[ES] El tratamiento de enfermedades neurológicas está muy limitado por la ineficiente penetración de los fármacos en el tejido cerebral dañado debido a la barrera hematoencefálica (BHE), lo que imposibilita mejorar la salud del paciente. La BHE es un mecanismo de protección natural para evitar la difusión de agentes potencialmente peligrosas para el sistema nervioso central. No obstante, la BHE se puede inhibir mediante ultrasonidos focalizados e inyección de microburbujas de forma segura, localizada y transitoria, una tecnología empleada mundialmente. La principal ventaja es su carácter no invasivo, siendo así muy atractiva y cómoda para el paciente. Normalmente, la zona cerebral enferma se trata en su parte central empleando un único foco. Sin embargo, enfermedades como el Alzheimer o el Parkinson requieren un tratamiento sobre estructuras de geometría compleja y tamaño elevado, situadas en ambos hemisferios cerebrales. Por tanto, la tecnología actual está muy limitada al no cumplir dichos requisitos. Esta tesis doctoral tiene como objetivo el desarrollo de una técnica novedosa, basada en hologramas acústicos, para resolver las limitaciones presentes en los tratamientos neurológicos empleando ultrasonidos. Se estudian las lentes acústicas holográficas impresas en 3D, que acopladas a un transductor mono-elemento, permiten el control preciso del frente de onda ultrasónico tanto para (1) compensar las distorsiones que sufre el haz hasta alcanzar el cerebro, como (2) focalizarlo simultáneamente en regiones múltiples y de geometría compleja o formando de vórtices acústicos, proporcionando así efectividad en tiempo y coste. Por ello, la investigación desarrollada en esta tesis abre un camino prometedor en el campo de la biomedicina que permitirá mejorar los tratamientos neurológicos, además de aplicaciones en neuroestimulación o ablación térmica del tejido. / [CA] El tractament de malalties neurològiques està molt limitat per la ineficient penetració del fàrmac en el teixit cerebral danyat a causa de la barrera hematoencefàlica (BHE), i així no és possible una millora de salut del pacient. La BHE és un mecanisme de protecció natural per a evitar la difusió d'agents potencialment perillosos per al Sistema Nervios Central. No obstant això, aquesta barrera es pot inhibir mitjancant una tecnologia emprada mundialment basada en ultrasons focalitzats i injeccio de microbombolles. El principal avantatge és el seu caràcter no invasiu, sent així molt atractiva i còmoda per al pacient, i permet obrir la BHE de manera segura, localitzada i transitòria. Normalment, la zona cerebral malalta es tracta en la seua part central, emprant un unic focus. No obstant això, malalties com l'Alzheimer o el Parkinson requereixen un tractament al llarg d'estructures de geometria complexa i grandària elevada, situades en tots dos hemisferis cerebrals. Per tant, la tecnologia actual està fortament limitada al no complir amb aquests requeriments. Aquesta tesi doctoral està enfocada a investigar i desenvolupar una tècnica nova, basada en hologrames acústics, per a solucionar les limitacions presents en els tractaments neurològics. Una lent acústica holograca de baix cost impresa en 3D acoblada a un transductor d'element simple permet el control precs del front d'ona ultrasònic punt per a (1) compensar les distorsions que pateix el feix en el seu camí cap al cervell, i (2) focalització simultània del feix en regions multiples i de geometria complexa, proporcionant aix un tractament efectiu en temps i cost. Per això, la investigació desenvolupada en aquesta tesi demostra la possibilitat de realitzar qualsevol tractament neurològic, a més d'aplicacions en la neuroestimulació o l'ablació tèrmica dins del camp biomèdic. / [EN] Treatments for neurological diseases are strongly limited by the inefficient penetration of therapeutic drugs into the diseased brain due to the blood-brain barrier (BBB), and therefore no health improvement can be achieved. In fact, the BBB is a protection mechanism of the human body to avoid the diffusion of potentially dangerous agents into the central nervous system. Nevertheless, this barrier can be successfully inhibited by using a worldwide spread technology based on microbubble-enhanced focused ultrasound. Its main advantage is its non-invasive nature, thus defining a patient-friendly clinical procedure that allows to disrupt the BBB in a safe, local and transient manner. Conventionally, the diseased brain structure has been targeted