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Le atassie ereditarie sono malattie rare geneticamente eterogenee. L'atassia di Friedreich (AF) rappresenta la forma più frequente, con una prevalenza nelle popolazioni caucasiche di 1:50.000. A causa della bassa prevalenza la selezione delle misure di outcome più sensibili è fondamentale per gli studi clinici. Finora sono state testate diverse misure di outcome, ma la maggior parte di esse richiede un numero considerevole di pazienti per studi con potenza adeguata. Lo scopo di questo progetto è stato analizzare le misure di outcome esistenti e valutare strumenti di nuova concezione per valutare la gravità e la progressione della AF. Un gruppo di 207 soggetti con AF afferenti alla Fondazione IRCCS Istituto Neurologico “Carlo Besta” è stato incluso nei seguenti studi: - Analisi delle proprietà statistiche della Scale for the Assessment and Rating of Ataxia (SARA) e della scala FARS. Abbiamo valutato le proprietà della scala SARA in 170 soggetti. Gli item che valutano arti inferiori, deambulazione e stazione eretta aumentano linearmente fino al punteggio SARA di 20-25 punti (su 40), quando si perde la deambulazione. Nei soggetti non deambulanti, gli item che valutano gli arti superiori aumentano più rapidamente. Nell’analisi longitudinale i soggetti con punteggi basali di SARA più bassi hanno maggiore velocità di progressione rispetto ai soggetti con punteggi più alti. Le prestazioni delle scale SARA e FARS sono state confrontate in 99 soggetti. Entrambe le scale hanno mostrato una buona consistenza interna, si sono dimostrate altamente correlate reciprocamente e con la durata della malattia. - Posturografia. Sono stati valutati 76 soggetti con AF. Le valutazioni effettuate al basale e al follow-up a 1 e 2 anni prevedevano 4 situazioni: posizione seduta, e in piedi in posizione naturale, in posizione a piedi uniti e in tandem. L'oscillazione degli assi X (medio-laterale) e Y (antero-posteriore) è stata registrata da un accelerometro posizionato sopra lo sterno. Le variabili estratte non si sono mostrate correlate con la durata della malattia e la gravità dell'atassia, ma con l'età di insorgenza dell'atassia. Nelle analisi longitudinali sono state identificate prestazioni significativamente peggiori durante la posizione dei piedi uniti a 1 anno di follow-up. - Wet biomarkers (in collaborazione con l'Università di Roma "Tor Vergata", Prof. F. Malisan). 53 soggetti con AF e 27 controlli sono stati inclusi nella studio che ha analizzato la proteina HAX-1 come potenziale biomarcatore. L'espressione di FXN e HAX-1 si è dimostrata co-regolata, sia a livello di mRNA che di proteine. Il microRNA hsa-miR223-3p è risultato elevato nei soggetti con AF e correlato negativamente con i livelli di mRNA e proteina di HAX-1. I livelli di hsa-miR223-3p sono risultati significativamente correlati con i parametri cardiaci. - Monitoraggio dell’attività nella vita reale. Abbiamo testato il monitoraggio remoto con Actigraph in 25 soggetti con AF e 13 controlli. Due Actigraph sono stati indossati dai soggetti (polso non dominante e vita) durante le ore di veglia per 7 giorni. L'attività giornaliera è stata riassunta come: spesa attiva media (Kcal), %attività, MET (Metabolic Equivalent of Task) e conteggio passi giornalieri. Le variabili di attività hanno differenziato soggetti con AF dai controlli. Il conteggio passi derivato dal dispositivo indossato in vita era la variabile più correlata ai parametri clinici. In conclusione, le scale cliniche rimangono essenziali per monitorare la progressione dell’AF e la conoscenza delle loro proprietà è fondamentale per le sperimentazioni cliniche. I biomarker e le misure di attività possono essere utili per valutare caratteristiche specifiche della malattia e forniscono informazioni più obiettive e di vita reale. L'uso combinato di scale cliniche e misure