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.

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The histopathological and immunohistochemical technique makes it possible to differentiate the subtypes of dementia by pre-established macroscopic and microscopic criteria. Banks brain, although recent, provide biological material quality for multidisciplinary research in normal subjects and with dementia. Objectives: To correlate clinical findings with neuropathological cases with dementia from the Brains Bank of Central of Brazil (BBCB); establish morphological patterns in macroscopic focal dementias; determine the prevalence of diagnosis of other types of dementia. Materials and Methods: Brain Study from autopsies of patients with neurodegenerative diseases of dementia clinic of the HC-UFG (Ethics Committee of the Protocol on research 0692007). The brains were processed following dissection and measurement protocol. Appropriate external and macroscopic morphological descriptions and coronal and sagittal sections were performed. Results: 15 brains, 9 female patients were studied, aged 10 to 89 years. The types of dementias found in BCBC were 5 cases of frontotemporal dementia (FTD), 3 Alzheimer's disease (AD), 3 patients had primary progressive aphasia (PPA) and corticobasal degeneration (CBD), 1 Huntington's disease, 1 disease Creutzfeldt-Jakob 1, Rasmussen's encephalitis and 1 depressive pseudodementia (Cotard’s syndrome). Described frontal gyrus supernumerary in 3 cases of CBD and 2 cases of FTD. Discussion: Most cases presented morphological pattern of the respective type of dementia according to the literature, except PPA with CBD. In BCBC material only 20% of AD cases were 27% and frontotemporal lobar degeneration (FTLD). Conclusion: The higher prevalence of dementia in BCBC was the type FTLD. The frontotemporal focal atrophy was the most observed type of change. The cases with FTD showed classic morphological patterns, while the PPA CBD was different standard literature. The BCBC will enable studies in various research areas. / O diagnóstico definitivo das demências é feito através de necropsia. Os exames anatomopatológico e imunoistoquímico possibilitam diferenciar os subtipos de demência por critérios macroscópicos e microscópicos pré-estabelecidos. Os bancos de cérebros, apesar de recentes, fornecem material biológico de qualidade para pesquisas multidisciplinares de indivíduos normais e com demência. Objetivos: Correlacionar aspectos clínicos com alterações neuropatológicas de casos com demências provenientes do Banco de Cérebros do Brasil Central (BCBC); estabelecer padrões morfológicos macroscópicos nas demências focais; verificar a prevalência do diagnóstico de outros tipos de demências. Materiais e Métodos: Estudo de cérebros provenientes de necropsias de pacientes com doenças neurodegenerativas do ambulatório de demências do HC-UFG (protocolo do Comitê de ética em pesquisa 0692007). Os cérebros foram processados seguindo protocolo de dissecção e mensuração. Foram realizadas as devidas descrições morfológicas e macroscópicas externas e dos cortes coronais e sagitais. Resultados: Foram estudados 15 cérebros, 9 de pacientes do sexo feminino, com idade entre 10 a 89 anos. Os tipos de demências encontrados no BCBC foram: 5 casos de demência frontotemporal (DFT), 3 de doença de Alzheimer (DA), 3 casos com afasia progressiva primaria (APP) e degeneração corticobasal (DCB), 1 de doença de Huntington, 1 de doença de Creutzfeldt-Jakob, 1 de encefalite de Rasmussen e 1 de pseudodemência depressiva (síndrome de Cotard). Foi observado giro frontal supranumerário nos 3 casos de DCB e em 2 casos de DFT. Discussão: A maioria dos casos apresentou padrão morfológico do respectivo tipo de demência de acordo com a literatura, exceto APP com DCB. No material do BCBC apenas 20% dos casos foram de DA e 27% degeneração lobar frontotemporal (DLFT). Conclusão: A maior prevalência de demências no BCBC foi do tipo DLFT. A atrofia focal frontotemporal foi o tipo de alteração mais observada. Os casos com DFT apresentaram padrões morfológicos clássicos, enquanto que os de APP com DCB apresentaram padrão diferente da literatura. O BCBC possibilitará a realização de estudos em várias linhas de pesquisa.

