The fused in sarcoma (FUS) protein has been shown to be a significant disease protein in a subgroup of patients with frontotemporal lobar degeneration (FTLD). Nevertheless, the mechanism underlying FUS associated FTLD is only poorly understood. Recent research has identified a large hexanucleotide repeat expansion in chromosome 9 open reading frame 72 (C9orf72), reinforcing the association between C9orf72 and FTLD. Moreover, an unusual histopathological change has been observed within the granule cell layer of the cerebellum in chromosome 9p-linked frontotemporal dementia with motor neuron disease. Whether this type of cerebellar pathology is a pathological marker for chromosome 9p-linked families remains unknown. The purpose of this study was to genetically, neuropathologically and biochemically characterize FUS and C9orf72 in FTLD, and also to investigate the association between the cerebellar pathology and chromosome 9p-linked families. The genetic sequencing study searching for potential genetic factors of FUS in FTLD failed to detect any pathogenic mutations or variations. Immunohistochemical study for FUS pathology in FTLD provided strong evidence for FUS being the specific pathological protein in all forms of FTLD-FUS. Immunoblotting for FUS in FTLD detected one novel disease-associated FUS aggregate (~37 kDa) in the urea fraction of atypical FTLD with ubiquitinated inclusions (aFTLD-U) frontal cortical samples, suggesting this unique protein product might be more associated with disease than the full-length protein itself. Immunohistochemical study of C9orf72 in FTLD detected a 'synaptic' staining in CA sectors, as the most prominent histological feature identified. Immunoblotting for C9orf72 protein demonstrated no distinctive bands among different diagnostic groups, in frontal and cerebellar cortical regions. The present study also confirmed the presence of cerebellar p62 neuronal cytoplasmic inclusions (NCI) in a proportion of FTLD-TDP cases. Although most of these cases showed an autosomally dominant pattern of inheritance, not all of them shared a common C9orf72 haplotype, or mutation in C9orf72.Much work is still needed to investigate the underlying pathogenesis of FTLD-FUS. Attention should still be given to identifying possible genetic risk factors in FUS using a large series of FTLD samples and searching for other possible proteins within the FUS immunoreactive neuronal inclusions. Moreover, the target protein within the cerebellar p62 NCI remains unknown, but it is clear that it is not C9orf72 protein.

No description available.

A esclerose lateral amiotrófica (ELA) é uma doença neurodegenerativa, progressiva de início tardio que afeta principalmente os neurônios motores (NM). As causas que levam os NM à morte são variadas e ainda sendo investigadas. A descoberta de alterações genéticas como uma possível causa de ELA deu início à uma nova era na investigação desta afecção. Atualmente existem mais de 30 genes associados com a doença, entre eles o FUS, um gene que frequentemente aparece mutado em casos familiais da doença. A proteína FUS normalmente se localiza predominantemente no núcleo, mas na maioria dos casos de mutações na FUS relacionadas à ELA, ela aparece retida no citoplasma. O presente estudo traz um paciente de ELA (P) portando a mutação p.R521H no gene FUS e três de seus irmãos (dos quais um é portador da mutação e não apresnta sinais clínicos de ELA, e os outros dois não apresentam mutações no FUS) dos quais foram obtidas amostras de sangue e biópsia de pele. O DNA extraído das amostras de sangue, foi submetido ao sequenciamento do tipo Sanger para verificar a presença, ou ausência, da mutação R521H na FUS. A partir dos fibroblastos dos participantes, foram derivadas linhagens de células tronco pluripotentes induzidas (iPSC). As iPSC produzidas passaram por ensaios a fim de indicar o estado de pluripotência e de indiferenciação destas linhagens. Nós investigamos a posição da proteína FUS nas linhagens de iPSC e de fibroblastos e há evidências que, assim como descrito na literatura, a proteína FUS aparece retida no citoplasma das linhagens do paciente e de seu irmão portador da mutação. Desta forma, o presente estudo associa dois irmãos com