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Stress-related psychological ill health has increased dramatically in Europe. A diagnosis equivalent to occupational burnout can be found in the Swedish version of the tenth edition of the “International Statistical Classification of Diseases and Related Health Problems" by the World Health Organization. The Swedish National Board of Health and Welfare lists treatment suggestions including a section of self-care that recommended something that could be translated to “a sensible diet” (“vettig kost”) without providing evidence for what could constitute a sensible diet. By using the hypothesis of burnout being a stress-mediated decrease in neurogenesis which in turn decrease the ability to cope with stress, this article systematically reviews the efficacy of dietary polyphenols on neurogenesis in rodents to evaluate if dietary polyphenols could constitute a part of a sensible diet for burnout patients. Dietary polyphenols significantly increased various parts of neurogenesis, in rodents subjected to stressors, in some cases demonstrating effect sizes comparable to antidepressants. Adverse effects have been observed in extremely high doses and young rodents not exposed to induced stressors with a putative high level of neurogenesis.

Alzheimer disease (AD) is a progressive neurodegenerative disorder pathologically characterized by deposition of ß-amyloid (Aß) peptides as plaques in the brain. Central to this AD pathology is mismetabolism of the amyloid precursor protein (APP). Recent studies suggest that flavonoids, a class of secondary plant metabolites, may be useful for the prevention and treatment of a variety of neurodegenerative diseases. The studies detailed herein, investigate the ability of two such classes of flavonoids, green tea derived catechins and 5,7-dihydroxyflavones, to modulate APP metabolism in "Swedish" mutant APP (APPsw) models of AD. Studies showed that green tea derived (-)-epigallocatechin-3-gallate (EGCG) effectively reduced Aß generation and resultant amyloidosis both in vitro and in vivo. In concert with these findings, EGCG markedly promoted non-amyloidogenic APP proteolysis via activation of the putative a-secretase, a-disintegrin-and-metalloprotease-10 (ADAM10). Furthermore, luteolin and various related 5,7-dihydroxyflavones, effectively reduced Aß generation and resultant amyloidosis both in vitro and in vivo, as well. Data revealed that luteolin decreased amyloidogenic γ-secretase APP proteolysis via presenilin-1 (PS1) carboxyl-terminal fragment (CTF) phosphorylation. Elucidation of these flavonoids' cellular/molecular mechanisms also revealed their potential for opposing neurofibrillary tangle (NFT) pathology, another hallmark of AD. These data raise the possibility that flavonoid administration to AD patients may prove to be viable and effective prophylactic strategy.

Les maladies inflammatoires du système nerveux central (SNC) sont caractérisées par l'altération de la barrière hémato-encéphalique induite par les cellules immunitaires et les cellules tumorales. Il est reconnu que les macrophages peuvent induire l'inflammation en infiltrant la BHE. Lors d'un dommage ou d'une infection du SNC, les macrophages dérivés du sang sont activés. Une fois activés, ceux-ci migrent au site infecté ou endommagé et libèrent des cytokines et médiateurs inflammatoires tels que IL1, TNFα, VEGF. Ces cytokines jouent un rôle essentiel dans l'inflammation du SNC puisqu'elles induisent des chimiokines tels que la sphingosine-1-phosphate (S1P), un sphingolipide fortement exprimé dans les glioblastomes et qui joue un rôle important dans la chimiotaxie et le trafic des cellules immunitaires. Nous avons étudié l'efficacité d'une molécule dérivée de notre diète possédant des propriétés chimiopréventives et anti-inflammatoires, l'épigallocatéchine gallate (EGCG), sur la régulation transcriptionnelle des récepteurs de la S1P à divers stades de différenciation des cellules promyélomonocytaires HL-60. Nous avons d'abord différencié les cellules promyélomonocytaires HL-60 en « macrophages-like » en utilisant un promoteur tumorigène et activateur de la protéine kinase C-Phorbol 12-myristate 13-acétate (PMA). Nous avons démontré que le PMA induit l'adhésion cellulaire et augmente l'expression des transcrits S1P1, S1P2 et S1P5. Nous avons ensuite constaté que les cellules adhérentes semblaient être sensibles à la S1P en induisant la phosphorylation d'ERK, de JNK et de P38 MAPK. Cependant, l'inclusion de l'EGCG avant la différenciation par le PMA inhibe l'induction de ces trois voies MAPK par la S1P. D'autre part, un traitement par l'EGCG au cours de la différenciation, c'est-à-dire simultanément avec le PMA, affecte seulement la voie P38 MAPK. De plus, les cellules différenciées « macrophages-like » devenaient insensibles à l'EGCG. Enfin, nous démontrons que seul le récepteur S1P2, parmi les récepteurs fortement induits par le PMA, diminue lors du prétraitement par l'EGCG. Nos résultats suggèrent que l'EGCG antagonise la réponse à la S1P dans les cellules prédifférenciées via l'inhibition de la signalisation induite par le PMA. Par conséquent, une réponse réduite à la S1P pourrait abroger la migration transendothéliale des monocytes vers le SNC, et prévenir la neuroinflammation, les infections cérébrales secondaires ou certaines pathologies cérébrales conséquentes à l'infiltration de cellules immunitaires. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Neuroinflammation, S1P, EGCG, macrophages, chimiotactisme, chimioprévention