funzionali è ideale per malattie eterogenee, come l'atassia. Hereditary ataxias are rare and genetically heterogeneous disorders. Friedreich ataxia (FRDA) represent the most frequent of these forms, with a prevalence in Caucasian populations is 1:50.000. Clinical trials are particularly challenging in rare disease, therefore the selecting the most sensitive outcome measures is critical for trial designs. FRDA is characterized by juvenile onset, progressive disequilibrium and cardiomyopathy. Several outcome measures have been tested, but the majority of them require a considerable number of patients for appropriately powered studies. The aim of this project was to analyze existing outcome measures and to evaluate newly developed tools that can be used to rate FRDA disease severity and progression. A group of 207 subjects with FRDA from the Ataxia clinic of the Fondazione IRCCS Istituto Neurologico “Carlo Besta”, were included in the following studies: - Analysis of statistical properties of the Scale for the Assessment and Rating of Ataxia (SARA) and the FRDA rating scale (FARS). We assessed the properties of SARA scale in 170 subjects. We evaluated the contribution of each item to the total score. Items evaluating lower limbs, walking and stance linearly increase up to SARA score of 20-25 points (out of 40), when ambulation is lost. In non-ambulatory subjects, items assessing upper limbs rapidly increase. In longitudinal analyses subjects with lower baseline scores progressed more rapidly than subjects with higher scores. Performances of SARA and FARS scales were compared in 99 subjects. Both scales displayed good internal consistency and were mutually highly correlated and also correlated with disease duration. - Posturography. Seventy-six subjects with FRDA were assessed. Evaluations carried out at baseline and at 1- and 2-years follow-up consisted of 4 different tasks: seated, natural stance, feet together stance and tandem stance. X (medio-lateral) and Y (antero-posterior) axis sway was recorded from accelerometer placed over sternum. Posturography variables were not correlated with disease duration and ataxia severity, but were significantly related to age at ataxia onset. In longitudinal analyses significantly worse performances were identified during Feet-together-stance at 1 year follow-up. - Wet biomarkers (in collaboration with the University of Rome "Tor Vergata”, Prof. F. Malisan). Fifty-three subjects with FRDA and 27 controls were included in the first part of the study that analyzed HS-1-associated protein X-1 (HAX-1) expression as a potential biomarker in FRDA. FXN and HAX-1 expression appeared to be co-regulated, both at mRNA and protein levels. The microRNAs hsa-miR223-3p resulted up-regulated in FRDA subjects, and negative correlated with HAX-1 mRNA and protein levels. hsa-miR223-3p levels were also significantly correlated with cardiac parameters in FRDA. - Real-life activity monitoring. We tested remote monitoring with Actigraph in a cohort of 25 subjects with FRDA and 13 healthy controls. Two Actigraphs were worn by subjects (non-dominant wrist and waist) during waking hours for 7 days. Daily activity was summarized as: average active expense (Kcal), %active, MET (Metabolic Equivalent of Task) score and daily step count. Activity variables differentiated FRDA from matched controls. Step count derived from waist worn device was the variables that correlated more with clinical parameters. In conclusion, clinical scales remain essential to monitor disease progression in FRDA and knowledge of their properties is crucial for interventional trials. Biomarkers and activity measures can be used to assess specific features of the disease, and provide a more objective, real-life information. The combined use of clinical scales and functional measures may be ideal for heterogeneous diseases, such as ataxia.

