Eicosapentaenoic acid (EPA) is a polyunsaturated fatty acid (PUFA) with neuroprotective and cardioprotective properties. It is thought that some of the actions of EPA may be attributed to its elongated metabolite, the PUFA docosapentaenoic acid (DPA). Docosahexaenoic acid (DHA) and arachidonic acid (AA) are bioactive PUFA ubiquitously expressed in neural tissues. EPA and AA can be converted by cyclooxygenase (COX) to prostanoids and by lipoxygenase (LOX) to hydroxy fatty acids. PUFA can also be converted to ethanolamides in the brain. These mediators are involved in physiological and pathological processes in many bodily systems. The purpose of this study was to examine the production of eicosanoids, hydroxy fatty acids and fatty acid ethanolamides in young and aged rat brain following EPA or DPA enriched diets. The effects of specific PUFA on human platelet eicosanoid production were also investigated as these mediators play a role in adhesion and aggregation. Liquid chromatography coupled to tandem mass spectrometry (LC/ESI-MS/MS) assays were developed and used to measure lipid mediators in rat brain and human platelets. Ageing in rat brain was accompanied with several changes in the prostanoid and hydroxy fatty acid profiles. Supplementing the diet with EPA or DPA at a daily dose of 200 mg/kg for 8 weeks prevented these changes and decreased levels of PGE2. DPA changed the profile of hydroxy fatty acids synthesised in the brain tissue of young animals. This study has shown that levels of eicosapentaenoylethanolamide (EPA-EA) increase in the brain as a result of ageing and that this is accompanied by an increase in levels of anandamide. Feeding aged animals EPA or DPA further increased the levels of EPA-EA but prevented any change in the level of anandamide. Niacin is used to treat hypercholesterolaemia although it is associated with an unpleasant PGD2 mediated skin flush. This exploratory study has shown that human platelets treated with niacin did not show any changes in their prostanoid and hydroxy fatty acid profiles. Platelets treated with EPA showed increased production of TXB3 and 12-HEPE. Niacin augmented the effects of EPA on human platelet mediator synthesis. Overall, this study has demonstrated that EPA can change brain and platelet lipid mediator synthesis and has provided evidence that could explain some of the neuroprotective and cardioprotective actions of this PUFA.

No / Drugs which upregulate astrocyte glutamate transport may be useful neuroprotective compounds by preventing excitotoxicity. We set up a new system to identify potential neuroprotective drugs which act through GLT-1. Primary mouse striatal astrocytes grown in the presence of the growth-factor supplement G5 express high levels of the functional glutamate transporter, GLT-1 (also known as EAAT2) as assessed by Western blotting and (3)H-glutamate uptake assay, and levels decline following growth factor withdrawal. The GLT-1 transcriptional enhancer dexamethasone (0.1 or 1 muM) was able to prevent loss of GLT-1 levels and activity following growth factor withdrawal. In contrast, ceftriaxone, a compound previously reported to enhance GLT-1 expression, failed to regulate GLT-1 in this system. The neuroprotective compound riluzole (100 muM) upregulated GLT-1 levels and activity, through a mechanism that was not dependent on blockade of voltage-sensitive ion channels, since zonasimide (1 mM) did not regulate GLT-1. Finally, CDP-choline (10 muM-1 mM), a compound which promotes association of GLT-1/EAAT2 with lipid rafts was unable to prevent GLT-1 loss under these conditions. This observation extends the known pharmacological actions of riluzole, and suggests that this compound may exert its neuroprotective effects through an astrocyte-dependent mechanism.

No / Flavonoids are plant-derived polyphenolic compounds with neuroprotective properties. Recent work suggests that, in addition to acting as hydrogen donors, they activate protective signalling pathways. The anti-oxidant response element (ARE) promotes the expression of protective proteins including those required for glutathione synthesis (xCT cystine antiporter, gamma-glutamylcysteine synthetase and glutathione synthase). The use of a luciferase reporter (ARE-luc) assay showed that the dietary flavan-3-ol (-)epicatechin activates this pathway in primary cortical astrocytes but not neurones. We also examined the distribution of NF-E2-related factor-2 (Nrf2), a key transcription factor in ARE-mediated gene expression. We found, using immunocytochemistry, that Nrf2 accumulated in the nuclei of astrocytes following exposure to tert-butylhydroquinone (100 microM) and (-)epicatechin (100 nM). (-)Epicatechin signalling via Nrf2 was inhibited by wortmannin implicating a phosphatidylinositol 3-kinase-dependent pathway. Finally, (-)epicatechin increased glutathione levels in astrocytes consistent with an up-regulation of ARE-mediated gene expression. Together, this suggests that flavonoids may be cytoprotective by increasing anti-oxidant gene expression.

