Дисертації з теми "Modulating aggregation"
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Shi, Mengying. "Functionalised Azamacrocycles for Modulating Amyloid Aggregation." Thesis, The University of Sydney, 2019. https://hdl.handle.net/2123/22075.
Повний текст джерелаFonseca, Ornelas Luis Eduardo Verfasser], Markus [Akademischer Betreuer] Zweckstetter, Tiago Fleming [Gutachter] Outeiro, Henning [Gutachter] Urlaub, Kai [Gutachter] Tittmann, Christian [Gutachter] [Griesinger, and Reinhard [Gutachter] Jahn. "Modulating the aggregation of alpha-synuclein and prion protein with small molecules. / Luis Eduardo Fonseca Ornelas ; Gutachter: Tiago Fleming Outeiro, Henning Urlaub, Kai Tittmann, Christian Griesinger, Reinhard Jahn ; Betreuer: Markus Zweckstetter." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2016. http://d-nb.info/1118846931/34.
Повний текст джерелаOliveira, Márcia Santos. "Modulation of α-synuclein aggregation and toxicity". Master's thesis, Faculdade de Ciências e Tecnologia, 2013. http://hdl.handle.net/10362/11195.
Повний текст джерелаIt is widely known that α-synuclein (aSyn) is an amyloidogenic protein prone to aggregation. This protein is found in specific inclusions named Lewy bodies in the surviving neurons of Parkinsons’s disease patients and other synucleinopathy brains. This aggregation process is greatly affected by different post-translational modifications, such as phosphorylation, acetylation, and glycation. Lately it was shown that aSyn oligomeric species are more toxic than the inclusion bodies. Heat shock proteins (HSPs) are molecular chaperones able to modulate the folding and refolding of proteins. Its overexpression in Parkinson’s disease models reduces and prevents aSyn aggregation. As the reduction of aSyn aggregation can lead to an eventual accumulation of oligomeric species which may cause cell damage, the main goal of this work is to better understand the role of HSPs in aSyn oligomer formation, clarifying which are the aSyn resulting species formed in the presence of HSPs. Moreover, as glycation is suggested to accelerate abnormal protein deposition, we aimed to investigate how HSPs interfere with the oligomerization process of glycated aSyn. In this study Hsp70 seemed to induce recombinant aSyn oligomerization, generating higher molecular weight species with no associated toxicity. On the other hand, Hsp27 reduced aSyn oligomerization in vitro possibly by inducing the formation of non-reactive small oligomers. MGO glycation increased protein aggregation and cell death. Interestingly, Hsp27 overexpression reversed glycated aSyn aggregation and its associated toxicity. These results demonstrate the importance of HSPs modulation as a possible target of Parkinson’s disease therapeutics.
Fontaine, Sarah. "Modulation of PrP aggregation and cellular prion transfer." Thesis, University of Bath, 2010. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.518303.
Повний текст джерелаTam, Stephen Jed. "Eukaryotic chaperonin-mediated modulation of polyglutamine aggregation and neurotoxicity /." May be available electronically:, 2007. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.
Повний текст джерелаGuivernau, Almazán Biuse 1988. "Modulation of Amyloid-β peptide aggregation and neurotoxicity in Alzheimer's disease". Doctoral thesis, Universitat Pompeu Fabra, 2016. http://hdl.handle.net/10803/585932.
Повний текст джерелаL’agregació del pèptid b-amiloide (Aβ) en forma d’oligòmers i fibres és un esdeveniment central en la patogènesi de la malaltia d’Alzheimer. Aquesta tesi pretén aprofundir en els coneixements actuals sobre la toxicitat causada per l’Aβ així com en l’impacte que tenen els canvis en l’agregació d’aquest, tots dos rellevants per la malaltia d’Alzheimer. Els nostres resultats indiquen que la nitrotirosinació de l’Aβ inhibeix la formació de fibres, afavorint l’estabilització d’oligòmers. Demostrem que els oligòmers d’Aβ nitrat s’uneixen a les dendrites, alterant la funció fisiològica dels receptors d’N-metil- D-aspartat (NMDAR) i provocant disfuncions neuronals i la mort cel·lular. A més, proposem un model d’assemblatge per a les fibres d’Aβ, segons el qual la nitrotirosinació interromp l’elongació de la fibra a causa de la desestabilització dels contactes entre protofibres. Addicionalment, utilitzant un cribratge genòmic en Saccharomyces cerevisiae, hem identificat nous moduladors de la toxicitat causada per Aβ, que podrien ser clau per al desenvolupament de noves estratègies terapèutiques de la malaltia Alzheimer.
