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Diabetic vasculopathy (DV) is the most important consequence of chronic hyperglycemia in patients with diabetes mellitus (DM). This thesis explores the interaction of blood, bone and kidney in the pathogenesis of DV by i) reviewing the current understanding of pathogenesis of macrovascular and microvascular diseases in DM to identify gaps in literature and generate hypotheses relating to various facets of DV ii) undertaking a series of prospective studies to examine these hypotheses iii) analysing the findings and integrating any new information obtained from the clinical studies into the current knowledge base and iv) generating hypotheses upon which future work might be based. The literature search was carried out with the aim of understanding current concepts of pathogenesis of DV and its potential modulators. The original reviews resulting from this process are presented in chapters 2 to 4. A series of pilot studies reported in chapters 7 to 11, were then carried out to interrogate hypotheses originating from this process. The first study was carried out in healthy individuals to define the biological variation of potential modulators of DV, namely erythropoietin (EPO), parathyroid hormone, 25 hydroxyvitamin D and 1, 25-dihydroxyvitamin D to facilitate the design and interpretation of subsequent studies. It revealed a wide biological variation of these modulators in the healthy population thus,emphasizing the need to have a control group in the subsequent study population. To examine whether tubulointerstitial dysfunction occurs before the onset of microalbuminuria, a measurement of the above mentioned parameters was carried out along with markers of tubulointerstitial injury in patients with type 1 and type 2 DM without microalbuminuria and in non-diabetic controls. It was found that tubulointerstitial dysfunction with low levels of EPO and 1, 25-dihydroxyvitamin D and higher excretion of tubular injury markers, occurs before the onset of microalbuminuria. Subsequently, diabetic and nondiabetic chronic kidney disease (CKD) patients with EPO deficiency anaemia were examined to study the effects of EPO therapy on the excretion of tubular injury markers. However, in these patient groups, we were unable to demonstrate an effect of EPO therapy on the markers of tubular injury in spite of a beneficial haematological response. To examine whether vascular calcification (VC) and bone mineral density (BMD) were linked in patients with diabetes mellitus and to explore their relationship to modulators of DV, an assessment of VC and BMD was undertaken in patients with type 2 DM with different degrees of proteinuria and normoalbuminuria. VC was assessed by CT scan and BMD by a DEXA scan. Modulators of DV were measured including serum Osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-b-ligand (RANKL). The findings were i) a high prevalence of VC and osteopenia in normoalbuminuric type 2 DM patients with normal serum creatinine ii) a weak inverse relationship between VC and osteopenia iii) proteinuric patients had worse VC but not osteopenia iv) weak relationships between OPG levels and both VC and osteopenia, masked by age in multivariate analysis. The final study examined the relationship between modulators of DV, including OPG and RANKL, and the degree of CKD. It was found that abnormalities of OPG and RANKL occur before the onset of microalbuminuria and progress with deterioration of renal function. Compared to nondiabetics, DM patients have higher OPG levels in the predialysis phase and lower levels in haemodialysis phase, a phenomenon that might indicate endothelial exhaustion in dialysis patients with DM. The derangements associated with DV seem to occur earlier than previously thought. Further work is required to untangle these complexities and to define the contribution of factors such as the adverse blood milieu, the vasculature, abnormal bone and mineral metabolism, and early tubulointerstitial damage. The findings from the studies reported here may help in the formulation of new hypotheses, which might contribute to future work in this area.

In der Entwicklung des peripheren Nervensystems formen Schwannzellen eine Myelinscheide um Axone mit einem Durchmesser von mehr als 1 μm durch die Bildung multipler kompakter Membranschichten. Voraussetzung einer optimalen Nervenleitgeschwindigkeit ist dabei ein physiologisches Verhältnis der Dicke der Myelinscheide zu dem jeweiligen Axondurchmesser. Eine zentrale Rolle spielt dabei der axonale EGF-like growth factor NRG1 Typ III, der ErbB2/3- Rezeptoren der Schwannzelle bindet. Der PI3K-AKT-Signalweg ist ein bekannter intrazellulärer Effektor des ErbB2/3-Rezeptors und wurde bereits mit dem Prozess der Myelinisierung in Verbindung gebracht. Um die spezifische Funktion des PI3K-AKT-Signalwegs in Schwannzellen zu erforschen, generierten wir mit Hilfe des Cre/LoxP-Systems Mausmutanten, die eine zellspezifische Inaktivierung des Gens Phosphatase and Tensin Homolog (Pten) in myelinisierenden Gliazellen aufweisen (Pten-Mutanten). Der Verlust der Lipidphosphatase PTEN führte zu einer Anreicherung ihres Substrates, des second messenger Phosphatidyl-(3,4,5)-Trisphosphat (PIP3), und damit zu einer gesteigerten Aktivität des PI3K-AKT-Signalwegs in den Schwannzellen der Pten-Mutanten. Wir beobachteten in den Pten-Mutanten eine ektopische Myelinisierung von unmyelinisierten C- Faser-Axonen sowie eine Hypermyelinisierung von Axonen bis 2 μm Durchmesser. Bei Axonen über 2 μm Durchmesser kam es zu Myelinausfaltungen und fokalen Hypermyelinisierungen (Tomacula) anliegend an Regionen des unkompakten Myelins (Paranodien und Schmidt- Lantermann-Inzisuren). Weiterhin bildeten die mutanten Remak-Schwannzellen unkompakte Membranwicklungen um nicht-myelinisierte C-Faser-Axone und um Kollagenfaserbündel aus („Remak-Myelin“). Sowohl in den Regionen unkompakten Myelins als auch in Remak- Schwannzellen konnte eine erhöhte Aktivität des PI3K-AKT-Signalwegs nachgewiesen werden. Vermutlich setzt die Anreicherung von PIP3 mit Überaktivierung des PI3K-AKT-Signalwegs in den mutanten Gliazellen einen zellautonomen Prozess der Umwicklung von Axonen in Gang. Die zusätzliche Bildung von „Remak-Myelin“ um Kollagenfasern, die keine Membranoberfläche besitzen, weist darauf hin, dass dieser Prozess nicht von einer bidirektionalen axo-glialen Kommunikation abzuhängen scheint. Die beobachteten Tomacula und Myelinausfaltungen zeigten Ähnlichkeiten mit Mausmodellen für hereditäre Neuropathien des Menschen, wie HNPP und CMT4B. Wir vermuten, dass PTEN im unkompakten Myelin unkontrolliertes Membranwachstum verhindert und dass eine gestörte Balance von Phosphoinositiden einen Pathomechanismus von tomaculösen Neuropathien darstellt. Somit identifizieren wir den PI3K-AKT-Signalweg als ein mögliches Ziel zukünftiger Therapiekonzepte für hereditäre Neuropathien des Menschen.

