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Resumo Fundamento A doença arterial coronariana (DAC) devido à isquemia miocárdica causa perda permanente de tecido cardíaco. Objetivos Nosso objetivo foi demonstrar o possível dano ao miocárdio em nível molecular através dos mecanismos de autofagia e apoptose em pacientes submetidos à cirurgia de revascularização miocárdica. Métodos Um grupo recebeu uma solução de cardioplegia Custodiol e o outro grupo uma solução de cardioplegia sanguínea. Duas amostras miocárdicas foram coletadas de cada paciente durante a operação, imediatamente antes da parada cardíaca e após a liberação do pinçamento aórtico. Foram avaliadas as expressões de marcadores de autofagia e apoptose. O nível de significância estatística adotado foi de 5%. Resultados A expressão do gene BECLIN foi significativa nos tecidos miocárdicos do grupo CS (p=0,0078). Os níveis de expressão dos genes CASPASE 3, 8 e 9 foram significativamente menores no grupo CC. Os níveis pós-operatórios de TnT foram significativamente diferentes entre os grupos (p=0,0072). As expressões dos genes CASPASE 8 e CASPASE 9 foram semelhantes antes e depois do pinçamento aórtico (p=0,8552, p=0,8891). No grupo CC, os níveis de expressão gênica de CASPASE 3, CASPASE 8 e CASPASE 9 não foram significativamente diferentes em amostras de tecido coletadas após pinçamento aórtico (p=0,7354, p=0,0758, p=0,4128, respectivamente). Conclusões Com nossos achados, acreditamos que as soluções CC e CS não apresentam diferença significativa em termos de proteção miocárdica durante as operações de by-pass. Abstract Background Coronary artery disease (CAD) due to myocardial ischemia causes permanent loss of heart tissue. Objectives We aimed to demonstrate the possible damage to the myocardium at the molecular level through the mechanisms of autophagy and apoptosis in coronary bypass surgery patients. Methods One group was administered a Custodiol cardioplegia solution, and the other group was administered a Blood cardioplegia solution. Two myocardial samples were collected from each patient during the operation, just before cardiac arrest and after the aortic cross-clamp was released. The expressions of autophagy and apoptosis markers were evaluated. The level of statistical significance adopted was 5%. Results The expression of the BECLIN gene was significant in the myocardial tissues in the BC group (p=0.0078). CASPASE 3, 8, and 9 gene expression levels were significantly lower in the CC group. Postoperative TnT levels were significantly different between the groups (p=0.0072). CASPASE 8 and CASPASE 9 gene expressions were similar before and after aortic cross-clamping (p=0.8552, p=0.8891). In the CC group, CASPASE 3, CASPASE 8, and CASPASE 9 gene expression levels were not found to be significantly different in tissue samples taken after aortic cross-clamping (p=0.7354, p=0.0758, p=0.4128, respectively). Conclusions With our findings, we believe that CC and BC solutions do not have a significant difference in terms of myocardial protection during bypass operations.

Il misfolding proteico e l’aggregazione di proteine sono problemi essenziali alla base di diversi disordini neurodegenerativi. Tali malattie sono caratterizzate dalla presenza di inclusioni di proteine misfoldate che possono interferire con la corretta proteostasi, portando alla perdita dell’attività neuronale cerebrale. Ciononostante, la formazione di aggregati proteici è un meccanismo messo in atto anche da cellule eucariote sane per proteggere il proprio proteoma. Tra tali siti di deposito, i Dendritic cells Aggresome-Like Induced Structures (DALISs) sono stati recentemente identificati in cellule dendritiche. Essi rappresentano dei depositi transienti per proteine difettive ubiquitinate ed antigeni, che si formano in risposta a diversi stress. La loro funzione non è ancora completamente compresa, specialmente dopo essere stati identificati anche in cellule non appartenenti al sistema immunitario. Ad oggi, nessuna indagine è stata condotta in altre cellule eucariote ma, fungendo da siti di deposito per proteine ubiquitinate, ci si aspetta di incontrarli anche in altri modelli cellulari. Il lievito Saccharomyces cerevisiae viene ampiamente utilizzato per la ricerca sugli aggregati cellulari, poiché presenta un ampio range di inclusioni dinamiche che sono stati dimostrate essere conservati tra diverse specie, nonostante il maggior grado di complessità nella composizione proteica e nelle funzionalità negli eucarioti multicellulari. Abbiamo identificato quattro diverse condizioni di stress in cui il lievito S. cerevisiae manifesta la formazione di corpi citoplasmatici: shock termico a 42°C, stress acuto da etanolo, deprivazione di azoto e carenza di glucosio. Abbiamo quindi sottoposto S. cerevisiae a questi stimoli di stress e investigato la formazione di inclusioni insolubili, concentrandoci sugli aggregati ubiquitinati. A questo fine, abbiamo