate and multivariate statistical analysis, in various tissues from three established rodent models of OA; meniscal transection (MNX), monosodium iodoacetate (MIA), and destabilization of the medial meniscus (DMM). The overall goal was to identify statistically differentiated lipids between controls versus OA rodents that may reflect changes in the pathophysiology of OA and associated pain. In addition, a global lipidomics workflow was developed by me, following the latest trends used within the wider metabolomics community, ensuring robustness and reproducibility in the identification of putative metabolite/lipid biomarkers for diseases. Experiments in this thesis using a targeted oxylipin liquid chromatography tandem mass spectrometry (LC-MS/MS) method showed that statistical significant changes in the levels of certain oxylipins were observed. More specifically, 11,12-DHET (mean concentration: 0.26 pmol/g in control, 0.54 pmol/g in MNX; p < 0.01), 14,15-DHET (0.46 pmol/g in control, 0.75 pmol/g in MNX; p < 0.05) and 8-HETE (5.46 pmol/g in control, 7.40 pmol/g in MNX; p < 0.05) were statistically increased in the MNX compared to control (sham) rats in ventral spinal cord in the MNX rat model of OA. These findings are supported by literature since these three lipids exhibit pro-inflammatory properties and thus are expected to increase in the OA group where inflammation is the main feature of OA. Regarding the MIA rat model levels of other oxylipins in synovial fluid were differentially expressed in the MIA compared to saline (control) rats. Arachidonic acid (AA), (272.3 pmol/g in control, 435.3 pmol/g in MIA; p < 0.05) was increased in the MIA-treated compared to saline-treated rats, while 9-HODE (4.42 pmol/g in control; 1.21 pmol/g in MIA; p < 0.05) was statistically decreased in the MIA compared to saline rats. Since AA has been reported to be released from membrane phospholipids in OA, the observation that AA is statistically increased in synovial fluid in MIA- compared to saline-treated rats bears strong significance. In addition, maps of oxylipins metabolism were generated to visualize the pathways underlying the changes of lipid concentrations in plasma between control and OA rats for both MNX and MIA rat models. Therefore, applying a targeted oxylipin LC-MS/MS method in different tissues of MNX and MIA rat models of OA is a successful approach and informative about changes in pathophysiology of OA, underlying significant alterations in oxylipins concentrations. Although the global lipidomics approach was able to measure different classes of lipids that might account for differences in plasma between MNX/MIA and sham/saline-treated rats, this approach exhibited weak MVA (multivariate analysis) models. In contrast to MNX and MIA rat models, the global LC-MS lipidomics profile in plasma from a DMM mouse model of OA exhibited excellent MVA models with good prediction scores. Twenty-six statistically significant lipids were identified, using the lipidomics workflow that I have developed, and when four of these lipids were used to build Receiver Operative Curves (ROC) the model produced high prediction (84%) power in separating sham from DMM mice. The identity of these four lipids was classified as being fatty acids (FAs), sterols, sphingolipids, and diacylglycerols (DAG). In addition, MS/MS experiments were performed to confirm the identity of significant lipids. Thus, it was shown herein that applying a global lipidomics LC-MS approach in plasma from the mouse DMM model, using only a small number of mice (15 in total), can be informative about significant changes in the “lipidome” in OA and can be used as a robust means of predicting OA in mice based on their global lipidomics profile. Lastly, correlation statistical analysis was applied between levels of lipids in the various tissues, pain behaviour, and histopathology parameters in the three rodent models of OA. Although many oxylipins/lipids levels were found to be statistically correlated with the aforementioned parameters, the most striking finding is that 9-HODE and AA were both found to be positively correlated with Weight Bearing (WB), a parameter of pain behaviour, in plasma and synovial fluid in the MIA rat model of OA. Since plasma reflects systemic inflammation and synovial fluid reflects local (inflammation) 9-HODE (p < 0.01 in plasma; p < 0.05 in synovial fluid) and AA (p < 0.01 in plasma and synovial fluid) are oxylipins that potentially depict systemic and local changes in WB differences, and subsequently in OA related pain. This finding is supported by literature since both AA and 9-HODE are both agonists of a pain receptor (i.e. transient receptor potential vanilloid 1, TRPV-1). Thus, it was proved in this thesis that correlation analysis can be used as an additional and complementary statistical tool in an effort to determine the role of lipids in OA pathogenesis in rodent models of OA. In conclusion, applying both targeted oxylipin LC-MS/MS and global lipidomics LC-MS analytical methods capable of measuring either oxylipins or the whole “lipidome” in vivo, have provided novel findings to support the involvement of these lipids in OA and associated pain.