Spinal muscular atrophy (SMA) and Charcot-Marie-Tooth disease type 2D (CMT2D)/distal SMA type V (dSMAV) are two incurable neuromuscular disorders that predominantly manifest during childhood and adolescence. Both conditions are caused by mutations in widely and constitutively expressed genes that encode proteins with essential housekeeping functions, yet display specific lower motor neuron pathology. SMA results from recessive inactivating mutations in the survival motor neuron 1 (SMN1) gene, while CMT2D/dSMAV manifests due to dominant point mutations in the glycyl-tRNA synthetase (GlyRS) gene, GARS. Using a number of different model systems, ranging from Caenorhabditis elegans to the mouse, this thesis aimed to identify potential novel therapeutic compounds for SMA, and to increase our understanding of the mechanisms underlying both diseases. I characterised a novel C. elegans allele, which possesses a point mutation in the worm SMN1 orthologue, smn-1, and showed its potential for large-scale screening by highlighting 4-aminopyridine in a screen for compounds able to improve the mutant motility defect. Previously, the gene encoding three isoforms of chondrolectin (Chodl) was shown to be alternatively spliced in the spinal cord of SMA mice before disease onset. I performed functional analyses of the three isoforms in neuronal cells with experimentally reduced Smn levels, and determined that the dysregulation of Chodl likely reflects a combination of compensatory mechanism and contributor to pathology, rather than mis-splicing. Finally, working with two Gars mutant mice and a new Drosophila model, I have implicated semaphorin-plexin pathways and axonal guidance in the GlyRS toxic gain-of-function disease mechanism of CMT2D/dSMAV.

Dentre as células ganglionares da retina existe uma pequena população de células que contem melanopsina e respondem diretamente à luz. Estas são as células ganglionares intrinsecamente fotossensíveis (ipRGCs), cujas funções são principalmente não visuais. Dentre as funções não visuais das ipRGCs sua influência na resposta pupilar dependente da luz foi o objeto central desta tese. Tanto a retina interna, através das ipRGCs, quanto a retina externa, através dos bastonetes e cones, fornecem uma informação neural que regula a resposta pupilar à luz (RPL). Este estudo avaliou a integridade das ipRGCs através do RPL em pacientes com glaucoma primário de ângulo aberto (GPAA), leve, moderado e avançado, e em pacientes com síndrome da apnéia obstrutiva do sono (SAOS), moderada e grave. Também foi avaliada a discriminação cromática e a sensibilidade ao contraste espacial de luminância (SC), a perimetria visual e a espessura da retina avaliada por tomografia de coerência óptica (OCT). Foram avaliados 98 participantes: 45 pacientes com GPAA ( 27, 18; idade média = 65,84 + 10,20), 28 pacientes com SAOS ( 14, 14; idade média = 52,93 + 7,13), e 25 controles ( 17, 8; idade média = 54,27 + 8,88). Após o exame oftalmológico foram avaliadas a SC de grades e a discriminação de cores através do Cambridge Colour Test (CCT). A avaliação do RPL foi feita apresentando-se flashes de 470 e 640 nm, de 1s de duração, em 7 luminâncias desde -3 até 2.4 log cd/m2 em um Ganzfeld Q450 SC (Roland Consult). O RPL foi registrado pelo sistema de eye tracker View Point System (Arrington Research Inc.). Os testes foram realizados em ambos os olhos, de forma monocular e no escuro. Para a comparação dos dados entre os grupos, utilizou-se um modelo de equações de estimação generalizada (GEE), para correção da dependência entre os dois olhos. O RPL dos pacientes com GPAA moderado e avançado apresentou redução significativa na amplitude do pico, dependente da severidade do glaucoma, nas diferentes luminâncias tanto para 470 nm quanto para 640 nm, evidenciando redução das contribuições dos cones e bastonetes ao RPL. As contribuições das ipRGCs ao RPL (avaliadas pela amplitude da resposta sustentada entre 6-8 s) foram também significativamente menores em GPAA moderado e avançado. No estado inicial do GPAA as contribuições das ipRGCs para o RPL encontram-se preservadas. No entanto, o GPAA parece afetar o processamento espacial desde o inicio da doença. Nos pacientes com GPAA leve foi observada uma perda acentuada nas faixas baixas de frequência espacial, compatível com prejuízo seletivo das células ganglionares do tipo M. A SC de pacientes com GPAA moderado e avançado mostrou perdas nas faixas baixas e altas de frequência espacial, apontando um prejuízo nas vias parvo- e margnocelulares. Uma perda significativa da discriminação de cores no eixo azul-amarelo foi observada em todos os estágios do GPAA. O RPL nos pacientes com SAOS está parcialmente preservado, não obstante, as respostas da amplitude do pico para o flash de 470 nm diminuem conforme aumenta a severidade da SAOS. As contribuições dos fotorreceptores da retina externa ao RPL, foram significativamente menores em algumas das luminâncias. Não foram observadas diferenças significativas de SC ou discriminação de cores nos pacientes com SAOS. Em conclusão, no estágio moderado e avançado do glaucoma tanto as contribuições das ipRGCs ao RPL quanto as vias M e P, se encontram mais afetadas do que no inicio do GPAA, quando a via parvocelular e as contribuições das ipRGCs ao RPL parecem estar mais preservadas / Among the retina ganglion cells there are a small population of cells containing melanopsin and which respond directly to light. They are the intrinsically photosensitive ganglion cells (ipRGCs), whose functions are mainly non-visual. Among these non-visual functions of the ipRGCs, their influence on the pupillary response as a function of light was the central subject of this thesis. Both the inner retina through the ipRGCs and the outer retina through the rods and cones, provide neural information that regulates the pupillary light response (PLR) to light. This study evaluated the integrity of ipRGCs through PLR in patients with Primary Open Angle Glaucoma (POAG), mild, moderate and advanced, and in patients with Obstructive Sleep Apnea Syndrome (OSAS), moderate and severe. We evaluated also the color discrimination and achromatic spatial contrast sensitivity (CS), visual perimetry and retinal thickness evaluated by Optical Coherence Tomography (OCT). 