University, Diss., 2005--Heidelberg.

The glial cell-line derived neurotrophic factor (GDNF) and neurturin (NTN) belong to a structurally related family of neurotrophic factors. NTN exerts its effect through a multi-component receptor system consisting of the GDNF family receptor alpha 2 (GFRα2), proto-oncogene RET and/or NCAM. GFRα2 is spliced into at least three isoforms, GFRα2a, GFRα2b and GFRα2c. The present study investigated the expression and functional differences of GFRα2 isoforms. These receptor isoforms are differentially expressed in specific human brain regions. Using Neuro2A model, GDNF and NTN promote neurite outgrowth via GFRα2a and GFRα2c, but not GFRα2b. These GFRα2 isoforms regulate different early response genes when stimulated with GDNF and NTN. Interestingly, using co-expression models, GFRα2b inhibits ligand induced neurites outgrowth of GFRα2a and GFRα2c, and also the related receptor, GFRα1a. More intriguingly, ligands activated GFRα2b was also able to attenuate neurite extension induced by an unrelated stimulation using retinoic acid. MAPK activation induced by GDNF was not attenuated by GFRα2b in a co-expression model, while the early response genes expression profile (up-regulation of FosB) was similar to that induced by GFRα2b alone. This study suggest that GFRα2b is not merely a dominant negative isoform, but signals through a yet to be determined mechanism to antagonize and inhibit neuritogenesis. Together, these data suggest a new paradigm for the regulation of growth factor signaling and neurite outgrowth via an inhibitory splice variant of the receptor. / Singapore-MIT Alliance (SMA)

Th.--Méd.--Nancy 1, 1984. N°: 78.

The glucagon-secreting α-cell is vital for the maintenance of glucose homeostasis and prevention of hypoglycaemia. Despite its importance many aspects of α-cell physiology are disputed. Thus, in this thesis, I aimed to elucidate several features of α-cell function - exploring how autonomic signals are integrated by the cell and how diabetes leads to its dysfunction. The autonomic response to hypoglycaemia results in increased acetylcholine and adrenaline in the islet vicinity, which stimulate glucagon secretion. The mechanisms underlying these effects were investigated using live [Ca²⁺]_i imaging and patch-clamp electrophysiology. Adrenaline was found to target the α-cell via a Î2-adrenergic mechanism, inducing TPC2-mediated Ca²⁺ release from the (endo)lysosomal stores, which triggered calcium-induced calcium release from the endoplasmic reticulum (ER). Acetylcholine also induced ER Ca²⁺-release via muscarinic G_q signalling. However, a component of the effect resulted from activation of a nicotinic pathway that evoked P/Q-type Ca²⁺ channel influx. The glucagon response to hypoglycaemia is lost in diabetes. To investigate the effect of hyperglycaemia on α-cell function at low glucose, the Fh1Î2KO type 2 diabetic mouse model was used. In these mice, prolonged hyperglycaemia led to blunted glucagon secretion at low glucose. Using live pH imaging, it was shown that this was caused by hyperglycaemia increasing flux through Na⁺ coupled glucose transporters (SGLTs), disrupting Na⁺-dependent acid extrusion and inducing cytoplasmic acidification. The resulting build-up of protons was speculated to compromise mitochondrial ATP production leading to the observed glucagon secretory defects. The effects of insulin-induced hypoglycaemia on δ-cell [Ca²⁺]i activity were also investigated. Increased SGLT2 transport and low [K⁺]_o, features of insulin-induced hypoglycaemia, were both shown to increase [Ca²⁺]i activity in δ-cells, stimulating somatostatin secretion and consequently suppressing glucagon secretion. Together these data suggest that glucagon secretion at low glucose is lost due to the combined effects of hyperglycaemia-driven intrinsic dysfunction and insulin-induced somatostatin secretion.