quadros clínicos discordantes mas que apresentam a mesma mutação e sinais moleculares patológicos semelhantes. As linhagens de iPSC obtidas são um rico material para o uso em pesquisas futuras sobre a ELA / Amyotrophic lateral sclerosis (ALS) is a late onset, progressive, neurodegenerative disease that primarily affects motor neurons (MNs). The causes behind motor neuron death are diverse and still under investigation. The discovery of genetic alterations as possible causes of ALS initiated a new era for ALS research. There are currently over 30 genes associated with the disease, among which is FUS, one of the most frequently mutated in familial cases. The FUS protein is predominantly located in the nucleus, but in most of the ALS-related FUS mutations this protein is dislocated to the cytoplasm. The present work investigates the molecular aspects of a specific FUS mutation, p.R521H. An ALS patient (P) harboring the mutation and three siblings (of which one is a non-affected carrier and two present no mutations in FUS) were analyzed using blood samples and skin biopsies. We extracted DNA from blood samples and submitted it to Sanger sequencing for confirmation of the presence, or absence, of the R521H FUS mutation. The fibroblasts obtained from these biopsies were used for iPSC derivation. Assays were performed to confirm the undifferentiated state and pluripotency for the four strains obtained. We investigated the FUS location in these strains, and there is evidence for FUS retention in the cytoplasm of cells harboring the mutation (as seen in recent literature). Thus, this work associates two siblings with the same pathogenic mutation, showing the same molecular pathological signal but with discording clinical phenotypes. The iPSC strains obtained here are a valuable resource for further ALS investigation

A esclerose lateral amiotrófica (ELA) é uma doença neurodegenerativa, progressiva de início tardio que afeta principalmente os neurônios motores (NM). As causas que levam os NM à morte são variadas e ainda sendo investigadas. A descoberta de alterações genéticas como uma possível causa de ELA deu início à uma nova era na investigação desta afecção. Atualmente existem mais de 30 genes associados com a doença, entre eles o FUS, um gene que frequentemente aparece mutado em casos familiais da doença. A proteína FUS normalmente se localiza predominantemente no núcleo, mas na maioria dos casos de mutações na FUS relacionadas à ELA, ela aparece retida no citoplasma. O presente estudo traz um paciente de ELA (P) portando a mutação p.R521H no gene FUS e três de seus irmãos (dos quais um é portador da mutação e não apresnta sinais clínicos de ELA, e os outros dois não apresentam mutações no FUS) dos quais foram obtidas amostras de sangue e biópsia de pele. O DNA extraído das amostras de sangue, foi submetido ao sequenciamento do tipo Sanger para verificar a presença, ou ausência, da mutação R521H na FUS. A partir dos fibroblastos dos participantes, foram derivadas linhagens de células tronco pluripotentes induzidas (iPSC). As iPSC produzidas passaram por ensaios a fim de indicar o estado de pluripotência e de indiferenciação destas linhagens. Nós investigamos a posição da proteína FUS nas linhagens de iPSC e de fibroblastos e há evidências que, assim como descrito na literatura, a proteína FUS aparece retida no citoplasma das linhagens do paciente e de seu irmão portador da mutação. Desta forma, o presente estudo associa dois irmãos com quadros clínicos discordantes mas que apresentam a mesma mutação e sinais moleculares patológicos semelhantes. As linhagens de iPSC obtidas são um rico material para o uso em pesquisas futuras sobre a ELA / Amyotrophic lateral sclerosis (ALS) is a late onset, progressive, neurodegenerative disease that primarily affects motor neurons (MNs). The causes behind motor neuron death are diverse and still under investigation. The discovery of genetic alterations as possible causes of ALS initiated a new era for ALS research. There are currently over 30 genes associated with the disease, among which is FUS, one of the most frequently mutated in familial cases. The FUS protein is predominantly located in the nucleus, but in most of the ALS-related FUS mutations