<p>UVOD: Neurokognitivni poremećaj udružen sa HIVinfekcijom (HIV associated neurocognitive disorder- HAND) javlja se u oko polovine pacijenata sa HIV-om. HAND obuhvata spektar neurolo&scaron;kih poremećaja koji variraju od asimptomatskog neurokognitivnog poremećaja(ANI), preko blagog kognitivnog o&scaron;tećenja, koje se naziva blagi kognitivni poremećaj (MND) do demencije udružene sa HIV-om (HAD). U evaluaciji i dijagnostici ovog poremećaja koriste se razne laboratorijske, kliničke i metode imidžinga, među kojima magnetno-rezonantni imidžing (MRI) zauzima posebno mesto u pogledu dijagnostike strukturnih poremećaja. Međutim, za dijagnostiku suptilnih supcelularnih neurobiohemijskih poremećaja neophodno je kori&scaron;ćenje magnetno-rezonantne spektroskopije (MRS). Klasično shvatanje promena u neurobiohemijskom profilu kod pacijenata sa HIV infekcijom uključuje: sniženje NAA (neuronskog markera) kao odraz neurodegeneracije, povi&scaron;enje Cho (markera razgradnje membrana) kao odraz inflamacije/ apoptoze, povi&scaron;enje mI (markera proliferacije mikroglije) kao odraz inflamacije i povi&scaron;enje Glx+Gln (markera glutaminergične ravnoteže) kao odraz ekscitotoksičnosti. Do sada u literaturi nije opisano kori&scaron;ćenje multivokselske MRS mozga na HIV+ pacijentima. CILJEVI: Ciljevi studije su bili: utvrditi da li postoje promene u odnosima koncentracija metabolita u mozgu dobijenih metodom multivokselske MRS u neurolo&scaron;ki asimptomatskih HIV+ pacijenata u poređenju sa zdravim kontrolnim ispitanicima,zatim da li postoje razlike između pacijenata na kombinovanoj antiretroviralnoj terapiji (cART) i pacijenata bez cART; utvrditi da li postoji i kakva je povezanost odnosa metabolita dobijenih MRS sa imunolo&scaron;kim parametrima HIV+ pacijenata i nivoom nadir CD4+ T-limfocita; i konačno, utvrditi da li postoji i kakva je povezanost dobijenih odnosa metabolita sa parametrima penetracije antiretroviralne terapije u centralni nervni sistem. ISPITANICI I METODE: U studiju je uključeno ukupno 114 ispitanika (32 HIV+ pacijenta na cART, prosečne starosti 41.97 godina (25-61); 28 HIV+ pacijenata bez cART, prosečne starosti 35.21 godina (24-52); 50 kontrolnih, zdravih subjekata prosečne starosti 36.56.godina (19-53)). Svi ispitanici su potpisali informisani pristanak za uče&scaron;će u studiji. Ispitivanje je odobreno od strane Etičke komisije Instituta za onkologiju Vojvodine, kao i Etičke komisije Medicinskog fakulteta Univerziteta u Novom Sadu. Kriterijum za uključenje pacijenata u studiju je bio prisustvo HIV infekcije. Kriterijumi za isključivanje pacijenata iz studije su bili: prisustvo aktivne oportunističke infekcije, prisustvo aktivnog neurolo&scaron;kog oboljenja, podatak o aktivnoj zloupotrebi narkotika, koinfekcija virusom hepatitisa B i C, prisustvo lezija bele ili sive mase i kontraindikacije za snimanje na aparatu za magnetnu rezonancu. 4 ispitanika su isključena iz ispitivanja. Svim ispitanicima urađeni su skrining neurokognitivni testovi, kao i rutinska laboratorijska ispitivanja (broj CD4+, CD3+ i CD8+ limfocita). Nakon toga, svim ispitanicima urađen je konvencionalni MRI praćen multivokselskom MRS supratentorijalne suprakalozalne bele mase. Ispitivano je ukupno 12 voksela (6 u sivoj i 6 u beloj masi), odnosno preko 7900 spektara. Određeni su pikovi karakterističnih metabolita. Na metodi dugog eha analizirana su tri glavna pika: NAA na 2.0ppm, Cho na 3.2ppm i tCr na 3.ppm, izražena preko odnosa koncentracija NAA/Cr i Cho/Cr. Na metodi kratkog eha analizirani su signali NAA, Cho, Cr te mI na 3.5ppm i Glx+Gln na 2.2-2.4ppm. Ovi signali su izraženi kroz odnose koncentracija NAA/Cr, Cho/Cr, mI/Cr i (Glx+Gln)/Cr. Za statističku obradu podataka kori&scaron;ćen je IBM SPSS ver. 21.0. Deskriptivna statistika je uključila određivanje srednje vrednosti, minimuma, maksimuma i standardne devijacije. Razlike između posmatranih grupa ispitanika za sve kontinuirane varijable ispitivane su jednofaktorskom analizom varijanse (ANOVA) sa naknadnim (post-hoc) testovima za koje je kori&scaron;ćena metoda po Tukey-ju. Rezultati su prikazani u vidu srednjih vrednosti, standardne devijacije, najvi&scaron;e i najniže izmerene vrednosti (maksimum i minimum), i za svaki ispitivani parametar pridružena je vrednost F i p. Veza između kontinuiranih varijabli je ispitivana pomoću koeficijenta Pirsonove linearne korelacije, uz prethodnu proveru zadovoljavanja uslova o homogenosti varijansi, normalnosti raspodele i linearnosti. Vrednosti p&lt;0.05 su uzimane kao statistički značajne. REZULTATI: Pokazano je da su HIV+ pacijenti na cART značajno stariji od druge dve grupe ispitanika. Nije pokazana značajna razlika u stepenu obrazovanja među grupama. Pokazano je da godine života statistički značajno utiču samo na koncentracije NAA/Cr, dok na odnose drugih metabolita ne utiču značajno. Utvrđeno je statistički značajno sniženje (p&lt;0.05) koncentracija NAA/Cr dobijenih metodom dugog eha između tri grupe ispitanika na svim posmatranim vokselima. Naknadnim analizama utvrđena je statistički značajna razlika na 10/12 voksela između HIV+ pacijenata sa cART i zdravih, kao i između HIV+ pacijenata bez terapije i zdravih, dok su se koncentracije NAA/Cr značajno razlikovale između HIV+ pacijenata sa i bez cART samo na jednom vokselu (duboka frontalna bela masa levo). Utvrđena je statistički