in its center, with a single focus. However, Alzheimer's or Parkinson's Diseases do require that ultrasound is delivered to entire, complex-geometry and large-volume structures located at both hemispheres of the brain. Therefore, current technology presents several limitations as it does not fulfill these requirements. This doctoral thesis aims to develop a novel technique based on using focused ultrasound acoustic holograms to solve the existing limitations to treat neurological diseases. In this dissertation, we study 3D-printed holographic acoustic lenses coupled to a single-element transducer that allow to accurately control the acoustic wavefront to both (1) compensate distortions suffered by the beam in its path to the brain, and (2) simultaneous focusing in multiple and complex-geometry structures or acoustic vortex generation, providing a time- and cost- efficient procedure. Therefore, the research carried out throughout this thesis opens a promising path in the biomedical field to improve the treatment for neurological diseases, neurostimulation or tissue ablation applications. / Acknowledgments to the Spanish institution Generalitat Valenciana, which funding grant allowed me to develop this doctoral thesis, and as well funded my research stay at Columbia University. The development of the entire thesis was supported through grant Nª. ACIF/2017/045. Particularly, the research carried out in Chapter 3 and Chapter 4 was possible thanks to and supported through grant BEFPI/2019/075. Action co-financied by the Agència Valenciana de la Innovació through grant INNVAL10/19/016 and by the European Union through the Programa Operativo del Fondo Europeo de Desarrollo Regional (FEDER) of the Comunitat Valenciana 2014-2020 (IDIFEDER/2018/022). / Jiménez Gambín, S. (2021). Transcranial Ultrasound Holograms for the Blood-Brain Barrier Opening [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/171373 / TESIS

Neurodegenerative diseases have become one of the most common causes of death worldwide over the last couple of decades, with increasing tendency. Amyotrophic lateral sclerosis (ALS) is the most common neurodegenerative disease affecting specifically spinal (lower) and cortical (upper) motor neurons in the spinal cord and brainstem, respectively. It is usually a late onset disorder (average age of onset in Germany is 61 years) and leads to death within 2-5 years after symptoms onset due to respiratory failure. To date, there is no cure for ALS and only two drugs have been approved for its treatment, which prolong the lifespan for up to six months or slow down disease progression in a subpopulation of patients. About 90 % of ALS cases are sporadic, while about 10 % are familial and hence caused by mutations in specific genes, among them fused in sarcoma (FUS), a DNA- and RNA-binding protein. Mutations in FUS account for roughly 5 % of familial cases and occur predominantly in its nuclear localization sequence (NLS), such as the FUS-P525L mutation. Neurons expressing this variant display a strong cytoplasmic mislocalization of FUS and hence a loss of its nuclear function. Among other pathological events, defects in axonal transport along microtubules have been observed early in disease progression in several models of FUS-ALS, indicating its role as a major hallmark of the disease. However, the mechanism of how transport is impaired within these neurons remains unknown to date. This study aimed at investigating two possible mechanisms how the FUS-P525L mutant variant affects microtubule-based axonal transport. First, it was analyzed whether FUS directly interacts with microtubules or motors and if the mislocalized, mutant variant alters this interaction. Secondly, cytoplasmic mislocalized FUS-P525L can no longer fulfil its regular role in the splicing of pre-mRNAs, among them the mRNA coding for the microtubule-associated protein tau. This reportedly leads to an increased ratio of translated tau isoforms containing four microtubule binding repeats (4R) to those containing three repeats (3R). 