L’asthme est une maladie inflammatoire chronique induite par des allergènes ou des substances environnementales irritantes et caractérisée par une hyperréactivité des voies respiratoires. Il existe un lien entre l’inflammation neurogène et l’inflammation immunitaire au niveau des voies respiratoires. Des études suggèrent que des facteurs neurotrophiques participent à l’apparition des symptômes de l’asthme.Chez la souris, la Neurturine (NTN) est un facteur neurotrophique qui serait capable de moduler les principaux symptômes liés à l’asthme via un récepteur spécifique, le GDNF Family Receptor alpha 2 (GFRalpha2) et son co-récepteur, la tyrosine kinase RET. Afin de déterminer par quels mécanismes la NTN peut influencer le niveau d’inflammation de la voie Th2, des souris sauvages et NTN-/- ont été comparées dans différents modèles d’asthme aigu ou chronique induits avec les allergènes ovalbumine ou du « House Dust Mite ». L’implication de la NTN au niveau de l’inflammation neurogène, de la régulation des cellules immunitaires et dans le remodelage des tissus a été évaluée. Son rôle anti-inflammatoire a également été testé in vitro.Les résultats obtenus suggèrent que la Neurturine est capable d’agir in vivo et in vitro comme médiateur anti-inflammatoire. / Allergic asthma is a chronic inflammatory disease in response to allergens and environmental irritants. The pathophysiology of asthma is defined by airway inflammation and airway hyperreactivity. Interestingly, it has been shown that there is a link between neurogenic and immune airway inflammation. Moreover studies suggest that neurotrophic factors participate in the pathogenesis of many features and symptoms of asthma.Neurturin (NTN) is a neurotrophic factor which could be involved in the modulation of many symptoms of asthma through the GDNF family receptor alpha 2 (GFRalpha2) and the proto-oncogene RET co-receptor. However, the underlying mechanisms remain unclear. By studying WT and NTN-/- mice after acute and chronic airway inflammation protocols induced by the allergens ovalbumin or house dust mite, we investigated how NTN is able to modulate the level of Th2 responses through neurogenic inflammation and immune cells’ regulation. We analysed its relationship with structural airway remodelling and we also tested the potential anti-inflammatory role of NTN in vitro.The achievements suggest that Neurturin acts in vivo and in vitro as an anti-inflammatory mediator.

Thesis is focused on special education - speech therapy and summarizes current findings about etiology, classification and symptomatology of aphasia, it also involves different approaches to the diagnosis and therapy. Thesis contains therapeutical materials, the use of which in practice is illustrated by case studies of five individuals with aphasia.

To form complex organisms characterized by different tissues with specialized functions, cells must acquire distinct identities during development. Yet, all the cells of an organism are equipped with the same genomic information. Elucidating the mechanisms that regulate the determination of a cell identity, i.e. the cell-fate commitment, is a main purpose in developmental biology. Numerous studies focused on genes that are activated or repressed at each stage of differentiation, identifying several key regulators of development. However, this approach ignores the transcript variability derived from alternative splicing, the transcriptional process by which different gene coding segments, i.e. exons, are combined giving rise to multiple transcripts and proteins from the same gene. With the advent of novel sequencing technologies, it is becoming clear that alternative splicing is widespread in higher organisms, regulates several processes and presents tissue- and cell-specificity. In mammals, the brain shows the highest degree of alternative splicing, with neurons expressing a high variety of splice variants. In this project I investigated whether and how alternative splicing could regulate cell-fate determination in the context of the embryonic development of the mouse neocortex, a highly complex structure presenting several different neuronal subtypes generated at specific time points. For this purpose, I analyzed transcriptome data of cells of the neurogenic lineage isolated from the developing mouse neocortex at subsequent stages of differentiation. I showed that the expression pattern of the proteins regulating splicing, i.e. the splicing factors, changes during neocortical development. By employing several bioinformatic tools, I described the splicing profile that characterizes each differentiation stage and, for the first time, I identified the splicing events that mark cell-fate commitment to a neurogenic identity. Alternative splicing mostly involved genes with a role in nervous system development, cell growth and signaling, mainly leading to the production of alternative protein isoforms. Splicing choices taken during the neurogenic commitment were kept throughout neurogenesis. Thus, exons that start to be included during cell-fate determination are always included in post-mitotic neurons. Exons gained during neurogenic commitment were characterized by strong features in their upstream intron, presented a general short length with an overrepresentation of microexons in the 3-27 nucleotides length range and showed an enrichment for binding motifs of the neural splicing factor nSR100. In vivo manipulation in the embryonic mouse neocortex