Gene transfer to the brain is a promising therapeutic strategy for a variety of neurodegenerative disorders including Parkinson‟s disease (PD). PD is the second most common neurodegenerative disease. Although many drugs have been developed and introduced into the market to provide symptomatic treatment, there is still no cure for PD. Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for injured nigrostriatal dopamine neurons and is currently being evaluated as a potential treatment for PD. Gene therapy allows localized, long-term and stable transgene expression after a single intervention to obtain a therapeutic effect. Regulatable promoters for transgene expression furthermore allow optimizing GDNF concentration to avoid undesirable biological activity and clinical side effects. In the first part of the study, an autoregulatory tetracycline-inducible recombinant adeno-associated viral vector (rAAV-pTetbidiON) utilizing the rtTAM2 reverse tetracycline transactivator (rAAV-rtTAM2) was used to conditionally express the human GDNF cDNA. Eight weeks after a single intrastriatal injection of the rAAV-rtTAM2-GDNF vector encapsidated into AAV serotype 1 capsids (rAAV2/1), the GDNF protein level was respectively 15 fold higherand undistinguishable from the endogenous level in doxycycline(Dox) treated and untreated animals. However, a residual GDNF expression in the uninduced animals was evidenced by a sensitive immunohistochemical staining. As compared to rAAV2/1-rtTAM2-GDNF, the rAAV2/1-rtTAM2-WPRE-GDNF vector harboring a woodchuck hepatitis post-transcriptional regulatory element, which increases and stabilizes the transgene transcript, expressed a similar concentration of GDNF in the induced state but a basal level ~2.5-fold higher than the endogenous striatal level. However, the distribution of GDNF in the striatum in induced state was more widespread using the rAAV2/1-rtTAM2-WPRE-GDNF vector as compared to rAAV2/1-rtTAM2- GDNF. As a proof for biological activity, for both vectors, downregulation of tyrosine hydroxylase (TH) was evidenced in dopaminergic terminals of Dox-treated but not untreated animals. In the second part of my study, functional (behavioural) and neurochemical changes mediated by delayed intrastriatal GDNF gene delivery in the partial Parkinson‟s disease rat model were investigated. The rAAV2/1-rtTAM2-WPRE-GDNF vector (3.5 108 viral genomes) was administered unilaterally in the rat striatum 5 weeks after intrastriatal injection of 6-hydroxydopamine (6-OHDA) which produces a partial and progressive lesion of the nigro-striatal dopaminergic pathway. Rats were treated with Dox or untreated from the day of vector injection until sacrifice at 4 or 14 weeks (continuous treatment). A sub-group was Dox-treated for 7 weeks (temporary treatment) then untreated until 14 weeks. In the absence of Dox, the GDNF tissue concentration was found to be equivalent to the endogenous level in 6-OHDA-lesioned rats. In the presence of Dox, it was ~10-fold higher. Dox-dependent behavioral improvements were demonstrated 4 weeks post-vector injection. At later time points, spontaneous partial recovery was observed in all rats, but no further improvement was found in Dox-treated animals. Moreover GDNF gene delivery only transiently improved dopaminergic function. Over the long term, TH was more abundant, but not functional, and the increase was lost when GDNF gene expression was switched off. The third part of my study consisted in the evaluation of the respective dose-range of therapeutical and undesirable effects of GDNF. Functional effects appeared after delivery of 3.5 108 viral particles which produced 200-300 pg/mg protein of GDNF in the lesioned rat striatum (see above). In order to evaluate the viral dose producing undesirable effects, we compared two different doses of vector: 3.5x108 and 4.4x109 viral genome. In the low dose group, the GDNF concentration in the striatum was ~300 pg/mg protein in the Dox-treated animals and equivalent to the endogenous level in untreated animals (~20 pg/mg protein). In contrast, in the high dose group, GDNF levels reached ~1200 pg/mg protein in induced animals but up to ~300 pg/mg protein in uniduced animals. In the low dose group, Dox-dependent downregulation of TH but no asymetrical behaviour was evidenced. In the high dose group, TH downregulation was observed in both Dox+ and Dox-rats. In addition, amphetamine-induced rotational behaviour was evidenced in Dox+ but not in Dox-rats. These data suggest that low doses of virus are sufficient to induce therapeutically-relevant but not undesirable functional effects of GDNF. Nevertheless,a neurochemical effect of GDNF (TH down-regulation) did appear at low dose. In order to understand the GDNF-induced motor asymmetry, we investigated the anatomical pattern of TH down regulation in striatum. Strikingly, there was a greater loss of TH labeling in striosomes than in the surrounding matrix. Receptors which are known to be differentially expressed in the striosomes i.e. µ-opioid receptor(MOR-1) and N-methyl-D-aspartic acid (NMDA) receptor 1 (NR1) as compared to the matrix were analyzed in the high-dose group of animals. MOR-1 was not affected by GDNF gene delivery. In contrast, NR1 was down regulated. The potential relationship between TH and NR1 down-regulation as well as other previously described neurochemical effects of GDNF (as enhancement of DA release and metabolism, of DA neurons excitability or of TH phosphorylation) and behavioural asymmetry remains to be clarified. As summary, our data suggest that behavioural and neurochemical effects of striatal delivery of GDNF can be controlled by Dox by using the autoregulatory rAAV2/1-TetON- GDNF vector, provided the dose range of gene delivery is carefully adjusted. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Le terme dégénérescence lobaire frontotemporale ou FTLD définit un groupe hétérogène de maladies neurodégénératives caractérisé par des troubles du langage, du comportement et/ou moteurs qui résultent principalement d’une dégénérescence du cortex frontal et temporal. Cette hétérogénéité tant au niveau clinique, génétique que neuropathologique rend cette pathologie très complexe et il existe aujourd’hui un véritable problème de diagnostic différentiel des FTLD. Le diagnostic final des FTLD repose ainsi sur l’examen neuropathologique, la nature des lésions observées et leurs constituants moléculaires. La caractérisation de ces lésions a permis d’établir une classification des FTLD qui ne cesse d’évoluer avec la découverte de nouveaux acteurs moléculaires. À l’instar de nombreuses maladies neurodégénératives, les FTLD sont caractérisées par la présence de protéines agrégées dans les régions cérébrales affectées. Cependant, contrairement à la maladie d’Alzheimer (MA), ces agrégats ne sont pas toujours constitués des mêmes protéines. Ainsi, approximativement 40% des cas de FTLD présentent des agrégats composés de protéines Tau hyper et anormalement phosphorylées, et forment le groupe FTLD-Tau. Lorsqu’aucune pathologie Tau n’est détectée, les patients présentent généralement des inclusions neuronales cytoplasmiques ou intranucléaires immunoréactives pour la protéine TDP-43 (transactive response DNA binding protein 43), et constituent la sous-classe FTLD-TDP. Plus rarement, la protéine FUS (Fused in Sarcoma, FTLD-FUS) ou des protéines liées au système ubiquitine protéasome peuvent également s’agréger (FTLD-UPS). La génétique représente également une composante majeure des FTLD avec 10 à 15% des cas correspondant à des formes héréditaires dominantes. Les premières mutations furent découvertes sur le gène MAPT. Le gène de la progranuline (GRN) fut ensuite identifié comme fréquemment associé aux FTLD. Plus récemment, une répétition anormale d’héxanucléotides GGGGCC au sein du gène C9ORF72 (chromosome 9 open reading frame 72) a été montrée comme étant responsable d’un grand nombre de cas familiaux de FTLD. De manière moins fréquente, d’autres gènes tels que VCP (valosin containing protein) ou CHMP2B (charged multivesicular body protein 2B) peuvent aussi être associés à des cas familiaux de FTLD. Des années avant la découverte des principaux acteurs moléculaires des FTLD, des études ont décrit une perte partielle ou totale des protéines Tau physiologiques dans le tissu cérébral. A l’origine, ce phénomène fut observé dans un groupe de démences appelées DLDH pour démences sans signe histopathologique distinctif (plus tard appelé FTLD-ni pour no inclusion). En 2006, la majorité de ces cas a été reclassée en tant que FTLD-U (présence de lésions immunoréactives pour l’ubiquitine). En revanche, aucune étude ne s’est intéressée à cette perte de Tau depuis celle de Zhukareva et collègues en 2003. Au regard des récentes avancées sur la compréhension de la base moléculaire et génétique des FTLD, la pertinence de cette perte de Tau reste ainsi encore à déterminer. Dans ce contexte, ce travail de recherche a pour principal objectif d’étudier l’expression des protéines Tau au sein du tissu cérébral d’individus sains ou atteints de différents troubles neurodégénératifs (MA, FTLD-Tau, FTLD-TDP-GRN, FTLD-TDP-C9ORF72, FTLD-TDP et FTLD-FUS sporadiques) en utilisant la technique d’immunoempreinte. De manière remarquable, nous avons mis en évidence une réduction significative de Tau, et ce, spécifiquement chez les patients FTLD-TDP-GRN. Ainsi, nos résultats démontrent que ces cas, appelés FTLD-TDP-GRNltau (pour low Tau protein level), caractérisés par une altération synaptique et une astrogliose très importante, pourraient constituer un groupe distinct dans la classification des FTLD [...] / FTLD is a clinical syndrome mainly characterized by progressive deterioration in behavior, personality and/or language resulting from progressive frontal and temporal degeneration. In addition, movement disorder can also be frequently observed. Given this phenotype variability, FTLD clinical diagnosis remains difficult and uneasy to establish with certainty.The final diagnosis relies on neuropathological examination of the brain, the characteristics of these brain lesions and their molecular basis. Indeed, as many neurodegenerative diseases, FTLD are characterized by the presence of protein aggregates in the affected brain regions. However, in contrast to the well-characterized nature of protein inclusions in Alzheimer’s disease (AD), proteinaceous aggregates in FTLD can be composed of different proteins. Thus, approximatively 