Karras, Stephanie. "Modulation der Tau-Aggregation durch Modifikation der Cystein-Reste im Tau-Protein." Doctoral thesis, Universitätsbibliothek Leipzig, 2017. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-219737.
Повний текст джерелаLanger, Carola Alexandra. "Molekulare Analyse der Huntingtin-Aggregation und deren Modulation durch das eukaryontische Chaperonin TRiC." Diss., lmu, 2007. http://nbn-resolving.de/urn:nbn:de:bvb:19-69757.
Повний текст джерелаAbelein, Axel. "Modulation of amyloid β peptide self-assembly : Aggregation mechanisms associated with Alzheimer's disease". Licentiate thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-89078.
Повний текст джерелаRose, J. "Molecular chaperone modulation of Parkin aggregation and function in a cell model of Parkinson's disease." Thesis, University College London (University of London), 2010. http://discovery.ucl.ac.uk/19810/.
Повний текст джерелаEvgrafova, Zhanna [Verfasser], Wolfgang H. [Gutachter] Binder, and Felix [Gutachter] Schacher. "Modulation of amyloid peptides aggregation by hydrophilic polymers / Zhanna Evgrafova ; Gutachter: Wolfgang H. Binder, Felix Schacher." Halle (Saale) : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2020. http://d-nb.info/121950825X/34.
Повний текст джерелаRichet, E. "The modulation of tau aggregation in a cell model of Alzheimer's disease by the proteasome adaptor protein NUB1." Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1355106/.
Повний текст джерелаAyala, Mariscal Sara Maria. "Modulation of Alzheimer's disease amyloid beta peptide aggregation by molecular chaperones, polyphosphates and metal ions, and their interplay." Thesis, Toulouse 3, 2018. http://www.theses.fr/2018TOU30108.
Повний текст джерелаAlzheimer's disease is the most frequent type of dementia. With an exponentially growing number of cases, understanding the underlying molecular events leading to this devastating condition is of crucial importance. Much evidence points to a disequilibrium in the production and degradation of amyloid beta (Aß), a normally physiological 42 amino acid peptide, as an early key event in Alzheimer's etiology. Whether Aß is overproduced or poorly degraded, the overall result is an abnormally large pool of peptide that gradually aggregates forming extracellular deposits of fibrils, called amyloid plaques, in specific brain regions. Hence, modulation of Aß aggregation process is one of the suggested approaches to control the evolution of Alzheimer's disease. Universally conserved molecular chaperones have been intensively studied for their capacity to prevent aggregation of disease-related proteins, and many of them have proven to efficiently modulate Alzheimer's Aß aggregation. In a scenario where chaperones are overexpressed or directly administered into the affected tissue, the universal conservation and the relatively poor client-specificity of generic chaperones can become a downside because of the risk of interaction with proteins other than the targeted one is not dismissible, and thus the consequences unpredictable. In the first part of this work, we looked upon a bacterial chaperone call SecB with an unusually robust holdase activity (i.e. it prevents early protein folding) as a promising modulator of Alzheimer's Aß peptide aggregation. [...]
Abelein, Axel. "Modulation of Alzheimer's amyloid β peptide self-assembly : Insights into molecular mechanisms of peptide aggregation associated with Alzheimer's disease". Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-114172.
Повний текст джерелаAt the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.
Lázaro, Diana [Verfasser], Tiago Fleming [Akademischer Betreuer] Outeiro, Markus [Gutachter] Zweckstetter, and Silvio [Gutachter] Rizzoli. "Genetic and Pharmacological Modulation of Alpha-Synuclein Aggregation / Diana Lázaro ; Gutachter: Markus Zweckstetter, Silvio Rizzoli ; Betreuer: Tiago Fleming Outeiro." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2017. http://d-nb.info/1137054301/34.