highlighted isoform-specific effects on neocortical development, strongly suggesting a causal relationship between alternative splicing choices and cell-fate commitment. Moreover, the higher cell-specificity offered by the present dataset, compared to similar studies, allowed a better understanding of previously identified splicing events that characterize the nervous system and the relationships between neural-specific splicing factors.:Table of Contents Abstract I Zusammenfassung III Table of Contents V List of Figures VII List of Tables IX Abbreviations X Gene abbreviations XII 1 Introduction 1 1.1 Neurogenesis during embryonic development 2 1.1.1 Formation and patterning of the neural tube 2 1.1.2 Neural progenitors in the dorsal telencephalon 6 1.1.3 Neurogenesis 8 1.1.4 Regulation of neurogenesis 10 1.1.5 A novel tool to investigate cell-fate determination in the central nervous system: the Btg2RFP/Tubb3GFP mouse line 13 1.2 Alternative splicing: an additional level of genomic regulation 15 1.2.1 The splicing reaction 16 1.2.2 What makes splicing alternative? 18 1.2.3 Regulation of alternative splicing 19 1.2.4 The challenge to detect splicing 23 1.2.5 New sequencing technologies reveal a high transcriptome complexity 29 1.2.6 Splicing in nervous system development 31 1.2.7 Aims of the project 36 2 Materials and methods 38 2.1 Materials 38 2.1.1 Bacteria, cells, mouse strains 38 2.1.2 Vector 38 2.1.3 Primers 38 2.1.4 Chemicals and buffers 41 2.1.5 Antibodies 42 2.1.6 Kits and enzymes 42 2.2 Methods 43 2.2.1 Animal experiments 43 2.2.2 Molecular biology 44 2.2.3 Immunohistochemistry 46 2.2.4 Bioinformatics 47 3 Results 53 3.1 Splicing factors are differentially expressed during neurogenic commitment and neurogenesis 53 3.2 Detection of alternative splicing 55 3.2.1 Isoform-switching 55 3.2.2 Exon usage and splicing events 57 3.3 Validation 62 3.3.1 The isoform switching method has a poor validation rate 62 3.3.2 Analysis at the exon level has a high rate of validation 65 3.4 Pattern and representation of splicing events 67 3.4.1 Splicing choices during neurogenic commitment define the splicing profiles of neurons 67 3.4.2 Splicing events: microexon inclusion characterizes neurogenic commitment 69 3.5 Alternative splicing changes the protein output of genes involved in neurogenesis 75 3.5.1 Spliced genes are involved in neurogenesis and signaling 75 3.5.2 Impact of alternative splicing on the proteome 77 3.6 Splicing regulation: neural exon features and splicing factor binding 79 3.6.1 Included neural exons are short and preceded by strong exon-definition features 79 3.6.2 Early included exons are enriched for nSR100 binding sites 85 3.7 The Btg2RFP/Tubb3GFP mouse line outperforms previous models for the study of cell-type-specific splicing in the brain 88 3.8 In vivo manipulation of splice variants 90 4 Discussion 94 4.1 The combination of different bioinformatic approaches allows an accurate identification of splicing events at the exon-level 95 4.2 Splicing choices during neurogenic commitment establish a neural signature characterized by microexon inclusion 97 4.3 Splicing during neocortical development leads to the generation of alternative protein isoforms in genes involved in neurogenesis and signaling 98 4.4 Neural exons are short and present strong features facilitating inclusion 101 4.5 Neural exons are prevalently regulated by nSR100 during neurogenic commitment 102 4.6 In vivo overexpression of splice variants highlights isoform-specific functions in neurogenic commitment 105 4.7 Concluding remarks and future perspectives 108 5 Supplementary figures 110 6 References 118 Acknowledgments 137 Anlange I 138 Anlange II 139

Background: Consecutive adult neurogenesis is a well-known phenomenon in the ventricular–subventricular zone of the lateral wall of the lateral ventricles (V–SVZ) and has been controversially discussed in so-called “non-neurogenic” brain areas such as the periventricular regions (PVRs) of the aqueduct and the fourth ventricle. Dopamine is a known modulator of adult neural stem cell (aNSC) proliferation and dopaminergic neurogenesis in the olfactory bulb, though a possible interplay between local dopaminergic neurodegeneration and induction of aNSC proliferation in mid/hindbrain PVRs is currently enigmatic. Objective/Hypothesis: To analyze the influence of chronic–progressive dopaminergic neurodegeneration on both consecutive adult neurogenesis in the PVRs of the V–SVZ and mid/hindbrain aNSCs in two mechanistically different transgenic animal models of Parkinson´s disease (PD). Methods: We used Thy1-m[A30P]h α synuclein mice and Leu9′Ser hypersensitive α4* nAChR mice to assess the influence of midbrain dopaminergic neuronal loss on neurogenic activity in the PVRs of the V–SVZ, the aqueduct and the fourth ventricle. Results: In both animal models, overall proliferative activity in the V–SVZ was not altered, though the proportion of B2/activated B1 cells on all proliferating cells was reduced in the V–SVZ in Leu9′Ser hypersensitive α4* nAChR mice. Putative aNSCs in the mid/hindbrain PVRs are known to be quiescent in vivo in healthy controls, and dopaminergic deficiency did not induce proliferative activity in these regions in both disease models. Conclusions: Our data do not support an activation of endogenous aNSCs in mid/hindbrain PVRs after local dopaminergic neurodegeneration. Spontaneous endogenous regeneration of dopaminergic cell loss through resident aNSCs is therefore unlikely.