deciso di includere anche il trattamento con puromicina come condizione di stress a cui esporre le cellule di lievito, poiché la puromicina è stata dimostrata indurre la formazione di DALIS in cellule dendritiche. Abbiamo dimostrato la formazione di aggregati ricchi di ubiquitina in cellule di lievito a seguito di stress acuto da etanolo, di deprivazione di azoto prolungata e di trattamento con puromicina. Abbiamo poi indagato la localizzazione cellulare degli aggregati ubiquitinati in cellule di S. cerevisiae grazie a microscopia a fluorescenza. Abbiamo osservato l’accumulo di aggregati ricchi di ubiquitina dalla forma rotonda, che sono disseminati nel cytosol e non colocalizzati con altre strutture cellulari. I corpi osservati rispecchiano le caratteristiche morfologiche riportate per i DALISs nelle cellule dendritiche. Abbiamo poi deciso di investigare come gli aggregati ricchi di ubiquitina indotti a seguito di stress acuto da etanolo vengano eliminati all’interno della cellula di lievito. Abbiamo dimostrato che l’accumulo di aggregati ricchi di ubiquitina è persistente nelle cellule mutanti atg1Δ rispetto alle cellule wild type, in cui il livello di proteine ubiquitinate decresce qualche ora dopo l’esposizione allo stress. Ciò suggerisce che il processo di autofagia è implicato nella rimozione degli aggregati ubiquitinati, poiché Atg1 è una chinasi che gioca un ruolo fondamentale nella regolazione di tale processo. D’altro canto, abbiamo osservato che le cellule mutanti cue5Δ mostravano un minor accumulo di ubiquitina durante l’intera durata del trattamento con etanolo, rispetto alle cellule wild type. La proteina di lievito Cue5 è una proteina che lega ubiquitina e che collabora con gli enzimi di coniugazione dell’ubiquitina Ucb4/Ubc5 e con l’ubiquitina ligasi Rsp5 per congiungere l’ubiquitina al suo substrato. Dato che abbiamo osservato una minor presenza di ubiquitina nelle cellule mutanti cue5Δ, suggeriamo che Cue5 possa avere un ruolo nell’ubiquitinazione stessa agli aggregati osservati in condizioni di stress acuto da etanolo. Protein misfolding and aggregation are major issues in neurodegenerative disorders. Such diseases are characterized by the presence of large inclusions of misfolded proteins which can interfere with the proper proteostasis, possibly leading to loss of neuronal activity. Nevertheless, the formation of protein aggregates is a conserved mechanism engaged also by healthy eukaryotic cells in response to external stimuli that alter their physiological environment. In this context, protein aggregates function as well-organized deposition sites which help the cell to protect its proteome in the waiting for the restoration of the physiological condition. Among the organized cytoplasmic deposition sites for proteins, Dendritic cell Aggresome-Like Induced Structures (DALISs) has recently been identified in dendritic cells. They represent transient deposits for ubiquitinated defective proteins and antigens, which are formed in response to several stresses. Their function is still not completely understood, especially since they were later characterized in non-immune cells as well. No investigation has ever been conducted in other eukaryotic cells, but since they act as deposition sites for ubiquitinated misfolded proteins, we can expect to identify them in other cellular models as well. The yeast Saccharomyces cerevisiae is widely used for the research on cellular aggregates, as it displays a range of different dynamic deposition sites that are demonstrated to be conserved between different species, despite the increased complexity in protein composition and function in multicellular eukaryotes. We identified four main stress conditions under which the yeast Saccharomyces cerevisiae manifests the formation of large cytoplasmic bodies, namely heat shock temperatures at 42°C, severe ethanol stress, nitrogen depletion and glucose starvation. Therefore, we subjected yeast cells to these stress conditions and investigated the formation of insoluble inclusions, with a particular focus on ubiquitin-rich protein aggregates. To this purpose, we decided to include puromycin treatment as a stress condition to which a puromycin-sensitive yeast strain should be exposed, as puromycin was demonstrated to induce the formation of DALISs in dendritic cells. We demonstrated the formation of ubiquitin-rich protein aggregates in S. cerevisiae cells following the exposure to severe ethanol stress, under prolonged nitrogen starvation, and following puromycin treatment. We further investigated the cellular localization of ubiquitinated aggregates of wild type S. cerevisiae cells by fluorescence microscopy. We observed that the accumulation of round-shaped ubiquitin-rich aggregates, which are disseminated in the cytosol and not colocalized with any cellular structure. Such structures reflect the characteristic morphology of DALIS reported in dendritic cells. We then