40% of FTLD cases display aggregates made of abnormally and hyperphosphorylated Tau proteins and constitute the FTLD-Tau subclass. However, most of FTLD brains are negative for Tau inclusions and exhibit neuronal cytoplasmic and/or nuclear inclusions immunoreactive for transactive response DNA binding protein 43 (TDP-43) and constitute the FTLD-TDP subclass). To a lesser extent, another protein called FUS (Fused in Sarcoma protein) is found in aggregates that are Tau and TDP-43 negative. This subclass is thus named FTLD-FUS. Finally, inclusions negative for Tau, TDP-43 or FUS are observed in rare cases of FTLD and associated with ubiquitin-proteasome system related proteins (FTLD-UPS).Gene mutations also play an important role in FTLD with 30 to 50% of patients reporting a positive family history of FTD and 10 to 15% of patients corresponding to dominantly inherited form. Firstly described are the MAPT mutations. Mutations in the progranulin gene GRN were then found to be the most frequent mutations associated with FTLD. More recently, two studies demonstrated that expanded hexanucleotide GGGGCC repeats in a noncoding region of the chromosome 9 open reading frame 72 (C9ORF72) gene was responsible for a large proportion of FTLD. Less frequently mutations in the valosin containing protein (VCP) gene or charged multivesicular body protein 2B (CHMP2B) gene are also found associated with FTLD.Prior to the discovery of the main molecular actors of FTLD, studies described a partial or total loss of soluble or physiological Tau protein expression in both grey and white matter. This loss of Tau was originally found in a subset of dementia called DLDH for Dementia Lacking Distinctive Histopathology (renamed later FTLD-ni for FTLD with no inclusion). In 2006, most of these cases were reclassified as FTLD-U (presenting with ubiquitin positive inclusions). However, additional investigation with specific regards to this loss of Tau expression has not been reported since Zhukareva et al. in 2003. With the progress in genetics and neuropathology of FTLD, the question of whether this reduction of Tau expression is seldom remains ill-defined.This work takes place in this context whose primary goal was to investigate human brain Tau protein expression in Control, AD, FTLD-Tau, FTLD-TDP-GRN, FTLD-TDP-C9ORF72, sporadic FTLD-TDP and sporadic FTLD-FUS brains using western blot analysis. Remarkably, we demonstrated a huge reduction of all six human brain Tau isoforms only in a subset of FTLD-TDP brains with mutation on the GRN gene. Thus, our data clearly suggest that these specific cases, referred to as FTLD-TDP-GRNltau (for low levels of Tau protein), could be part of the current classification as a distinct entity with more severe synaptic dysfunction and astrogliosis. Beside this, we also performed a comparative proteomic study between the different FTLD sub-classes in order to find common physiopathological mechanisms.

Abstract Earlier studies on lumbar degenerative imaging findings in magnetic resonance imaging (MRI) have been done mainly in adult populations and the associations of degenerative findings with low back pain (LBP) are controversial. Only a few studies have involved adolescents and young adults. Heritability has been acknowledged as the only explicit risk factor of disc degeneration (DD). This study investigated the prevalence and environmental determinant of lumbar degenerative findings in MRI and their association with low back symptoms among young adults. The data were based on two physical assessments, three questionnaires and one lumbar MRI that were executed on members of the Oulu Back Study (n=558), a subsample of Northern Finland Birth Cohort 1986, between 16 and 21 years of age. Prevalences of lumbar DD (54%), bulging (25%), protrusion (18%) and Schmorl’s node (17%) were high, whereas other degenerative findings were rare among young adults. Males had higher prevalence of DD and Schmorl’s nodes than females. DD and herniations were associated with low back symptoms. On the other hand, symptoms were present among subjects without DD or other abnormal findings on MRI. Of the environmental determinants, high body mass index and MRI-based obesity measurements of visceral adiposity were associated with lumbar DD among males. Waist circumference and smoking showed a comparable association with DD among males, but the level of physical activity was not associated with DD in either gender. Low back symptoms are more common among young adults with a higher degree of DD or presence of disc herniation. Smoking and overweight are associated with lumbar DD among young male adults. / Tiivistelmä Aikaisempia tutkimuksia magneettikuvantamisella (MK) todetuista lannerangan rappeumamuutoksista ja niiden yhteyksistä alaselkäkipuun on tehty lähinnä aikuisväestöllä ja tulokset ovat ristiriitaisia. Vain muutamia tutkimuksia on tehty alle 25-vuotiailla. Välilevyrappeuman mahdollisista riskitekijöistä vain perimästä on vahvaa näyttöä. Tässä tutkimuksessa tarkasteltiin MK:lla todettujen lannerangan rappeumamuutosten esiintyvyyttä, niihin vaikuttavia ympäristötekijöitä ja yhteyttä alaselkäoireisiin nuorilla aikuisilla. Tutkimuksen aineisto perustui kahteen kliiniseen