In this research, we aimed to develop thin-film devices on a polymer substrate and an alternative 3D-printed device with macroelectrodes for treating gastrointestinal (GI) conditions. First, the fabrication of thin-film devices was demonstrated on a softening thiol-ene/acrylate polymer utilizing titanium nitride (TiN) as electrode material. This was achieved by utilizing cleanroom fabrication processes such as photolithography, wet and dry etching. The functionality of the device was shown by performing electrochemical characterization tests, mainly cyclic voltammetry, electrochemical impedance spectroscopy, and voltage transient. We synthesized a novel thiol-ene/acrylate polymer based on 1,3,5-triallyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione (TATATO), trimethylolpropanetris (3-mercaptopropionate) (TMTMP), and polyethylene glycol diacrylate (PEGDA). We show that this stretchable shape memory polymer substrate is well suited for cleanroom processes. Finally, for the high throughput of the wearable devices with electrodes size 10 mm in diameter, we implemented single electrode fabrication using printed circuit boards (PCBs) and depositing gold (Au) and TiN on the plated side of PCBs utilizing the sputtering tool. This step was followed by the assembly of those single electrodes on the flexible 3D printed device. We showed that the TiN electrode material performed better in terms of charge storage capacity and charge injection capacity than the widely used stainless steel electrode material for wearables.

During evolution, the mammalian cerebral cortex has undergone a considerable increase in size and complexity. The emergence of the cortical structure begins during embryonic development when neural stem cells initially undergo proliferative division to expand their pool and then switch to neurogenic division, generating differentiating progenitors that will give rise to neurons. Although the intrinsic molecular mechanisms instructing the switch from proliferative to neurogenic division have been well-studied, most work to date has focused on gene expression. However, as a consequence of transcriptional and post-transcriptional regulation, different transcripts can arise from a single gene. In particular, the process of alternative splicing occurs at a high frequency in the nervous system and can lead to changes in protein output regardless of gene expression. In the past years, the role of post- transcriptional mechanisms in neuronal maturation and function have been extensively investigated, mostly focusing on the function of specific isoforms or RNA binding proteins. Yet, the role of alternative splicing in generating transcript and protein diversity during neurogenic commitment is still unknown. Therefore, I used a combination of different RNA sequencing technologies and bioinformatic tools to reveal the transcript and protein diversity of proliferating progenitors, differentiating progenitors, and neurons isolated from double reporter mouse line. I identified widespread isoform diversity and many novel transcripts amongst expressed genes in the developing cortex. To date, this analysis represents the most comprehensive characterization of full-length transcript diversity at different stages of the neurogenic lineage in the developing mouse cortex. The resulting transcriptome annotation was used to quantify changes in exon inclusion across cells of the neurogenic lineage and identified alternative splicing events potentially involved in neurogenic commitment. These alternative splicing events were enriched in the coding sequence of isoforms, indicating that they might be relevant for protein diversity generation in the developing cortex. During neurogenesis, alternative splicing events were less enriched in regions that could disrupt or strongly affect protein structure and function, such as transmembrane regions, active sites, and domains. Interestingly, my results indicated that alternative splicing enables increased functional diversity by modulating protein-protein interaction sites and signaling properties of proteins. Still, further studies are required to delineate the causal relationship between these alternative splicing choices and cell-fate commitment. Applying a similar approach to other mammalian species, including humans, has the potential to uncover species-specific innovations and conserved features that underlie evolutionary cortex expansion. Moreover, understanding the function of isoforms during neural development could provide important insights into the molecular mechanisms involved in the onset of neurodevelopmental disorders. Therefore, the higher cell-specificity offered by the present dataset, compared to similar studies, allowed not only a better understanding of transcript and protein diversity generated by alternative splicing in the nervous system and highlighted potential functional consequences, but also shed light on the advantages of applying such strategy to address different biological questions.