As doenças neurodegenerativas (DN) estão entre as principais causas de mortalidade e morbidade nos países ocidentais. Não há ainda um tratamento definitivo para estas neuropatias, mas estudos têm indicado mecanismos comuns de toxicidade que incluem disfunção mitocondrial, estresse oxidativo, apoptose e neuroinflamação. Adicionalmente, o efeito benéfico da neuroplasticidade induzida por fatores neurotróficos no retardamento ou inibição do processo neurodegenerativo também tem sido sugerido por muitos estudos. O beta-cariofileno é um sesquiterpeno bi-cíclico encontrado no óleo essencial de algumas plantas, e que possui efeito anti-inflamatório e antioxidante. Assim, este composto possui características e é capaz de induzir efeitos que o tornam um potencial candidato ao tratamento/prevenção dos processos envolvidos na neurodegeneração. Apesar disso, pouco se sabe sobre os efeitos e os mecanismos de ação do beta-cariofileno no processo de degeneração neuronal. Então, neste estudo, avaliou-se o efeito do beta-cariofileno em modelos celulares (PC 12) de neurotoxicidade que mimetizam in vitro os mecanismos moleculares envolvidos nas doenças de Parkinson, Huntington e Alzheimer, os quais, para efeitos práticos, denominaremos de \"modelos celulares de Parkinson, Huntington e Alzheimer\". Estes modelos são induzidos experimentalmente pela neurotoxina dopaminérgica iodeto de 1-metil 4-fenil piridina (MPP+), pela neurotoxina mitocondrial ácido 3-nitropropiônico (3NP) e pelo peptídeo neurotóxico B-amiloide (AB42), respectivamente. O beta-cariofileno apresentou efeitos benéficos nestes três modelos de neurotoxicidade, e adicionalmente induziu neuritogênese e a expressão de proteínas neurotípicas no modelo neuronal. Este é o primeiro estudo a demonstrar tais efeitos do beta-cariofileno. / Neurodegenerative diseases (ND) are among the leading causes of mortality and morbidity in Western countries. There is not a definitive treatment for these neuropathies, but studies have indicated common mechanisms of toxicity that include mitochondrial dysfunction, oxidative stress, neuroinflammation and apoptosis. Additionally, the beneficial effect of the neuroplasticity induced by neurotrophic factors on the retardation or inhibition of neurodegeneration has also been suggested by several studies. Beta-caryophyllene is a bicyclic sesquiterpene found in essential oils of some plants, and possesses anti-inflammatory and antioxidant effects. Thus, this compound has characteristics and is capable of inducing effects that make it a potential candidate for treatment / prevention of the processes involved in neurodegeneration. Despite this, little is known about the effects and mechanisms of action of beta-caryophyllene in the neuronal degeneration process. Then, this study evaluated the effect of beta-caryophyllene in cellular models of neurotoxicity (PC 12) that mimic in vitro the molecular mechanisms involved in Parkinson\'s, Huntington\'s and Alzheimer\'s diseases, which, for practical purposes, we will denominate \"Cellular models of Parkinson\'s, Huntington\'s and Alzheimer\'s diseases.\" These models are experimentally induced by the dopaminergic neurotoxin 1-methyl iodide, 4-phenyl pyridine (MPP+), by the mitochondrial neurotoxin 3-nitropropionic acid (3NP) and the neurotoxic peptide B-amyloid (AB42), respectively. Beta-caryophyllene showed beneficial effects on these three models of neurotoxicity, and additionally induced neuritogenesis and the expression of neurotypic proteins in the neuronal model. This is the first study to demonstrate such effects of beta-caryophyllene.