98 participants were evaluated, 45 patients with POAG ( 27 18; mean age = 65.84 + 10.20), 28 with OSAS ( 14 14; mean age = 52.93 + 7.13) and 25 controls ( 17 8; mean age = 54.27 + 8.88). After the ophthalmological exam it was evaluated the contrast sensitivity and color discrimination measures using the Cambridge Colour Test (CCT). Pupil responses were elicited by Ganzfeld (Q450 SC, Roland Consult) presentation of 1-sec flashes of 470- and 640-nm at 7 luminance from -3 to 2.4 log cd/m2. PLR was measured with the eye tracker system View Point (Arrington Research Inc.). The tests were performed monocularly, on both eyes, in a darkened room. In order to compare data across groups, we used a General Estimating Equations (GEE) to adjust for within subject inter-eye correlations. Patients with moderate and advanced POAG had a significantly decreased PLR that depends on the severity of the glaucoma, for both the 470- and 640-nm stimuli, making evident the reduction of the contributions of the cones and rods to the PLR. The contributions of ipRGCs to PLR (assessed by the amplitude of the sustained response between 6 8 sec) were also significantly lower in patients with moderate and advanced POAG. In the initial and mild stages of POAG the contribution of ipRGCs to the PLR is preserved. However, POAG appears to affect spatial processing from the early stages of the disease. Mild-POAG patients showed a marked loss in the low spatial frequency bands, compatible with selective loss of magnocellular ganglion cells. The CS of patients with moderate and advanced POAG showed losses at both low and high spatial frequencies, suggesting a loss in both parvo- and margnocellular channels. A significant loss of color discrimination along the blue-yellow axis was observed in all stages of POAG. The PLR in patients with OSAS is partially preserved, however the peak amplitude responses for the 470-nm flash decreased with increased severity of OSAS. The contributions of the photoreceptors of the outer retina to the PLR were significantly lower at some of the luminance. Significant differences in CS or color discrimination were not observed in patients with OSAS. In conclusion, in moderate and advanced stages of glaucoma, both the contributions of ipRGCs to PLR as well as the M- and P channels, were found more affected than at the beginning of POAG, in contrast the parvocellular channel and the contributions of ipRGCs on the PLR would be more preserved

Diabetiker entwickeln nach durchschnittlich 10 Jahren Folgeschäden. Die Polyneuropathie mit nachfolgender Osteoarthropathie des Fußes ist eine meist zu spät erkannte und suboptimal versorgte Komplikation. Nicht rechtzeitig erkannte Schäden führen zu Ulcera, sekundären Infektionen, Osteomyelitiden und Amputationen. Fragestellung: Ist eine Ganganalyse incl. Posturographie in der Lage, eine evtl. bestehende Polyneuropathie zu verifizieren, um eine frühzeitige orthopädische Schuhversorgung vornehmen zu können? Material und Methoden: Es wurden Diabetiker (n = 73) mit (mittels Messung der Nervenleitgeschwindigkeit NLG) nachgewiesener Neuropathie und eine gesunde Vergleichsgruppe (n = 38) ohne Neuropathie am Ganganalysesystem GANGAS untersucht. Ergebnisse: Die Allgemeinparameter relative Geschwindigkeit, Schrittlänge und Kadenz, sowie die Belastungsparameter Fersen-, Mittelfuß-, Vorfußbelastung und Belastung beim Auftritt und Abstoß zeigten keine wesentlich signifikanten Unterschiede im Gruppenvergleich. Dagegen zeigte die Posturographie (apparative Untersuchung der Schwankung des Druckschwerpunktes beim Romberg-Test, welche durch die Länge und Geschwindigkeit beschrieben wurde) signifikante Unterschiede zwischen beiden Patientengruppen. So lag z.B. der Median der Weglänge des Druckschwerpunktes beim Test mit geschlossenen Augen bei den Diabetikern bei 21,27 cm und in der Vergleichsgruppe bei 15,4 cm (p = 0,007). Die Bewegungsgeschwindigkeit des Druckschwerpunktes beim Test mit geschlossenen Augen betrug im Median 1,33 cm/sec bei den Diabetikern und 0,96 cm/sec in der gesunden Vergleichsgruppe (p = 0,006). Beim Test mit offenen Augen ergab sich kein signifikanter Unterschied im Gruppenvergleich. Klinische Relevanz: Eine Instabilität des Ganges der Patienten mit diabetischer Neuropathie und entsprechenden Folgeschäden des Fußes läßt sich durch die Ganganalyse mit Standardparametern nach vorliegendem Datenmaterial nicht nachweisen. Die Posturographie ist eine einfache, zeitlich mit geringem Aufwand verbundene Methode, die mit statistischer Signifikanz die subjektiven Kriterien der klinisch-orthopädischen Frühdiagnostik sinnvoll ergänzen und damit die Verdachtsdiagnose eine diabetischen Fußes objektiv nachweisen kann, bevor bleibende Schäden am Fuß entstanden sind. / Every diabetic patient after about ten years develops secondary changes in different tissues. Polyneuropathy with consecutive osteoarthropathy of the foot often is recognized too late and suboptimal treated. Diabetic disorders recognized too late may lead to ulcera, secondery infections and even amputations. Question: Is standard gait analysis including posturography able to verify diabetic neuropathy in order to supply the patient with adapted orthopadic shoeware in time? Materials and methods: A group of diabetics (n=73) with proved neuropathy by measuring nerve conduction velocity and a healthy control group (n=38) without neuropathy were examined by the gait-analysis-system GANGAS. Results: The general parameters: relative speed, length of step and cadence as well as the loading parameters of the heel, middle foot and forefoot during treading and repulsion show no significant differences between both groups. Posturography however (apparative examination of the elongation of focal point of pressure (FPP) according to the Romberg test, described by length and speed) shows significant differences between both groups. For diabetics making the test with closed eyes the mean value of walking length of the FPP was 21,27 cm, for the healthy control persons it was only 15,4 cm (p = 0,007). Asimulary results were found for the speed of movement of FPP during the test with closed eyes: mean value in diabetics 1,33 cm/s and for the healthy control persons 0,96 cm/s (p = 0,006). During this test with open eyes there was no significant difference between both groups. Clinical Relevance: Walking instability of patients with diabetic neuropathy and corresponding sequential damage of the foot could not be proved by the current data with standard parameters. Posturography however is able to give additional information with statistical significance to the subjective criteria of the clinical-orthopedic early diagnosis and verify the diagnosis of a beginning diabetic foot before severe disorders have occured.