Résumé : Contrairement à ce que l'on pourrait croire, les douleurs chroniques ne constituent pas uniquement des symptômes, mais bien une pathologie à part entière. La pharmacopée actuelle ne permettant pas de les soulager efficacement, il y a maintenant un besoin de les considérer dans leur spécificité lors de la recherche de nouvelles thérapies. Dans cette optique, nous avons étudié le rôle des chimiokines et de leurs récepteurs dans le contrôle de la douleur. Connu pour leur rôle dans la réponse immunitaire, nous avons en particulier investigué le rôle du couple ligand-récepteur CCL2-CCR2 dans la régulation des douleurs d’origine arthritique. Nous avons d'abord évalué l'effet analgésique de notre molécule antagoniste du récepteur CCR2, l'INCB3344, dans un modèle de douleur inflammatoire où elle renverse d'environ 50% les douleurs provoquées et les douleurs spontanées lorsqu'administrée par voie spinale. Nous avons également pu observer que bloquer l'activation de CCR2 au niveau de la moelle épinière limite non seulement la neuroinflammation spinale, mais permet également de réduire la sévérité de l'atteinte inflammatoire périphérique en limitant le transport rétrograde de la substance P. Nous avons ensuite appliqué nos observations sur un modèle de douleur arthritique plus près de la réalité clinique des patients souffrant d'arthrite rhumatoïde. Nous y avons reproduit la majorité de nos effets analgésiques suite à une administration spinale. Le traitement est cependant demeuré sans effet sur l'œdème périphérique. Parallèlement à cela, l'administration périphérique du composé, sur une base de deux bolus par jour ou en libération continue par des mini-pompes osmotiques, n'a eu que de très faibles effets analgésiques, mais s'est révélé avoir un impact marquant sur l'œdème périphérique et le gain de poids des animaux. Finalement, c'est en combinant l'INCB3344 avec de l'ibuprofène qu'il a été possible d'avoir un impact positif sur le plus grand nombre de paramètres associés à la douleur chronique. Ces résultats suggèrent donc qu'un antagoniste du récepteur CCR2 possède un potentiel analgésique intéressant, d'autant plus lorsqu'il s'agit de douleur d'origine inflammatoire puisqu'il permet d'agir à la fois sur l'hypersensibilité nociceptive et sur la source même de la douleur inflammatoire. Ce potentiel devient d'autant plus intéressant que de le combiner à un anti-inflammatoire non-stéroïdien (l'ibuprofène) améliore l'efficacité des deux composés. // Abstract : Contrary to popular beliefs, chronic pain is not only a set of symptoms, but a bona fide pathology that the drugs currently available are not sufficient to efficiently relieve. There is thus a need to modify our approach to discover new analgesic agents, taking into consideration the specific physiopathology of chronic pain. With this in mind, we investigated the role of chemokines and their receptors in the modulation of pain. Also known for their participation in the immune response, we focused on the CCL2-CCR2 ability to regulate arthritic pain. We first evaluated the analgesic properties of INCB3344, a specific antagonist of the CCR2 receptor, in a model of inflammatory pain. It reverses both provoked and spontaneous pain by 50% when administered i.t. We also observed that spinally blocking CCR2 limited the expression of proinflammatory mediators. It also reduced peripheral inflammation by preventing peripheral transport of SP. We then translated our findings in a model of arthritic pain, closer to the clinical reality of patients with rheumatoid arthritis. Spinal administration of INCB3344 had similar analgesic actions, but did not altered peripheral inflammation. On the other hand, peripheral administration of INCB3344, either by subcutaneous injection or by continuous release assured by an osmotic pump, had almost no analgesic effects, but significantly reduced peripheral inflammation and reduced the weight loss. By combining INCB3344 with a daily administration of ibuprofen, we were however able to reduce both pain hypersensitivity and the severity of the peripheral inflammation. Taken together, these results suggest that CCR2 antagonism has promising analgesic properties; especially for inflammatory or arthritic pain as it can acts both on the sensibilized nociceptive network and on the peripheral source of the inflammatory pain. This become even more interesting as its mechanism is at least not completely redundant with those of classic non-steroidal anti-inflammatory drugs, which allow the combination of both class of molecule to yield even larger effect.