this protein is dislocated to the cytoplasm. The present work investigates the molecular aspects of a specific FUS mutation, p.R521H. An ALS patient (P) harboring the mutation and three siblings (of which one is a non-affected carrier and two present no mutations in FUS) were analyzed using blood samples and skin biopsies. We extracted DNA from blood samples and submitted it to Sanger sequencing for confirmation of the presence, or absence, of the R521H FUS mutation. The fibroblasts obtained from these biopsies were used for iPSC derivation. Assays were performed to confirm the undifferentiated state and pluripotency for the four strains obtained. We investigated the FUS location in these strains, and there is evidence for FUS retention in the cytoplasm of cells harboring the mutation (as seen in recent literature). Thus, this work associates two siblings with the same pathogenic mutation, showing the same molecular pathological signal but with discording clinical phenotypes. The iPSC strains obtained here are a valuable resource for further ALS investigation

Submitted by João Arthur Martins (joao.arthur@ufpe.br) on 2015-03-10T19:14:48Z No. of bitstreams: 2 DISSERTAÇÃO Hugo Albuquerque.pdf: 7422850 bytes, checksum: 3133c927702ab38bd7ee89b9c3f5cc73 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Approved for entry into archive by Daniella Sodre (daniella.sodre@ufpe.br) on 2015-03-10T19:43:36Z (GMT) No. of bitstreams: 2 DISSERTAÇÃO Hugo Albuquerque.pdf: 7422850 bytes, checksum: 3133c927702ab38bd7ee89b9c3f5cc73 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-10T19:43:36Z (GMT). No. of bitstreams: 2 DISSERTAÇÃO Hugo Albuquerque.pdf: 7422850 bytes, checksum: 3133c927702ab38bd7ee89b9c3f5cc73 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2013-08-20 / A fus~ao de imagens tem-se destacado na area de processamento de imagens, atrav es da ideia de combinar duas ou mais imagens que apresentem caracter sticas distintas (provenientes de diferentes sensores) ou alguma degrada c~ao (regi~oes desfocadas, mal iluminadas, com baixo contraste). O foco e uma propriedade intimamente relacionada com a qualidade da imagem. Em algumas situa c~oes, n~ao e poss vel manter simultaneamente todas as regi~oes da paisagem bem focadas. Uma alternativa e utilizar a fus~ao de imagens para combinar os focos diferentes de uma foto em apenas uma imagem com todas as regi~oes melhor focadas. Este trabalho apresenta uma ideia de utilizar diferentes t ecnicas de fus~ao de imagens em conjunto para compor o resultado nal. Para tanto, as imagens de entrada s~ao divididas em blocos, nos quais s~ao aplicados os algoritmos de fus~ao escolhidos. Em seguida, escolhe-se a t ecnica que atuou melhor em cada bloco, atrav es de uma m etrica de qualidade de imagem (como a PSNR (Peak Signal to Noise Ratio)), formando um mosaico com os melhores resultados. Dois algoritmos existentes na literatura envolvendo a fus~ao de imagens no dom nio da frequ^encia baseados em foco s~ao descritos: Contraste no dom nio da DCT (Transformada Discreta de Cosseno) e Frequ^encia Espacial. Modi ca c~oes s~ao feitas em ambas as t ecnicas a m de melhorar os crit erios de sele c~ao para cada bloco, por meio da de ni c~ao da escolha de um bloco inteiro ou dos pixels (ou coe cientes) individualmente. A qualidade da imagem nal e avaliada utilizando m etricas conhecidas, como a PSNR, RMSE (Root Mean Square Error), SSIM (Structural Similarity), entre outras. Tanto para as melhorias quanto para a combina c~ao de algoritmos, os resultados s~ao equivalentes ou superiores quando comparados aos das t ecnicas originais. Em caso de igualdade da qualidade, o algoritmo melhorado possui execu c~ao mais r apida que o original, justi cando assim o seu uso. O tempo de execu c~ao de cada algoritmo e avaliado, mostrando que as t ecnicas de combina c~ao aumentam o tempo de processamento, pois os algoritmos s~ao executados sequencialmente. Nesse caso, a utiliza c~ao de paralelismo pode acelerar a execu c~ao. Uma vez que a combina c~ao adiciona um processamento extra, estudos de viabilidade podem ser realizados para cada caso, a m de considerar a real necessidade do aumento da qualidade da imagem fundida, em detrimento dos recursos a serem utilizados.