Introdução: A síndrome SPOAN é uma doença neurodegenerativa, de transmissão genética autossômica recessiva, até o momento reconhecida apenas no Brasil, que caracteriza-se por: paraplegia espástica, de início nos primeiros anos de vida e caráter progressivo; atrofia óptica congênita; neuropatia periférica sensitivo-motora axonal, de início a partir da primeira década de vida; sobressaltos à estimulação sonora, disartria, deformidades de coluna e pés e sinais extra piramidais. A sua caracterização foi feita por nosso grupo, que avaliou clinicamente 71 indivíduos, originários do Rio Grande do Norte. Estudo de ligação mapeou o locus responsável pela síndrome SPOAN em uma região de 2 Mb no cromossomo 11q13. O gene responsável pela síndrome SPOAN permanece desconhecido. A síndrome SPOAN é considerada uma forma complicada de paraplegia espástica. A associação entre neuropatia e paraplegia espástica está relacionada à perda progressiva de axônios longos e tem sido relatada em algumas formas complicadas de neuropatias e paraplegias espásticas hereditárias. Casuística e métodos: Foi realizada a avaliação de 27 pacientes, 20 mulheres, com idade variando entre 4 e 58 anos. Todos os indivíduos compartilhavam o mesmo fenótipo (paraplegia espástica, atrofia de nervo óptico e neuropatia periférica) e tinham o mesmo haplótipo 11q13. Pacientes com história de diabetes mellitus ou alcoolismo foram excluídos do estudo. A avaliação neurológica incluiu a pesquisa dos escores modificados de sintomas e comprometimento neuropáticos. A força muscular foi testada e graduada conforme a escala MRC (Medical Research Council). Foi realizada a pesquisa da sensibilidade dolorosa, térmica, tátil, vibratória e artrestésica. O trofismo foi avaliado pela presença de deformidades na coluna e atrofia nos membros inferiores. Os reflexos profundos e o cutâneo plantar também foram analisados. Os estudos da condução nervosa foram realizados em um aparelho portátil Nicolet - Viking Quest, (Viasys, USA). Para os estudos de condução motora foram analisados os nervos axillar, mediano, ulnar, femoral, tibial e fibular direito. A condução sensitiva foi analisada nos nervos mediano, ulnar, radial, sural e fibular direito. O reflexo H e as ondas F foram avaliados com técnicas padrão. Alguns testes não puderam ser realizados devido à intensa atrofia e deformidades esqueléticas. O coeficiente de correlação de Pearson foi calculado entre a idade e os parâmetros, velocidade de condução, latência e amplitude. Valores de P < 0,05 foram considerados estatisticamente significantes. Resultados: Avaliação clínica: Todos os pacientes obtiveram escore de sinais neuropáticos graves e demonstraram déficit de força e atrofia distal. Deformidades dos pés estavam presentes em todos os pacientes e deformidades na coluna, em 58%. Os reflexos profundos dos membros superiores estavam exaltados em 92% dos casos e o reflexo patelar, em 63%. O reflexo Aquileu estava ausente em todos os pacientes. Todas as modalidades de sensibilidade foram afetadas, principalmente nos membros inferiores. Os dados do exame de sensibilidade na paciente de 4 anos foram desconsiderados. Estudo da condução nervosa sensitiva: Os SNAPs dos nervos mediano, sural e fibular estavam ausentes em todos os pacientes. SNAPs do nervo ulnar estavam ausentes em 96% da amostra e do nervo radial, em 80%. Estudo da condução nervosa motora: As latências motoras dos nervos axilar e femoral estavam normais em todos os pacientes. As amplitudes dos CMAPs estavam reduzidas em 15 e 52% da amostra nos nervos mediano e ulnar, respectivamente. Velocidades de condução estavam reduzidas em 50 e 41% desta casuística nos nervos mediano e ulnar, respectivamente. Velocidades de condução estavam acima de 80% do limite inferior da normalidade, em todos os nervos, exceto em 1 paciente que apresentou redução de 27% no nervo ulnar. Entretanto, este mesmo paciente apresentou amplitude menor que 2mV. Ondas F apresentavam aumento da latência, de acordo com a altura, em 100% dos casos. CMAPs estavam ausentes em 93 e 84% da amostra nos nervos fibular e tibial, respectivamente. Reflexo H estava ausente em 88% dos pacientes. Não houve correlação entre idade e a velocidade de condução, latência e amplitude dos nervos mediano e ulnar. Discussão: O estudo da condução nervosa neste grupo preencheu critérios para uma neuropatia primária axonal. Nenhum paciente apresentou bloqueio de condução ou dispersão temporal. As alterações encontradas na velocidade de condução provavelmente se devem à perda de fibras nervosas de condução rápida. Fenótipos SPOAN-like foram descritos em famílias com mutações nos genes C12orf65, TFG e OPA1. No entanto, não existem detalhes sobre a condução nervosa nestes pacientes. Neuropatia axonal de início tardio foi relacionada à SPG55 e DOA (dominant optic atrophy), enquanto neuropatia axonal e desmielinizante com leve comprometimento sensitivo foi descrita na família com mutação no gene TFG. Conclusão: Os pacientes com a síndrome SPOAN apresentam uma acentuada neuropatia axonal, sensitivo motora. As alterações encontradas na condução nervosa dos pacientes com síndrome SPOAN não são específicas, no entanto, resultados normais excluem esta condição em adultos. A paciente mais jovem desta casuística já apresentava alterações ao exame, o que pode sugerir um início precoce da neuropatia. Entretanto, não temos dados suficientes para afirmar que este seja um achado comum a todos os pacientes SPOAN / Introduction: SPOAN syndrome (Spastic Paraplegia, Optic Atrophy and Neuropathy) is a progressive neurodegenerative disorder of autosomal recessive inheritance described by our group in a large inbred family from Northeastern Brazil. The clinical picture is characterized by non-progressive congenital optic atrophy, progressive spastic paraplegia, axonal neuropathy, auditory startles, dysarthria, spinal and foot deformities and also extrapyramidal signs. Linkage studies mapped the responsible