INTRODUÇÃO: Há pouco tempo atrás não havia ferramenta específica para avaliar os sintomas do trato urinário inferior de pacientes com doenças neurológicas. Diversos instrumentos foram desenvolvidos e recomendados para avaliação de sintomas urinários em pacientes com disfunção do trato urinário inferior, porém, por não captarem a especificidade da bexiga neurogênica, apresentam perguntas inaplicáveis ou insuficientes para avaliar os sintomas desta população. Em 2014 foi introduzido o Neurogenic Bladder Symptom Score (NBSS) específico para pacientes com diagnóstico de bexiga neurogênica. Contém questões sobre incontinência urinária, sintomas e métodos de esvaziamento vesical, complicações associadas à disfunção miccional e qualidade de vida. OBJETIVO: este estudo se propôs a realizar a adaptação cultural e validação do questionário de sintomas NBSS para o português do Brasil. MÉTODOS: O questionário de sintomas foi adaptado culturalmente conforme diretrizes internacionais, seguindo os passos de: tradução, harmonização, retrotradução, revisão do comitê de especialistas, e teste da versão pré-final. A versão adaptada para o Brasil foi aplicada em pacientes com diagnóstico de bexiga neurogênica por lesão medular ou esclerose múltipla, que compareceram para consultas médicas com fisiatra ou urologista. Os mesmos pacientes responderam novamente o questionário num intervalo de 7 a 14 dias (reteste). A confiabilidade foi avaliada pela consistência interna, através do alfa de Cronbach, e pela estabilidade (teste-reteste), avaliada pelo Coeficiente de Correlação Intraclasse. Pacientes que não retornaram para a reaplicação do questionário, ou que relataram mudança nos sintomas do trato urinário inferior durante este período, foram excluídos da análise de estabilidade. A validade de critério foi avaliada pela aplicação da versão brasileira do questionário de qualidade de vida Qualiveen-SF e o resultado foi mensurado através do Coeficiente de Correlação Intraclasse. A validade de conteúdo foi determinada pela avaliação e julgamento do comitê de especialistas. A validade de construto foi obtida pela comparação entre o NBSS e a avaliação da função global da bexiga, através da análise de variância. RESULTADOS: Um total de 68 pacientes (57 homens - 83,8%) com idade média de 38,9 (± 14,7) anos participaram do estudo. Observou-se boa consistência interna da versão em português, com alfa de Cronbach de 0,813. A estabilidade (teste/reteste) também foi alta, com um Coeficiente de Correlação Intraclasse de 0,8596 [0,7586-0,9202] (p < 0,0001). Na validade de critério, o Coeficiente de Correlação Intraclasse revelou moderada correlação entre a versão em português do NBSS e o questionário Qualiveen-SF (r=0,6552 [0,423-0.8151]; p < 0,0001). CONCLUSÕES: Os resultados do estudo demonstraram que a versão brasileira do NBSS - Neurogenic Bladder Symptom Score, apresenta boa consistência interna, estabilidade e capacidade de medir o construto correto. O processo de adaptação cultural e validação do questionário NBSS para língua portuguesa, em pacientes com disfunção neurogênica do trato urinário inferior, foi concluído. O instrumento encontra-se apto para uso clínico e em pesquisa / INTRODUCTION: Until recent times, there was no specific tool to evaluate the symptoms of the lower urinary tract of patients with neurological diseases. Several instruments have been developed and recommended for evaluation of urinary symptoms in patients with lower urinary tract dysfunction, but because they do not capture the specificity of the neurogenic bladder, they present questions that are inapplicable or insufficient to evaluate the symptoms of this population. In 2014 it was introduced the Neurogenic Bladder Symptom Score (NBSS), specific for patients with a neurogenic bladder diagnosis. It contains questions about urinary incontinence, symptoms and methods of bladder emptying, complications associated with voiding dysfunction and quality of life. OBJECTIVE: This study aimed to perform the cultural adaptation and validation of the NBSS questionnaire for the Brazilian Portuguese. METHODS: The symptom questionnaire was culturally adapted according to international guidelines, following the steps of: translation, synthesis, back-translation, expert committee review, and pre-final version test. The version adapted for Brazil was applied in patients with neurogenic bladder diagnosis due to spinal cord injury or