4R tau isoforms are known to have a stronger binding affinity towards microtubules and may hence impair transport more severely by acting as a roadblock for motor proteins. Towards this end, this study investigated whether an increase in 4R:3R tau isoform ratio is sufficient to impair microtubule based transport. Axonal transport was reconstituted in vitro using a kinesin-1-dependent microtubule gliding assays, in which microtubules are propelled by surface-immobilized kinesin-1 motors. The assay was modified and optimized to operate sensitively and robust in the presence of complex solutions such as whole cell lysates and the microtubule gliding velocity analyzed as a measure for motility of the underlying motors. To determine the direct interaction of FUS variants with kinesin-1 or microtubules, recombinant human wildtype FUS-GFP and FUS-P525L-GFP was added to the assay. In addition, ALS patient-specific induced pluripotent stem cells (iPSCs) expressing the same FUS variants were differentiated towards spinal motor neurons and their cell lysates applied to this assay in order to determine whether FUS variants need endogenous adaptors or interaction partners to interfere with kinesin-1 motility on microtubules. Further, to investigate the interference of tau isoforms with kinesin-1 motility, recombinant human 2N3R tau-GFP and 2N4R tau-mScarlet was purified from insect cells and added to the modified kinesin-1-dependent microtubule gliding assay, either individually or combined at different ratios. In addition, the binding of these tau variants to microtubules was assessed. The kinesin-1-dependent microtubule gliding assays was modified to operate sensitively and robustly in the presence of β-glycerophosphate (to inhibit endogenous phosphatases in whole cell lysates), and methylcellulose (to prevent microtubule detachment from kinesin-1 motors due to presence of β-glycerophosphate). Under these conditions, neither recombinant human FUS-GFP nor endogenous FUS-GFP variants in lysates of spinal motor neurons bound to microtubules or interfered with kinesin-1 motility. In contrast, both tau isoforms used in the present study bound to microtubules and impaired kinesin-1 motility, while 2N4R tau-mScarlet was a much more potent inhibitor of microtubule gliding and displayed a 20-fold stronger binding affinity to microtubules compared to 2N3R tau-GFP. Interestingly, increasing ratios of 4R:3R tau isoforms impaired kinesin-1-dependent microtubule gliding. In addition, the presence of 2N4R tau-mScarlet strongly prevented 2N3R tau-GFP from binding to microtubules. This study provides evidence that neither wildtype FUS nor the FUS-P525L variant directly interfere with axonal transport by interacting with kinesin-1 motors or microtubules. Further, the present data suggests that neither FUS variant impedes kinesin-1 motility on microtubules by interacting with endogenous adaptor proteins present in cell lysates of iPSC-derived spinal motor neurons. Therefore, it is proposed that axonal transport defects are not directly caused by interaction of cytoplasmic mislocalized FUS with the motors or microtubules, but rather arise as a consequence of other pathological events triggered by mutant FUS variants. In particular, this study demonstrates that an increased ratio of 4R:3R tau isoforms is sufficient to impair kinesin-1 motility on microtubules due to increased decoration of microtubules with 4R tau isoforms, preventing 3R tau isoforms from binding to microtubules. This strongly suggests that an increased ratio of 4R:3R tau isoforms, since FUS no longer regulates splicing of tau pre-mRNA upon its cytoplasmic mislocalization, may be sufficient to cause or contribute to the axonal transport defects observed early in FUS-ALS pathology. / Neurodegenerative Erkrankungen sind in den letzten Jahrzehnten mit zunehmender Tendenz zu einer der häufigsten Todesursachen weltweit geworden. Amyotrophe Lateralsklerose (ALS) ist die häufigste neurodegenerative Erkrankung, die spezifisch spinale (untere) und kortikale (obere) Motoneuronen im Rückenmark bzw. im Hirnstamm betrifft. Es handelt sich in der Regel um eine spät einsetzende Krankheit (das mittlere Erkrankungsalter in Deutschland beträgt 61 Jahre) und führt innerhalb von 2-5 Jahren nach Auftreten der Symptome zum Tod aufgrund von Atemversagen. Bisher gibt es keine Heilung für ALS und es wurden nur zwei Medikamente für die Behandlung zugelassen, die die Lebensdauer um bis zu sechs Monate verlängern oder das Fortschreiten der Krankheit bei einer Subpopulation von Patienten verlangsamen. Ungefähr 90% der ALS-Fälle sind sporadisch, während ungefähr 10% familiär sind und daher durch Mutationen in bestimmten Genen verursacht werden, darunter fused in sarcoma (FUS), einem DNA- und RNA-bindenden Protein. Mutationen in FUS machen etwa 5% der familiären Fälle aus und treten überwiegend in der Kernlokalisierungssequenz (NLS) auf, wie