Повний текст джерелаWattin, Marion. "Modulation des mécanismes de Contrôle Qualité des Protéines dans la dystrophie musculaire de Duchenne." Thesis, Lyon, 2017. http://www.theses.fr/2017LYSE1323/document.
Повний текст джерелаVarious studies have highlighted the importance of Protein Quality Control (PQC), including protein refolding (molecular chaperones) and degradation (autophagy, proteasome) mechanisms in inherited muscle disorders such as Ullrich Congenital Muscular Dystrophy (UCMD), Duchenne Muscular Dystrophy (DMD) or Emery-Dreifuss Muscular Dystrophy (EDMD); however, to date, no extensive study has been conducted on these mechanisms in a same model, in muscle cells before muscle differentiation. Thus, we were interested in PQC mechanisms functionality and their interconnection in human immortalized myoblasts from healthy donors or patients suffering from DMD. We observed an increase of protein aggregation in DMD cells. This phenomenon is accompanied by a deregulation of sequestration mechanisms by molecular chaperones, reflected by the modulation of HSPB5 and HSPB8 expression. Degradation mechanisms are also deregulated; indeed, we observed on one hand a decrease of proteasome enzymatic activity and multiubiquitinated proteins UPS-adressing molecules and on the other hand, an increase of NF?B transcription factor’s activity, involved in autophagy, and of BAG3/HSPB8 complexes, leading to an increase of the autophagic flux. These PQC defects reflect the existence of a protein aggregation stress in myoblasts coming from DMD patients. In this context, pharmacological modulation of PQC in these cells could represent a new therapeutic strategy for Duchenne Muscular Dystrophy
Karras, Stephanie [Verfasser], Max [Akademischer Betreuer] Holzer, Thomas [Gutachter] Arendt, and Daniel [Gutachter] Huster. "Modulation der Tau-Aggregation durch Modifikation der Cystein-Reste im Tau-Protein / Stephanie Karras ; Gutachter: Thomas Arendt, Daniel Huster ; Betreuer: Max Holzer." Leipzig : Universitätsbibliothek Leipzig, 2017. http://d-nb.info/124031700X/34.
Повний текст джерелаCarboni, Eleonora Verfasser], Paul [Akademischer Betreuer] Lingor, Tim [Gutachter] [Salditt, and Tiago Fleming [Gutachter] Outeiro. "Alpha-synuclein aggregation: visualization by X-ray techniques and its modulation by iron / Eleonora Carboni ; Gutachter: Tim Salditt, Tiago Fleming Outeiro ; Betreuer: Paul Lingor." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2017. http://d-nb.info/1139491512/34.
Повний текст джерелаRichter, Luise [Verfasser]. "Einfluss niedermolekularer Substanzen auf die Homointeraktion des Amyloid-Vorläuferproteins : das GxxxG-Motiv als Zielstruktur für die Modulation der Bildung und Aggregation von Aβ / Luise Richter". Berlin : Freie Universität Berlin, 2011. http://d-nb.info/1026358655/34.
Повний текст джерелаFrey, Margo Tilley. "Development of a Substrate with Photo-Modulatable Rigidity for Probing Spatial and Temporal Responses of Cells to Mechanical Signals: A Dissertation." Digital WPI, 2008. https://digitalcommons.wpi.edu/etd-dissertations/337.
Повний текст джерелаCarvoeiro, Daniela Claudino. "Modulating immune responses by targeting protein aggregation." Master's thesis, 2019. http://hdl.handle.net/10773/28429.