značajna razlika u smislu sniženja Cho/Cr odnosa dobijenih metodom dugog eha u 5/12 voksela, sa pojedinačnim vokselima koji su prikazivali razlike između grupa. Na metodi kratkog eha utvrđeno je značajno sniženje odnosa koncentracija NAA/Cr kod HIV+ pacijenata samo na tri voksela, pri čemu nisu prikazane značajne razlike između dve grupe pacijenata sa HIV infekcijom (sa i bez cART). Rezultati odnosa koncentracija Cho/Cr između tri gurpe pacijenata dobijeni metodom kratkog eha slični su rezultatima dobijenim na metodi dugog eha (statistički značajna razlika dobijena je na 5/12 voksela). &Scaron;to se tiče odnosa koncentracija mI/Cr, uočeno je značajno povi&scaron;enje ovog odnosa kod HIV+ pacijenata u odnosu na zdrave na 6/12 voksela. Prikazano je statistički značajno povi&scaron;enje ovog markera kod pacijenata bez cART u odnosu na pacijente sa cART samo u regiji levog dorzalnog anteriornog cinguluma. Statistički značajno povi&scaron;enje (Glx+Gln)/Cr odnosa je prikazano u regiji zadnjeg cinguluma desno kod pacijenata na terapiji u odnosu na pacijente bez terapije, dok na drugim vokselima nije prikazana značajna razlika. Vokseli 4, 7 i 10 su dali najvi&scaron;e informacija(supkortikalna bela masa frontalno levo, dorzalni prednji cingulum levo te parijetalni supkorteks leve cerebralne hemisfere), sa prikazanim značajnim razlikama u bar 4 odnosa metabolita. Prikazana je značajna pozitivna korelacija nadir CD4 + broja limfocita sa koncentracijama NAA/Cr, a negativna sa odnosima Cho/Cr i mI/Cr, &scaron;to čini nadir CD4+ najboljim serolo&scaron;kim prediktorom neurodegenerativnog o&scaron;tećenja. Pokazana je pozitivna korelacija indeksa penetracije lekova u monocite (ME) sa odnosima NAA/Cr i negativna korelacija indeksa penetracije lekova u centralni nervni sistem (CPE) sa Cho/Cr i mI/Cr. Do&scaron;li smo do zakljulka da je ME indeks bolji marker neurodegenerativnog odgovora a CPE indeks bolji u monitoringu kontrole inflamacije. ZAKLJUČAK: Smatra se da HIV virus uzrokuje prerano starenje mozga&scaron;to je prvenstveno posledica direktnog o&scaron;tećenja nervnih ćelija samim virusom (preko viralnih proteina, indukovanih citokina i hemokina). Pokazali smo da su neuronski gubitak i neurodegeneracija proces koji zahvata celokupan volumen mozga, dok su procesi inflamacije i proliferacije mikroglije svedeni na tačno određene regione, pretežno sive mase. Visoka senzitivnost multivokselske 1H-MRS sa kori&scaron;ćenjem senzitivnih povr&scaron;inskih kalemova omogućava mapiranje metabolita sa prostornom rezolucijom. MRS može dati ključni uvid u promene koncentracija metabolita mozga tokom razvoja infekcije od akutne i primarne do hronične. Vrlo brzo nakon serokonverzije, dolazi do detektabilnih promena u metabolitima mozga u smislu neuronskog o&scaron;tećenja i inflamacije. U na&scaron;em istraživanju su po prvi put analizirani rezultati protonske multivokselske MRS bele i sive mase velikog mozga u suprakalozalnom regionu. Utvrđeno je da postoje difuzne, ali ipak visoko regionalno-zavisne promene u odnosima neurometabolita kod pacijenata koji dobijaju antiretroviralnu terapiju i kod pacijenata koji je ne dobijaju, u poređenju sa zdravim kontrolnim ispitanicima (odgovarajućim po polu i starosti). Dodatne studije sa posmatranjem apsolutnih koncentracija neurometabolita, kao i longitudinalne studije u koje su uključeni HIV+ pacijenti u različitim fazama bolesti, su neophodne za dalje i bolje razumevanje neuropatogeneze HAND-a. MRS se pokazala uspe&scaron;nom u detekciji efikasnosti određenih terapijskih opcija. Dva postojeća indeksa za procenu efikasnosti antiretroviralne terapije (CPE, ME) odvojeno pogađaju dva puta neuropatogeneze kognitivnog poremećaja, sa različitim uspehom u sveobuhvatnoj proceni efekta i efikasnosti terapije. U budućnosti je potrebna njihova pojedinačna modulacija ili kreiranje jedinstvenog indeksa, koji bi obuhvatio i efikasnost prolaza leka kroz hematoencefalnu barijeru i dejstvo na latentni rezervoar HIV-a u ćelijama monocitno-makrofagne loze.</p> / <p>INTRODUCTION: HIV associated neurocognitive disorder- HAND appears in about half of the HIV+ patients. HAND represents a spectrum of neurological disorders varying from asymptomatic neurocognitive impairment (ANI), over mild neurocognitive disorder (MND) to HIV associated dementia (HAD). For evaluation and diagnostics of this disorder, many laboratory, clinical and imaging methods are used, first of all magnetic resonance imaging (MRI). Nevertheless, for detecting subtle subcellullar neurobiochemical disorders, the use of magnetic resonance spectroscopy (MRS) is necessary. Classical pattern of neurobiochemical changes in HIV infection consist of: decrease in NAA (neuronal marker) depicting neurodegeneration, increase in Cho (metabolism on membrane marker) depicting inflammation/ apoptosis, increase in mI (marker of microglial proliferation) depicting inflammation and increase in Glx+Gln (glutaminergic balance marker) depicting the effect of excytotoxicity. To the best of our knowledge, this is the first study using multivoxel MRS of the brain in HIV+ patients. AIMS: The aims of this study were: to show whether there are differences in metabolites&#39; ratios on multivoxel MRS in neurologically asymptomatic HIV+ patients compared to control subjects; whether there are differences in metabolites&#39; ratios