One of the major hallmarks of Parkinson’s disease (PD) is the deposition of intracellular Lewy body inclusions. α-Synuclein is a small protein that accumulates and aggregates to form Lewy bodies. Recent studies uncovered variation of α-synuclein mRNA 3’ untranslated region (UTR), but the role of this region in regulating the α-synuclein expression is poorly understood. 3’UTR is a target region for RNA binding proteins and microRNAs (miRs) in regulating protein translation from the mRNA transcript. Leucine-rich repeat kinase 2 (LRRK2) is a key regulator of miR-mediated translational repression and is frequently mutated and causally associated with PD. We hypothesize that LRRK2 regulates α-synuclein expression post-transcriptionally via binding of miR to α-synuclein mRNA’s 3’UTR. We have found that α-synuclein mRNA with short 3’UTR has similar protein expression level to that of long 3’UTR in the absence of LRRK2 in both HEK-293 FT cells and primary hippocampal neurons. However, LRRK2 wild-type and disease mutant G2019S increased α-synuclein protein expression. In particular, an increase of 2-fold was observed for the short 3’UTR transcript, which is significantly greater than the increase for the long isoform. These data suggest differential effects of LRRK2 on α-synuclein depending on the length of 3’UTR. The short 3’UTR of the α-synuclein transcript has a binding site for miR-7; whereas, that of the long isoform has binding sites for miR-7 and miR-153. We discovered that these differential effects of LRRK2 on α-synuclein are dependent on the binding of miR-7 and miR-153 to the 3’UTR of the isoforms. Specifically, miR-7 is a stronger mediator in regulating α-synuclein translation compared to miR-153, leading to an approximately 30% inhibition of α-synuclein protein expression. Our studies have also shown that the effects of LRRK2 on regulating α-synuclein protein expression are dependent on LRRK2 kinase activity. Gain-of-kinase-function mutation, G2019S, leads to a greater increase of α-synuclein protein expression compared to wild-type; whereas, inhibition of LRRK2 kinase function decreases its effect on α-synuclein protein expression. These findings highlight novel mechanisms regulating the expression of α-synuclein involving LRRK2, miRs-7 and -153. These results highlight miRs as potential targets for reducing levels of α-synuclein in PD.

Two types mechanisms that control gene expression involve cis-regulatory factors and trans-regulatory factors. Cis-acting regulatory RNAs include targeted messenger RNA (mRNA) specificity and AU-rich elements (AREs). AU-rich mRNAs are a subcategory of mRNAs that have AREs in their 3'-Untranslated Regions (UTRs). These ARE-genes have been observed to correlate with rapid mRNA decay patterns. They comprise approximately 12% of all transcripts and are known to encode for a group of proteins that have involvement in the inflammatory response. Trans-acting regulatory mechanisms are micro RNAs (miRNAs) in eukaryotes, and small RNAs (sRNA) in prokaryotes. Misregulation of these mechanisms can lead to many disease states if rapid mRNA decay does not occur, leading to tumorigenesis, and eventually, different types of cancer. In this project, the TNF-α ARE was studied in both serum-positive and quiescent G0 conditions in order to analyze whether the translation of the gene differed in any respect due to the binding of a known miRNA called miR-130a. Additionally, both serum-positive and one-day serum-starved quiescent G0 conditions were analyzed for eIF5B and FXR1 levels to analyze whether there was a correlation between the two proteins.