tutkimukseen, kolmeen kyselyyn ja yhteen MK:een, jotka toteutettiin Pohjois-Suomen syntymäkohortti 1986:een kuuluville Oulun selkätutkimuksen koehenkilöille (n=558) 16-21 vuoden iässä. Lannerangan välilevyrappeumalla (54 %), välilevyn pullotuksilla (bulge; 25 %), sellaisilla välilevyn pullistumilla jotka eivät läpäisseet selkärangan takimmaista pitkittäissidettä (protruusio; 18 %) sekä päätelevyn läpi suuntautuvilla välilevyn pullistumilla (Schmorlin keräset; 17 %) oli korkea esiintyvyys nuorilla aikuisilla, kun taas muut kuvantamislöydökset olivat harvinaisia. Välilevyrappeuma ja Schmorlin keräset olivat yleisempiä miehillä. Sekä välilevyrappeuma että pullistumat olivat yhteydessä alaselkäoireisiin molemmilla sukupuolilla, mutta kaikilla oireisilla ei todettu poikkeavia löydöksiä MK:ssa. Ympäristötekijöistä korkea kehon painoindeksi ja MK:sta mitatut rasvan määrää mittaavat muuttujat olivat miehillä yhteydessä välilevyrappeumaan. Miehillä vyötärönympärys ja tupakointi olivat heikommin yhteydessä välilevyrappeumaan, kun taas liikunta-aktiivisuus ei ollut kummallakaan sukupuolella yhteydessä rappeumaan. Alaselkäoireet ovat yleisempiä nuorilla aikuisilla, joilla on vaikea-asteisempi välilevyrappeuma tai välilevyn pullistuma. Tupakointi ja ylipaino ovat yhteydessä lannerangan välilevyrappeumaan nuorilla aikuisilla miehillä.

Dehydroepiandrosterone, a C-19 steroid, is found endogenously with the highest circulating serum levels. It is converted to important steroids such as the sex hormones oestrogen and testosterone. DHEA has come under the spotlight as a purported “fountain of youth” due to its well-characterised age-related decline. The supplementation of DHEA in both the elderly and those with a pathophysiological deficiency has been shown to be of benefit, particularly with regard to wellbeing and depression. The role of DHEA in the periphery has not been elucidated beyond its role as a precursor hormone in sex steroid biosynthesis, though it has been established as a neuroactive neurosteroid, capable of exerting neuroprotective effects in the brain. Since the importance of free radicals in aging and neurodegeneration is well established, investigations were conducted on the ability of DHEA to inhibit free radical generation or scavenge existing free radicals. DHEA was able to significantly inhibit quinolinic acid-induced lipid peroxidation, a measure of membrane damage, over a range of concentrations, although the reduction did not appear to be dose-dependent. This was observed in both in vitro and in vivo studies. Thus, the ability of a compound to reduce the degree of lipid peroxidation may indicate its value as a neuroprotectant. However, DHEA did not significantly reduce cyanide induced generation of the superoxide free radical, suggesting that DHEA is not an effective free radical scavenger of the superoxide anion and that the reduction in lipid peroxidation does not occur through a scavenging mechanism. Apoptosis is a physiological process which is necessary for development and homeostasis. However, this form of programmed cell death can be initiated through various mechanisms and too much apoptotic cell death results in deleterious effects in the body. DHEA was shown not to induce apoptosis. Even the lowest concentration of DHEA investigated in this thesis shows a remarkable decrease in the degree of apoptosis caused by intrahippocampal chemical insult by the neurotoxin quinolinic acid. Cresyl violet was used to visualise tissue for histological examination which revealed that DHEA is able to preserve the normal healthy morphology of hippocampal cells which have been exposed to quinolinic acid. Cells maintained their integrity and showed little evidence of swelling associated with necrosis. Organ culture studies were performed by assessing the impact of DHEA on several pineal metabolites. The study revealed that DHEA exerted an effect on the metabolism of indoleamines in the pineal gland. Melatonin, the chief pineal hormone, did not appear to be affected while the concentrations of N-acetylserotonin, serotonin and methoxytryptamine showed significant alterations. Thus, the neuroprotective mechanism of DHEA does not appear to be mediated by an increase in the presence of melatonin. The biological importance of metal ions in neurodegeneration is also well established and thus the potential interaction between DHEA and metal ions was considered as a mechanism of action. Spectroscopic and electrochemical analyses were performed to determine whether DHEA is able to interact with metal ions as a ligand. These reveal that DHEA does not form a strong bond with the metals investigated, namely copper (II) and iron (III), but that a weak interaction is evident. These investigations were conducted in a rodent model, which has neither large amounts of endogenous DHEA, nor the enzymatic infrastructure present in humans. Thus, the theory that DHEA exerts its effects through downstream metabolic products is unlikely. However, these investigations reveal that there is merit in the statement that DHEA itself is a neuroprotective molecule, and confirm that the further investigation of DHEA is an advisable strategy in the war against neurodegeneration and aging.