decided to investigate how these ethanol-induced ubiquitin-rich protein aggregates are cleared inside the yeast cell. We demonstrated that the accumulation of ubiquitin-rich aggregates is persistent in atg1Δ mutant cells, whereas in wild type cells the level of ubiquitinated proteins decrease a few hours after the exposure to the stress. This suggests that autophagy is implied in the clearance of ubiquitinated aggregates, as Atg1 is a kinase which plays a crucial role in the regulation of this process. On the other hand, we observed that cue5Δ mutant cells displayed a minor accumulation of ubiquitin throughout the whole treatment with ethanol, compared to wild type cells. The yeast protein Cue5 is a ubiquitin-binding protein that collaborates with Ubc4/Ubc5 ubiquitin-conjugating enzymes and the Rsp5 ubiquitin ligase for the conjugation of ubiquitin to its substrate. Since we observed a lower presence of ubiquitin in cue5Δ mutant cells, we propose that Cue5 might have a role in the conjugation of ubiquitin itself to the aggregates under severe ethanol stress.

O cancro é uma das principais causas de morte no mundo. As células cancerígenas beneficiam de processos celulares como a autofagia para sobreviver a condições de stress no seu microambiente, como privação de nutrientes, baixos níveis de energia e hipóxia. Em vários tipos de tumor a via de PI3K/AKT está hiperactiva, o que permite que a cinase mTOR, regulador da proliferação celular, apoptose e regulador negativo da autofagia, esteja ativo. Na quimioterapia convencional são utilizados inibidores de mTOR e PI3K/AKT, que consequentemente ativam a autofagia, o que resulta na sobrevivência das células cancerígenas e resistência aos tratamentos. A proteína TRIB2 é uma pseudocinase descrita como um oncogene que favorece a ativação da via de PI3K/AKT. É um membro da família TRIBBLES que inclui TRIB1 e TRIB3. Previamente foi publicado por diferentes grupos de investigação que a sobreexpressão de TRIB3 parece ter em vários contextos tumorais uma ação oposta à exercida por TRIB2, conferindo características de gene supressor de tumor. No contexto autofágico, TRIB3 parece suprimir a indução da autofagia conferindo às células acumulação de fatores promotores de tumor. Tendo em conta estes dados, propusemos estudar o papel de TRIB2 no processo autofágico. Sabendo que TRIB2 e TRIB3 parecem exercer em determinadas circunstâncias papéis antagónicos, a nossa hipótese é que tumores com elevados níveis de TRIB2 levariam a um aumento no fluxo autofágico. Os nossos dados demonstraram que células com sobreexpressão de TRIB2 apresentam níveis reduzidos de expressão de genes autofágicos. A análise de autofagossomas e autolisossomas por microscopia confocal permitiu concluir que a dinâmica do fluxo autofágico é menor em células que sobreexpressam TRIB2. Quando analisámos o impacto da presença de TRIB2, quando a autofagia está bloqueada, verificaram-se níveis elevados de morte celular, o que indica que células com TRIB2 são mais vulneráveis ao bloqueio da autofagia. Assim, concluímos que a nossa hipótese é refutada e que TRIB2 inibe o fluxo autofágico. O nosso estudo contribui para o conhecimento de TRIB2 na autofagia, e vem reforçar a importância deste biomarcador tumoral na terapêutica de tumores com elevados níveis de TRIB2. No entanto é necessário explorar o mecanismo pelo qual a proteína TRIB2 inibe a autofagia. Em suma, pacientes com elevados níveis de TRIB2 podem beneficiar de compostos que inibam diretamente a via de PI3K/AKT e o fluxo autofágico. Cancer is one of the main causes of death in the world. Cancer cells benefit from cellular processes such as autophagy to survive stressful conditions in their microenvironment, such as nutrient deprivation, low energy levels, and hypoxia. In several tumor types, the PI3K/AKT pathway is hyperactivated, which allows the mTOR kinase, a major regulator of cell proliferation, apoptosis, and negative regulator of autophagy, to be active. In conventional chemotherapy, mTOR and PI3K/AKT inhibitors are used, which consequently activate autophagy, resulting in cancer cell survival and resistance to treatments. The TRIB2 protein is a pseudokinase described as an oncogene that contributes to PI3K/AKT pathway activation. It is a member of the TRIBBLES family, which also includes TRIB1 and TRIB3. Previously, different research groups reported that TRIB3 overexpression appears to have the opposite effect of TRIB2 in several tumor contexts, conferring tumor suppressor gene characteristics. TRIB3 appears to suppress autophagy induction in the autophagic context by allowing cells to accumulate tumorpromoting factors. Taking these data together, we proposed to study the role of TRIB2 in the autophagic process. It has been proposed that TRIB2 and TRIB3 may have antagonistic roles under certain circunstances, our hypothesis is that tumors with high levels of TRIB2 would lead to an increase in autophagic flux. Our data