Drug delivery systems in the size range of ~ 10-250 nm are enabling tools for site-specific targeting and controlled release applications. To take advantage of these capabilities, various nanocarriers e.g., micelles, dendrimers, liposomes, nanoparticles, nanocapsules, nanotubes, and nanogels, have been designed for drug delivery. Specifically, micelle-based drug carrier systems have emerged as promising tools for site-specific delivery and controlled release applications. Despite several advantages over conventional drugs, some limitations of micelle-based drug delivery have also been reported. These drawbacks include low stability in vivo, poor penetration, modest accumulation in tumour tissues, and inadequate control over drug release. To overcome these limitations, stimuli-responsive or smart polymeric nanocarriers have been developed for drug delivery and tumour therapy, previously. The most well-known internal stimuli in cancerous regions include higher acidity associated with dysregulated metabolism in tumour tissues, elevated levels of glutathione in the cytosol and nucleus of cancer cells, and altered degradative enzymes in the lysosomes, and reactive oxygen species in the mitochondria. These intrinsic microenvironments can be exploited as internal stimuli to attain active drug release in the tumour tissues or cancer cells. In particular, the reducing potential inside the cancer cells is considerably higher than found in the extracellular environment and bloodstream. Such varying redox potential can be exploited for tumour-specific drug delivery and controlled release applications. Various types of redox-responsive micelles have been developed, previously. Generally, redox-responsive micelles have disulfide linkages that undergo rapid cleavage in the presence of reducing agents in the intracellular components, however, are stable at oxidising extracellular environment. The redox-responsive disulfide bridges can be incorporated into nanocarriers by placing multiple disulfide bonds in the hydrophobic backbone or by conjugating therapeutic agents to the side chain of the polymer via a disulfide linker. Another strategy to construct redox-responsive linkages is to crosslink the polymeric nanocarriers with a disulfide crosslinker. Studies have shown that polymeric micelles can dissociate, especially upon administration when they are diluted below their critical micelle concentration. The stability of polymeric micelles can be enhanced by chemical crosslinking. Various types of crosslinked micelles can be prepared subjected to the localisation of the crosslinking, e.g. shell crosslinked micelles, and core crosslinked micelles. Introducing redox-responsive bridges through disulfide crosslinkers may not only provide stability to nano-carriers against dilutions during circulation, but also render them responsive to reducing conditions. Specifically, redox-responsive core-crosslinked micelles have demonstrated good stability and better ‘stealth’ properties, nevertheless, the hydrophobic core of most of the existing core-crosslinked micelles have been based on non-degradable polymers such as polyacrylamide or polyacrylate. The non-degradable constituent of the block copolymer may cause complications in clinical applications. Therefore, reduction-responsive core-crosslinked micelles comprising entirely of biologically inert or biocompatible and biodegradable polymers would be better candidates for drug delivery and controlled release application. To overcome these limitations, micelles based on polyesters (a class of aliphatic biodegradable polymers) can used for drug delivery application. In the last few decades, various FDA approved aliphatic polyesters e.g. poly(lactic-co-glycolic acid) (PLGA), poly(ε-caprolactone), and poly(lactic acid), have been intensively studied to exploit their potential in drug, gene and protein delivery and controlled release applications. Nevertheless, most of these polyesters lack functional groups, making it difficult to incorporate redox-responsive linkages to core-crosslink their micelles. To address these issues, we have synthesised functional biodegradable and biocompatible block copolymers based on methoxypoly(ethyleneglycol)-b-poly(-caprolactone-co--azido--caprolactone) (mPEG-b-poly(CL-co-N3CL)). The pendent chloro groups of the block copolymer were converted into azides using nucleophilic substitution reaction to obtain mPEG-b-poly(CL-co-N3CL) block copolymer as a precursor of reactive polymeric micelles. The synthesised polymers were characterised by NMR, FT-IR and size exclusion chromatography (SEC). Micelles were prepared using the dialysis method and methotrexate (an anticancer drug) was loaded into the hydrophobic core of the reactive micelles. Micelles were subsequently crosslinked by a redox-responsive bis-alkyne ethyl disulfide crosslinker. The size distributions and morphology of core-crosslinked micelles were assessed using dynamic light scattering (DLS) and transmission electron microscopy. The drug release studies were performed under simulated non-reducing and reducing conditions. Cellular uptake studies in human breast cancer cells (MCF7 cells) were performed using Oregon-green loaded core-crosslinked micelles. The MTX-loaded core-crosslinked micelles were assessed for their cytotoxicity in human breast cancer cells by MTT assays. The apoptosis inducing potential of MTX-loaded core-crosslinked micelles was analysed using Hoechst/PI assays and was further probed by annexin-V/PI assays. The data from these studies indicate that drug release from these crosslinked micelles can be controlled and that redox-responsive micelles are more effective carriers for MTX than non-cross-linked analogues in the cell lines tested. In another strategy, a multifunctional amphiphilic block copolymer based on -amine-PEG-b-poly(CL-co-N3CL) was synthesised and subsequently was used to conjugate methotrexate on the hydrophilic block for receptor mediated targeting of breast cancer cells. Cellular uptake studies revealed 2.3-fold higher uptake of MTX-conjugated micelles as compared with un-conjugated micelles. The blank micelles showed low cytotoxicities in breast cancer cells, however, MTX-conjugated micelles exhibited greater antitumor activity in contrast to the free-MTX. We hypothesise that these functional micelles could be potentially powerful nanocarriers for stimuli-responsive controlled release, active tumour targeting, and cancer therapy.