Alcohol abuse is a very common practice (just like in many other parts of the world) in Nkonkobe Municipality, Eastern Cape Province, South Africa. This is associated with liver disease. An ethnobotanical survey of plants used for the treatment of alcohol-induced liver damage in Nkonkobe Municipality was conducted. During the survey and also from information gathered in the literature, Pelargonium reniforme Curtis, was prominently mentioned, among other plants, as the species used generally for the treatment of alcohol-induced liver damage. This project was designed to evaluate the effects of the plant on alcohol-induced liver damage, including its antioxidant and antimicrobial properties. It also involves safety evaluation studies to determine if the plant is safe for consumption. Studies using rats of the Wistar strain were carried out to determine the protective and curative effects of P. reniforme on alcohol-induced liver damage. Results obtained showed that the plant extract can protect the liver cells as well as enhance recovery from tissue damage. The plant also showed good antimicrobial and antioxidant activity and this further validates its use in the treatment of liver diseases. Safety evaluation studies of the extract were carried out by investigating the effects of the oral administration on some haematological and biochemical parameters in male Wistar rats. The results obtained from the study suggest that the plant extract is not toxic at the doses used and is therefore safe for medicinal uses. The results of the various bioassays carried out in this project have justified the traditional uses of P. reniforme for the treatment of alcohol-induced liver damage.

Osteoarthritis (OA) is the leading cause of disability in North American and has major economic consequences for society. People with knee OA experience the worst quality of life, among musculoskeletal conditions, with function and mobility being influenced by symptoms such as pain and stiffness. However, the impact of OA symptoms varies due to intrinsic and extrinsic factors, leading many researchers to employ biopsychosocial and other population health frameworks to study the disease. These population health approaches have not been adopted when studying knee OA outcomes in bioarchaeology, where a limited biological lens prevails due to the sole reliance on skeletal remains. The purpose of this research was to explore methods for identifying the impairment potential of knee OA in archaeological populations using a clinical sample and population health approaches. Clinical studies have the advantage of assessing not only the biological implications of knee OA but also the functional outcomes. By creating a knee OA grading system applicable for both MRI and dry bone femora samples (Clinical Archaeological Osteoarthritis Score) a link between clinical and archaeological populations was proposed. Using this link to infer functional deficits onto archaeological populations using population health frameworks, a theoretical analysis was performed with two populations; the 17th century Huron and the 19th century Inuit from the Igloolik region of Nunavut. The results demonstrated the increased impairment potential of knee OA in the Inuit population versus the Huron population, produced by contrasting factors captured by the determinants of health, including social and physical environments.

La Dystrophie Musculaire de Duchenne (DMD) est la plus fréquente et la plus sévère des maladies dégénératives du muscle. Elle se caractérise par une dégénérescence progressive des fibres musculaires due à l’absence de dystrophine fonctionnelle dans les muscles. Actuellement, le rôle physiologique de la dystrophine n’est pas clairement établi et il n’existe pas encore de traitement curatif pour cette maladie. La difficulté de mettre en évidence la fonction de la dystrophine et la physiopathologie de la DMD est en partie expliquée par la complexité moléculaire et cellulaire du muscle des modèles vertébrés utilisés dans les études actuelles. Notre équipe de recherche a développé un modèle de DMD chez le nématode Caenorhabditis elegans. Dans ce modèle, la mutation du gène de la dystrophine, provoque une dégénérescence progressive des muscles conduisant à une paralysie des animaux adultes. Nous utilisons ce modèle afin d’étudier la fonction de la dystrophine et les mécanismes impliqués dans la dégénérescence musculaire chez le nématode. Ce travail de thèse porte sur deux nouveaux acteurs de la dégénérescence musculaire dépendante de la dystrophine : la protéine DYC‐1 et son principal partenaire ZYX‐1. Ce travail présente la caractérisation de ces deux protéines et étudie leurs fonctions dans le muscle. Par ailleurs, ce travail de thèse présente les premiers résultats d’un projet de microscopie électronique ayant pour but de caractériser en détail les évènements subcellulaires du processus dégénératif au cours du cycle de vie du nématode dystrophique. À plus long terme, les études chez le nématode permettront de proposer de nouvelles hypothèses quant aux mécanismes moléculaires et cellulaires de la dégénérescence musculaire / Duchenne Muscular Dystrophy (DMD) is the most prevalent