Phenolic antioxidants reduce the effect of oxidative stress within cells. They are found in a various fruits, vegetables and as food additives to reduce spoilage. Consumption of antioxidants by humans has been linked with increased lifespan and reduced incidence of cancer and cardiovascular disorders (Cabrera et al. 2006; Kuriyama 2008). In cultured mammalian cells however, some of these phenolic antioxidants have been reported to generate reactive oxygen species (ROS), leading to chromosomal breakage (Long et al. 2007; Long & Halliwell 2001). It is clear then, that amongst this group of compounds, in vitro toxicological study is not a reliable prediction of human hazard. It is for this reason that the work described in this thesis was undertaken: the principal aim was to gain a better understanding of the reasons underlying this contradiction. It has been suggested that excessive ROS generated in vitro might be a result of the higher levels of oxygen (~20%) compared to (1-7%) in vivo: (Yusa et al. 1984; Turrens et al. 1982). With clearer understanding, new experimental approaches might be taken to highlight or reduce positive in vitro genotoxicity test results that might be considered misleading. A diverse set of test compounds was first chosen. It included polyphenolic (PPA), monophenolic (MPA) and non-phenolic antioxidants (NPA), in addition to mechanistically characterised oxidants, genotoxins and cytotoxic, non-genotoxins as controls. Genotoxicity was assessed in vitro using the GADD45a, GFP reporter assay and in silico using Derek Nexus™. Amongst the 19 antioxidants assessed, the 11 of 12 of PPAs, 0 of 4 MPAs and 1 of 3 NPAs (ethoxyquin) produced positive results in vitro and 8 of 12 PPAs generated alerts of at least plausible genotoxicity in silico. To discover whether these results were the result of cellular hyperoxia-promoted generation of physiologically irrelevant ROS in cells, genotoxicity was reassessed in the presence of 1 and 5% oxygen. This reduced oxygen exposure had no effect upon the qualitative result for any of the assessed compounds and a negligible effect upon the dose at which any positive result was produced. An assessment of the ability of antioxidants to generate potentially genotoxic ROS within cells was carried out using the intracellular fluorescent dye, dichlorofluorescin diacetate (DCFH-DA). 10 of 12 PPAs, 0 of 4 MPAs and 1 of 3 NPAs (ethoxyquin) were shown to increase the level of ROS within TK6 human lymphoblastoid cells within 4 hours of compound exposure. Within this same timeframe, the mitochondrial membranes in cells treated with 10 of 12 PPAs, 2 of 4 MPAs and 1 of 3 NPAs (ethoxyquin) were shown to become depolarised using JC-1 dye. It was unclear however, whether mitochondrial membrane depolarisation was a cause or a consequence of ROS generation within the cells. In order to assess whether the increase in intracellular ROS led to an increase in oxidised DNA within treated cells, 8-oxoguanine (8-OG) was quantified using a FITC conjugated anti8-OG antibody. This assessment revealed that levels of the oxidised base were only increased in cells exposed to two of the 12 PPAs (quercetin and resorcinol). The level of 8OG detected was lower than the vehicle control for cells treated with 10 of the 15 antioxidants. One interpretation of this is that these agents induce the repair pathway for oxidative damage, which leads to a lower level of oxidised DNA bases in the genome. The results showed that while a large proportion of PPAs produce genotoxic results in vitro and lead to increased levels of ROS, the amount of oxidised DNA is not higher in treated cells. This would suggest the presence of a different mechanism for the observed genotoxicity.