between patients on combined antiretroviral therapy (cART) and therapy-naive ones; whether there are correlations between matebolites&#39; ratios and immunological parameters in HIV+ patients as well as with nadir CD4+ count; whether there are correlations between metabolites&#39; ratios with parameters of drugs&#39; penetration in central nervous system (CNS). SUBJECTS AND METHODS: Overall of 114 subjects were enrolled in the study (32 HIV+ paients on cART, average age 41.97 years (25-61); 28 HIV+ patients off cART, average age 35.21 years (24-52); 50 control subjects, average age 36.56 years (19-53)). All the subjects signed the informed consent. The study was ethically approved by Ethical committee of Vojvodina Oncology Institute and Ethical committee of Faculty of Medicine, University of Novi Sad. Inclusion criteria for HIV+ subjects were: the presence of HIV infection. Exclusion criteria included: active opportunistic infection, active neurological illness, usage of drugs of abuse, hepatitis B or C coinfection, presence of both white or grey matter lesions, and contraindications that apply for magnetic resonance (MR) examination. 4 subjects were excluded from the study due to the presence of white matter lesions (3 HIV+ and one control subject). Each patient performed International HIV Dementia Scale (IHDS), a screening test for evaluation of global cognitive status in HIV-infected patients. Baseline study laboratory variables were assessed (CD4+ T-lymphocyte count and plasma HIV RNA, nadir CD4+ counts and CD4+ T-cell counts at the moment of MR scan. Conventional MRI scan was followed by multivoxel MRS with both long and short echo. We analyzed 12 voxels (6 in grey and 6 in white matter) with overall of over 7900 spectra. Finally, we analyzed following dominant signals: on the long echo tCr (creatine plus phosphocreatine) at 3.0 ppm, NAA (N-acetyl-aspartate) at 2.0 ppm and Cho (choline containing compounds) at 3.2ppm (ratios of NAA/Cr and Cho/Cr were assessed); on the short echo tCr, NAA, Cho, (Glx+Gln) at 2.2-2.4ppm and mI (myoinositol) at 3.5ppm (ratios of NAA/Cr, Cho/Cr, (Glx+Gln)/Cr and mI/Cr were assessed. All statistical calculations were performed using IBM SPSS software (version 21.0, Chicago, IL, USA). Descriptive statistics included determination of mean values, minimum, maximum and standard deviation. Among-group differences (HIV infected subjects versus healthy controls) in acquired metabolite ratios were evaluated using ANOVA with post hoc Tukey test to determine the differences between separate groups. Due to a known impact of age and education on the NAA concentrations, differences in NAA/Cr ratios among groups were tested using ANCOVA, with age as a covariate variable. Testing relationships between continuous variables was performed using Pearson linear correlation. Statistical significance was set at value p&lt;0.05. RESULTS: We showed that HIV+ patients on therapy were significantly older than the other two groups of patients. There was no significant difference in the level of education. We confirmed that the age significantly affects the level of NAA/Cr only.There was significant decrease (p&lt;0.05) in NAA/Cr level on long echo MRS among three groups on all the observed voxels. Post hoc analysis showed that there was significant difference in 10/12 voxels between HIV+ patients on cART and healthy controls and between HIV+ patients off cART and controls, while NAA/Cr differed significantly between HIV+ patients on and off cART in only one voxel (deep frontal white matter on the left). There was decrease in Cho/Cr levels on long echo MRS in 5/12 voxels among three groups. On short echo MRS, we showed decrease in NAA/Cr level in 3/12 voxels, while there were no differences between two groups of HIV+ patients. Results of short echo MRS in the means of Cho/Cr resembled long echo MRS. There was significant increase in mI/Cr level in HIV+ patients in 6/12 voxels compared to healthy controls, while there was difference in only one voxel between HIV+ patients on and off therapy (dorsal part of anterior cingulate on the left). Significant increase in (Glx+Gln)/Cr level was present between HIV+ patients on and off therapy in the region of right posterior cingulate. Voxels 4, 7 and 10 were the most informative ones (subcortical frontal white matter on the left, dorsal part of left anterior cingulate and right posterior cingulate), showing significant differences in 4 metabolites&#39; ratios. We showed positive correlation between nadir CD4+ count and NAA/Cr and negative correlation between nadir CD4+ count and Cho/Cr, and nadir CD4+ count and mI/Cr, which made nadir CD4+ count the best serological predictor of neurodegeneration. Positive correlation was showed between monocyte efficacy (ME) index and NAA/Cr, while negative correlation was present between CNS penetration efficacy (CPE), Cho/Cr and mI/Cr. We concluded that ME better depicted neurodegenerative process while CPE was better in monitoring of inflammation. CONCLUSIONS: HIV causes premature ageing of the brain, in the means of cognition, attention, working memory and executive function. These effects are due to direct affection of neurons by virus per se (viral proteins, induced cytokines and chemokynes). We showed tha neuronal loss and neurodegeneration affect the whole volume of