Objective To assess neurofilaments as neurodegenerative biomarkers in serum of patients with Friedreich's ataxia. Methods Single molecule array measurements of neurofilament light (NfL) and heavy chain (pNfH) in 99 patients with genetically confirmed Friedreich's ataxia. Correlation of NfL/pNfH serum levels with disease severity, disease duration, age, age at onset, and GAA repeat length. Results Median serum levels of NfL were 21.2 pg/ml (range 3.6-49.3) in controls and 26.1 pg/ml (0-78.1) in Friedreich's ataxia (p = 0.002). pNfH levels were 23.5 pg/ml (13.3-43.3) in controls and 92 pg/ml (3.1-303) in Friedreich's ataxia (p = 0.0004). NfL levels were significantly increased in younger patients (age 16-31 years, p < 0.001) and patients aged 32-47 years (p = 0.008), but not in patients of age 48 years and older (p = 0.41). In a longitudinal assessment, there was no difference in NfL levels in 14 patients with repeated sampling 2 years after baseline measurement. Levels of NfL correlated inversely with GAA1 repeat length (r = - 0.24, p = 0.02) but not with disease severity (r = - 0.13, p = 0.22), disease duration (r = - 0.06, p = 0.53), or age at onset (r = 0.05, p = 0.62). Conclusion Serum levels of NfL and pNfH are elevated in Friedreich's ataxia, but differences to healthy controls decrease with increasing age. Long-term longitudinal data are required to explore whether this reflects a selection bias from early death of more severely affected individuals or a slowing down of the neurodegenerative process with age. In a pilot study over 2 years of follow-up-a period relevant for biomarkers indicating treatment effects-we found NfL levels to be stable.

In der vorliegenden Arbeit wurden Untersuchungen an Rückständen von thermisch abgebauten, flammgeschützten Polymeren vorgenommen, mit dem Ziel, die Struktur und den Phasenbestand der eingebauten Flammschutzmittel und der Polymere sowie deren Wechselwirkungen als Funktion der Temperatur und umgebenden Atmosphäre (N2 und Luft) zu charakterisieren. Ein wichtiges Werkzeug, das Informationen über den amorphen Zustand der Abbauprodukte und deren thermisch bedingte Phasenumwandlungen in andere amorphe oder kristalline Strukturen sowie Aussagen über die Nahordnungen der betrachteten Kernspinsorte liefert, stellt in dieser Arbeit der Einsatz der Festkörper-NMR-Spektroskopie dar. Hierbei sind neben Einzelimpuls- (SP), rotor-synchronisierte Spin-Echo- (RSE) und Kreuzpolarisationstechniken (CP) auch REDOR- (Rotational echo double resonance) und TRAPDOR- (Transfer of population in double resonance) Messungen zur Anwendung gekommen. Zusätzlich konnten aus den 11B- und 31P-NMR-Experimenten quantitative Aussagen über den relativen Borat- und Phosphor bzw. Phosphat-Anteil im festen Rückstand getroffen werden, wobei insbesondere für die 31P-Kerne eine quantitative Erfassung der kristallinen und amorphen Phosphatphasen durchgeführt wurde. Im ersten System wurden die Flammschutzmittel roter Phosphor (Prot) und Mg(OH)2 in HIPS kombiniert. Aus den Ergebnissen umfangreicher NMR-Experimente konnte abgeleitet werden, dass der größte Teil des eingesetzten Prot hauptsächlich in amorphen (Mg-Ortho-, -Di-, -Ketten- und Ringphosphaten) und weniger in kristallinen Phosphatphasen verbleibt. Zudem konnte für den Parameter der Temperatur und aus der Verfügbarkeit von Sauerstoff (N2-Atmosphäre/Luft) einen deutlicher Einfluss auf den Abbauprozess und die Bildung der Phosphatphasen (kristallin/amorph) nachgewiesen werden. Aus dem Vergleich der Ergebnisse der Temperversuche mit den Ergebnissen der Verbrennungsversuche im Cone Calorimeter konnte ein anaerober Abbauweg bestätigt werden. In einem zweiten System wurden die thermischen Reaktionen zwischen den Flammschutzadditiven BDP und Zinkborat sowie ihren Einfluss auf den thermischen Abbau eines PC/ABS-Blends untersucht. Der thermisch belastete Rückstand wird unabhängig von der Atmosphäre von amorphen Phosphatgruppen dominiert. Dabei konnten die während der Temperprozesse gebildeten Verbindungen α Zn3(PO4)2 und BPO4 als Folge einer Festphasenreaktion zwischen den eingesetzten Flammschutzadditiven identifiziert werden, wobei das α Zn3(PO4)2/BPO4 Verhältnis als Indikator für einen aeroben bzw. anaeroben