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 187 pages. Includes vita. Includes bibliographical references.

Les maladies à expansion de polyglutamine sont un ensemble de troubles neurodégénératives héréditaires se développant lorsqu’il y a répétitions de trinucléotides CAG dans les gènes causatifs au-delà d’un certain seuil. L’expansion des répétitions de trinucléotides CAG entraîne des désordres neurologiques héréditaires précoces, dont de multiples formes d’ataxie spinocérébelleuse (SCA). Parmi celles-ci, le type le plus commun et dominant est l’ataxie spinocérébelleuse de type 3 (SCA3), aussi connue sous le nom de la maladie de Machado-Joseph (MMJ). Ce dernier est un désordre neurologique progressif autosomique dominant. Le gène causatif de MMJ est ATXN3 (ATAXINE-3). Plusieurs études récentes suggèrent une association entre ce gène et la modulation du stress du réticulum endoplasmique (RE). Lors de ce travail de maîtrise, des souches transgéniques de C. elegans exprimant les formes sauvage et mutante du gène ATXN3 humain ont été générées. Les résultats suggèrent des phénotypes importants chez la souche transgénique mutante associés à la pathologie humaine: défaut de motilité, longévité réduite et profil neurodégénératif considérable. Ceci dit, ces résultats nous ont poussé à vouloir déterminer si l’utilisation des composés chimiques, connus en tant que modulateurs du stress du RE et possédant des rôles neuroprotecteurs, sont capables de restaurer les phénotypes notés. Les composés utilisés, c’est-à-dire le Bleu de Méthylène, le Salubrinal et le Guanabenz, ont démontré une capacité de corriger les phénotypes rapportés dans la souche transgénique mutante. De plus, ces composés ont aussi été en mesure de prévenir une augmentation du niveau du stress oxydatif et de la réponse au stress du RE exhibé chez les vers mutants. Par le développement de nouveaux modèles C. elegans pour la MMJ, où il y a expression du gène ATXN3 complet dans les motoneurones, il a été possible de trouver qu’une modulation chimique du stress du RE peut réduire considérablement la neurodégénérescence et par conséquent, être une possible nouvelle approche thérapeutique pour traiter cette pathologie. / Polyglutamine expansion diseases are a class of dominantly inherited neurodegenerative disorders that develop when a CAG repeat in the causative genes is unstably expanded above a certain threshold. The expansion of trinucleotide CAG repeats causes hereditary adult-onset neurodegenerative disorders such as multiple forms of spinocerebellar ataxia (SCA). The most common dominantly inherited spinocerebellar ataxia is the type 3 (SCA3) also known as Machado-Joseph disease (MJD), an autosomal dominant, progressive neurological disorder. The gene causing MJD is ATXN3 (ATAXIN-3): MJD is caused by an abnormal CAG trinucleotide repeat expansion in the ATXN3 gene. Several recent studies have shown that this gene is associated with endoplasmic reticulum (ER) stress. In this study, we generated transgenic C. elegans strains expressing wild type or mutant human ATXN3 genes and tested them for recovery of locomotor phenotype, lifespan and neurodegeneration phenotypes upon treatment with compounds known to modulate ER stress and having neuroprotective roles. We observed differences between both transgenic lines and found that the motility defects, the reduced lifespan and the neurodegeneration can be rescued by methylene blue, guanabenz and salubrinal. These compounds were also able to prevent the oxidative stress and the ER stress response induced by mutant transgenic worms. We introduce novel C. elegans models for MJD based on the expression of full-length ATXN3 in GABAergic motor neurons. Using these models we discovered that chemical modulation of the ER unfolded protein response reduced neurodegeneration and could be a new therapeutic approach for the treatment of MJD.