and one of the most severe muscular dystrophy. DMD is due to the absence of functional dystrophin in cardiac and skeletal muscle cells, this lack leads to a progressive muscle degeneration of contractile fibres. Currently, the physiological role of dystrophin is not yet clearly established and curative treatments for DMD are not yet available. The lack of knowledge about dystrophin function and DMD physiopathology can be partly attributed to the complexity of vertebrate muscle, and the absence of a simple model that emulates the human pathology. Our research team developed a model of muscle degeneration in the nematode Caenorhabditis elegans. In this model, the mutation of the dystrophin gene produces a progressive muscle degeneration leading to the paralysis of the adult worms. We use this model for investigating the role of dystrophin and the mechanisms of muscle degeneration in C. elegans. This PhD work concerns two new actors of dystrophin‐dependant muscle degeneration: The DYC‐1 protein and its main interactor ZYX‐1. This study aims to characterise these proteins and to study their muscle functions. Moreover, this PhD work presents preliminary results of an in depth characterisation of subcellular processes of muscle degeneration in dystrophic worms by electron microscopy. Our aim is to visualise first events and to observe the progression of degeneration until the death of muscle cell. These molecular and cellular approaches aims to get new insights in the mechanisms underlying muscle degeneration in order to propose new hypotheses for the understanding of DMD

The purpose of this dissertation was to understand how genetics, socioeconomic status (SES), and lifestyle factors influence the development of age-related macular degeneration (AMD), glaucoma, and diabetic retinopathy in an adult population in Dallas County. Two hundred fifty-three older adults participated in this study as the sample. Crosstabulation and binary logistic regression were utilized to analyze the data. Results indicated a disparity among participants' test scores, visual health status, and perceptions of their visual impairment and highlighted the fact that many seniors are not educated about age-related retinal disorders. Furthermore, variables reaching statistical significance were consistent with the literature included race/ethnicity, age, having a family history of both AMD and diabetes, frequency of eye exams, and level of education. The results not consistent with the literature as affecting visual health included health insurance, access to health care, body weight, and smoking status. Recommendations for future study included applied research focusing on determining risk factors, raising awareness, educating, and providing early detection of these diseases among low to middle income Caucasian, African American, and Hispanic older adults.

Orientadores: Jacqueline Mendonça Lopes de Faria, José Butori Lopes de Faria / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T10:54:36Z (GMT). No. of bitstreams: 1 Duarte_DiegoAndreazzi_D.pdf: 28811769 bytes, checksum: 726e2f631efd3a14fedaf1f8148784aa (MD5) Previous issue date: 2014 / Resumo: A retinopatia diabética (RD) é uma doença devastadora que está entre as maiores causas de cegueira entre pessoas na idade adulta em todo o mundo. Considerada multifatorial e progressiva, a RD afeta células neurais e gliais, e também elementos vasculares da retina. Sabe-se que diversas vias estão envolvidas na patogênese da RD, no entanto, os mecanismos que levam a exacerbação da inflamação e morte de células gliais/neurais o que caracteriza a neurodegeneração da retina, ainda permanecem desconhecidos. Diante disso, a redução desses fatores tem sido extensivamente estudada como alvo no combate a RD. As Sirtuínas, histonas desacetilases dependentes de nicotinamida adenina dinucleotídeo (NAD+), atuam em resposta a vários estresses e atualmente tem sido relacionadas às importantes funções moleculares na regulação de várias doenças. Considerado um redox sensível, a SIRT1 pode estar reduzida em condição de doença, o que agravar ainda mais a situação patológica. No entanto, não se sabe ao certo o mecanismo de modulação e/ou atuação da SIRT1 frete às doenças neurodegenerativas, tais como a RD. No Artigo I, foram avaliados os possíveis efeitos protetores do cacau rico em polifenóis na retina diabética. As células Müller da retina (rMC-1) foram expostos por 24h à glicose normal (NG), alta glicose (HG) ou peróxido de hidrogênio (H2O2) e submetidas ao tratamento com cacau na presença ou não de um inibidor da SIRT1 ou siRNA. O estudo animal foi desenvolvido em ratos experimentalmente diabéticos induzidos por estreptozotocina e randomizado para receber tratamentos com cacau com baixa, intermediária, ou elevada dose de polifenol (0,12 mg; 2,9 mg; 22,9 mg/kg/dia) por gavagem durante 16 semanas. As células expostas a H2O2 ou HG apresentaram aumento de proteína acídica fibrilar glial (GFAP) e acetil-RelA/p65 e diminuição da atividade/expressão da SIRT1. Estes efeitos foram anulados pelo cacau, que diminuiu a produção de espécies reativas de oxigênio e reduziu a ativação da poli(ADP-ribose) polimerase-1 (PARP-1); melhorou