Paraoxonase 1 (PON1) is an enzyme synthesised in the liver that is associated with High Density Lipoprotein (HDL) and increasingly with a number of human diseases, including cardiovascular disease and cancer. PON1 is (i) a lactonase, hydrolysing aliphatic and aromatic lactones, (ii) a phosphotriesterase, acting on certain organophosphates and (iii) a peroxidase that combats lipid oxidation. The aim of this study was to investigate the extent to which PON1 might impact on the levels of lactone-induced and oxidative DNA damage. Initially, the plasmid DNA (pDNA) nicking assay was used to show that of the 12 lactones and homocysteine (HC) examined, only alpha angelica lactone (AAL), Furanone (FUR) and HC caused DNA single strand breaks (SSB) under physiological conditions. Further studies indicated that AP sites were not involved suggesting that DNA-phosphotriesters were responsible. AAL-reacted pDNA bound the damage sensing protein, Atl1, and AAL, HC and HC thiolactone (HCTL) -reacted calf thymus DNA inhibited the activity of the DNA repair protein, MGMT, both indicating the presence of O6-alkylguanines in DNA, although this could not be confirmed using MALDI-ToF MS analyses of tryptic digests of MGMT incubated with lactone-reacted DNA. The inhibition of rPON1 by these lactones and HC was determined using paraoxon as a substrate and two groups were identified comprising lactones that caused reductions in PON1 activity of (i) ˃15% (e.g. HCTL, and AAL) and (ii) ˂10% (e.g. FUR, and HC). The pDNA nicking assay then showed that only AAL and FUR induced DNA single strand breaks. PON1 itself nicked pDNA, and bound to group 1 lactone-reacted pDNA by an unknown mechanism, both effects not previously reported, but, with the possible exception of AAL, did not increase the extent of plasmid nicking. The MTT, cell viability assay, indicated that all of the lactones with the exception of γ-BL (IC50 >12 mM) were to some degree toxic in HepG2 cells with AAL being the most toxic (IC50 1.0 ±0.03 mM). It was not possible to quantify PON1 activity in HepG2 cells and agents that are reported to change the expression levels of PON1 had no detectable impact on the toxicity of AAL, γ-VL, FUR or MBL, so any possible effect of PON1 could not be determined. The neutral Comet assay showed that AAL and HCTL generated the highest levels of DNA double strand breaks and DNA fragmentation in HepG2 cells, and that this effect was greatly enhanced for most of the lactones by the addition of rPON1. The impact of PON1 on oxidative stress was investigated using serum samples collected as part of a previous case-control study of lung cancer. Initially, an in-house developed ELISA assay to quantify 8-hydroxydeoxyguanosine (8-OHdG) levels had insufficient sensitivity and poor reproducibility. Hence 8OHdG levels were measured using kits along with PON1 levels and other serum parameters including HDL-C, LDL-C, TG and apoAI in serum from 112 patients with lung cancer and 249 patients without. No correlation was found between serum level of PON1 activity and level of 8-OHdG in patients with lung cancer, however a negative non-significant correlation was found between PON1 and 8-OHdG in control. The level of 8-OHdG was significantly higher in lung cancer patients than in controls and in the controls, the OGG1 wild type genotype correlated with reduced levels of 8-OHdG in serum. These studies showed that certain lactones, are toxic and DNA damaging and this can be increased by PON1, suggesting that any association between PON1 and human disease will be substrate dependent and may be PON1 genotype-dependent.