the brain while inflammation and glial proliferation affect restricted areas predominantly in grey matter. High sensitivity of multivoxel MRS with use of sensitive surface coils enables metabolite mapping with high spatial resolution. MRS can give essential data on metabolites&#39; changes during the evolution of the infection from acute, over primary to chronic. Early after seroconversion, metabolites&#39; changes can be detected (neuronal dysfunction and inflammation).To the best of our knowledge, this is the first study using multivoxel MRS of the brain in HIV infection in human population, analyzing data from supracallosal grey and white matter. We showed the presence of diffuse but regionally highly specific changes in metabolites&#39; ratios in patients on cART and off cART, compared to age and gender matched healthy controls. Additional studies with absolute concentrations of metabolites, as well as longitudinal studies with HIV+ patients in different stages of the disease, are necessary for better understanding of neuropathogenesis of HAND. We showed that MRS can be useful tool in evaluation of therapy regimens efficacy. Two available indices for evaluation of cART efficacy target two separate pathways of cognitive disorder pathogenesis, with different reliability in evaluation of effect and efficacy of applied therapy. In the future, their modulation or creation of new index is needed, in order to include drug delivery through the blood-brain barrier as well as the effect on latent reservoir of HIV in monocyte/macrophage cells.</p>

For many dominant diseases, conventional treatment options are limited. This makes them attractive candidates for gene therapy, which may be directed to specifically silence a disease-causing allele. However, many mutations are not easily amenable to this technique, including nucleotide repeat expansions, which cause numerous neurodegenerative diseases such as Huntington’s disease and several Spinocerebellar Ataxias. Combined gene knockdown and replacement (K&R) may present a more practical approach for such conditions, whereby the gene of interest is subject to mutation-independent non-allele-specific silencing and concurrently replaced with a functional copy. Artificial mimics of naturally occurring intronic microRNAs are theoretically ideal for this purpose, since they can be nested within the replacement gene. This thesis investigates the development of mimics of two different intronic miRNA systems (mirtron miR-1224 and the miR-106b cluster) to silence Ataxin-7 and to be incorporated into novel single K&R constructs for testing in vitro. Artificial mirtrons and intronic miRNAs were successfully developed and shown to silence Ataxin-7 mRNA in numerous cell lines. An RNAi-resistant gene was developed and mirtrons could be successfully incorporated as introns. Patient-derived fibroblasts and iPSC-derived neuronal cells were investigated as models for testing of gene silencing therapies. This work suggests that mirtron-based K&R is achievable, and warrants further investigation.

Parkinson's Disease affects more than 1 million people in the United States with 60,000 new cases being diagnosed each year. Currently, there is no cure for Parkinson's Disease, but there are several treatment options available. Currently the most popular surgical option is Deep Brain Stimulation. Microelectrode recording helps identify nuclei as the microelectrode passes through them. While the firing frequencies of the target nuclei are well defined, other nuclei are not. This study will attempt to characterize the Zona Incerta, which is the structure directly above the Subthalamic Nucleus, a target nucleus. Characterization of the firing frequency of the Zona Incerta will help aid Deep Brain Stimulation procedures. Looking at the Interspike Intervals for 25 files showed that the average firing frequency is 11.6Hz. A file recorded in the STN was used for comparison and to validate the methods used. This yielded an average firing frequency of 37.5Hz.

Mesenchymal stem cells (MSC) possess a multilineage differentiation potential and have the ability to regulate reactivity of the immune system. They are usually isolated and expanded from the bone marrow, adipose tissue or umbilical cord. MSC represent promising cell population for the treatment of some severe diseases, such as amyotrofic lateral sclerosis (ALS), due to the combination of regenerative and immunomodulatory properties. The aim of this study is to compare MSC from ALS patients and healthy donors in their phenotype, proliferative activity and mainly their immunomodulatory properties. The assessment of impact of the disease on the properties of MSC is important for their autologous use in clinical trials. In this study we used MSC isolated from bone marrow of 14 ALS patients and 15 patients undergoing mostly orthopedic surgery as control group. We also used MSC stimulated for 24 hours by poinflammatory cytokines. Cells were compared in terms of immunophenotype, differentiation in adipocytes and osteoblasts, metabolic activity, expression of selected genes for immunomodulatory molecules and for inhibition of lymphocyte proliferation. Further experiments were focused on evaluation of immunomodulatory properties of MSC. The effect of MSC on peripheral blood mononuclear cells stimulated...