Abbauprozess dient, der für die Feuerrückstände eindeutig einen anaeroben Abbau liefert., This study aims at the structural characterisation of embedded flame retardants and polymers. Analysis includes determination of the phase contents as well as description of their mutual interactions as a function of their thermal history in different atmospheres (air and nitrogen). Solid-state NMR spectroscopy is the most important tool applied in this thesis. This technique enables structural information about the amorphous state of the decomposed products, determination of the thermal and thermo-oxidative conversion in different amorphous and crystalline structures, and predictions about the short-range order of the observed nuclei. In this context measurements of single pulse (SP), rotor-synchronised spin echo (RSE) and cross polarisation (CP) experiments, as well as REDOR (Rotational echo double resonance) and TRAPDOR- (Transfer of population in double resonance) have been conducted. Additionally, 11B SP and 31P RSE experiments have been used to study quantitatively the borate and both phosphorus and phosphate contents in the solid residues. Particular emphasis was put on quantifying crystalline and non-crystalline phosphate phases. The first system consists of a combination of the flame retardants red phosphorus (Pred) and Mg(OH)2 in HIPS. From the results of extensive NMR experiments it was deduced that most of inserted red phosphorus remains in amorphous phosphates phases (ortho, pyro, and chain/ring phosphates) besides some crystalline phosphate phases. Decomposition characteristics such as temperature and the availability of oxygen (N2 atmosphere/air) show a significant influence on the decomposition process and the formation of phosphate phases (crystalline/amorphous). By comparing the results of the annealing processes to the results of the cone calorimeter measurements an anaerobic decomposition way was confirmed. In a second system, thermal reactions between the two flame retardants BDP and zinc borate as well their influence on the thermal decomposition of a PC/ABS blend were investigated. The thermally residues are independently of the atmosphere dominated by amorphous phosphate units. During the thermal decomposition the formation of α-Zn3(PO4)2 and BPO4 could be identified as solid reaction products of the inserted flame retardants. In addition, the α Zn3(PO4)2/BPO4 ratio served as an indicator of aerobic or anaerobic decomposition processes, proving an anaerobic decomposition mechanism for the fire residues.

Journal of neurology -, - (2020). doi:10.1007/s00415-020-09722-6, Published by Springer37620, Berlin

Parkinson’s disease is a common neurodegenerative disease that can be treated with pharmacological or surgical therapy. Subthalamic nucleus (STN) deep brain stimulation is a commonly used surgical option. A reported side effect of STN-DBS is weight gain: the aim of our study was to find those factors that determine weight gain, through one year-long observation of 32 patients that underwent surgery in our centre. During the follow-up, we considered: anthropometric features, hormonal levels, motor outcome, neuropsychological and quality of life outcomes, therapeutic parameters and electrodes position. The majority (84%) of our patients gained weight (6.7 kg in 12 months); more than a half of the cohort became overweight. At 12th month, weight gain showed a correlation with dyskinesias reduction, electrodes voltage and distance on the lateral axis. In the multivariate regression analysis, the determinants of weight gain were dyskinesias reduction and electrodes position. In this study, we identified dyskinesias reduction and distance between the active electrodes and the third ventricle as determining factors of weight gain after STN-DBS implantation in PD patients. The first finding could be linked to a decrease in energy consumption, while the second one could be due to a lower stimulation of the lateral hypothalamic area, known for its important role in metabolism and body weight control. Weight gain is a common finding after STN-DBS implantation, and it should be carefully monitored given the potential harmful consequences of overweight.