os níveis intracelulares de NAD+ e consequentemente aumentou da atividade da SIRT1. As retinas dos ratos diabéticos exibiram os primeiros marcadores de retinopatia acompanhada pela eletrorretinografia prejudicada. A presença de diabetes levou a ativação da PARP-1 e diminuição dos níveis de NAD+, resultando em comprometimento da SIRT1. O aumento na acetilação do RelA/p65 levou na hiperexpressão do GFAP. A administração oral de cacau polifenol restaurou as alterações acima referidas. Este estudo revelou que o cacau enriquecido com polifenóis teve efeito protetor da retina diabética restabelecendo a via da SIRT-1. No Artigo II, foi investigado o possível efeito terapêutico de células derivadas de animais saudáveis (Dock7 m +/+ Leprdb db/m) e diabéticos (BKS.Cg-Dock7 m +/+ Leprdb/J, db/db) na retinopatia diabética (RD). Os camundongos db/db (espontaneamente diabéticos) com 8 semanas de idade foram randomizados para receber uma única injeção intravenosa de PBS ou células early outgrowth (EOCs) de doadores db/m ou db/db. Quatro semanas mais tarde, os animais foram sacrificados e os olhos enucleados. Para estudo in vitro, o meio condicionado das EOCs (EOC-CM) foi gerado a partir do cultivo de EOCs de animais db/m e db/db. As células rMC-1 foram expostas por 24h a NG ou HG e submetidas ao tratamento com db/m ou db/db EOC-CM, em presença ou não de um inibidor farmacológico (EX527) ou gênico (siRNA) da SIRT1. Nos ratos diabéticos, houve um aumento de marcadores de RD e do dano oxidativo, acompanhado por uma diminuição da proteína SIRT1 e seguido pelo aumento da acetilação da lisina-310 do complexo p65-NFkB. A terapia celular com EOCs reduziu significativamente todas as alterações mencionadas acima. As rMC-1 expostas a HG apresentaram aumento da expressão de GFAP, fator de crescimento do endotélio vascular e Nox4, acompanhado pelo aumento dos níveis de espécies reativas de oxigênio e acetil-lisina-310-p65-NFkB. Além disso, a expressão/atividade da SIRT1 foram reduzidas em ambiente diabético. O tratamento com EOC-CM impediu todas estas alterações. Este estudo demonstra que a capacidade parácrina das EOCs, na secreção de fatores, é eficaz no restabelecimento da via de SIRT1 retina, e assim, proteger a retina dos insultos diabéticos. Em resumo, a presente tese fornece evidências que tanto a administração oral do cacau enriquecido com polifenóis quanto à terapia celular com EOCs, conferem neuroproteção da retina aos insultos do diabetes. Portanto, intervenções que modulem a atividade das sirtuínas são promissoras no tratamento farmacológico da retinopatia diabética / Abstract: The diabetic retinopathy (RD) is a devastating disease and the principal cause of blindness among people in adulthood worldwide. The RD is considered a multifactorial and progressive disease, affecting neuronal and glial cells, and also vascular elements of the retina. It is known that several pathways are involved in the pathogenesis of RD, however, the mechanisms that lead to exacerbation of inflammation and death of glial/neuronal cell, characterizing retinal neurodegeneration, remain unknown. Therefore, the reduction of these factors have been extensively studied as a therapeutic target against RD. Sirtuin 1 (SIRT1), a family of histone deacetylase enzyme, acts in response to various stresses and, currently, has been related to important molecular functions in the regulation of various diseases. Considered a redox-sensitive, SIRT1 may be reduced under disease condition, whereby aggravate the pathological situation. However, is not known the mechanism of modulation/activity of SIRT1 in neurodegenerative diseases, such as RD. In the article I, were studies the possible protective effects of cocoa in the diabetic retina were assessed. rMCs exposed to NG, HG or H2O2 were submitted to cocoa treatment in the presence or absence of SIRT-1 inhibitor and siRNA. The experimental animal study was conducted in streptozotocin-induced diabetic rats and randomized to receive low, intermediate, or high polyphenol cocoa treatments via daily gavage for 16 weeks (i.e., 0.12 mg/kg/day, 2.9 mg/kg/day, or 22.9 mg/kg/day of polyphenols). The rMCs exposed to HG or H2O2 exhibited increased GFAP and acetyl-RelA/p65 and decreased SIRT1 activity/expression. These effects were cancelled out by cocoa, which decreased ROS production and PARP-1 activity, augmented the intracellular pool of NAD+, and improved SIRT1 activity. The rat diabetic retinas displayed the early markers of retinopathy accompanied by markedly impaired electroretinogram. The presence of diabetes activated PARP-1 and lowered NAD+ levels, resulting in SIRT1 impairment. This augmented acetyl RelA/p65 had the effect of upregulated GFAP. Oral administration of polyphenol cocoa restored the above alterations in a dose-dependent manner. This study reveals that cocoa enriched with polyphenol improves the retinal SIRT-1 pathway, thereby protecting the retina from diabetic milieu insult. In the article II, were investigated the possible therapeutic effect of cells derived from control (db/m) and spontaneously diabetic (db/db) mice on diabetic retinopathy. The db/db mice with 8 weeks of age were randomized to receive a unique