locus for the syndrome to a 2Mb region on chromosome 11q13. The gene responsible for SPOAN syndrome remains elusive. Materials and Methods: This is a cross sectional study which was conducted from 2009 to 2011. We evaluated 27 patients (20 females), with a0ges ranging from 4 to 58 years. All patients shared the same phenotype (spastic paraplegia, optic atrophy and peripheral neuropathy) and had the same 11q13 haplotype in homozygosis. Patients with history of diabetes mellitus or alcoholism were excluded from this study. All patients were evaluated by the same clinical researcher (SA). Neurological evaluation included determination of modified neuropathy symptoms (NSS) and neuropathy disability (NDS) scores. Motor strength was assessed using MRC scale. Sensibility assessment included small-fiber (pain and temperature) and large-fiber modalities (vibration-128Hz diapason, 10g monofilament and joint position sense). Spine deformities and atrophy in the lower limbs were observed. We also evaluated osteotendineous reflexes and cutaneous plantar reflexes. Nerve conduction studies were performed using a portable Nicolet - Viking Quest, (Viasys,USA). Motor conduction studies included axillary, median, ulnar, femoral, tibial and fibular nerves on the right side. Sensory nerve action potentials of median, ulnar, sural and superficial fibular nerves were recorded using a bar electrode of 3 cm and standard fixed distances. Tibial H-reflex was evaluated with standard technique. Minimal F wave latencies were obtained from ulnar and tibial nerves. A few tests could not be done in every patient due to severe deformities. We calculated Pearson\'s correlation coefficients between age and nerve conduction parameters, including velocities, latencies and amplitudes. P values <0.05 were considered statistically significant. Results: Clinical data: Neuropathic symptoms such as pain and paresthesias were rare. All patients had signs of severe neuropathy. All subjects demonstrated weakness and atrophy that were more significant distally than proximally. Foot deformities were present in all patients and spine deformities were seen in 58%. Upper limb deep tendon reflexes were exalted in 92% and patelar reflex in 63%. Ankle reflex was absent in all patients. In one patient, who was 4 years-old, sensory evaluation was inconsistent and the results were not considered. In all the other ones, sensory modalities were affected and occurred predominantly in the lower limbs. Electrodiagnostic data: Sensory nerve conduction: Median nerve SNAP was absent in all 27 patients. Ulnar nerve SNAPs were absent in 96%, whereas radial nerve SNPAs were absent in 80%. Superficial fibular and sural SNAPs were absent in all patients. Motor nerve conduction: The motor latencies of axillary and femoral nerves were normal in all patients. CMAP amplitudes were reduced in 15% of the median nerves and in 52% of the ulnar nerves. Conduction velocities (CV) were reduced in 50% of the median nerves and in 41% of the ulnar nerves. CV was above 80% of the lower normal limit for all nerves, except for one patient who showed a 27% reduction of ulnar CV, but had also a CMAP amplitude of less than 2 mV. F waves were prolonged according to the height in 100%. Only one patient who presented significant motor CV reduction of the ulnar nerve. CMAPs were absent in 93% of the fibular nerves and in 84% of the tibial nerves. A single fibular nerve showed more than 20% of CV reduction, but also had severely reduced CMAP amplitude. H reflex was absent in 88% of the patients. There was no correlation between age and neurophysiological parameters, such as median or ulnar CV, latencies or CMAP amplitudes. Discussion: Nerve conduction studies in this group fulfill criteria for primary axonal neuropathy. No patient showed conduction block or temporal dispersion. Abnormalities seen in CV and F waves are probably related to loss of fast conduction fiber nerves. We could not demonstrate correlation between age and nerve conduction parameters, including velocities, latencies and amplitudes. SPOAN-like phenotype has been found in families with mutations in C12orf65, TFG and OPA1 genes, however there is no detailed report on nerve conduction studies in these conditions. Axonal neuropathy is also described in SPG55 and DOA plus, but usually with a later onset than on SPOAN syndrome. Peripheral neuropathy is also described in the family with mutation in TFG gene, but this presents a different pattern characterized as a mixed axonal demyelinating neuropathy with mild sensory involvement. Although the nerve conduction abnormalities seen in SPOAN syndrome are not specific, normal results seem to rule out this condition, at least in adult patients. The younger patient in our series was 4-years-old, and her neurophysiological study was severely abnormal, suggesting an early-onset neuropathy. However, we do not have a comprehensive study of several young patients to support that this feature is

Este estudo mostra como o rolamento do pé de pacientes com polineuropatia diabética pode ser melhorado com exercícios para pés e tornozelos, visando a recuperação muscular e articular comprometidos pela doença. Um ensaio clínico randomizado, paralelo, com um braço de crossover, e avaliador cego, foi conduzido. Cinquenta e cinco pacientes com polineuropatia diabética foram randomizados e alocados para o grupo controle (n=29) e grupo intervenção (n=26). A intervenção foi aplicada por 12 semanas, 2 vezes por semana, por 40 a 60 minutos cada sessão. As variáveis primárias foram definidas como as que descrevem o rolamento do pé: pressão plantar em seis regiões plantares de interesse. As variáveis secundárias foram a cinética e cinemática de tornozelo no plano sagital, e as medidas clínicas da função de pés e tornozelo (teste de função muscular manual, testes funcionais), de sinais e sintomas da polineuropatia diabética, exame físico dos pés e teste de confiança e