A neurodegeneração é um processo onde ocorre morte celular progressiva. O tráfego neuronal anterógrado e retrógado, e entre os compartimentos é essencial para a viabilidade celular. As proteínas Rabs pertencem à família de pequenas GTPases, com funções de tráfego de vesículas e organelas, para realizarem sua função as proteínas Rab podem recrutar proteínas motoras como as KIF 1B e KIF 5, responsáveis pelo transporte anterógrado mitocondrial. A associação do distúrbio do tráfego intracelular com doenças neurodegenerativas tem sido tema de estudos recentes. Com isso o objetivo do presente trabalho é analisar a expressão das proteínas Rab, bem como estudar as proteínas motoras que podem contribuir para o esclarecimento sobre os distúrbios no tráfego intracelular que antecedem a formação de agregados proteicos envolvidos em neurodegeneração. Para tanto, utilizou-se o modelo de tratamento com rotenona para indução de agregados em Ratos Lewis idosos que foram expostos a rotenona durante 4 semanas, em seguida foram avaliados os níveis de expressão das proteínas Rab no hipocampo, substância negra e locus coeruleus, por western blotting. Foram analisados também os níveis de expressão das proteínas motoras KIF1B e KIF5 antes e durante a formação de agregados proteicos, em culturas de células, de ratos Lewis neonatos, do hipocampo, substância negra e locus coeruleus tratadas com rotenona por 24 horas ou 48 horas nas concentrações de 0,1nM, 0, 3nM e 0,5nM. Foi observado diminuição dos níveis de expressão das proteínas Rab 1 nas regiões do hipocampo e locus coeruleus. Houve aumento de expressão das Rab 4,5 e 6 no hipocampo, porém na substância negra a expressão da Rab 1 aumentou e da Rab 6 diminuiu. Já no locus coeruleus in vivo a Rab 6 aumentou, mas as Rab 1, 5 e 11 diminuíram sua expressão. Já a expressão da KIF 5 aumentou com o tratamento de 0,1nM de rotenona e diminuiu após 0,5nM do xenobiótico por 48 horas in vitro, na mesma região. Na substância negra aumentaram as KIFs 1B e 5 após o tratamento com 0,5nM por 48 horas in vitro, mas diminuíram as KIF 1B e 5 após o tratamento com 0,3nM por 24 horas e KIF 5 após o tratamento com 0,1nM por 48 horas. Esses resultados permitem concluir que a expressão de proteínas importantes para o tráfego mitocondrial e de vesículas encontram-se alteradas e fazem parte dos eventos intracelulares que antecedem a neurodegeneração / Neurodegeneration is a process that leads to progressive cell death. The anterograde and retrograde neuronal traffic as well as the traffic between compartments are essential for cell viability. The Rab proteins belong to the small GTPases family with function of vesicles and organelle trafficking. Rab proteins can recruit motor proteins such as KIF 1B and KIF 5 that are responsible for anterograde mitochondrial transport. The association of intracellular traffic disturb with neurodegenerative diseases have been theme of recent studies. Thereat the objective of this study is analyze the expression of Rab and motor proteins that can contribute for the understanding about the disturb of the intracellular traffic that precedes protein aggregation involved in neurodegeneration. For this purpose it was employed the model of rotenone treatment for induction of aggregation in aged Lewis rats that were exposed to rotenone during 4 weeks in order to evaluate Rabs expression. The levels of motor proteins KIF 1B and KIF 5 expression were evaluated before and during the formation of protein aggregates in hippocampus, substantia nigra and locus coeruleus cell cultures of neonates Lewis rats, exposed to rotenone for 24 hours or 48 hours in the concentrations of 0.1nM, 0.3nM or 0.5nM. It was observed decreased levels of Rab 1 expression in hippocampus and locus coeruleus. Rabs 4,5 and 6 were increased in the hippocampus, but in the substantia nigra the expression of Rab 1 increased and Rab 6 decreased. In the locus coeruleus the Rab 6 increased, but Rabs 1, 5 and 11 decreased. The expression of KIF 5 increased after 0.1nM of rotenone and decreased after the exposure to 0.5nM of for 48 hours in cultured cell from the locus coeruleus. In the substantia nigra the KIF1B and KIF 5 increased after treatment with 0.5nM for 48 hours in vitro, but these protein decreased after treatment with 0.3nM for 24 hours in vitro, and KIF 5 after treatment with 0.1nM for 48 hours. These results allow us conclude that the expression of important proteins for the mitochondrial and vesicles traffic are altered and participate of intracellular events that precede the neurodegeneration

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.