Amyotrophic lateral sclerosis (ALS) is a devastating adult onset neurodegenerative disorder characterized by the selective degeneration of upper and lower motor neurons. Patients typically die from respiratory failures within 2-5 years after diagnosis. One of the milestones in ALS research is the discovery Fused in Sarcoma (FUS), an ALS causative gene. FUS is an RNA/DNA-binding protein and predominantly resides in the nucleus. Majority of the FUS mutations are located in the C-terminus and causing aberrant misdistribution to the cytoplasm. Currently, only a few binding partners of FUS are known, which makes it difficult to speculate on the function and interaction of the protein. In this study, we conducted a kinome-wide RNAi screen to identify kinases that affect the localization of FUS. A dual specificity protein kinase named CDC2-like kinase (CLK1) from the screen was found to be responsible for in post-translational modification of FUS and affects the localization of FUS in the nucleus. The identification of CLK1 as FUSmodifying kinase is consistent with roles ascribed to both in the binding and regulation of RNA.

Nanoparticles have the potential to revolutionize medicine, but many obstacles complicate the translation of nanoparticles from the bench to the clinic. A deeper understanding of nanoparticle synthesis parameters that influence nanoparticle size, drug loading, and surface chemistry is needed to accelerate the design of efficacious therapeutic nanoparticle systems. In this work, organic and inorganic nanoparticles were prepared with hydrodynamic diameters below 200 nm for applications in cancer treatment and immunology. Hydrophobic ion pairing was applied to enhance the loading capacity of drugs and peptides in polyester and polysaccharide nanoparticles systems. Polyester nanoparticles were successfully functionalized with streptavidin-Cy3, interferon gamma (IFN-γ), and CX3CL1. Poly(methacrylic acid), chitosan, and polyinosinic-polycytidylic acid (poly(I:C)) were successfully adsorbed to the surfaces of nanoparticles to enhance particle stability and targeting. Iron-based coupling media capable of eliminating ~ 90% of the water signal from an acoustic coupling bath during gradient echo magnetic resonance imaging (MRI) thermometry was successfully designed using magnetic iron oxide nanoparticles to improve the clinical efficacy of MRI-guided focused ultrasound surgery (MRI-FUS). While the critical nanoparticle design criteria may change depending on the biomedical application, fundamental concepts of nanoparticle design and synthesis can be applied across applications. The projects presented here help to bridge the knowledge gap regarding the use of flash nanoprecipitation (FNP) for nanoparticle synthesis. FNP is a scalable nanoparticle fabrication method that produces small, well-defined nanoparticle populations through rapid, turbulent mixing of multiple solvent streams. This work elucidates nanoparticle design concepts that can be applied across a wide variety of biomedical applications. / Doctor of Philosophy / Cancer remains a critical public health issue worldwide because many promising therapies never make it from the lab into the hospital. Many chemotherapeutic drugs are hindered by poor solubility and serious, undesirable side effects. In the past few decades, new production techniques have been developed to create carriers for these drugs to help overcome these obstacles. These carriers can be made from a variety of materials including metals and biodegradable polymers. In fact, it is even possible to create "smart" carriers that react to their environment to travel within the body or release the drugs they contain. Understanding how to design these carriers for different biomedical applications is critical. This work shows how carriers made from metal or polymer can be designed to exhibit desirable characteristics for use in biomedical applications ranging from vaccines to cancer treatment. Various ways to modify the surfaces of these carriers to tailor them for different applications are presented. This work provides valuable information that can help drive the next generation of biomedical innovation.