Повний текст джерелаA agregação de proteínas é um processo que pode ser induzido por uma grande variedade de stresses celulares, incluindo privação de aminoácidos, infeção viral, stress do retículo endoplasmático, lipopolissacarídeos, e stress oxidativo. Alterações na proteostase parecem ter um impacto na resposta inflamatória, mas os mecanismos subjacentes a este impacto são ainda pouco conhecidos. Esta tese tem por objetivo analisar de que forma a agregação de proteínas influencia a resposta das células dendríticas humanas, em particular das células dendríticas plasmocitóides (pDCs). Foram utilizados inibidores de autofagia e do proteossoma para manipular o número de agregados na linha celular de pDCs, CAL-1. A inibição do proteossoma em pDCs induziu a formação de agregados constituídos por p62 de grandes dimensões, e levou à secreção de IL-1β e morte celular, de forma irreversível e específica destas células. Os mesmos efeitos não foram observados após inibição da autofagia nem se verificaram numa outra linha celular, monocítica. Para estudar o mecanismo por detrás do aumento da resposta inflamatória após a inibição do proteossoma, foram criadas células knockout para o p62 usando o sistema CRISPR/Cas9. Os nossos resultados sugerem que nem o p62 nem o inflamassoma de NLRP3 são necessários para a indução da morte celular por inibição do proteossoma em pDCs. De forma geral, concluímos que a inibição do proteossoma induz uma resposta inflamatória específica para pDCs. Propomos que esse efeito deve ser tido em consideração quando se utilizem inibidores de proteossoma como potenciais fármacos para o tratamento de distúrbios mediados por pDCs e, portanto, mais estudos devem ser feitos para esclarecer o efeito da inibição do proteossoma em pDCs.
Mestrado em Biomedicina Molecular
Gibson, Todd Jonathan. "Modulating protein aggregation with targeted, surface tension-modifying compounds." 2005. http://catalog.hathitrust.org/api/volumes/oclc/65648185.html.
Повний текст джерелаFonseca, Ornelas Luis Eduardo. "Modulating the aggregation of alpha-synuclein and prion protein with small molecules." Thesis, 2016. http://hdl.handle.net/11858/00-1735-0000-002B-7C8F-3.
Повний текст джерелаLázaro, Diana. "Genetic and Pharmacological Modulation of Alpha-Synuclein Aggregation." Thesis, 2017. http://hdl.handle.net/11858/00-1735-0000-0023-3EAB-F.
Повний текст джерелаKim, Jin Ryoun. "Characterization of beta-amyloid aggregation and its modulation." 2004. http://www.library.wisc.edu/databases/connect/dissertations.html.
Повний текст джерелаPallitto, Monica M. "Kinetic characterization and modulation of [beta]-amyloid aggregation and toxicity." 2001. http://www.library.wisc.edu/databases/connect/dissertations.html.
Повний текст джерелаKarras, Stephanie. "Modulation der Tau-Aggregation durch Modifikation der Cystein-Reste im Tau-Protein." Doctoral thesis, 2016. https://ul.qucosa.de/id/qucosa%3A14350.
Повний текст джерелаCarboni, Eleonora. "Alpha-synuclein aggregation: visualization by X-ray techniques and its modulation by iron." Doctoral thesis, 2016. http://hdl.handle.net/11858/00-1735-0000-0023-3EF2-D.
Повний текст джерелаSroka, Kamila. "Huntingtin toxicity is ameliorated by BAG1 through modulation of its aggregation, degradation and subcellular distribution." Doctoral thesis, 2008. http://hdl.handle.net/11858/00-1735-0000-0006-B4DB-D.
Повний текст джерелаLanger, Carola Alexandra [Verfasser]. "Molekulare Analyse der Huntingtin-Aggregation und deren Modulation durch das eukaryontische Chaperonin TRiC / Carola Alexandra Langer." 2007. http://d-nb.info/984675264/34.
Повний текст джерелаSroka, Kamila [Verfasser]. "Huntingtin toxicity is ameliorated by BAG1 through modulation of its aggregation, degradation and subcellular distribution / submitted by Kamila Sroka." 2008. http://d-nb.info/1005151059/34.
Повний текст джерелаBarone, Paul. "Modulation of single-walled carbon nanotube fluorescence via enzyme reaction, controlled aggregation and hydrogel swelling towards a continuous glucose sensor /." 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3362724.
Повний текст джерелаSource: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3651. Adviser: Michael S. Strano. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.
Borges, Alice da Cruz Madeira. "Modulation of the aggregation pathway of a model variant form of human phenylalanine hydroxylase: towards the development of a new class of pharmacological chaperones for the treatment of phenylketonuria." Master's thesis, 2020. http://hdl.handle.net/10451/47666.