beispielsweise die FUS-P525L Mutation. Neuronen, die diese Mutante exprimieren, zeigen eine starke zytoplasmatische Fehllokalisierung von FUS und damit einen Verlust seiner Funktionen im Zellkern. Neben anderen pathologischen Ereignissen wurden in mehreren FUS-ALS Modellsystemen Defekte im Mikrotubuli-basierenden axonalen Transport früh im Krankheitsverlauf beobachtet, was auf seine Rolle als eines der Hauptmerkmale dieser Krankheit hindeutet. Der Mechanismus, wie der Transport innerhalb dieser Neuronen beeinträchtigt wird, ist jedoch bis heute unbekannt. Ziel dieser Studie ist es, zwei mögliche Mechanismen zu untersuchen, wie das mutierte FUS-P525L Protein den axonalen Transport entlang von Mikrotubuli beeinflusst. Zunächst wurde analysiert, ob FUS direkt mit Mikrotubuli oder Motorproteinen interagiert und ob zytoplasmatische fehllokalisierte FUS-P525L Protein diese Interaktion verändert. Ferner kann zytoplasmatische fehllokalisiertes FUS-P525L seine reguläre Rolle beim Spleißen von Prä-mRNAs nicht mehr erfüllen, darunter die mRNA, die für das mit Mikrotubuli-assoziierte Protein Tau kodiert. Dies führt zu einem erhöhten Verhältnis von translatierten Tau-Isoformen, die vier Mikrotubuli-Bindestellen (4R) enthalten, zu solchen mit drei Bindestellen (3R). Es ist bekannt, dass 4R-Tau-Isoformen eine stärkere Bindungsaffinität zu Mikrotubuli im Vergleich zu 3R-Tau-Isoformen aufweisen und daher den Transport stärker beeinträchtigen können, indem sie als Hindernis für Motorproteine agieren. In dieser Studie wurde daher untersucht, ob eine Erhöhung des Verhältnisses von 4R:3R-Tau-Isoform ausreicht, um den Mikrotubuli-basierenden Transport zu beeinträchtigen. Der axonale Transport wurde in vitro unter Verwendung eines Kinesin-1-gestuerten Mikrotubuli Motilitätsassay rekonstruiert, bei welchem Mikrotubuli von darunterliegenden oberflächenimmobilisierte Kinesin-1 Motorproteinen transportiert werden, also über die Oberfläche gleiten. Der Assay wurde modifiziert und optimiert, um in Gegenwart komplexer Lösungen wie Ganzzelllysaten sensitiv und robust zu funktionieren, und die Gleitgeschwindigkeit der Mikrotubuli wurde als Maß für die Motilität der darunterliegenden Motoren analysiert. Um die direkte Wechselwirkung von FUS-Varianten mit Kinesin-1 Motorproteinen oder Mikrotubuli zu bestimmen, wurde dem Assay rekombinantes menschliches Wildtyp-FUS-GFP und FUS-P525L-GFP hinzugegeben. Zusätzlich wurden ALS-patientenspezifische, induzierte pluripotente Stammzellen (iPSCs), welche dieselben FUS-Varianten exprimieren, zu spinalen Motoneuronen differenziert und ihre Zelllysate in diesem Assay angewendet, um zu bestimmen, ob FUS-Varianten endogene Adapter oder Interaktionspartner für die Interaction mit Kinesin-1 oder Mikrotubuli benötigen. Um den Einfluss von Tau-Isoformen auf die Kinesin-1 Motilität zu untersuchen, wurde rekombinantes menschliches 2N3R Tau-GFP und 2N4R Tau-mScarlet aus Insektenzellen aufgereinigt und dem modifizierten Kinesin-1-gesteuerten Mikrotubuli Motilitätsassay entweder einzeln oder in unterschiedlichen Verhältnissen kombiniert hinzugegeben. Zusätzlich wurde die Bindung dieser Tau-Varianten an Mikrotubuli analysiert. Der Kinesin-1-gesteuerte Mikrotubuli Motilitätsassay wurden so modifiziert, dass er in Gegenwart von β-Glycerophosphat (zur Hemmung endogener Phosphatasen in Ganzzelllysaten) und Methylcellulose (zur Verhinderung der Ablösung von Mikrotubuli von Kinesin-1 Motoren aufgrund von β-Glycerophosphat) empfindlich und robust funktioniert. Unter diesen Bedingungen zeigten weder rekombinantes menschliches FUS-GFP noch endogene FUS-GFP-Varianten in Lysaten von spinalen Motoneuronen eine Wechselwirkung mit Mikrotubuli und beeinträchtigten auch nicht die Kinesin-1 Motilität. Im Gegensatz dazu banden beide in der vorliegenden Studie verwendeten Tau-Isoformen an Mikrotubuli und beeinträchtigten die Kinesin-1-Motilität, wobei 2N4R Tau-mScarlet das Gleiten von Mikrotubuli viel stärkerer beeinträchtigte und eine 20-fach stärkere Bindungsaffinität zu Mikrotubuli im Vergleich zu 2N3R Tau-GFP zeigte. Ferner beeinträchtigten steigende Verhältnisse von 4R:3R Tau-Isoformen über Kinesin-1 gleitende Mikrotubuli, während die Präsenz von 2N4R Tau-mScarlet die Bindung von 2N3R Tau-GFP an Mikrotubuli stark verminderte. Diese Studie liefert Hinweise darauf, dass weder Wildtyp-FUS noch die FUS P525L-Variante den axonalen Transport direkt beeinflussen, da sie nicht mit Kinesin-1 Motorproteinen oder Mikrotubuli interagieren. Ferner legen die