La dysautonomie familiale (FD) est une neuropathie héréditaire provoquée par des mutations au sein du gène IKBKAP, la plus commune d'entre elles induisant un épissage alternatif de l'exon 20 au sein de du pré-ARNm de façon tissu-spécifique. L'épissage aberrant est particulièrement prononcé dans les tissus nerveux, conduisant à la dégénerescence progressive des neurones sensoriels et autonomes. La spécificité de la perte des cellules nerveuses dans la FD est mal comprise, par manque d'un modèle approprié. Afin de mieux comprendre les mécanismes moléculaires de l'épissage des ARNm d'IKBKAP, nous avons utilisé un modèle original : les cellules souches olfactives ecto-mesenchymateuses (hOE-MSC) de patients FD. Les hOE-MSC sont pluripotentes et ont la capacité de se différencier en diverses lignées cellulaires, y compris les neurones et les cellules gliales.Nous avons confirmé la présence du transcrit exempt de l'exon 20 d'IKBKAP dans les hOE-MSC de FD et nous avons observé une expression significativement inférieure de la somme des transcrits IKBKAP chez ces patients, du fait de la dégradation d'une partie des isoforme aberrants. Cette réduction est correlée avec une réduction d'expression de la protéine traduite à partir du transcrit d’IKBKAP possèdant l’exon 20, IKAP/hELP1. Nous avons localisé IKAP/hELP1 dans différents compartiments cellulaires, y compris le noyau, ce qui soutient des rôles multiples de cette protéine. Nous avons confirmé que la kinétine, une cytokinine, améliorait le taux de transcrit incluant l'exon 20 et rétablissait des niveaux normaux d'IKAP/hELP1 dans les hOE-MSC de FD. Par ailleurs, nous avons pu modifier le rapport d'épissage d'IKBKAP en augmentant ou en réduisant le ratio WT (inclusion de l'exon 20) : MU (saut de l'exon 20) respectivement, en produisant des sphères flottantes, ou en engageant les cellules vers une différentiation neurale. Les sphères et les cellules différenciées ont été étudiées au niveau pan-génomique, ce qui a permis d'identifier le développement du système nerveux comme étant le processus le plus affecté chez les FD. De plus, nous soulignons le rôle de la kinétine comme un probable régulateur de facteurs d'épissage contribuant à la restauration d'un épissage correct d'IKBKAP.Les hOE-MSC isolées de patients FD représentent une nouvelle approche pour modéliser la pathologie et mieux comprendre l'expression génétique et les approches thérapeutiques possibles de la FD. En outre, elles offrent une application originale à la compréhension d'autres maladies génétiques neurologiques. / Familial dysautonomia (FD) is a hereditary neuropathy caused by mutations in the IKBKAP gene, the most common of which results in variable tissue-specific mRNA splicing with skipping of exon 20. Defective splicing is especially severe in nervous tissue, leading to incomplete development and progressive degeneration of sensory and autonomic neurons. The specificity of neuron loss in FD is poorly understood due to the lack of an appropriate model system. To better understand and modelize the molecular mechanisms of IKBKAP mRNA splicing, we collected human olfactory ecto-mesenchymal stem cells (hOE-MSCs) from FD patients. hOE-MSCs have a pluripotent ability to differentiate into various cell lineages, including neurons and glial cells.We confirmed IKBKAP mRNA alternative splicing in FD hOE-MSCs and observed a significant lower expression of both IKBKAP transcripts and IKAP/hELP1 protein in FD cells resulting from the degradation of the transcript isoform skipping exon 20. We localized IKAP/hELP1 in different cell compartments, including the nucleus, which supports multiple roles for that protein. Moreover, we showed that kinetin improved exon 20 inclusion and restores a normal level of IKAP/hELP1 in FD hOE-MSCs. Furthermore, we were able to modify the IKBKAP splicing ratio in FD hOE-MSCs, increasing or reducing the WT (exon 20 inclusion):MU (exon 20 skipping) ratio respectively, either by producing free-floating spheres, or by inducing cells into neural differentiation. Spheres forming cells and lineage neuroglial progenitors were investigated at the genome-wide level, and we confirmed that nervous system development was the most altered process in FD. More, we highlight kinetin role as a putative regulator of splicing factors which contribute to restore a correct splicing of IKBKAP.hOE-MSCs isolated from FD patients represent a new approach for modeling FD to better understand genetic expression and possible therapeutic approaches. This model could also be applied to other neurological genetic diseases.