La microscopie non-linéaire résolue en polarisation est un outil puissant pour accéder à des informations structurelles dans les assemblages biomoléculaires. Les interactions non-linéaires entre matière et lumière induisent des processus complexes où des champs électromagnétiques cohérents interagissent avec les dipôles de transitions moléculaires. Le contrôle de la polarisation des champs électromagnétiques excitateurs et l’étude des réponses non-linéaires induites procurent de riches informations sur la distribution angulaire des molécules présentes dans le volume focal de l’objectif du microscope. Dans cette thèse, nous appliquons cette sensibilité à la polarisation à plusieurs modalités de microscopie cohérentes sans marquage (diffusion cohérente Raman anti-Stokes (CARS), diffusion Cohérente stimulée (SRS)) et à la fluorescence à deux photons (2PEF) afin d’obtenir des informations quantitatives sur la forme de la distribution moléculaire et l’orientation des lipides dans les membranes artificielles, ainsi que dans les membranes biologiques telles que la myéline des tissus de la moelle épinière. Avec cette technique, nous adressons une question fondamentale sur le comportement des ensembles lipidiques dans les membranes et sur l’effet d’autres molécules telles que le cholestérol et les marqueurs fluorescents. Nous démontrons que le CARS résolu en polarisation permet d’accéder à de fines informations sur l’organisation des lipides dans les membranes de la myéline, en deçà de la limite de diffraction. / Polarization resolved nonlinear microscopy is a powerful tool to image structural information in biomolecular assemblies. Nonlinear interaction between light and matter lead to complex processes where coherent combinations of optical fields couple to assemblies of molecular transition dipoles. Controlling polarized optical fields and monitoring nonlinear induced signals in a medium can nevertheless bring rich information on molecular orientational organization within the focal spot of a microscope objective. In this PhD thesis we apply this polarization sensitivity to different label-free optical coherent techniques (coherent anti-Stokes Raman scattering (CARS), stimulated Raman scattering (SRS)) and to two-photon fluorescence (2PEF) to retrieve quantitative information on the static molecular distribution shape and orientation of lipids in model membranes and biological membranes such as myelin sheaths in spinal cord tissues. With this technique, we address fundamental questions about lipid packing behavior in membranes, and how it can be affected by other molecules such as cholesterol and the insertion of fluorescent lipid probes. We demonstrate that polarization resolved CARS give access to fine details on lipids arrangement in myelin sheaths, at a sub-diffraction scale. In the context of experimental autoimmune encephalomyelitis disease (EAE) we show, that even at the stage of disruption of the myelin envelope during the demyelination process, lipids multilayers reveal strong capability to preserve their macroscopic self-assembly into highly organized structures, with a degree of disorganization occurring only at the molecular scale.

>Magister Scientiae - MSc / This study focused on the synthesis of a series of novel tricycloundecane derivatives and evaluation of these compounds for neuroprotection using the fluorescent ratiometric calcium assay that indicates the ability of the test compounds to inhibit NMDA receptors and VGCC. The cycloaddition reaction between p-benzoquinone and monomerised dicyclopentadiene yielded tricycloundeca- 4,9-diene-3,6-dione which was used as the base structure and further derivatised. These derivatives were conjugated with benzylamine to form a series of imines and amines. A total of 10 compounds were synthesised for evaluation of inhibition of calcium influx through NMDA receptor channels and voltage-gated calcium channels. The structures were confirmed using NMR, IR and MS. On the proton NMR, the characteristic AB-quartet system was observed in the region of 1-2 ppm for all the compounds and the aromatic moiety was observed between 6.5-7.5 ppm for the novel polycyclic amines. These, with other functional groups, were used to confirm the individual structures

The onset of a progressive, fatal illness such as Motor Neuron Disease (MND) inevitably results in physical and communication disabilities that impinge on the individuals’ ability to remain functionally independent. The loss of speech as a result of dysarthria, a motor speech disorder, is one of the most profound changes that the person with MND will experience. The decline in the individuals’ speech intelligibility, that negatively influences communication effectiveness, implies that in 80% of cases alternative and augmentative communication (AAC) strategies are required to support the daily communication needs of individuals with MND. The dyadic nature of chronic illness implies that multiple aspects of one of the most important adult relationships, marriage, will be affected. Roles and responsibilities performed by each member of the couple will continually change as the disease progresses. The emotional trauma of adjusting to the unavoidable alteration in their relationship elicits strong emotions such as guilt, anger and frustration. Communication is one of the most constructive ways of dealing with these emotions. The ability of spouses to convey their innermost thoughts, feelings and intimacy through communicative interaction is vitally important in marital communication. The aim of this study was to compare how persons with MND and their spouses perceive changes in their marital communication in relation to the deteriorating speech of persons with MND. Fourteen couples divided into two participant groups, persons with MND and spouses, participated in this non-experimental correlational research study. Data was collected during three visits at six-monthly intervals over a 12 month period. At each of these visits both participant groups completed a variety of objective and subjective measures, of which twenty percent were interrated by independent raters. Results confirmed the inevitable decline in speech intelligibility of persons with MND across the disease progression. The persons with MND did not report a change in their perception of marital communication although their spouses indicated a statistically significant decrease between the first and last visits. Interestingly, there was no statistically significant relationship between the deteriorating speech of persons with MND and the couples’ perception of marital communication, confirming that marital communication was not influenced by decreased speech intelligibility. / Thesis (PhD)--University of Pretoria, 2010. / Centre for Augmentative and Alternative Communication (CAAC) / unrestricted