<p>Glavni cilj ove doktorske disertacije je određivanje hemijskog sastava, biolo&scaron;kih i funkcionalnih karakteristika novih proizvoda od zove. Dobijanje proizvoda na bazi zove zasniva se na primeni tradicionalnih i savremenih tehnolo&scaron;kih procesa proizvodnje. Iskori&scaron;ćenje prirodnog potencijala zove započeto je primenom tradicionalne i savremene (liofilizacija) tehnike su&scaron;enja. U cilju dobijanja visoko-vrednih ekstrakata ploda i cveta zove primenjene su tradicionalna (maceracija) i savremene (ultrazvučna i mikrotalasna) ekstrakcione tehnike sa dva ekstragensa (50% etanol i voda). Dobijanje matičnog soka od plodova zove podrazumevalo je primenu tradicionalnog načina ceđenja, dok je vino od plodova zove dobijeno po standardnom postupku proizvodnje vina. Vino je izloženo različitim temperaturnim tretmanima u različitom vremenskom periodu (60 &deg;C u toku 5 minuta, 60 &deg;C u toku 10 minuta, 70 &deg;C u toku 5 minuta i bez toplotnog tretmana) u cilju evaluacije biolo&scaron;ke aktivnosti dobijenog proizvoda. Etarsko ulje ploda i cveta zove je dobijeno hidrodestilacijom. Ispitivanje efikasnosti primenjenih tehnolo&scaron;kih postupaka su&scaron;enja i ekstrakcije je zasnovano na određivanju biolo&scaron;kih i funkcionalnih karakteristika dobijenih ekstrakata ploda i cveta zove. U ispitivanim ekstraktima ploda i cveta zove dominantne fenolne kiseline su hlorogenska i protokatehinska kiselina, a rutin i kvercetin-3-O-heksozid su dominantna flavonoidna jedinjenja. Biolo&scaron;ke i funkcionalne karakteristike su ispitane primenom različitih in vitro antioksidativnih, neuroprotektivnih, antitirozinaznih i antidijabetogenih testova. Primenom liofilizacije kao savremene tehnike su&scaron;enja i mikrotalasne ekstrakcije kao savremene ekstrakcione tehnike povećava se biopotencijal ispitivanih ekstrakata. Matični sok od plodova zove kao potencijalno novi funkcionalni proizvod je analiziran u cilju definisanja hemijskog, fitohemijskog i nutritivnog sastava, biolo&scaron;kog potencijala i senzorskih karakteristika. Ispitivanja dobijenog vina su bila usmerena na utvrđivanje optimalnih uslova za proizvodnju voćnog vina. Definisanjem hemijskog i fitohemijskog sastava i evaluacijom biopotencijala vina određen je optimalan temperaturni profil za dobijanje jednog od novih funkcionalnih proizvoda. Na osnovu utvrđenog hemijskog sastava etarsko ulje ploda i cveta zove se pokazalo kao potencijalno novi prirodni agens za održavanje svežine i produženja roka trajanja prehrambenih proizvoda. Zova je nesumnjivo samonikla biljna vrsta koja u budućnosti osnovano može biti polazna sirovina za kreiranje i dobijanje novih prehrmabenih proizvoda na domaćem i inostranom trži&scaron;tu.</p> / <p>The main goal of this doctoral dissertation is to determine the chemical composition, biological and functional characteristics of new elderberry products. Elderberry products were obtained via traditional and modern technological processes. The exploitation of the natural potential of the elderberry started with the application of traditional and modern (lyophilization) drying techniques. To obtain high-value extracts of fruits and flowers, traditional (maceration) and modern (ultrasonic and microwave) extraction techniques with two solvents (50% ethanol and water) were applied. Obtaining the juice from the elderberry fruits implied the application of the traditional cold pressing method, whereas wine from the elderberry fruits was obtained in accordance with the standard procedure of wine production. The wine was exposed to different temperature treatments in different periods (60&deg;C for 5 minutes, 60&deg;C for 10 minutes, 70&deg;C for 5 minutes and without heat treatment) to evaluate the biological activity of the product. The essential oil of the fruit and flower was obtained by hydrodistillation. The examination of the efficiency of the applied technological procedures of drying and extraction is based on observing the biological and functional characteristics of the obtained extracts of the said fruits and flowers. In the examined fruit and flower extracts, the dominant phenolic acids are chlorogenic and protocatechuic acid, while rutin and quercetin-3-O-hexoside are the dominant flavonoid compounds. Biological and functional characteristics were examined using various in vitro antioxidant, neuroprotective, antityrosinase, and antidiabetic tests. The application of lyophilization and microwave extraction (as modern drying and extraction techniques) increased the biopotential of the analyzed extracts. Elderberry juice, a potentially new functional product, was analyzed to define the chemical, phytochemical and nutritional composition, biological potential, and sensory characteristics. The wine was tested in order to determine the optimal conditions for the production of fruit wine. By defining the chemical and phytochemical composition and evaluating the biopotential of wine, the optimal temperature profile for obtaining one of the new functional products was determined. Based on the obtained chemical composition, it is determined that the essential oil of fruits and flowers is a potentially new natural agent for maintaining freshness and extending the shelf life of food products. Without a doubt, elderberry is a wild plant species that could be used in the future as the starting material for creating and obtaining new food products on the domestic and foreign markets.</p>