demonstrated that cells overexpressing TRIB2 have reduced levels of autophagic gene expression. Analysis of autophagosomes and autolysosomes by confocal microscopy allowed us to conclude that autophagic flux dynamics are lower in cells overexpressing TRIB2. When we analyzed the impact of TRIB2 presence when autophagy is blocked, high levels of cell death were observed, indicating that cells with more TRIB2 are more vulnerable to autophagy blockage. However, we conclude that our hypothesis is refuted and that TRIB2 inhibits autophagic flux. Our study contributes to the knowledge of TRIB2 in autophagy and reinforces the importance of this tumor biomarker in the therapy of tumors with high levels of TRIB2. However, it is necessary to explore the mechanism by which the TRIB2 protein inhibits autophagy. In summary, patients with high levels of TRIB2 may benefit from compounds that directly inhibit the PI3K/AKT pathway and autophagic flux.

Dissertação de mestrado em Ciências da Saúde, Os adoçantes tornaram-se uma escolha eletiva para substituir o açúcar, particularmente para indivíduos obesos ou para aqueles que não podem tolerar açúcar nas suas dietas, tais como os diabéticos. Os adoçantes apresentam o mesmo sabor doce que o açúcar, no entanto os efeitos por eles promovidos não são bem definidos ou completamente compreendidos. Os adoçantes são classificados como naturais e sintéticos/artificiais e são utilizados para fins terapêuticos, clínicos, e como um substituto palatável do açúcar. Embora muitos adoçantes tenham a sua utilização aprovada por agências internacionais, faltam ainda estudos que os liguem ao envelhecimento, que é um processo biológico complexo e multifatorial determinado pela combinação de fatores genéticos e ambientais. O principal objetivo deste trabalho foi estudar o impacto que a suplementação com adoçantes (sucralose, ciclamato, sacarina, xilitol e stevia) tem no envelhecimento celular. Para atingir os objetivos propostos, o envelhecimento cronológico da levedura Saccharomyces cerevisiae foi utilizada como modelo de envelhecimento celular. Os resultados apresentados demonstraram que as células têm uma resposta diferente de acordo com o adoçante usado. Enquanto que o xilitol e a sacarina inibem o crescimento celular, o ciclamato de sódio ou a stevia estimulam o crescimento das células. Adicionalmente, também demonstramos que a suplementação com ciclamato de sódio ou stevia induz uma extensão da longevidade, que foi acompanhada por um aumento da autofagia, sem grandes alterações no perfil do ciclo celular e acumulação de espécies recativas de oxigénio. As análises por HPLC mostraram que as células não são capazes de metabolizar nenhum dos adoçantes, sugerindo que provavelmente estão a sinalizar a ativação de vários mecanismos. Globalmente, estes resultados demonstram a importância destes estudos e abrem novas perspetivas sobre o efeito destes adoçantes no envelhecimento celular., Sweeteners have become an elective choice to substitute sugar, particularly for those who may struggle with obesity or cannot tolerate sugar in their diets, such as diabetics. Sweeteners present the same sweet taste as sugar, nevertheless the effects promoted are not well defined or completely understood. Sweeteners are classified into natural and synthetic/artificial and are used for therapeutic and clinical purposes, and as a palatable substitute for sugar. Although many sweeteners have their use approved by international health agencies, there are still lacking studies linking them to aging, which is a complex, multifactorial biological process determined by the combination of genetic and environmental factors. The main objective of this work was to study the impact that supplementation with sweeteners (sucralose, cyclamate, saccharin, xylitol, and stevia) have on chronological aging. To achieve the proposed aims, the chronological lifespan (CLS) of the yeast Saccharomyces cerevisiae was used as an aging cellular model. The results presented demonstrated that cells have a different response according to the sweetener. While xylitol and saccharin inhibit cell grow, sodium cyclamate or stevia stimulate it. Furthermore, sodium cyclamate or stevia supplementation are associated with extended CLS, which was accompanied with increased autophagy, without major alterations on cell cycle profile and accumulation of reactive oxygen species. Furthermore, HPLC analyses showed that cells are not able to metabolize none of the sweeteners, suggesting that they are probably signaling the activation of several mechanisms. Overall, these results open new perspectives for the effects of sweeteners on cellular aging., The work presented in this thesis was performed in the Life and Health Sciences Research Institute (ICVS), University of Minho, Financial support was provided by National funds, through the Foundation for Science and Technology (FCT) - project UIDB/50026/2020 and UIDP/50026/2020.