Повний текст джерелаPhenylketonuria (PKU; OMIM #261600) is an autosomal recessive disorder, caused mostly by missense mutation in the PAH gene, which encodes human phenylalanine hydroxylase (hPAH).Human PAH is a nonheme, homotetrameric, iron-containing enzyme, which catalyzes the conversion of LPhenylalanine (L-Phe) into L-Tyrosine, by para-hydroxylation of the aromatic side chain in the presence of the co-factor (6R)-L-erytro-5,6,7,8-tetrahydrobiopterin (BH4), non-hemic iron and molecular oxygen (O2). Presently, classical PKU patients must follow restricted diets in order to control L-Phe levels in the blood. Compliance is difficult and cognitive and social development impaired. PKU can be classified as a misfolding disease, since the majority of the PKU causing mutations result in misfolded hPAH variants which, in the cellular context, will form soluble aggregates targeted for degradation, leading to a loss-of-function phenotype. Therefore, pharmacological chaperone therapy imposes a viable alternative to PKU treatment. By stabilizing the variant protein, and avoiding its degradation, rescue of enzyme residual activity may be attained, improving patient’s quality of life by increasing dietary LPhe tolerance and alleviating some of the dietary restrictions imposed. Among the high number of PAH variants already identify, the p. G46S is considered an excellent model to the in vitro study of the hPAH aggregation process, as it presents a high tendency to selfassociate and form non-amyloid fibrils, when overexpressed in E.coli, and to be rapidly degraded in eukaryotic systems, representing the most severe form of Classical PKU phenotype. In the presence of maltose binding protein (MBP) added N-terminally to p.G46S, it is possible to obtain soluble metastable tetramers with a normal enzyme activity rate. Upon MBP tag cleavage with Factor Xa protease the hPAH p.G46S aggregates in a process efficiently followed in vitro. Our aim was to identify, among an in-house compound library, molecules able to modulate this behavior in vitro, and to characterize the underlying mechanism by studying the full-length (FL) and a truncated form of p.G46S representing the unstable N-terminal regulatory domain (N1-120) harboring the allosteric site. For this, the p.G46S forms were expressed in a prokaryotic system in fusion with the MBP tag and purified. The rate of self-association of the isolated FL tetramers and N1-120 dimers (variant and wild-type) were studied by real-time turbidimetry, upon cleavage of the MBP tag, in the presence and absence of studied molecules. Following in vitro studies, molecules with a positive impact on p.G46S aggregation were submitted to in cellullo studies to evaluate their influence on enzyme activity and stability. From the aggregation assays, five molecules had positive effects on the self-association pattern of the FL p.G46S, either by inhibiting aggregation or by delaying it. From those molecules, two (C4 and C14) also inhibited the wild-type and p.G46S RD1-120 aggregation, and one (C17) only impacted the aggregation of the wild-type RD1-120, suggesting that these molecules bind to the unstable N-terminal domain and particularly C4 and C14 will be interacting with helix a1 of the ACT domain, the main driver of p.G46S-RD1-120 instability while C17 is acting on a different region. In cellullo assays showed that C17 and C14 could improve enzyme activity when compared to p.G46S in the absence of compounds (control). Enzyme content in cell lysates, however, was at the same level as the p.G46S control, thus suggesting that these two compounds are promoting a stabilization of the enzymatic reaction, allowing it to present higher activity levels than it normally would have (activity chaperone), instead of acting as stabilizers of the protein helping it to avoid the cell protein control quality mechanisms (pharmacological chaperone). Although further studies were not carried out due to the restrictions and contingency plans applied by the Research institutions to respond to SARS-CoV-2_COVID-19 pandemics, here we present possible hit structures for the development of a new class of pharmacological/activity chaperones, specifically designed for the treatment of classical PKU.