equilíbrio em atividades da marcha. Os efeitos de tempo (baseline e 12 semanas), de grupo (controle e intervenção) e de interação foram calculados por meio de ANOVAs casewise 2 fatores, e para as comparações intragrupo do grupo intervenção (baseline, 12 semanas e 24 semanas) foram usadas ANOVAs para medidas repetidas. As variáveis não paramétricas foram comparadas entre grupos por meio de testes de Mann-Whitney e entre os tempos de intervenção por meio do teste de Wilcoxon. Adotou-se um ? de 5% para diferenças estatísticas e o coeficiente d de Cohen para descrição do tamanho do efeito da intervenção. Após 12 semanas de exercícios, observou-se mudanças positivas no rolamento do pé. Houve uma suavização do contato do calcanhar no apoio inicial, refletido pelo aumento do tempo do pico de pressão e da integral do pico de pressão. O médio-pé aumentou sua participação no rolamento observado pela diminuição da velocidade média do deslocamento do centro de pressão e aumento da integral do pico de pressão. O antepé lateral passou a realizar o apoio no solo antecipadamente em relação ao antepé medial, que previamente à intervenção aconteciam concomitantemente, e esse resultado foi evidenciado pela antecipação do tempo do pico de pressão em antepé lateral após a intervenção. A ação de hálux e dedos também aumentou (aumento de integral do pico de pressão e picos de pressão), em uma patologia marcada pela diminuição do contato do hálux e desenvolvimento de dedos em garra, que diminui o contato dos dedos com o solo. O grupo controle apresentou algumas pioras com relação à função muscular e parâmetros cinéticos e cinemáticos de tornozelo, enquanto que o grupo intervenção mostrou melhora na função de muitos grupos musculares, em testes funcionais e no pico de momento extensor na fase de aplainamento do pé. Apesar do protocolo de intervenção ter sido construído de modo a permitir que o paciente incorpore os exercícios na sua rotina diária, a aderência a este tipo de intervenção deve ser estudada, já que grande parte das variáveis retornaram ao baseline após o período de follow up. Ações preventivas são fundamentais para diminuir as complicações devastadoras da neuropatia diabética / This study shows how the foot rollover process during gait of patients with diabetic polyneuropathy can be improved with exercises for foot and ankle, aiming at the recovery of the muscles and joints affected by the disease. A clinical trial randomized, parallel, one arm of crossover, with blind assessment was conducted. Fifty-five patients with diabetic polineuropathy were randomly allocated to the control group (n = 29) and intervention group (n = 26). The intervention was applied for 12 weeks, twice a week, for 40 to 60 minutes per session. The primary variables were defined as those that describe the foot rollover: plantar pressure in 6 plantar areas of interest. The secondary variables were kinetic and kinematics of the ankle in the sagittal plane were calculated, and the clinical measures of foot and ankle function (manual muscle function testing, functional testing), signs and symptoms of diabetic polyneuropathy, physical examination of the feet and balance and confidence test in gait activities. The time effects (baseline and 12 weeks), group effects (control and intervention groups) and interaction effects were calculated using casewise two factos ANOVAs, and for intragroup comparisons of intervention group (baseline, 12 weeks and 24 weeks) it was used ANOVAs for repeated measures. The nonparametric variables were compared between groups using Mann-Whitney tests and between periods of assessment using Wilcoxon test. We adopted an ? of 5% for statistical differences and the Cohen\'s d coefficient for description of the effect size. After 12 weeks ofexercises, there were positive changes in the foot rollover process. There was a softening of heel contact in initial contact, reflected by the increase in time to peak pressure and the pressure time integral. The midfoot increased its participation observed by the decrease in speed of displacement of the center of pressure and increased pressure time integral. The lateral forefoot contact was earlier relative to the medial forefoot, that occurred at the same time before intervention, observed by the early time to peak pressure of lateral forefoot after the intervention. The participation of the hallux and toes also increased (increase of pressure time integral and peak pressure), in a pathology that is marked by decreased contact of the hallux and development of claw toes, which reduces contact of the toes with the ground. CG showed some worsening in relation to muscle function and kinematic and kinetic parameters of the ankle, while the IG showed improvement in the function of many muscles groups, functional tests and peak extensor moment during the forefoot contact. The intervention protocol was constructed to allow the patient to incorporate exercise into their daily routine, but adherence to treatment should be studied and motivational strategies need to be applied, since most of the variables returned to baseline after the follow up period (12 weeks after the intervention). Preventive actions are critical to reducing the devastating complications of diabetic neuropathy