Parkinson’s disease; Autophagy; Lysosome Enfermedad de Parkinson; Autofagia; Lisosoma Malaltia de Parkinson; Autofàgia; Lisosoma GBA gene variants were the first genetic risk factor for Parkinson’s disease. GBA encodes the lysosomal enzyme glucocerebrosidase (GBA), which is involved in sphingolipid metabolism. GBA exhibits a complex physiological function that includes not only the degradation of its substrate glucosylceramide but also the metabolism of other sphingolipids and additional lipids such as cholesterol, particularly when glucocerebrosidase activity is deficient. In the context of Parkinson’s disease associated with GBA, the loss of GBA activity has been associated with the accumulation of α-synuclein species. In recent years, several hypotheses have proposed alternative and complementary pathological mechanisms to explain why lysosomal enzyme mutations lead to α-synuclein accumulation and become important risk factors in Parkinson’s disease etiology. Classically, loss of GBA activity has been linked to a dysfunctional autophagy–lysosome system and to a subsequent decrease in autophagy-dependent α-synuclein turnover; however, several other pathological mechanisms underlying GBA-associated parkinsonism have been proposed. This review summarizes and discusses the different hypotheses with a special focus on autophagy-dependent mechanisms, as well as autophagy-independent mechanisms, where the role of other players such as sphingolipids, cholesterol and other GBA-related proteins make important contributions to Parkinson’s disease pathogenesis. This work was supported by the Fondo de Investigación Sanitaria-Instituto de Salud Carlos III (Spain)-FEDER (PI20/00728), Fundación BBVA (NanoERT), Fundació La Caixa (HR22-00602), E.P. was supported by a VHIR doctoral fellowship.

Autophagy; Genetic variants; Multiple myeloma Autofagia; Variantes genéticas; Mieloma múltiple Autofàgia; Variants genètiques; Mieloma múltiple Multiple myeloma (MM) arises following malignant proliferation of plasma cells in the bone marrow, that secrete high amounts of specific monoclonal immunoglobulins or light chains, resulting in the massive production of unfolded or misfolded proteins. Autophagy can have a dual role in tumorigenesis, by eliminating these abnormal proteins to avoid cancer development, but also ensuring MM cell survival and promoting resistance to treatments. To date no studies have determined the impact of genetic variation in autophagy-related genes on MM risk. We performed meta-analysis of germline genetic data on 234 autophagy-related genes from three independent study populations including 13,387 subjects of European ancestry (6863 MM patients and 6524 controls) and examined correlations of statistically significant single nucleotide polymorphisms (SNPs; p < 1 × 10−9) with immune responses in whole blood, peripheral blood mononuclear cells (PBMCs), and monocyte-derived macrophages (MDM) from a large population of healthy donors from the Human Functional Genomic Project (HFGP). We identified SNPs in six loci, CD46, IKBKE, PARK2, ULK4, ATG5, and CDKN2A associated with MM risk (p = 4.47 × 10−4−5.79 × 10−14). Mechanistically, we found that the ULK4rs6599175 SNP correlated with circulating concentrations of vitamin D3 (p = 4.0 × 10−4), whereas the IKBKErs17433804 SNP correlated with the number of transitional CD24+CD38+ B cells (p = 4.8 × 10−4) and circulating serum concentrations of Monocyte Chemoattractant Protein (MCP)-2 (p = 3.6 × 10−4). We also found that the CD46rs1142469 SNP correlated with numbers of CD19+ B cells, CD19+CD3− B cells, CD5+IgD− cells, IgM− cells, IgD−IgM− cells, and CD4−CD8− PBMCs (p = 4.9 × 10−4−8.6 × 10−4) and circulating concentrations of interleukin (IL)-20 (p = 0.00082). Finally, we observed that the CDKN2Ars2811710 SNP correlated with levels of CD4+EMCD45RO+CD27− cells (p = 9.3 × 10−4). These results suggest that genetic variants within these six loci influence MM risk