multiple sclerosis, who attended for medical appointments with physiatrist or urologist. The same patients answered the questionnaire again within 7 to 14 days (retest). Reliability was assessed by internal consistency, using Cronbach\'s alpha, and stability (test-retest), as assessed by Intraclass Correlation Coefficient. Patients who did not return for questionnaire reapplication or who reported changes in lower urinary tract symptoms during this period were excluded from the stability analysis. The criterion validity was evaluated by applying the Brazilian version of the Qualiveen-SF quality of life questionnaire and the result was measured using the Intraclass Correlation Coefficient. The validity of content was determined by the evaluation and judgment of the committee of experts. The construct validity was obtained by the comparison between the NBSS and the evaluation of the global function of the bladder, through analysis of variance. RESULTS: A total of 68 patients (57 men - 83.8%) with an average age of 38.9 (± 14.7) years participated in the study. There was good internal consistency of the Portuguese version, with Cronbach\'s alpha of 0.813. Stability (test / retest) was also high, with a Intraclass Correlation Coefficient of 0.8596 [0.7586-0.9202] (p < 0.0001). For criterion validity, the Intraclass Correlation Coefficient revealed a moderate correlation between the Portuguese version of the NBSS and the Qualiveen-SF questionnaire (r = 0.6552 [0.423-0.8151]; p < 0.0001). CONCLUSIONS: The results of the study demonstrated that the Brazilian version of the NBSS - Neurogenic Bladder Symptom Score shows good internal consistency, stability and ability to measure the correct construct. The process of cultural adaptation and validation of the NBSS questionnaire for Portuguese language in patients with neurogenic lower urinary tract dysfunction was concluded. The instrument is suitable for clinical and research use

INTRODUÇÃO: Líquen plano pilar (LPP) e alopecia frontal fibrosante (AFF) são alopecias cicatriciais linfocíticas crônicas, caracterizadas pela destruição permanente da unidade pilossebácea. Neuropeptídeos como a substância P (SP) e o peptídeo relacionado ao gene da calcitonina (CGRP) têm sido implicados no metabolismo lipídico das glândulas sebáceas e na manutenção do estado inflamatório de diversas doenças. OBJETIVOS: 1. Quantificar e comparar a expressão dos neuropeptídeos SP e CGRP em amostras do couro cabeludo (áreas afetadas e aparentemente não afetadas) e séricas de pacientes com LPP e AFF, em relação a indivíduos sadios, utilizando a técnica de ELISA. 2. Analisar áreas afetadas e aparentemente não afetadas de pacientes com LPP e AFF através da imunofluorescência direta (IFD). MÉTODO: 20 pacientes (10 com LPP e 10 com AFF) e 11 indivíduos sadios foram submetidos a biópsias com punch de 4mm do couro cabeludo e coleta de amostras sanguíneas. Pacientes foram submetidos a biópsias das áreas afetadas e aparentemente não afetadas do couro cabeludo, as quais foram pareadas com amostras da região anterior e posterior do couro cabeludo dos indivíduos-controle. As amostras dos pacientes foram enviadas para análise histopatológica, IFD e teste de ELISA para SP e CGRP. As amostras dos controles foram submetidas à análise histopatológica e aos mesmos testes de ELISA. Sintomas (dor, prurido, queimação e formigamento) e sinais inflamatórios (eritema difuso, eritema peripilar e descamação peripilar) na região afetada dos pacientes também foram avaliados. Este estudo foi realizado nas Universidades de São Paulo (BRA) e de Minnesota (EUA), entre os anos de 2012 e 2014. RESULTADOS: A análise histopatológica evidenciou infiltrado perifolicular linfocítico típico em 70% das áreas aparentemente não afetadas do couro cabeludo de pacientes com LPP e AFF, além de fibrose e depósitos de mucina perifoliculares. Em relação à IFD, o resultado se mostrou positivo em 50% das amostras das áreas afetadas e em 40% das áreas aparentemente não afetadas dos pacientes com LPP, em comparação a 40% e 20% nos casos de AFF, respectivamente. No teste de ELISA, pacientes do grupo LPP e infiltrado histopatológico de moderado a intenso na área afetada, demonstraram maior expressão de SP na área afetada, em comparação àquela aparentemente não afetada (P=0,046). Já pacientes do grupo AFF com o mesmo grau histopatológico de inflamação, demonstraram maior expressão de SP na área aparentemente