Les paraplégies spastiques héréditaires (PSH) font partie d’un groupe plus large de pathologies neurodégénératives associant une spasticité. J’ai exploré la variabilité clinique et moléculaire de ces pathologies à l’aide d’une cohorte de familles soudanaises. Nous avons recruté 41 familles soudanaises [337 individus/106 atteints de PSH]. J’ai extrait l’ADN génomique et constitué une banque. Le criblage de gènes candidats a été réalisé dans 4 familles en fonction du phénotype des patients. La technologie de séquençage de nouvelle génération (SNG) appliquée à 74 gènes de PSH a ensuite été appliquée aux 37 cas restants. Enfin, le séquençage de l’exome a permis de rechercher les gènes en cause dans les cas négatifs. Dans certains cas, des études fonctionnelles ont été utilisées afin de valider l’effet biologique des mutations. J’ai pu identifier la cause génétique dans 17 familles. Dans 12 familles, la mutation concernait un gène de PSH connu. Dans 3 familles, un nouveau gène a été identifié. 5 gènes candidats restent à départager dans 2 familles. Il est à noter que parfois, de multiple mutations ou maladies génétiques ségrégaient dans nos familles, dans la même branche ou dans des branches séparées. La complexité de ces familles fortement consanguines a rendu l’analyse des données du SNG difficile. Une autre particularité a été l’hétérogénéité clinique associée à des mutations du même gène entre patients de la même famille ou en comparaison avec la littérature. Ce travail est la première étude à grande échelle de patients soudanais avec PSH et rapporte de nouveaux gènes en cause, prérequis pour mieux comprendre dans le futur les mécanismes sous-jacents. / Hereditary spastic paraplegias (HSP), a heterogeneous group of spastic neurodegenerative disorders which impose diagnostic challenges. I explored the clinical varieties and genetic pathways of spastic neurodegeneration in a familial Sudanese cohort. We recruited 41 Sudanese families [337 individuals/106 HSP patients]. I have established a genomic DNA bank and when necessary, skin biopsies and fibroblasts were also obtained. A phenotype-based candidate gene approach was followed in 4 families. A targeted next generation sequencing (NGS) for 74 HSP-related genes was the main screening strategy in all-remaining 37 families. Whole exome sequencing (WES) was done in search for novel mutations in new genes in families with negative screening results. Occasionally, functional studies were conducted when feasible and relevant. I identified the genetic cause in 17/41 families. In 12 families, the mutated genes were known HSP genes. In 3 families, novel genes were identified mutated. 5 candidate genes segregated with disease in 2 other families with more experiments needed to conclude. Analysis of the NGS screening panel and of WES data imposed certain challenges as multiple genetic disorders were sometimes found running in parallel in the same/different branches of highly inbred families. We could expand the phenotypic heterogeneity of these disorders due to clinical differences observed between Sudanese patients and patients of other origins even when caused by mutations by the same gene/variant. This is the first genetic screening in a large set of HSP families in Sudan. It describes new causative genes, paving the way for further deciphering of the underlying mechanisms.

Beta-N-methylamino-L-alanine (BMAA) is a non-protein amino acid produced by many cyanobacteria, and thought to induce neurotoxic effects through excitotoxicity, contributing to neurodegenerative diseases such as Amyotrophic Lateral Sclerosis/Parkinsonism-dementia complex (ALS-PDC) and Alzheimer’s. The ubiquitous nature of cyanobacteria, and evidence of biomagnification through our food web, creates a dire need for the development of an analytical platform that will provide accurate identification and quantification of BMAA amounts in our ecosystem and potential food supply. The present study evaluated the ability of a MALDI-ToF-MS method to detect and quantify BMAA in a variety of biological matrices. Through validation procedures, it was demonstrated that this MALDI-ToF-MS method provided comparable data to currently accepted analytical methods, specifically LC-MS/MS. Further, the development of said method reduced sample preparation and data acquisition time (1-2 seconds per sample), while providing high throughput analysis and eliminating the need for derivatization, chromatographic separation, and modification of amino acids.