Depuis sa première description par James Parkinson dans son essai sur la paralysie agitante, la maladie de Parkinson (PD) a été reconnue comme une maladie du système moteur identifié par une tétrade de symptômes, à savoir : akinésie, rigidité musculaire, tremblement au repos et instabilité posturale. Ces symptômes sont liés à la perte de la dopamine (DA) dans le striatum après la dégénérescence neuronale dans la substance noire (SN). Il est de plus en plus reconnu que les symptômes non moteurs et peut-être non dopaminergiques inévitablement émergent et s'aggravent au cours de la progression de la maladie. Les perturbations du sommeil sont parmi les principaux symptômes non moteurs et ont été reconnus comme marqueurs précliniques de la maladie. Les modèles de régulation du sommeil ont insisté sur deux processus distincts : un mécanisme de contrôle du sommeil, ou homéostat sommeil, et un oscillateur circadien. L'oscillateur circadien, basé dans le noyau suprachiasmatique (NSC) est responsable de la tendance à dormir pendant certaines phases du cycle de 24 heures et la consolidation du sommeil et de réveil en épisodes distincts. L'homéostat sommeil est chargé de surveiller et de réagir à la nécessité pour le sommeil, provoquant l'envie de dormir à dépendre sur les montants avant du sommeil ou de l'éveil. Alors que les perturbations dans les circuits et les processus homéostatiques impliqués dans la régulation du sommeil-éveil comportement sont documenté dans la maladie de Parkinson, l'implication potentielle des altérations du système circadien n'ont pas été étudiés en détail. Le but de ma thèse est d'étudier les modifications dans le système circadien en utilisant deux modèles animaux de PD : la souris et le primate non-humain / Since the first description by James Parkinson in his essay on the shaking palsy, Parkinson’s disease (PD) was recognized as a motor disease identified by a tetrad of symptoms, namely; akinesia, muscular rigidity, resting tremor and postural instability. These symptoms are known to be related to loss of dopamine (DA) in the striatum following neural degeneration in the substantia nigra (SN). It is increasingly recognized that non-motor and perhaps non-dopaminergic related symptoms inevitably emerge and worsen during disease progression. Sleep disruption is one of the major non-motor symptoms and has been suggested as a preclinical marker of the disease. Models of sleep regulation have emphasized two distinct processes: a sleep-control mechanism, or sleep homeostat, and a circadian oscillator. The circadian oscillator, based in the suprachiasmatic nucleus (SCN), is responsible for the tendency to sleep during certain phases of the 24-hour cycle and the consolidation of sleep and wake into distinct episodes. The sleep homeostat is responsible for monitoring and reacting to the need for sleep, causing the urge to sleep to depend on prior amounts of sleep or wakefulness. While disruptions in the circuitry and the homeostatic processes involved in the regulation of sleep-wake behaviour is will documented in PD, the potential involvement of alterations of the circadian system have not been studied in detail. The aim of my thesis is to investigate alterations in the circadian timing system using two animal models of PD: the mouse and the non-human primate. Taken together, the studies show that disturbances of circadian functions occur after MPTP treatment in the non-human primate but not in the mouse model of PD. These results emphasize the limitations of the MPTP-treated mouse model of PD for the study of non-motor symptoms, and reinforce previous studies that question the adequacy of this model to replicate cardinal motor features of the disease. In contrast, results in the non-human primate model stress the importance of dopaminergic degeneration in the circadian organisation of behavioral sleep wake cycle in PD

Introdução. O transplante de células é possibilidade terapêutica promissora para muitas doenças neurológicas. Nos últimos anos, a possibilidade do isolamento de células-tronco dos tecidos adultos, por exemplo da medula-óssea, atrai a atenção da comunidade científica, estratégia que minimiza os problemas éticos relativos ao uso de tecido fetal para implantes visando ao tratamento de doenças neurológicas. Entretanto, a eficiência da transdiferenciação de células-tronco mesenquimais em neurônios, bem como os mecanismos envolvidos nesse processo, permanecem desconhecidos. A obtenção de neurônios maduros ocorreu somente em sistemas de co-cultura, o que induz a questão se a diferenciação representa um potencial das células per si, ou se é possível somente devido à fusão com neurônios maduros. Objetivos. No presente trabalho, pretendeu-se verificar o potencial de as células-tronco mesenquimais tornarem-se neurônios e esclarecer os possíveis mecanismos envolvidos nesse processo. Material e métodos. Células-tronco mesenquimais foram isoladas de 20 doadores voluntários normais e caracterizadas por análise de separação celular ativada por fluorescência. A multipotencialidade foi investigada ao se diferenciar as células em condrócitos e osteócitos. A capacidade de auto-renovação foi confirmada pelo ensaio de incorporação de BrdU. Ulteriormente, as células foram diferenciadas por uma semana em meio contendo AMPc, IBMX, ou combinação de ambos, e os resultados foram comparados com o cultivo em meio básico. Diferentes bloqueadores de Ca2+ ou inibidores de PKA foram usados como tentativa de se impedir a diferenciação, ocorrência que foi mensurada com imunocitoquímica para NF-200 (marcador de neurônios maduros). O registro eletrofisiológico por meio de patch clamp foi usado para se confirmar o fenótipo neuronal. As figuras foram configuradas em microscopia confocal. Para análise estatística foi utilizada ANOVA com teste post-hoc. Resultados. As células isoladas expressaram CD90, 105, 44 e 13 mas foram negativas para CD34 e 45. Isto significa que não são de origem hematopoiética; 98,74 ± 