La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative dont les premiers symptômes apparaissent généralement vers 60 ans. Elle affecte sélectivement le système moteur et provoque une paralysie progressive amenant au décès du patient par défaillance respiratoire en quelques années. À ce jour, il n’existe aucun traitement curatif, d’où la nécessité de comprendre la physiopathologie de la SLA. Bien que de nombreuses altérations dans le muscle aient été mises en évidence, sa contribution dans la SLA reste à définir. Nous avons montré que FUS est enrichi dans les noyaux sous-synaptiques de façon dépendante de l’innervation. De plus, FUS se lie au promoteur des récepteurs de l’acétylcholine et induit leur transcription de façon dépendante d’ERM. Le mutant FUS, quant à lui, est enrichi dans les noyaux extra-synaptiques et entraîne une toxicité musculaire responsable de l’altération de la jonction neuromusculaire (JNM). Au-delà de la JNM, FUS active MEF2A, de façon dépendante de PRMT1 afin de réguler les fonctions mitochondriales et la différenciation musculaire. La toxicité musculaire de FUS joue donc un rôle clé dans la physiopathologie de la SLA. / Amyotrophic lateral sclerosis is a neurodegenerative whose first symptoms generally appear around age 60. It is characterized by progressive motor neuron degeneration, paralysis and leading to death due to respiratory failure in a few years. Currently, there is no cure so the understanding of ALS physiopathology is necessary. Although many alterations in the muscle have been highlighted, its contribution in ALS remains to be defined. We showed that FUS is enriched in subsynaptic nuclei and this enrichment depended on innervation. Besides, FUS binds directly acetylcholine receptors (AchR) promoter and is required for Ermdependent induction of AChR expression. Conversely, mutant FUS is enriched on extra-synaptic nuclei and induce muscle intrinsic toxicity responsible for neuromuscular junction (NMJ) alteration. Beyond NMJ, FUS is required for muscle mitochondrial function and muscle differentiation through PRMT1-dependent MEF2A activation. Thus, FUS muscular toxicity plays a key role in the ALS physiopathology.

La sclérose latérale amyotrophique (SLA) et la démence fronto-temporale (DFT) sont deux maladies qui constituent un continuum clinico-pathologique. La mutation de FUS, une protéine nucléaire à fonctions multiples, provoque des cas familaux de SLA, et ces mutations provoquent une redistribution sub-cellulaire de FUS, du noyau vers le cytoplasme. Certains cas de DFT présentent une telles distribution anormale en l’absence de mutations de FUS. Il n’est pas connu si la maladie est provoquée par une perte de la fonction nucléaire de FUS et/ou un gain de fonction cytoplasmique.Nous avons généré et caractérisé une lignée de souris exprimant une forme cytoplasmique de FUS (Fus-ΔNLS). La localisation exclusive de FUS dans le cytoplasme provoque la mort des motoneurones via un gain de fonction dans les motoneurones eux-mêmes. Une localisation cytoplasmique partielle de FUS est suffisante pour développer un phénotype de la SLA et de DFT. Les mécanismes élucidés permettront de comprendre les bases des SLA/DFT. / Amyotrophic lateral sclerosis (ALS) and Frontotemporal dementia (FTLD) are now considered as a unique clinicopathological spectrum referred to as ALS/FTLD. Cytoplasmic aggregation of the physiologically nuclear FUS protein is a hallmark feature of a subset of ALS/FTLD. It remains unknonwn whether the critical pathogenic event relies on a loss of FUS normal nuclear functions, a toxic gain of function of FUS in the cytoplasm, or a combination of both.To answer this question we have generated a conditional mouse model expressing truncated FUS without nuclear localization signal - FusΔNLS. Our data showed that complete cytoplasmic mislocalization of truncated FUS protein within spinal motor neurons is a major determinant of motor neuron degeneration via toxic gain of function. A partial mislocalization of truncated FUS protein was sufficient to trigger key features of ALS and of FTLD.These studies allowed the elucidation of mechanisms underlying FUS role in ALS/FTLD, and will hopefully lead to development of therapies for these devastating diseases.