não afetada, em comparação à área afetada (P=0,050). No teste de ELISA para CGRP, pacientes com LPP e inflamação histopatológica de leve a ausente na área afetada, tiveram maior expressão deste neuropeptídeo na área aparentemente não afetada, em comparação à área afetada (P=0,048). Por outro lado, pacientes com AFF que tinham o mesmo grau de inflamação histopatológica, a expressão deste neuropeptídeo foi favorecida na área afetada, em relação àquela aparentemente não afetada (P=0,050). Todas as amostras séricas dos pacientes e controles e do couro cabeludo dos indivíduos-controle tiveram resultados indetectáveis para SP e CGRP no teste de ELISA. Nenhuma relação entre sinais e sintomas inflamatórios e expressão de SP e CGRP no teste de ELISA foi vista. CONCLUSÃO: O acometimento das áreas aparentemente não afetadas do couro cabeludo de pacientes com LPP e AFF ao exame histopatológico, sugere que ambas as doenças possam acometer de forma difusa esta região. Apesar da semelhança dos achados histopatológicos entre pacientes com LPP e AFF, resultados antagônicos dos neuropeptídeos encontrados no teste de ELISA apontam para mecanismos fisiopatogênicos distintos. A inflamação neurogênica poderia explicar a sintomatologia e contribuir para a patogênese destas doenças / INTRODUCTION: Lichen planopilaris (LPP) and frontal fibrosing alopecia (FFA) are primary lymphocytic cicatricial alopecias characterized by permanent destruction of the pilossebaceous unit. Neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP) are related to lipid metabolism in sebaceous glands and to the maintenance of many inflammatory chronic disorders. OBJECTIVES: 1. Quantify SP and CGRP expression in affected and in normal-appearing scalp areas and serum samples from patients with LPP and FFA, and compare to healthy controls using ELISA technique. 2. Compare affected and normal-appearing areas from patients with LPP and FFA, using direct immunofluoresce (DIF) technique. METHODS: Twenty patients (10 with LPP and 10 with FFA) and eleven healthy controls underwent 4mm-punch biopsies and blood extraction. Patients collected samples from affected and normal-appearing scalp areas, and controls collected from anterior and posterior scalp areas. Patients samples were sent to histopathologic examination, DIF and ELISA tests for SP and CGRP detection. Control samples were sent to histopathologic examination and to the same ELISA tests. Symptoms (pain, burning, itching and tingling) and signs of inflammation (diffuse erythema, perifollicular erythema and perifollicular scale) were also assessed. This study was done at the Universities of São Paulo (Brazil) and Minnesota (USA), between 2012 and 2014. RESULTS: Normal-appearing scalp areas from patients with LPP and FFA showed lymphocytic perifollicular typical inflammation in 70% of the cases, as well as perifollicular fibrosis and mucin deposits. DIF test was positive in 50% of the affected areas and in 40% of normalappearing areas from patients with LPP, comparing to 40% and 20% in the FFA group, respectively. In SP ELISA test, affected areas from patients with LPP that had histopathologic moderate or intense infiltrate showed more expression of SP in the affected scalp, comparing to normal-appearing areas (P=0,046). However, affected areas from patients with FFA that showed the same degree of histopathologic infiltrate had higher expression of SP in normalappearing scalp, comparing to affected scalp (P=0.050). In CGRP ELISA test, affected scalp from patients with LPP that had histopathologic mild or irrelevant infiltrate showed increased CGRP expression in normal-appearing scalp areas, comparing to affected scalp (P=0,048). Althought, affected areas with the same degree of histopathologic inflammation from patients with FFA had more CGRP, comparing to normal-appearing scalp (P=0,050). All serum samples and scalp samples from controls had undetectable results in SP and CGRP ELISA tests. No clinical relationship was found among symptoms, signs of inflammation, and neuropeptide expression. CONCLUSION: Normal-appearing scalp areas can show histopathologic inflammation suggesting that both LPP and FFA can be more generalized processes affecting the scalp. Although both diseases share similar histopathologic findings, the opposite results in the ELISA test point that these diseases may have diverse pathogenic mechanisms. Neurogenic inflammation possibly play an important role in the pathogenesis of both LPP and FFA and may explain the symptomatic scalp some patients refer