A fenilcetonuria (PKU; OMIM #261600) e uma doenca autossomica recessiva, causada na sua maioria por mutacoes missense no gene PAH, que codifica a fenilalanina hidroxilase humana (hPAH). A hPAH e uma enzima homotetramerica, que catalisa a conversao da L-fenilalanina (L-Phe) em Ltirosina (L-Tyr), por para-hidroxilacao da cadeia lateral aromatica. Para que esta reacao ocorra, e necessaria a presenca do seu co-factor natural (6R)-L-erytro-5,6,7,8-tetrahidrobiopterina (BH4), de ferro nao-hemico e oxigenio molecular (O2). As mutacoes missense no gene PAH originam na sua maioria a expressao de variantes de hPAH conformacionalmente alteradas (misfolded), no contexto celular, tem tendencia a formar agregados soluveis que rapidamente se tornam alvos das vias celulares de degradação proteica, levando a um fenotipo patologico de perda de funcao. A PKU e entao considerada uma doença conformacional, na qual o equilibrio normal dos estados folded« unfolded e alterado, deslocando-se preferencialmente para o estado unfolded. Consequentemente, as proteinas resultantes misfolded são reconhecidas, levando a que a maquinaria celular de controlo de qualidade e degradacao proteica atue no sentido de acelerar a degradacao de proteinas misfolded devido a sua instabilidade e tendencia para agregacao. Nos doentes PKU, quando nao tratados o mais rapidamente possivel apos diagnostico, a função alterada da hPAH variante resulta na acumulacao de L-Phe para niveis neurotoxicos, causando alteracoes neurologicas, convulsoes, atraso no desenvolvimento, problemas comportamentais, etc. O tratamento disponivel assenta basicamente em dietas restritivas e suplementos alimentares desde o diagnostico (preferencialmente dentro dos primeiros dias de vida) a fim de controlar os niveis de L-Phe em circulacao no sangue. Atualmente, as guidelines internacionais indicam que a restricao dietetica deve ser mantida durante a vida e que os niveis de L-Phe circulante devem ser mantidos abaixo de 600 μM (considerando niveis normais de circulacao no sangue ≈120 μM). A aquiescencia para com esta terapeutica e dificil, principalmente durante a adolescencia e nos adultos, e o desenvolvimento cognitivo e social sao prejudicados. Ao longo dos anos tem-se vindo a estudar diferentes terapeuticas alternativas, de forma a que seja possivel controlar os niveis de L-Phe circulantes e seja possivel um alivio nas restricoes dieteticas. A terapeutica baseada na administracao de chaperones propoem-se como uma alternativa viavel, uma vez que a PKU e atualmente reconhecida como uma doenca conformacional. Ao estabilizar a enzima misfolded e a sua degradacao iminente, permite-se que a enzima esteja presente e mantenha a sua atividade residual. Um leve aumento da capacidade de processamento da L-Phe poderá entao levar a um aumento da qualidade de vida do doente, aliviando algumas das restricoes dietéticas impostas. Ate a data, foram descritas tres abordagens diferentes para a terapia por chaperones: modulacao de chaperones moleculares a fim de resgatar proteinas com folding incorreto (reguladores de proteostase); chaperones quimicos e chaperones farmacologicos com o objetivo de estabilizar a proteina alterada, evitando-se a degradacao pelos sistemas de controlo de qualidade proteica. Entre as varias variantes causadoras de PKU, a p.G46S e considerada como um excelente modelo para o estudo in vitro do processo de agregacao da hPAH, uma vez que apresenta uma elevada tendencia para se autoassociar e formar fibrilhas nao amiloides, quando sobre-expressa em E.coli, e para ser rapidamente degradada em sistemas eucariotas, representando um fenotipo classico de PKU. Assim, e na presença da proteina de ligacao a maltose (maltose binding protein - MBP) adicionada a N-terminal da cadeia polipeptidica da p.G46S, e possivel obter tetrameros soluveis, metastaveis e com uma taxa de atividade enzimatica normal. Apos a clivagem da MBP com a protease Factor Xa (FXa), e entao possivel seguir in vitro e de forma eficiente o processo de agregacao da proteina hPAH p.G46S. Tendo em consideracao as caracteristicas descritas da p.G46S, o objetivo do presente estudo e o de identificar, de entre uma biblioteca de compostos desenhados e sintetizados pelo Grupo de Quimica Bioorganica do Instituto de Investigacao do Medicamento (iMed.ULisboa), moleculas capazes de modular o comportamento auto-associativo in