RNA-bindende Proteine spielen eine zentrale Rolle in der posttranskriptionellen Kontrolle von mRNAs, die zwischen Transkription und Abbau von mRNAs stattfindet. RNA-bindende Proteine beeinflussen Spleißen, Export, Stabilität, Lokalisierung und Translation von mRNAs. FUS, EWSR1 und TAF15 gehören zu der Familie der FET Proteine. Diese wirken an verschiedenen zellulären Prozessen wie Transkription, Spleißen und der Prozessierung von miRNAs mit. Translokationen und Mutationen der FET Proteine führen zu verschiedenen Krankheiten. FUS spielt eine Rolle bei den neurodegenerativen Krankheiten frontotemporale Lobärdegeneration (FTLD) und amyotrophe Lateralsklerose (ALS). In dieser Arbeit wurde die mithilfe von photoaktivierbaren Ribonukleotiden UV-Licht induzierte Quervernetzung und Immunpräzipitation (PAR-CLIP) Methode genutzt, um die RNA-Bindestellen von FUS, EWSR1 und TAF15, einer ALS-verursachenden FUS Mutante und einem anderen, mit ALS in Verbindung stehenden Protein, TARDBP, zu bestimmen. Die RNA-Bindestellen der FET-Proteine lagen größtenteils in Introns. Passend dazu konnte durch knockdown der FET Proteine eine Rolle von FUS und EWSR1 im Spleißen von mRNAs validiert werden. Dem Ubiquitin-Proteasom-System zugehörige RNAs waren unter den sowohl von FUS als auch TARDBP gebundenen mRNAs überrepräsentiert. Dies bestätigt die Annahme, dass Störungen in der Proteindegradation die ALS-Pathogenese beeinflussen. Zusätzlich konnte gezeigt werden, dass FUS und TAF15 bevorzugt UAC-reiche, einzelsträngige RNA-Sequenzen binden. Sequenzierung von mRNAs nach Depletion von FUS, EWSR1 und TAF15 in HEK293-Zellen zeigte einen stabilisierenden Effekt der FET-Proteine auf gebundene mRNAs. Desweiteren scheinen die FET Proteine durch Interaktion mit Promotor-assoziierten, nicht-kodierenden RNAs die Transkription zu beeinflussen. / Post-transcriptional regulation of gene expression takes place at multiple levels between transcription and decay of the mRNA. RNA-binding proteins play a key role in orchestrating splicing, export, stability, localization and translation of mRNAs. FUS, EWSR1 and TAF15 constitute the FET protein family which participates in multiple levels of cellular function. FET proteins have been implicated to function in various cellular processes including transcription, pre-mRNA splicing and miRNA processing. Translocations and mutations in FET proteins lead to diverse pathologies. FUS is involved in neurodegenerative diseases like frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). In this study, Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) was used to determine RNA-targets and binding sites of FUS, EWSR1 and TAF15, an ALS-causing FUS mutant and another ALS-related protein, TARDBP. The identified binding sites of FET proteins were mainly intronic, supporting the involvement of FUS and EWSR1 in splicing, which was validated by FET protein knockdown. Comparison of FUS and TARDBP RNA targets revealed that ubiquitin-proteasome related gene categories were overrepresented, further illustrating that aberrations in protein degradation are implicated in the pathogenesis of ALS. In addition, it was shown that FUS and TAF15 proteins preferentially bind UAC rich, single-stranded RNA sequences. mRNA sequencing after FUS, EWSR1 and TAF15 depletion in HEK293 cells revealed a stabilizing effect on their targets. Interestingly, FET proteins also seem to influence transcription by interaction with promoter-associated noncoding RNAs. In summary, we identified the RNA-targets and binding sites of all human FET proteins in comparison with an ALS-causing FUS mutant and TARDBP. Functional studies revealed an involvement of FET proteins in mRNA stabilization, splicing and transcriptional regulation.