vorliegenden Daten nahe, dass keine der FUS-Varianten die Kinesin-1 Motilität auf Mikrotubuli durch Wechselwirkung mit endogenen Adapterproteinen behindert, die in Zelllysaten von iPSC-differenzierte spinalen Motoneuronen vorhanden sind. Dies legt nahe, dass axonale Transportdefekte nicht durch direkte Wechselwirkung von zytoplasmatisch fehllokalisiertem FUS Protein mit Motorproteinen oder Mikrotubuli verursacht werden, sondern als Folge anderer pathologischer Ereignisse auftreten, die durch mutierte FUS-Varianten entstehen. Insbesondere zeigt diese Studie, dass ein erhöhtes Verhältnis von 4R:3R Tau-Isoformen ausreicht, um die Kinesin-1 Motilität auf Mikrotubuli zu behindern. Dies geschieht vermutlich aufgrund der erhöhten Bindung von 4R Tau-Isoformen an Mikrotubuli, weil dadurch die Bindung von 3R Tau-Isoformen an Mikrotubuli verhindert wird. Dies deutet stark darauf hin, dass ein erhöhtes Verhältnis von 4R:3R Tau-Isoformen, verursacht durch die fehlende regulatorische Beteiligung von FUS am Spleißen von Tau-Prä-mRNA aufgrund der zytoplasmatischen Fehllokalisation von FUS, wahrscheinlich zu den axonalen Transportdefekten beiträgt, die früh in der FUS-ALS-Pathologie beobachtet wurden.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-02-05T22:02:41Z No. of bitstreams: 1 PatriciaRibeiroRomano_DISSERT.pdf: 747260 bytes, checksum: 8d99b66a0fdadd0d33b31d559fbf4b19 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-02-15T21:42:45Z (GMT) No. of bitstreams: 1 PatriciaRibeiroRomano_DISSERT.pdf: 747260 bytes, checksum: 8d99b66a0fdadd0d33b31d559fbf4b19 (MD5) / Made available in DSpace on 2016-02-15T21:42:45Z (GMT). No. of bitstreams: 1 PatriciaRibeiroRomano_DISSERT.pdf: 747260 bytes, checksum: 8d99b66a0fdadd0d33b31d559fbf4b19 (MD5) Previous issue date: 2015-03-23 / Este trabalho investigou o impacto provocado por eventos de fus?es e aquisi??es (F&As) horizontais, nos retornos das a??es das empresas participantes e concorrentes com rela??o ? cria??o ou destrui??o de valor para essas firmas no Brasil, no per?odo de 2001 a 2012. Para tal, utilizou-se, inicialmente, a metodologia do estudo de eventos ? que estimou altera??es anormais ocorridas nos pre?os das a??ese, posteriormente, realizaram-se an?lises de regress?es m?ltiplas - cujas vari?veis independentes possu?ram algumas motiva??es que poderiam gerar o aumento ou a diminui??o de valor das firmas. Os resultados do estudo de evento atestaram que, ao utilizar subper?odos para os dados antes e ap?s o per?odo de crise, os efeitos foram diferentes para as empresas alvo - antes negativos, depois positivos. Com rela??o ?s empresas adquirentes e concorrentes, os resultados foram constantes. Os retornos foram pr?ximos ? zero para as firmas compradoras, j? para as concorrentes foram negativos. Ademais, os resultados das regress?es em rela??o ?s firmas adquirentes mostraram que as firmas investiram em processos de F&As para obterem um aumento ainda maior da sua efici?ncia. Al?m disso, esta pesquisa indicou que o endividamento da firma adquirente exerceu um papel importante para a cria??o de valor em aquisi??es quando ela possuiu um Q de Tobin mais elevado. Em rela??o aos resultados das firmas alvo, este trabalho concluiu que as pequenas empresas, quando fazem um an?ncio de fus?o ou aquisi??o, obt?m um melhor retorno do que as grandes empresas. / This study investigated the impact caused by events horizontal mergers and acquisitions (M&As) horizontal, in the stock returns of the participating companies and competitors regarding the creation or destruction of value for those firms in Brazil, from 2001 to 2012. For this, first was used the event study methodology to estimate abnormal returns in stock prices; after was conducted an analysis multiple regression. The results of the event study showed that using sub-periods for the data, before and after the crisis period, the effects were different for the target-before negative, after positive. Regarding the acquirer and competitors, the results were constant. For acquirer firms, the returns were close to zero, while for the competitors were negative. Furthermore, the regression results regarding the bidder showed that firms invested in processes of M&As to obtain a further increase its efficiency. Furthermore, this study indicated that the leverage of the bidder plays is important for creating value in acquisitions, when they has a higher Tobin?s Q. The results of target firms showed that a small firm had a better return than large firm did.