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O diagnóstico da hanseníase neural pura baseia-se em dados clínicos e laboratoriais do paciente, incluindo a histopatologia de espécimes de biópsia de nervo e detecção de DNA de Mycobacterium leprae (M. leprae) pelo PCR. Como o exame histopatológico e a técnica PCR podem não ser suficientes para confirmar o diagnóstico, a imunomarcação de lipoarabinomanana (LAM) e/ou Glicolipídio fenólico 1 (PGL1) - componentes de parede celular de M. leprae foi utilizada na primeira etapa deste estudo, na tentativa de detectar qualquer presença vestigial do M. leprae em amostras de nervo sem bacilos. Além disso, sabe-se que a lesão do nervo na hanseníase pode diretamente ser induzida pelo M. leprae nos estágios iniciais da infecção, no entanto, os mecanismos imunomediados adicionam severidade ao comprometimento da função neural em períodos sintomáticos da doença. Este estudo investigou também a expressão imuno-histoquímica de marcadores envolvidos nos mecanismos de patogenicidade do dano ao nervo na hanseníase. Os imunomarcadores selecionados foram: quimiocinas CXCL10, CCL2, CD3, CD4, CD8, CD45RA, CD45RO, CD68, HLA-DR, e metaloproteinases 2 e 9. O estudo foi desenvolvido em espécimes de biópsias congeladas de nervo coletados de pacientes com HNP (n=23 / 6 BAAR+ e 17 BAAR - PCR +) e pacientes diagnosticados com outras neuropatias (n=5) utilizados como controle. Todas as amostras foram criosseccionadas e submetidas à imunoperoxidase. Os resultados iniciais demonstraram que as 6 amostras de nervos BAAR+ são LAM+/PGL1+. Já entre as 17 amostras de nervos BAAR-, 8 são LAM+ e/ou PGL1+. Nas 17 amostras de nervos BAAR-PCR+, apenas 7 tiveram resultados LAM+ e/ou PGL1+. A detecção de imunorreatividade para LAM e PGL1 nas amostras de nervo do grupo HNP contribuiu para a maior eficiência diagnóstica na ausência recursos a diagnósticos moleculares. Os resultados da segunda parte deste estudo mostraram que foram encontradas imunoreatividade para CXCL10, CCL2, MMP2 e MMP9 nos nervos da hanseníase, mas não em amostras de nervos com outras neuropatias. Além disso, essa imunomarcação foi encontrada predominantemente em células de Schwann e em macrófagos da população celular inflamatória nos nervos HNP. Os outros marcadores de ativação imunológica foram encontrados em leucócitos (linfócitos T e macrófagos) do infiltrado inflamatório encontrados nos nervos. A expressão de todos os marcadores, exceto CXCL10, apresentou associação com a fibrose, no entanto, apenas a CCL2, independentemente dos outros imunomarcadores, estava associada a esse excessivo depósito de matriz extracelular. Nenhuma diferença na frequência da imunomarcação foi detectada entre os subgrupos BAAR+ e BAAR-, exceção feita apenas às células CD68+ e HLA-DR+, que apresentaram discreta diferença entre os grupos BAAR + e BAAR- com granuloma epitelioide. A expressão de MMP9 associada com fibrose é consistente com os resultados anteriores do grupo de pesquisa. Estes resultados indicam que as quimiocinas CCL2 e CXCL10 não são determinantes para o estabelecimento das lesões com ou sem bacilos nos em nervo em estágios avançados da doença, entretanto, a CCL2 está associada com o recrutamento de macrófagos e com o desenvolvimento da fibrose do nervo na lesão neural da hanseníase. / The diagnosis of pure neural leprosy (PNL) is based on clinical and laboratory data, including the histopathology of nerve biopsy specimens and detection of M. leprae DNA by polymerase chain reaction (PCR). Given that histopathological examination and PCR methods may not be sufficient to confirm diagnosis, immunolabeling of lipoarabinomanan (LAM) and/or phenolic glycolipid 1 (PGL1) M. leprae wall components were utilized in the first step of this investigation in an attempt to detect any vestigial presence of M. leprae in AFB- nerve samples. Furthermore, its well known that nerve damage in leprosy can be directly induced by Mycobacterium leprae in the early stages of infection; however, immunomediated mechanisms add gravity to the impairment of neural function in symptomatic periods of the disease. Therefore, this study also investigated the immunohistochemical expression of immunomarkers involved in the pathogenic mechanisms of leprosy nerve damage. These markers selected were CXCL10, CCL2 chemokines and CD3, CD4, CD8, CD45RA, CD45RO, CD68, HLA-DR, metalloproteinases 2 and 9 in nerve biopsy specimens collected from leprosy (23) and nonleprosy patients (5) suffering peripheral neuropathy. Twenty-three PNL nerve samples (6 AFB+ and 17 AFB-PCR+) were cryosectioned and submitted to LAM and PGL1 immunohistochemical staining by immunoperoxidase; 5 nonleprosy nerve samples were used as controls. The 6 AFB-positive samples showed LAM/PGL1 immunoreactivity. Among the 17 AFB- samples, only 8 revealed LAM and/or PGL1 immunoreactivity. In 17 AFB-PCR+ patients, just 7 had LAM and/or PGL1-positive nerve results. In the PNL cases, the detection of immunolabeled LAM and PGL1 in the nerve samples would have contributed to enhanced diagnostic efficiency in the absence of molecular diagnostic facilities. The results of the second part of this study showed that CXCL10-, CCL2-, MMP2- and MMP9-immunoreactivities were found in the leprosy nerves but not in nonleprosy samples. Immunolabeling was predominantly found in recruited macrophages and Schwann cells composing the inflammatory cellular population in the leprosy-affected nerves. The immunohistochemical expression of all the markers, but CXCL10, was associated with fibrosis; however, only CCL2 was, independently from the other markers, associated with this excessive deposit of extracellular matrix. No difference in the frequency of the immunolabeling was detected between the AFB+ and AFB- leprosy subgroups of nerves, exception made to some statistical tendency to difference in regard to CD68+ and HLA-DR+ cells in the AFB- nerves exhibiting epithelioid granuloma. MMP9 expression associated with fibrosis is consistent with previous results of this research group. The findings conveys the idea that CCL2 and CXCL10 chemokines at least in advanced stages of leprosy nerve lesions are not determinant for the establishment of AFB+ or AFB- leprosy lesions, however, CCL2 is associated with macrophage recruitment and fibrosis.