0,43% das células incorporaram BrdU em 24 horas. Após o isolamento, foi possível diferenciá-las em condrócitos ou osteócitos. Em situação controle, não foram evidenciadas células positivas para NF200. Por outro lado, ocorreu positividade em 10,75% ± 1,35 (p<0,0001) das células sob IBMX e, em 15,18% ± 1,12, sob a combinação cAMP e IBMX (p<0,0001). Foram registradas correntes de Na+ e K+ dependentes de voltagem, mas não potenciais de ação. A diferenciação foi inibida com PKAi (5,73% ± 0,42, p<0,0001), nifedipina (5,79% ± 0,98, p<0,0001), Ni2+ (7,06% ± 1,68, p<0,0001) e Cd2+ (0 ± 0, p<0,0001). Discussão. Isolou-se uma população de células-tronco estromais da medula-óssea de seres humanos que se mostrou multipotencial e auto-renovável. O aumento da concentração de AMPc no meio elevou a concentração de neurônios para 15%. A diferenciação parece depender da via PKA mas também envolve a concentração intracelular de Ca2+. Conclusão. O correto entendimento de como as células-tronco mesenquimais diferenciam-se pode contribuir para aumentar a eficácia do método e, talvez um dia, tornar possível o uso dessa ferramenta no campo clínico. / Introduction. Cell transplantation has been considered a promising therapeutic approach for many neurological diseases. The possibility of isolation of stem cells from adult tissues, i.e. bone marrow, has attracted the attention of the scientific community in the recent years. This strategy is interesting on avoiding the ethical issues regarding the use of fetal tissue for neural implants. Moreover, the efficiency of the transdifferentiation of mesenchymal stem cells (MSCs) into neurons, and the mechanisms involved in this process remain largely unknown. The obtention of mature neurons was described only in coculture systems, what raised the question if the differentiation is a potential of the cells itself, or if it is possible only due to fusion with mature neurons. Objectives. In the present investigation, we aimed to verify the potential of MSCs to differentiate into neurons, and also to clarify the possible mechanisms involved on it. Material and methods. MSCs were isolated from 20 healthy human subjects and characterized by FACS-analysis. Multipotentiality was addressed by differentiating them into chondrocytes and osteocytes. The self-renewal capacity was confirmed with BrdU-incorporation assay. Afterwards, cells were differentiated for 1 week in a medium containing cAMP, IBMX, or a combination of both, and the results were compared with cells treated in basal-medium condition. Different Ca2+-blockers and PKA-inhibitor peptide were used on an attempt to impair differentiation, which was quantified with NF-200 immunostaining (a marker of mature neurons). Patch-clamp recording was used to confirm neuronal phenotype. Pictures were taken in confocal microscope. For statistical analysis ANOVA with a post-hoc test was used. Results. The isolated cells expressed CD90, 105, 44, and 13, but were negative for CD34 and 45, meaning that they were non-hematopoiethic; 98.74 ± 0.43 % of them incorporated BrdU in 6hs. After isolation, they differentiated into chondrocytes and osteocytes. In a control situation, no NF200 positive cell was seen. On the other hand, 10.75% ± 1.35 (p<.0001) of positivity was seen under IBMX and 15.18% ± 1.12 in the combination of cAMP with IBMX (p<.0001). Na+ and K+-voltage gated currents were recorded. Differentiation was impaired with PKAi (5.73% ± 0.42, p<.0001), nifedipin (5.79% ± 0.98, p<.0001), Ni2+ (7.06% ± 1.68, p<.0001), and Cd2+ (0 ± 0, p<.0001). Discussion. We were able to isolate a population of stromal stem cells from the bone marrow of human subjects, since they were multipotential and self-renewable. Increasing the concentration of cAMP raised the percentage of neurons up to 15%. The differentiation seems to be dependent on the PKA pathway, but also involved the intracellular concentration of Ca2+. Conclusions. The complete understanding of how MSC differentiate can contribute to increase the efficiency of the method and thus make possible to use this powerful tool in the clinical practice.

A estreptozotocina (STZ) é utilizada como modelo de indução do Diabetes, e mais recentemente, sua injeção intracerebroventricular (icv) tem sido utilizada como modelo animal de DA. Nosso objetivo neste trabalho é o de avaliar os efeitos causados por doses subtóxicas de STZ em uma linhagem de neuroblastoma sobre a cascata intracelular associada à sinalização de insulina. Os resultados confirmam a doação espontânea de NO pela STZ e sugerem que a droga é capaz de modular a cascata intracelular associada ao receptor de insulina devido à doação do radical livre. O perfil de produção de EROs induzido por STZ pode ser a causa da sua neurotoxicidade. Por fim, foi visto que o tratamento com STZ, bem como a indução de resistência à insulina, é capaz de impedir a formação de neuritos pelo tratamento com NGF. Dessa forma, este trabalho contribui para a elucidação dos mecanismos pelos quais a injeção icv de STZ pode causar algumas características de toxicidade neuronal semelhantes à DA. / Streptozotocin (STZ) has been used as an animal model for Diabetes and, more recently, its intracerebroventricular (icv) injection as an AD animal model. Our objective involves the assessment of the effects caused by subtoxic doses of STZ in a neuroblastoma cell line upon insulin signaling associated intracellular cascade. Results confirm STZ spontaneous NO donation and suggest that the drug is capable of modulating the intracellular cascade associated to insulin receptor due to the spontaneous donation of the free radical. Therefore, EROs production may play an important role in the mechanism linked to STZ induced neurotoxicity. Ultimately, it has been shown that STZ treatment, as well as insulin resistance alone, is capable of impair neurite outgrowth by NGF treatment. This work contributes for the elucidation of the mechanisms by which STZ icv injection may cause features similar to DA.