Parkinson’s disease is a neurodegenerative disorder characterized by the loss of motor skills and cognitive impairment. The hallmark of this disease is the presence of Lewy bodies in the substantia nigra of the brain, where the accumulation of alpha-synuclein amyloid fibrils lead to the death of dopaminergic neurons. Understanding the factors influencing AS aggregation and developing effective strategies for its inhibition is of paramount importance for developing potential therapeutic interventions. Previous studies suggest that flavonoids in green and black tea have neuroprotective properties that decrease the fibrillization rate of AS. This thesis investigates the inhibitory effects of two flavonoids coming from green and black tea, namely: EGCG, TFDG, and HSA on AS aggregation, shedding light on their potential as therapeutic candidates. The study employed a combination of biochemical and biophysical experimental techniques to elucidate the inhibitory mechanisms of EGCG, TFDG, and HSA on AS aggregation. Initial experiments involved the characterization of AS fibrillation kinetics using ThT assays, TEM, and CD. Results revealed that flavonoids exhibited similar inhibitory effects on AS aggregation, with more than 90% inhibitory potency. Interestingly, when aggregated AS was exposed to EGCG or TFDG, the amyloid fibrils changed conformation and formed non-toxic amorphous oligomers. The 13C-detected NMR experiments, adapted to probe the AS dynamic conformation and interactions at the atomic level at physiologically relevant conditions, further provided insights into the binding interactions between flavonoids and AS, revealing interaction with hydrophobic residues involved in the inhibition process. Furthermore, the role of HSA, a major protein component of the blood plasma, in modulating α-synuclein aggregation in the presence of tea-derived flavonoids was investigated. The study demonstrated, in line with the previous reports, that HSA can significantly suppress AS fibrillation, and moreover, the presence of HSA further enhances the flavonoids’ inhibitory effect. My findings provide valuable atomic level mechanistic insights into the inhibitory effects of EGCG and TFDG on alpha-synuclein aggregation. The comprehensive spectroscopic and biophysical investigation provides a solid foundation for further developing flavonoid-based inhibitors, subsequently drug candidates blocking the AS toxic oligomers formation and aggregation.

Indiana University-Purdue University Indianapolis (IUPUI) / The degradation of the resin-dentin interface after restoration placement is multifactorial and can be attributed in part to matrix metalloproteinases (MMPs) enzymes associated with recurrent and secondary caries progression. This dissertation aimed to synthesize and characterize the effects of Epigallocatechin-3-gallate (EGCG) from green tea extract as an MMP-inhibitor loaded into a dental adhesive using slow therapeutic compound release nanotubes as a reservoir to allow sustained and slow release. Loading efficiency and drug release were evaluated using a UV-vis spectrometer. The effects on the degree of conversion (DC), polymerization conversion (PC), and Vickers Micro-Hardness (VHN) tests were performed. MMP mediated β-casein (bCN) cleavage rate was used to determine the potency of the eluates contained EGCG to inhibit MMP-9 activity. The results indicated that HNTs could hold about 21.35% (±4.2%) of the EGCG used in the encapsulation process. The addition of 7.5% HNT or 7.5% EGCG-encapsulated HNT adhesive groups did not alter the curing efficiency indicated by the degree of conversion, polymerization conversion, and surface hardness results compared to the control group (p> 0.05). A statistically significant influence of adding HNTs was found to slow down the EGCG release measured up to 8 weeks (p< 0.05). There was a significant decrease in the degradation of β-casein mediated by pre-activated MMP-9 exposed to eluates from EGCG adhesives compared to non-EGCG adhesive groups (p< 0.05). The results suggested that using HNTs for EGCG encapsulating can remedy the negative impact of EGCG on the adhesive’s polymerization and still have the MMP-inhibitory effect and longer release period. Dentin adhesive containing EGCG-encapsulated HNT may contribute to the long-term preservation of restorations through slow and controlled release to maintain the dentin-resin interface's integrity by inhibiting MMP activity.

In both Type 1 diabetes (T1D) and Type 2 diabetes (T2D), inadequate beta-cell mass and beta-cell dysfunction lead to impaired insulin secretion, and ultimately worsen glycemic control. Green tea has drawn wide attention due to its possible health-promoting properties, including enhancement of beta-cell function. We assessed the acute and relative long-term effects of epigallocatechin gallate (EGCG) on insulin secretion and synthesis from clonal beta-cells (INS1E cells), rat islets, and human islets, using 0.1, 1, or 5 µM. We determined if EGCG decreased blood glucose in healthy rats acutely, using 50 or 150 mg/kg body weight (BW), and after 12 days of supplementation in drinking water, using 0.1% and 0.5%. In the in vitro studies, EGCG significantly potentiated glucose-stimulated insulin secretion (GSIS) in rat islets (at 0.1, 1, and 5 µM) and human islets (at 1 µM), and elevated insulin content within INS1E cells (at 0.1, 1, and 5 µm) and human islets (at 1 µM), (P<0.05). Nutritional supplementation of EGCG (0.5% in drinking water) for 12 days in healthy rats significantly increased insulin synthesis, compared to that of controls, from 0.2 ± 0.02 to 1.4 ± 0.2 ng/mg protein, without alteration of insulin secretion in isolated islets (P<0.05). These findings demonstrate that EGCG may play a role in the regulation of pancreatic beta-cell function, thereby contributing to an anti-diabetic effect of this agent. / Master of Science