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O diagnóstico da hanseníase neural pura baseia-se em dados clínicos e laboratoriais do paciente, incluindo a histopatologia de espécimes de biópsia de nervo e detecção de DNA de Mycobacterium leprae (M. leprae) pelo PCR. Como o exame histopatológico e a técnica PCR podem não ser suficientes para confirmar o diagnóstico, a imunomarcação de lipoarabinomanana (LAM) e/ou Glicolipídio fenólico 1 (PGL1) - componentes de parede celular de M. leprae foi utilizada na primeira etapa deste estudo, na tentativa de detectar qualquer presença vestigial do M. leprae em amostras de nervo sem bacilos. Além disso, sabe-se que a lesão do nervo na hanseníase pode diretamente ser induzida pelo M. leprae nos estágios iniciais da infecção, no entanto, os mecanismos imunomediados adicionam severidade ao comprometimento da função neural em períodos sintomáticos da doença. Este estudo investigou também a expressão imuno-histoquímica de marcadores envolvidos nos mecanismos de patogenicidade do dano ao nervo na hanseníase. Os imunomarcadores selecionados foram: quimiocinas CXCL10, CCL2, CD3, CD4, CD8, CD45RA, CD45RO, CD68, HLA-DR, e metaloproteinases 2 e 9. O estudo foi desenvolvido em espécimes de biópsias congeladas de nervo coletados de pacientes com HNP (n=23 / 6 BAAR+ e 17 BAAR - PCR +) e pacientes diagnosticados com outras neuropatias (n=5) utilizados como controle. Todas as amostras foram criosseccionadas e submetidas à imunoperoxidase. Os resultados iniciais demonstraram que as 6 amostras de nervos BAAR+ são LAM+/PGL1+. Já entre as 17 amostras de nervos BAAR-, 8 são LAM+ e/ou PGL1+. Nas 17 amostras de nervos BAAR-PCR+, apenas 7 tiveram resultados LAM+ e/ou PGL1+. A detecção de imunorreatividade para LAM e PGL1 nas amostras de nervo do grupo HNP contribuiu para a maior eficiência diagnóstica na ausência recursos a diagnósticos moleculares. Os resultados da segunda parte deste estudo mostraram que foram encontradas imunoreatividade para CXCL10, CCL2, MMP2 e MMP9 nos nervos da hanseníase, mas não em amostras de nervos com outras neuropatias. Além disso, essa imunomarcação foi encontrada predominantemente em células de Schwann e em macrófagos da população celular inflamatória nos nervos HNP. Os outros marcadores de ativação imunológica foram encontrados em leucócitos (linfócitos T e macrófagos) do infiltrado inflamatório encontrados nos nervos. A expressão de todos os marcadores, exceto CXCL10, apresentou associação com a fibrose, no entanto, apenas a CCL2, independentemente dos outros imunomarcadores, estava associada a esse excessivo depósito de matriz extracelular. Nenhuma diferença na frequência da imunomarcação foi detectada entre os subgrupos BAAR+ e BAAR-, exceção feita apenas às células CD68+ e HLA-DR+, que apresentaram discreta diferença entre os grupos BAAR + e BAAR- com granuloma epitelioide. A expressão de MMP9 associada com fibrose é consistente com os resultados anteriores do grupo de pesquisa. Estes resultados indicam que as quimiocinas CCL2 e CXCL10 não são determinantes para o estabelecimento das lesões com ou sem bacilos nos em nervo em estágios avançados da doença, entretanto, a CCL2 está associada com o recrutamento de macrófagos e com o desenvolvimento da fibrose do nervo na lesão neural da hanseníase. / The diagnosis of pure neural leprosy (PNL) is based on clinical and laboratory data, including the histopathology of nerve biopsy specimens and detection of M. leprae DNA by polymerase chain reaction (PCR). Given that histopathological examination and PCR methods may not be sufficient to confirm diagnosis, immunolabeling of lipoarabinomanan (LAM) and/or phenolic glycolipid 1 (PGL1) M. leprae wall components were utilized in the first step of this investigation in an attempt to detect any vestigial presence of M. leprae in AFB- nerve samples. Furthermore, its well known that nerve damage in leprosy can be directly induced by Mycobacterium leprae in the early stages of infection; however, immunomediated mechanisms add gravity to the impairment of neural function in symptomatic periods of the disease. Therefore, this study also investigated the immunohistochemical expression of immunomarkers involved in the pathogenic mechanisms of leprosy nerve damage. These markers selected were CXCL10, CCL2 chemokines and CD3, CD4, CD8, CD45RA, CD45RO, CD68, HLA-DR, metalloproteinases 2 and 9 in nerve biopsy specimens collected from leprosy (23) and nonleprosy patients (5) suffering peripheral neuropathy. Twenty-three PNL nerve samples (6 AFB+ and 17 AFB-PCR+) were cryosectioned and submitted to LAM and PGL1 immunohistochemical staining by immunoperoxidase; 5 nonleprosy nerve samples were used as controls. The 6 AFB-positive samples showed LAM/PGL1 immunoreactivity. Among the 17 AFB- samples, only 8 revealed LAM and/or PGL1 immunoreactivity. In 17 AFB-PCR+ patients, just 7 had LAM and/or PGL1-positive nerve results. In the PNL cases, the detection of immunolabeled LAM and PGL1 in the nerve samples would have contributed to enhanced diagnostic efficiency in the absence of molecular diagnostic facilities. The results of the second part of this study showed that CXCL10-, CCL2-, MMP2- and MMP9-immunoreactivities were found in the leprosy nerves but not in nonleprosy samples. Immunolabeling was predominantly found in recruited macrophages and Schwann cells composing the inflammatory cellular population in the leprosy-affected nerves. The immunohistochemical expression of all the markers, but CXCL10, was associated with fibrosis; however, only CCL2 was, independently from the other markers, associated with this excessive deposit of extracellular matrix. No difference in the frequency of the immunolabeling was detected between the AFB+ and AFB- leprosy subgroups of nerves, exception made to some statistical tendency to difference in regard to CD68+ and HLA-DR+ cells in the AFB- nerves exhibiting epithelioid granuloma. MMP9 expression associated with fibrosis is consistent with previous results of this research group. The findings conveys the idea that CCL2 and CXCL10 chemokines at least in advanced stages of leprosy nerve lesions are not determinant for the establishment of AFB+ or AFB- leprosy lesions, however, CCL2 is associated with macrophage recruitment and fibrosis.