京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第22797号 / エネ博第411号 / 新制||エネ||79(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 片平 正人, 准教授 小瀧 努, 教授 森井 孝 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

Amyotrophic lateral sclerosis (ALS) is an incurable and fatal neurodegenerative disease characterized by motor neuron loss. Although pathological mutations exist in >15 genes, the mechanism(s) underlying ALS are unknown. FUS is one such gene and encodes the nuclear RNA-binding protein (RBP), fused in sarcoma (FUS), which actively shuttles between the nucleus and cytoplasm. Intriguingly, nearly half of the ALS mutations identified in FUS cause this protein to mislocalize, suggesting that FUS localization is relevant to disease. Here, we found that excitotoxicity, a neuronal stress caused by aberrant glutamate signaling, induces the rapid redistribution of FUS and additional disease-linked RBPs from the nucleus to the cytoplasm. As excitotoxicity is pathologically associated with ALS, it was notable that the nuclear egress of FUS was particularly robust. Further, ALS-FUS variants that predominantly localize to the nucleus also undergo redistribution. Thus, we sought to understand the purpose underlying FUS translocation and the potential relevance of this response to disease. As calcium dysregulation is strongly associated with neurodegenerative disorders, we examined the contribution of calcium to FUS egress. In addition to global changes to nucleocytoplasmic transport following excitotoxic insult, we observed that FUS translocation caused by excitotoxicity is calcium mediated. Moreover, we found that dendritic expression of Gria2, a transcript encoding an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit responsible for regulating calcium permeability, is FUS-dependent under conditions of stress. Together, these observations support the premise that FUS has a normal function during excitotoxic stress and that glutamatergic signaling may be dysregulated in FUS-mediated ALS.

Approximately 80% of all patients with ALS suffer from dysphagia sooner or later during the course of the disease. It is important to find patients with dysphagia in an early stage since weight loss and malnutrition, which dysphagia contributes to, are negative prognostic factors. The main purpose of this study was to identify early signs of dysphagia in patients with ALS, by investigating which clinical evaluation tools that discovers dysphagia in an early stage and see how the swallowing difficulties progress over time. Another purpose of the study was to evaluate the participants’ responses in a self-evaluation questionnaire (EAT-10) and how they correlate with findings on fiberoptic endoscopic evaluation of swallowing (FEES). Eleven participants with ALS were examined with FEES and non-instrumental tests one to four times over the course of one year. Only five participants were examined three or more times and because of that it is not possible to draw conclusions about the progression of dysphagia. Nine of 11 participants showed signs of dysphagia at the first examination. Test of lip strength was the clinical evaluation tool that identified most patients with dysphagia, followed by swallow capacity test. In an exploratory analysis, both of these tests showed good correlation with results on FEES. Patient responses on EAT-10 were also strongly correlated with the results on FEES.