Depuis le début des années 2000, la connaissance précise du kinome a entraîné l’émergence de nouvelles stratégies thérapeutiques ciblant des protéines kinases impliquées dans de nombreuses pathologies en oncologie et dans les maladies du système nerveux central. Afin de cibler les kinases d’intérêts identifiées au sein de ces travaux, nous avons effectué, dans une démarche orientée vers la diversité moléculaire, la synthèse de nouveaux hétérocycliques a fort potentiel de valorisation. Nous nous sommes appuyé sur la création et la fonctionnalisation de bicycles à 5 ou 6chaînons de type [6-5] ou [5-5], ces espèces chimiques représentant la voie d’accès à des inhibiteurs compétitifs du substrat naturel des kinases, l’ATP. Nous avons dans un premier temps travaillé autour des imidazo[1,2-b]pyridazines puis des[1,2,4] triazolo[4,3-b]pyridazines, scaffold plus original, pour concevoir des inhibiteurs plus actifs et spécifiques de la kinase HASPIN, nouvelle cible prometteuse en oncologie.Nous avons ensuite poursuivi les études précédentes du laboratoire sur les imidazo[2,1-b][1,3,4]thiadiazoles. Nous basant sur une méthodologie de synthèse bien développée, nous avons créé une librairie de composés dirigés contre les kinases DYRK1A et CLK1 impliquées dans les processus de neuro dégénération, notamment dans la maladie d’Alzheimer. Ainsi, au travers d’analogues des imidazothiadiazoles originaux, nous avons proposé des méthodologies de synthèses de ces nouveaux hétérocycles permettant des pharmaco modulations aisées.Ces divers projets de chimie médicinale ont pu être entrepris de façon à améliorer les connaissances des relations structure-activité, et concevoir de nouveaux inhibiteurs puissants des kinases HASPIN, DYRK1A etCLK1. / Since the early 2000s, precise knowledge of kinome has induced the emergence of novel therapies targeting kinases involved in several kinds of pathologies in oncology and nervous central systems disorders.In order to target original kinases of interest identified in this work, we have developed diversity-oriented synthesis to create new high-valuable heterocycles. We have focused our efforts on the design and functionalization of [6-5] or [5-5] fused ring bicycles. Those chemical species representing a great pathway tocreate competitive inhibitors of ATP; the natural substrate of kinases.First-of-all, we have worked on imidazo[1,2-b]pyridazines and then on [1,2,4]triazolo[4,3-b]pyridazinesscaffolds, to create more active and selective HASPIN kinase inhibitors, a new hot-target in oncology.Then, we have pursued previous laboratory studies on imidazo[2,1-b][1,3,4]thiadiazoles. Based on a well-built methodology, we have synthesized severals DYRK1A and CLK1 kinases inhibitors involved in neurodegenerative disorders, as Alzheimer’s disease. Thus, through original imidazothiadiazoles analogues,we have proposed synthetic methodologies to design these novel heterocycles allowding esay pharmacomodulations.These medicinal chemistry projects have been undertaken to improved knowledge of structure-activityrelashionship, and providing novel strong inhibitors of HASPIN, DYRK1A or CLK1 kinases.

La carence en vitamine B12 est fréquente chez les sujets âgés et produit un vieillissement cérébral par des mécanismes malconnus. La vitamine B12 joue un rôle majeur dans les régulations épigénomiques dépendantes de la S-adénosyl méthionine (SAM). Nous avons établi un modèle de cellules neuronales dopaminergiques NIE115 carencé en vitamine B12 par l'expression stable d'une protéine chimère : la transcobalamine-oléosine (TO) réduisant la disponibilité cellulaire en B12, la SAM et la prolifération cellulaire. La protéine chimère oléosine-transcobalamine (OT) ne lie pas la B12 et constitue un contrôle. Les cellules TO ont une diminution B12-dépendante de la déacétylase SIRT1 (sirtuin1) et un stress du réticulum endoplasmique (RE) avec une augmentation des transducteurs transmembranaires, une diminution des protéines chaperonnes et une augmentation des marqueurs pro-apoptotiques. La diminution de l'expression de SIRT1 déclenche le stress du RE en réponse au stress nutritionnel. Cette diminution produit une augmentation de HSF1 acétylé diminuant l'expression des protéines chaperons. L'ajout de B12, des activateurs de SIRT1 et HSF1, la surexpression de SIRT1 et HSF1 réduisent le stress du RE. Dans les cellules contrôles, le traitement par la thapsigargin, l'inhibition de SIRT1 et HSF1 induisent également un stress du RE réversible en présence de B12. Le traitement des cellules OT par Adox (inhibiteur des méthyltransférases) induit les mêmes effets que la carence. En conclusion, la carence en B12 induit un stress du RE via la diminution de SIRT1 et l'augmentation de HSF1 acétylé, l'ajout de B12 induit des effets neuro-protecteurs sur les cellules soumises à un stress du RE. Ces résultats suggèrent d'évaluer les effets des agonistes de SIRT1 sur les complications cérébrales de la carence / Vitamin B12 deficiency is common in elderly population and produces neurodegenerative disorders by elusive mechanisms. B12 is a key determinant for the S-adenosyl methionine-dependent epigenomic regulations. We have established a B12-deficient cell model via the stable expression of transcobalamin-oleosin chimera (TO), which impairs cellular availability of vitamin B12, reduces SAM level and cell proliferation. Since the expression of oleosin transcobalamin chimera (OT) does not modify the phenotype of the transfected cells, these cells serve as control cells. TO cells present a B12-dependant decrease of deacetylase SIRT1 (sirtuin1) and an endoplasmic reticulum stress (ER stress) reflected by the increased expression of ER stress tranducers, decreased chaperon proteins and increased pro-apoptotic markers. We propose that the decreased expression of SIRT1 triggers cell response to nutritional stress through ER stress. This decrease results in a greater acetylation of heat-shock factor protein 1 (HSF1) and thus reducing the expression of heat shock proteins (HSP). Adding B12, SIRT1, or HSF1 activators as well as overexpressing SIRT1 or HSF1 reduce ER stress. In OT cells, thapsigargin treatment or impairing SIRT1 and HSF1 leads to B12-reversible ER stress. Treating OT cells with AdoX, an inhibitor of methyltransferase activities, produces effects similar to those observed in cells with decreased B12 availability.In summary, the impaired cellular availability of vitamin B12 induces ER stress by increasing HSF1 acetylation through a decreased SIRT1 expression and adding vitamin B12 produces neuro-protective effects in cells subjected to prior ER stress. These results suggest evaluating the effects of SIRT1 agonists on cerebral complications due to a B12 deficiency