intravenous injection of PBS or 0,5x105 db/m EOCs or 0,5x105 db/db EOCs. Four weeks later, the animals were euthanized and the eyes enucleated. For in vitro study, EOC-CM was generated from db/m and db/db EOCs cultures. rMCs were exposed for 24h to NG or HG combined or not with db/m or db/db EOC-CMs. In diabetic rats, there was an increase of DR and oxidative damage markers, accompanied by decrease in SIRT1 protein followed by lysine-310-p65-NF?B acetylation. The treatment with cells from db/m significantly reduced all the above-mentioned, but interestingly the treatment with cells from db/db mice fully restored the above alterations to normal levels. rMCs exposed to HG displayed GFAP and VEGF expression up regulated, accompanied by increase in Nox4 expression and ROS levels, and acetyl-lysine-310-p65-NF?B. SIRT1 protein expression and activity were markedly reduced in diabetic milieu conditions. The treatment with both EOC-CMs prevented all these abnormalities, but db/db EOC-CM fully restored to NG conditions. This study demonstrates that endocrine capacity of EOCs is effective in improving retinal SIRT1 pathway thus protecting the retina from diabetic milieu insult. In summary, compelling novel evidence is provided herein that either through oral administration of polyphenol enriched cocoa or cell therapy with EOCs, conferred retinal neuroprotection against diabetic insults in animal models. The identification of SIRT-1 as a potential therapeutic target in the treatment of diabetic retinopathy may provide new perspective in the pharmacological treatment of this diabetic complication / Doutorado / Clinica Medica / Doutor em Clínica Médica

Made available in DSpace on 2016-01-26T18:55:43Z (GMT). No. of bitstreams: 1 Matheus Parra.pdf: 726860 bytes, checksum: fe316e1bd808b08f513b45b186081bcc (MD5) Previous issue date: 2015-02-11 / The objective was to evaluate the effect of probiotic on renal function in intoxicated Wistar rats by potassium dichromate (K2Cr2O7). We used 80 male Wistar rats, that were 21 to 25 days old, randomly divided into two treatments (n = 40 rats/treatment). In the DK treatment the animals consumed 0, 12, 24 and 36 mg of K2Cr2O7 added to the diet and in the DK+P treatment the rats consumed 0, 12, 24 and 36 mg of K2Cr2O7 in the diet with 0.2% probiotic. Those animals consumed their respective diets for 90 days, then, they were euthanized by exsanguination and blood samples were taken to evaluate serum creatinine and urea levels and histopathological analysis of the kidneys were realized. Serum creatinine levels in the DK+P treatment was significantly (p<0.05) lower in relation to the DK treatment in all dichromate doses analyzed. Serum urea levels did not differ significantly (p>0.05) between the DK and DK+P treatments. There was no significant difference (p>0.05) in serum creatinine and urea concentrations in the rats that consumed 0 and 12 mg of dichromate and probiotic, but in the other animals serum creatinine and urea increased significantly (p<0.05) along with increasing doses of the dichromate in both experimental treatments. The rats in DK and DK+P treatments showed hydropic degeneration in renal tubules at all doses of K2Cr2O7. The DK treatment rats showed interstitial nephritis. It was concluded that supplementation with the probiotic was beneficial for glomerular filtration in rats intoxicated with low doses of potassium dichromate, but did not prevent the tubular hydropic degeneration in Wistar rats intoxicated by potassium dichromate. / Objetivou-se avaliar o efeito de probiótico na função renal de ratos Wistar intoxicados por dicromato de potássio (K2Cr2O7). Utilizou-se 80 ratos Wistar, machos com 21 a 25 dias, divididos aleatoriamente em dois tratamentos (n=40 ratos/tratamento). No tratamento DK os animais consumiram 0, 12, 24 e 36 mg de K2Cr2O7 adicionado na dieta e o tratamento DK+P os ratos consumiram 0, 12, 24 e 36 mg de K2Cr2O7 na dieta com 0,2% de probiótico. Esses animais consumiram suas respectivas dietas durante 90 dias, foram eutanasiados por exsanguinação e colhidas amostras de sangue para realização de dosagem sérica de creatinina e uréia e realizou-se nos rins análise histopatológico. A creatinina sérica do tratamento DK+P foi significativamente (p<0,05) menor em relação ao tratamento DK em todas as doses de dicromato estudadas. A uréia sérica não diferiu significativamente (p>0,05) entre os tratamentos DK e DK+P. Não houve diferença significativa (p>0,05) na concentração sérica de creatinina e uréia dos ratos que consumiram 0 e 12 mg de dicromato e probiótico, mas dos demais animais a creatinina e uréia sérica aumentou significativamente (p<0,05) juntamente com as doses crescentes de dicromato estudadas em ambos os tratamentos experimentais. Os ratos dos tratamentos DK e DK+P apresentaram degeneração hidrópica nos túbulos renais em todas as doses estudadas de K2Cr2O7. Os ratos do tratamento DK apresentaram nefrite intersticial. Conclui-se que a suplementação com probiótico foi benéfica para a filtração glomerular nos ratos intoxicados com baixa dose de dicromato de potássio, mas não evitou a degeneração hidrópica tubular nos ratos Wistar intoxicados pelo dicromato de potássio.