Indiana University-Purdue University Indianapolis (IUPUI) / Human traumatic spinal cord injuries (SCI) are primarily incomplete contusion or compression injuries at the cervical spinal level, causing immediate local tissue damage and a range of potential functional deficits. Secondary damage exacerbates initial mechanical trauma and contributes to function loss through delayed cell death mechanisms such as apoptosis and autophagy. As such, understanding the dynamics of cervical SCI and related intracellular signaling and death mechanisms is essential. Through behavior, Western blot, and histological analyses, alterations in phosphatase and tensin homolog (PTEN)/phosphatidylinositol-3-kinase (PI3K) signaling and the neuroprotective, functional, and mechanistic effects of administering the protein tyrosine phosphatase (PTP) inhibitor, potassium bisperoxo (picolinato) vanadium ([bpV[pic]) were analyzed following cervical spinal cord injury in rats. Furthermore, these studies investigated the combination of subacute Schwann cell transplantation with acute bpV(pic) treatment to identify any potential additive or synergistic benefits. Although spinal SC transplantation is well-studied, its use in combination with other therapies is necessary to complement its known protective and growth promoting characteristics. v The results showed 400 μg/kg/day bpV(pic) promoted significant tissue sparing, lesion reduction, and recovery of forelimb function post-SCI. To further clarify the mechanism of action of bpV(pic) on spinal neurons, we treated injured spinal neurons in vitro with 100 nM bpV(pic) and confirmed its neurprotection and action through inhibition of PTEN and promotion of PI3K/Akt/mammalian target of rapamycin (mTOR) signaling. Following bpV(pic) treatment and green fluorescent protein (GFP)-SC transplantation, similar results in neuroprotective benefits were observed. GFP-SCs alone exhibited less robust effects in this regard, but promoted significant ingrowth of axons, as well as vasculature, over 10 weeks post-transplantation. All treatments showed similar effects in forelimb function recovery, although the bpV and combination treatments were the only to show statistical significance over non-treated injury. In the following chapters, the research presented contributes further understanding of cellular responses following cervical hemi-contusion SCI, and the beneficial effects of bpV(pic) and SC transplantation therapies alone and in combination. In conclusion, this work provides a thorough overview of pathology and cell- and signal-specific mechanisms of survival and repair in a clinically relevant rodent SCI model.

No / The alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR) subunit GluR2, which regulates excitotoxicity and the inflammatory cytokine tumour necrosis factor alpha (TNFalpha) have both been implicated in motor neurone vulnerability in amyotrophic lateral sclerosis/motor neurone disease. TNFalpha has been reported to increase cell surface expression of AMPAR subunits to increase synaptic strength and enhance excitotoxicity, but whether this mechanism occurs in motor neurones is unknown. We used primary cultures of mouse motor neurones and cortical neurones to examine the interaction between TNFalpha receptor activation, GluR2 availability, AMPAR-mediated calcium entry and susceptibility to excitotoxicity. Short exposure to a physiologically relevant concentration of TNFalpha (10 ng/mL, 15 min) caused a marked redistribution of both GluR1 and GluR2 to the cell surface as determined by cell surface biotinylation and immunofluorescence. Using fura-2-acetoxymethyl ester microfluorimetry, we showed that exposure to TNFalpha caused a rapid reduction in the peak amplitude of AMPA-mediated calcium entry in a PI3-kinase and p38 kinase-dependent manner, consistent with increased insertion of GluR2-containing AMPAR into the plasma membrane. This resulted in a protection of motor neurones against kainate-induced cell death. Our data therefore, suggest that TNFalpha acts primarily as a physiological regulator of synaptic activity in motor neurones rather than a pathological drive in amyotrophic lateral sclerosis.