Base teórica: Disfunções do trato urinário inferior são sintomas não motores comuns na Doença de Parkinson (DP) e incluem a Síndrome da Bexiga Hiperativa Neurogênica (SBHN), caracterizada pela urgência miccional, com ou sem urgeincontinência, acompanhada de aumento da frequência urinária e noctúria. A estimulação do nervo tibial posterior (ENTP) é uma das modalidades de tratamento disponíveis para o tratamento da SBHN. Objetivo: Determinar e comparar os efeitos do tratamento com ENTP em pacientes com DP e sintomas de SBHN e a manutenção dos resultados em 1 e 3 meses após o término do tratamento. Métodos: Ensaio-clínico, duplo-cego, randomizado, controlado e comparado com placebo. A pesquisa foi realizada com mulheres com DP e sintomas de SBHN no Hospital de Clínicas de Porto Alegre. Para o tratamento com ENTP domiciliar por 12 semanas as pacientes foram divididas em dois grupos: grupo ENTP e grupo ENTP sham/placebo. A avaliação da resposta pré e pós-tratamento foi realizada através de formulário específico, questionários de avaliação da incontinência urinária e qualidade de vida (OAB-V8 e KHQ) e de um diário miccional (DM) de 24 horas. Após, o fim do tratamento foi feito seguimento dos resultados para avaliação da melhora subjetiva em 30 e 90 dias. Resultados: O grupo ENTP apresentou uma diminuição da noctúria, número de episódios de urgência micciional e urge-incontinência, número de uso de proteções para incontinência, pontuação OAB-V8 e em sete domínios do KHQ (p <0,001). Embora, o grupo controle também tenha apresentado melhora dos sintomas, o grupo ENTP apresentou uma melhora superior no final do tratamento nas medidas do DM, OAB-V8 e na maioria dos domínios do KHQ. A ETNTP foi considerada um tratamento efetivo para SBHN em 93,3%, enquanto 33,3% dos tratados com placebo também melhoraram (p = 0,002). No seguimento de 30 e 90 dias, 53,3% e 33,31%, respectivamente, do grupo ENTP relataram que mantinham- se melhores dos sintomas da SBHN. Conclusão: a ENTP foi um tratamento efetivo para as pacientes com DP e SBHN. Nossa hipótese de superioridade clínica do grupo ENTP foi confirmada e a melhora subjetiva foi considerada positiva, mesmo que parcialmente em 30 e 90 dias após fim do tratamento. / Blackround: Lower urinary tract dysfunctions are common non-motor symptoms in Parkinson's disease (PD) and include Neurogenic Overactive Bladder Syndrome (NOBS), characterized by urinary urgency, with or without urge incontinence, accompanied by increased urinary frequency and nocturia . Posterior Tibial Nerve Stimulation (PTNS) is one of the treatment modalities available for the treatment of NOBS. Objective: To determine and compare the effects of PTNS treatment in patients with PD and NOBS symptoms and to maintain long-term results (1 and 3 months). Methods: Controlled, randomized, double-blind and compared with placebo clinical trial. The research was carried out with women with PD and symptoms of NOBS at the Hospital de Clínicas de Porto Alegre. For treatment with PTNS at 12 weeks, patients were divided into two groups: PTNS group and PTNS sham/placebo group. The evaluation of the pre- and post-treatment response was through a specific form, questionnaires to evaluate incontinence and quality of life (OVA-V8 and KHQ), and a voiding diary. After the end of the treatment, the results were followed up to evaluate the subjective improvement in 30 and 90 days. Results: The PTNS group presented a decreased nighttime urinary frequency, number of urgency and urinary incontinence episodes, number of incontinence protection, OAB-V8 and 7 domains of KHQ (p<0.001). Although the control group also showed improvement of the symptoms, the ENTP group presented a superior improvement at the end of the treatment in DM, OAB-V8 and most KHQ domains. PTNS was considered an effective treatment for OAB in 93.3%, while 33.3% of those treated with placebo was considered a responder (p=0.002). After 30 and 90 days, 53.3% and 33.31%, respectively, of the ENTP group reported that they maintained better SBHN symptoms. Conclusion: PTNS was an effective treatment for patients with PD and NOBS. Our hypothesis of clinical superiority of the ENTP group was confirmed and the subjective improvement was considered positive, even if partially at 30 and 90 days after the end of the treatment.!