Teblad innehåller en mängd substanser som på olika sätt kan påverka biologiska processer i människan. I föreliggande arbete har en sammanställning gjorts utifrån vetenskapliga artiklar över teets antagna effekter på människans immunsystem .Främst har den mest studerade komponenten i te, epigallocatechin gallate, EGCG, undersökts. De variabler som studerats i immunförsvaret är neutrofiler, tumörnekrosfaktor-α, interferon-γ, interleukin-1β, interleukin-2, interleukin-5, interleukin-6, interleukin-8, interleukin-10 ,interleukin-12, interleukin-13, eikosanoidmetabolismen, lymfocyter, makrofager, dendritiskaceller och T-cellsdifferentiering. Sammafattningsvis föreföll te hämma följande: Neutrofilaktiviteten, TNF-α-bildningen, IFN-γ-aktiviteten, stimuli-inducerad IL-β-bildning, IL-8-aktivering och IL-10-bildning. EGCG visades öka dendritiska cellers apoptos och ändra dedenderitiska cellernas morfologi och minska deras förmüga att stimulera T-cellproliferationen. EGCG-behandling av celler ledde till ökad makrofagaktivitet, ökad produktion av T-regceller kontra andra T-cellslinjer. Tebehandling ökade bildningen av IL-12 ochIL-13. EGCGs effekt på IL-6 var tvetydig; enligt en del artiklar påverkade EGCG ej IL-6, medan det enligt andra artiklar antingen ökade eller minskade IL-6-bildningen. Trots att resultaten ej var entydiga kunde slutsatsen att EGCG (och därmed te) utövade immunsupprimerande effekt dras men att det sannolikt krävs betydligt högre koncentrationer än de som är fysiologiskt uppnåbara (1 μM i plasma och 3 mM i tarmen) genom reguljärt tedrickande för att få en avgörande effekt på människans immunsystem. EGCGs hämmnade effekt på immunsystemet kan dock vara av intresse i högre – farmakologiska – koncentrationer genom att EGCG skulle kunna nyttjas som komplement eller alternativ till för närvarande nyttjade immunförtryckande läkemedel som ciklosporin och glukokortikoider.

Les polyphénols sont des métabolites secondaires d’origine végétale. Ces substances naturelles connues pour leurs pouvoirs antioxydants et anti-radicalaires, contribuent à la protection de la santé humaine notamment contre les maladies cardiovasculaires et neurodégénératives, mais également contre certains cancers et diabètes. Dans certains cas, ces effets biologiques bénéfiques pour la santé pourraient également être liés à une interaction spécifique polyphénol-protéine peu étudiée à ce jour par manque d’outils moléculaires adaptés. Les travaux effectués au cours de cette thèse ont consisté à concevoir, à synthétiser et à évaluer des sondes moléculaires polyvalentes porteuses de polyphénols comme substrats d’affinité pour l’analyse des interactions polyphénol-protéine. Dans ce contexte, de nombreuses sondes arborant différents types de polyphénols ont été synthétisées. Ces différentes sondes pourront être utilisées en protéomique chimique du type "Affinity-Based Protein Profiling" (ABPP) pour identifier au sein d’un mélange complexe de protéines, une protéine ayant une affinité spécifique pour un polyphénol donné. Ces mêmes sondes permettront également d’étudier de manière qualitative les interactions d’un polyphénol avec une protéine donnée en temps réel par la technique de résonance plasmonique de surface (SPR). / Polyphenols are plant secondary metabolites. These natural substances, known for their antioxidant and anti-free radical properties, generally contribute to the protection of human health not only against cardiovascular and neurodegenerative diseases, but also against certain cancers and diabetes. In some cases, these beneficial biological effects could also be related to specific polyphenol-protein interactions. However, studying this type of interactions has suffered from the lack of adequate molecular tools. The work carried out during this thesis has included designing, synthesizing and evaluating modulable polyfunctional molecular probes carrying polyphenols as affinity substrates to analyze polyphenol-protein interactions. In this context, various probes harboring different kinds of polyphenols were synthesized. These probes could be used in chemical proteomics following an “Affinity-Based Protein Profiling” approach (ABPP) to identify a protein within complex protein mixtures, which has a specific affinity with a given polyphenol. These probes will also allow studying the interactions of a polyphenol with a given protein in real time in a qualitative way by surface plasmon resonance (SPR).