The aim of this project was to prepare, optimize, characterize and compare a subcutaneous/oral delivery system for insulin. The effect of various low molecular weight chitosans (LMWCs) on the stability of insulin, using USP HPLC methods, was investigated. Insulin was found to be stable in a polyelectrolyte complex (PEC) consisting of insulin and LMWC in the presence of Tris-buffer at pH 6.5. In the presence of LMWC, the stability of insulin increased with decreasing molecular weight of LMWC; 13 kDa LMWC was the most efficient molecular weight for enhancing the physical and chemical stability of insulin. The bioactivity of insulin in the PEC was assessed using enzyme-linked immunosorbent assay (ELISA) testing; results showed that insulin is still functionally active in the presence of chitosan. Solubilization of the PEC in a reverse micelle formulation (RMF) and administration to diabetic rats resulted in an oral delivery system for insulin with acceptable bioactivity. The effect of reduced (GSH) and oxidized (GSSG) glutathione on the bioactivity of insulin was studied. A PEC of insulin with low molecular weight chitosan (13 kDa) was prepared and characterized. The PEC was then solubilized, in the presence and absence of GSH and GSSG, in a reverse micelle consisting of oleic acid and two surfactants (labrasol and plurol). The in vitro and in vivo performances of the reverse micelle formulations (RMFs) were evaluated in rats. At pH 6.5 the association efficiency of the PEC was 76.2%. In vitro insulin release from the RMs was negligible at pH 1.2 and was markedly increased at pH 6.8. The hypoglycemic activity of insulin in the PEC was reduced when administered via the subcutaneous route, regardless of the GSH content. On the other hand, the presence of GSSG significantly enhanced hypoglycaemia. When the RMF was administered via the oral route, the presence of GSH had no effect on the hypoglycemic activity of insulin compared with the GSH free system. However, the presence of GSSG in the oral preparation increased the hypoglycemic activity of insulin; probably by inhibiting insulin degradation, thereby prolonging its effect. Thus, incorporation of GSSG in the RMF reduces blood glucose levels in rats and protects insulin from degradation. The effect of glucosamine HCl (GlcN⋅HCl) on the bioactivity of insulin, administered via subcutaneous (SC) and oral routes, in rats was also investigated. The oral insulin delivery system (IC-RM) was prepared by solubilizing insulin-chitosan (13 kDa) polyelectrolyte complex (IC-PEC) in a RM system consisting of oleic acid, PEG-8 caprylic/capric glycerides and polyglycerol-6-dioleate. The blood glucose levels were measured using a blood glucose meter. The results revealed that the extent of hypoglycemic activity of SC insulin was GlcN⋅HCl dose dependent when they were administered simultaneously. A significant reduction in blood glucose level (p < 0.05) was found for the insulin:GlcN⋅HCl at mass ratios of 1:10 and 1:20, whereas lower ratios (e.g. 1:1 and 1:4) showed no significant reduction. Furthermore, enhancement of the action of SC insulin was achieved by oral administration of GlcN⋅HCl for five consecutive days prior to insulin injection (p < 0.05). For oral insulin administration via the IC-RM system, the presence of GlcN-HCl increased the hypoglycemic activity of insulin (p < 0.05). The relative pharmacological availabilities (PA) were 6.7% and 5.4% in the presence and absence of GlcN⋅HCl (i.e. the increase in the relative PA was about 23% due to the incorporation of GlcN⋅HCl in the IC-RM system), respectively. The aforementioned findings offer an opportunity to incorporate GlcN⋅HCl in oral insulin delivery systems in order to enhance a reduction in blood glucose levels.

The last two decades have seen an increased drive to administer parenteral nutrition (PN) to patients in their home environments, thereby reducing associated hospital costs and improving patient quality of life. The occurrence of deranged nutritional biochemistry results has baffled PN experts for years because PN additives are marketed for the general needs of patients and PN is tailored to each patient’s requirements (both formulation and regimen). This thesis documents the investigations into HPN population characteristics, the extent of nutritional abnormalities (deficiencies and excesses) in a cohort of LT PN patients in Wales. Both cross-sectional and longitudinal retrospective study designs were employed alongside small-scale laboratory efforts to investigate stability of vitamin D in PN additives using High Performance Liquid Chromatography (HPLC). Characteristics of the HPN population in Wales were shown to be variable in terms of PN requirements for a predominantly female sample population (2:1); in whom 78.6% of patients received PN for indications relating to short bowel syndrome (SBS). A database analysis of micronutrient test results revealed a high prevalence of deficiencies of vitamin D and selenium, as well as excesses of manganese and water-soluble vitamins; which can lead to clinically relevant effects in patients. The sample population was shown to have impaired bone health since first receiving PN; respective sites of the femoral neck and total hip presented 58% and 60.8% of patients had osteopenia, while 28% and 19.6% had osteoporosis. Evidence in the literature links these clinical outcomes of metabolic bone disease (MBD) to patients’ inadequate vitamin D status. A final study exploring the adequacy of the trace element (TE) preparation Additrace®, found it lacking in selenium and excessive in manganese for the general requirements of the PN population. Clinician-directed supplementation of PN outside of Additrace® was associated with better micronutrient status in patients and more test results within range.