My study was designed to identify the relationships between climate variables, such as precipitation and temperature, and δ 18 O values of tree ring &alpha-cellulose extracted from exactly dated tree rings of Pterocarpus angolensis growing in the arid to semiarid Mzola region of western Zimbabwe. This species is known to be sensitive to seasonal variation in rainfall. In this region, the wet season occurs during the austral summer from mid November to early April followed by a dry winter season from around June through October. Overall, the total annual rainfall exhibits a high degree of spatial and temporal variation with a mean of less than 600 mm per year. I applied the Modified Brendel technique to isolate &alpha-cellulose from raw wood samples extracted from two P. angolensis trees and measured the α-cellulose δ 18 O values using continuous flow isotope ratio mass spectrometry. I developed a 30-year (1955-1984) &alpha-cellulose δ 18 O chronology and correlated it with tree-ring width, meteoric water δ 18 O values, monthly and seasonal precipitation totals, and mean seasonal temperature. The δ 18 O values of meteoric water for this region were obtained from the Global Network of Isotopes in Precipitation (GNIP) and correlated with the δ 18 O values of tree ring &alpha-cellulose. The strongest correlations were observed between &alpha-cellulose δ 18 O values and February total precipitation (r = -0.49, p = 0.006) and to a lesser degree total wet season (NDJFMA) precipitation, In particular, unusually rainy wet seasons were significantly correlated (r = -0.79, P = 0.007) with &alpha-cellulose δ 18 O. This relationship is consistent with 18 O-depleted values measured in summer precipitation during periods of high rainfall, which is most likely the result of the isotopic amount effect reported in tropical regions. I also identified a positive correlation (r = 0.49, p = 0.03) between &alpha-cellulose δ 18 O and the δ 18 O values of meteoric water, and investigated the possibility of an isotopic temperature effect for δ 18 O in meteoric water, which also may be reflected in the δ 18 O values in tree ring &alpha-cellulose. The strongest correlation with mean temperature was observed during the wet summer season (r = 0.56, p = 0.01). My results suggest that the δ 18 O values of P. angolensis tree rings can be used as natural indicators of paleoclimate in southern Africa.

The Oxidative Hydrothermal Dissolution (OHD) process was developed at Southern Illinois University to convert solid organic material to low-molecular-weight, water-soluble products using small amounts of dissolved oxygen in liquid water at high temperature and pressure. The process is environmentally friendly; it does not involve the use of solvents or catalysts, and there is little emission of CO2 and no emission of NOx or SOx. Previous studies of the effects of OHD on organic matter have focused on coal, coal waste, and biomass. This study explores the application of OHD to organic-rich shales, providing a baseline investigation into how highly aliphatic materials react under OHD conditions, what types of products are created during the process, whether the products are economically valuable, and whether they provide novel structural and biomarker information that complements typical bulk organic matter characterization methods. Furthermore, typical oil shale utilization methods are plagued by environmental concerns akin, in some respects, to the environmental concerns associated with the coal industry. The successful application of OHD to these materials would provide a cleaner, more efficient way to process oil shale, resulting in an aqueous product that can be transported through pipelines and refined using conventional processing technology. To examine how OHD affects oil shale kerogen, a series of partial conversion experiments were conducted on an Alpha Torbanite sample at varied temperatures, reaction times, and oxidant inputs. Using a fixed-bed type reactor, over 90% carbon conversion was achieved in just 10-12 minutes under relatively mild reaction conditions (300°C) with little gas production, approximately twice as fast as OHD coal conversion. GC-MS analyses of the product distributions for these experiments demonstrate that they do not change significantly under varying reaction conditions, which can be adjusted for maximum carbon conversion. A sample of each type of oil shale (torbanite: Alpha Torbanite, lamosite: Green River Formation, marinite: New Albany Shale, tasmanite: Tasmanian tasmanite, kuckersite: Decorah Formation, cannel: Cannel King) was reacted and analyzed for product distributions. All of the oil shales produced a complex mixture of aliphatic carboxylic acids, dicarboxylic acids, keto-acids, aromatic acids, and poly-acids, some of which include phenolic structures. These products include materials that are useful as chemical feedstocks for the manufacture of plasticizers, nylons, polymers, lubricants, nylons, paints, and a variety of other materials, most of which are currently produced from petroleum derived precursors. Results of OHD biomarker analysis were not comparable to conventional solvent extraction results. With the exception of the Green River sample which did produce favorable results, analyzed steranes and hopanes were not present in measurable/identifiable amounts in the OHD products. It is unclear whether the biomarker peaks are buried under unresolved products or if the biomarkers are oxidizing/degrading under OHD conditions. However, a comparison of OHD product distributions to pyrolysate product distributions demonstrates that this method provides novel information regarding the original macromolecular structure of the kerogen. Since OHD converts a larger fraction of the original carbon, this approach may provide a more complete/correct representation of the initial structure than conventional methods.

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