vitro, e caracterizar o mecanismo subjacente atraves do estudo da proteina inteira (Full lenght; FL) e de uma forma truncada da p.G46S representando o domínio regulador instavel N-terminal (RD1-120) da hPAH e que contem o local de ligacao alosterico ao substrato L-Phe . Para tal, as formas de p.G46S (FL e RD1-120) foram expressas num sistema procariota como proteinas de fusao com a MBP e posteriormente purificadas. A taxa de auto-associacao dos tetrâmeros FL isolados e dos dimeros RD1-120 foram estudados por turbidimetria em tempo real, apos a clivagem da MBP, na presenca (100 μM) e ausencia das moleculas estudadas. Apos estudos in vitro, as moléculas com efeito inibidor na agregacao da p.G46S foram sujeitas a estudos in celullo com a finalidade de avaliar a sua influencia na atividade e estabilidade enzimatica na celula. Desta forma, células embrionicas do rim humano (human embryonic kidney cells), HEK293T, foram transfetadas com vetores de expressao eucariotas capazes de expressar a proteina p.G46S. Como controlo do ensaio celulas HEK293T foram tambem transfetadas com vetor de expressao contendo o gene normal da hPAH (wild type; WT). Para monitorizacao da estabilidade e variacao na tendencia de agregacao da proteína nas celulas eucariotas, foram realizados ensaios de imunocitoquimica. Adicionalmente, avaliou-se de que forma a funcao biologica celular da p.G46S seria afetada pela presenca dos compostos atraves da medicao da atividade enzimatica em lisados celulares. Dos ensaios de agregacao observou-se que cinco moleculas (C4, C10, C12, C14 e C17) tiveram efeitos positivos sobre o padrao de auto-associacao da forma FL da p.G46S, quer inibindo a agregacao, quer atrasando-a. Destas moleculas, duas (C4 e C14) atrasaram tambem a agregacao das formas truncadas representantes do RD (p.G46S e WT), enquanto uma (C17) apenas diminuia a agregacao da forma WT RD1-120, sugerindo que a interacao destas moleculas com a enzima ocorrera no dominio regulador N-terminal e que em particular as moléculas C4 e C14 deverao interagir com a helice a1 do dominio ACT promotora da instabilidade proteica. Estas cinco moleculas foram submetidas a estudos de imunocitoquimica, que, infelizmente, foram pouco conclusivos, devido a sobre-expressao tanto da proteina p.G46S como da WT, que impediu a discriminacao de agregados proteicos na celula. Desta forma, nao foi possivel obter resultados conclusivos acerca da capacidade de os compostos modularem a agregacao da proteina in cellullo. Para a avaliacao da modulacao da atividade biologica da proteina na celula pelos compostos em estudo (50 μM), realizaram-se ensaios na presenca de 0,5% DMSO e de 50 μM de C6 como controlos negativos. Como esperado, em ambos os ensaios a atividade enzimatica foi reduzida (» 15%), quando comparada com a hPAH WT. Estes resultados encontram-se em concordancia com os dados obtidos por analise de Western blot, sendo que os niveis intracelulares da proteina alvo se encontravam tambem reduzidos, quando comparados com a WT. Dos compostos que melhoravam a agregacao de p.G46S in vitro, tanto o C14 como o C17 mostraram ser capazes de melhorar a atividade da proteina variante, quando comparado com o ensaio controlo (p.G46S/0,5% DMSO). No entanto, para corroborar os pressupostos assumidos nas experiencias in vitro, ensaios adicionais deverao ser realizados, uma vez que se verificaram diferencas significativas entre os ensaios de atividade independentes, efetuados nas células HEK293T. Ao contrario do que seria de esperar, observou-se nos immunoblots, tanto para C14 como para C17, uma quantidade de proteina p.G46S tambem reduzida em comparacao com a hPAH WT, sugerindo que estes compostos poderao estar a estabilizar o processo enzimatico de modo a que esta tenha maior capacidade para realizar a sua funcao biologica (chaperone de atividade), ao inves de estabilizar a conformacao proteica, evitando a sua degradacao pela maquinaria de controlo de qualidade e degradacao proteica celular (chaperone farmacologico). Embora nao tenha sido possivel efetuar ensaios adicionais devido as restricoes impostas pelo plano de contingencia de combate a pandemia SARS-CoV-2_COVID-19, o presente estudo serviu como base para a identificacao de estruturas hit para o desenvolvimento de uma nova classe de chaperones farmacologicos ou chaperones de atividade direcionados para tratamento da PKU classica.