Nucleocytoplasmic transport (NCT) declines during aging and in the context of age-dependent neurodegenerative diseases. However, the mechanisms underlying NCT decline in the disease are poorly understood. FUS is an RNA binding protein that shuttles between the nucleus and cytoplasm and is actively involved in NCT. Mutations in FUS cause amyotrophic lateral sclerosis (ALS), a fatal and incurable motor neuron disorder. We sought to understand the disease mechanism underlying FUS-induced NCT decline in ALS. Here, I uncovered NCT-related defects in motor neurons derived from human induced pluripotent stem cells (iPSCs) harboring an ALS-linked FUS mutation. Importantly, these NCT defects were rescued by genetically correcting the FUS mutation in iPSCs. To gain insight into how expression of mutant FUS causes nuclear pore defects, I demonstrated an altered localization where FUS and nucleoporins (Nups) interact in situ within patient-derived human neurons. Moreover, FUS became aggregation-prone when interacting with Nup62 in vitro, and RNA further alleviated their aggregation propensity. Importantly, NCT-related defects and neuronal toxicity induced by ALS-FUS were ameliorated by modulating Nup expression in vivo. Collectively, these findings implicate aberrant Nup interactions in the pathogenic mechanism of ALS-FUS, and direct targeting the gain-of-function protein interactions could be therapeutic for multiple causes of neurodegeneration.

No description available.

Focused ultrasound (FUS) is a non-invasive technique producing a variety of biological effects by either thermal or mechanical mechanisms of ultrasound interaction with the targeted tissue. FUS could bring benefits, e.g., tumour sensitisation, immune stimulation, and targeted drug delivery, but investigation of FUS effects at the cellular level is still missing. New techniques are commonly tested in vitro on two-dimensional (2D) monolayer cancer cell culture models. The 3D tumour model—spheroid—is mainly utilised to mimic solid tumours from an architectural standpoint. It is a promising method to simulate the characteristics of tumours in vitro and their various responses to therapeutic alternatives. This study aimed to evaluate the effects of FUS on human prostate and glioblastoma cancer tumour spheroids in vitro. The experimental follow-up enclosed the measurements of spheroid integrity and growth kinetics, DNA damage, and cellular metabolic activity by measuring intracellular ATP content in the spheroids. Our results showed that pulsed FUS treatment induced molecular effects in 3D tumour models. With the disruption of the spheroid integrity, we observed an increase in DNA double-strand breaks, leading to damage in the cancer cells depending on the cancer cell type.

Orderliness, speed, and rhythm in biochemistry are vital for cellular function. In order to achieve this, cells implement compartmentalization via several methods, one of which is the formation of membrane-less compartments. These compartments, often referred to as “biomolecular condensates”, are understood to be formed by separation of proteins and other biomolecules into dense and dilute phases. While the formation of the resulting protein-rich condensates is fundamental for spatiotemporal organization of biochemistry within the cell, a vast majority of proteins found to phase separate in vitro do so at a concentration an order of magnitude above their endogenous expression levels. Recently, a theoretical study has shown that membrane binding of phase separating proteins can result in phase separation spatially occurring at the membrane well below bulk saturation concentrations. However, much remains unknown about the formation mechanism and function of these condensates. To that end, for my doctoral project, I used a synthetic system composed of supported lipid bilayers decorated with lipid-bound NTA(Ni) to allow for coordination and thus membrane binding of the well-characterized protein FUS via a C-terminal His-tag. Through this model system I found that 2D phase separation of FUS could occur an order of magnitude below the experimentally determined bulk saturation concentration. FUS was able to form dense and dilute phases in 2D and the transition point to form these phases could be controlled by modulating buffer conditions. Additionally, membrane-bound FUS condensates were able to further recruit FUS from the bulk to form a multilayer of protein through a prewetting transition. With this characterization of 2D phase separation of FUS, I then explored a physiologically relevant protein in the form of PAR-3, a fundamental protein of the PAR polarity system, which is necessary for the establishment of polarity in the C. elegans zygote. I found that full-length PAR-3 was able to phase separate under physiological salt conditions with a Csat of 100nM. Further, I identified a C-terminal predicted prion-like domain to act as a driver for phase separation. Additionally, I determined PAR-3’s affinity and specificity for PI(4,5)P2 and found that it could form 2D condensates upon binding to the membrane at physiological concentrations. Furthermore, these condensates were able to recruit PAR-6 alone and PAR-6 in complex with PKC-3 to the membrane, ultimately resulting the reconstitution of the anterior PAR complex which is known to exist in a condensed clustered form in vivo. Taken together, this work provides insight into a mechanism where phase separation can be locally triggered by membrane binding under sub-saturation concentration, offering a robust and potentially universal mechanism by which cells can spatially control phase separation and pattern cellular membranes.