through the modulation of specific subsets of immune cells, as well as vitamin D3−, MCP-2−, and IL20-dependent pathways. This work was supported by the European Union’s Horizon 2020 research and innovation program, N° 856620 and by grants from the Instituto de Salud Carlos III and FEDER (Madrid, Spain; PI17/02256 and PI20/01845), Consejería de Transformación Económica, Industria, Conocimiento y Universidades and FEDER (PY20/01282), from the CRIS foundation against cancer, from the Cancer Network of Excellence (RD12/10 Red de Cáncer), from the Dietmar Hopp Foundation and the German Ministry of Education and Science (BMBF: CLIOMMICS [01ZX1309]), and from National Cancer Institute of the National Institutes of Health under award numbers: R01CA186646, U01CA249955 (EEB). This work was also funded d by Portuguese National funds, through the Foundation for Science and Technology (FCT)—project UIDB/50026/2020 and UIDP/50026/2020 and by the project NORTE-01-0145-FEDER-000055, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF).

A ataxia espinocerebelosa tipo 2 (SCA2) é uma doença hereditária autossómica dominante que, em conjunto com outras oito doenças neurodegenerativas, forma um grupo de doenças, denominado por doenças de poliglutaminas. Estas são causadas por uma expansão anormal do trinucleótido CAG (citosina-adenina-guanina), que leva à formação de uma proteína anormal, o que afeta a homeostase celular. A SCA2 foi descrita pela primeira vez em 1971 e é causada por um aumento de mais de 31 repetições CAG no gene ATXN2. Este codifica uma proteína ataxina-2 anormal que não é eficientemente degradada, acumulando-se e levando à formação de agregados tóxicos, presentes principalmente no citoplasma. Esses agregados levam a diferentes mecanismos de patogénese, como a disfunção da autofagia, que é responsável pela degradação e reciclagem de proteínas. Ainda não existe cura para a SCA2, porém atualmente existem várias estratégias terapêuticas em estudo, como a redução do número de agregados através da ativação da autofagia pela cordicepina. Assim, o presente estudo teve como objetivo estudar a autofagia na SCA2 e perceber se a indução desta pode vir a ser uma futura estratégia terapêutica. Os resultados demostraram uma disfunção da via autofágica em células Neuro-2A transfectadas com formas mutantes de ataxina-2, pela verificação de um aumento dos níveis de SQSTM1 e de uma diminuição dos níveis de LC3B-II, e no tecido cerebral post-mortem de pacientes com SCA2, pela observação de um aumento dos agregados de ataxina-2 e de uma acumulação anormal de marcadores autofágicos. Além disso, demonstraram ainda que a administração de cordicepina leva à diminuição da perda neuronal e à redução da agregação da proteína ataxina-2. Assim e apesar de serem necessários mais estudos complementares este estudo demonstrou que a regulação positiva da autofagia pode ser uma futura estratégia terapêutica para o tratamento da doença SCA2. Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant inherited disease, which together with eight other neurodegenerative diseases, forms a group of diseases, called polyglutamine diseases. These are caused by an abnormal expansion of the CAG (cytosine-adenine-guanine trinucleotide), which leads to the formation of an abnormal protein, that affects cellular homeostasis. SCA2 was first described in 1971 and is caused by an increase of more than 31 CAG repeats in the ATXN2 gene. This encodes for an abnormal ataxin-2 protein that is not efficiently degraded, accumulating and leading to the formation of toxic aggregates, present mainly in the cytoplasm. These aggregates lead to different pathogenesis mechanisms, such as autophagy dysfunction, responsible for degradation and recycling of proteins. There is still no cure for SCA2, but there are currently several therapeutic strategies under study, such as reducing the number of aggregates through the activation of autophagy by cordycepin. Therefore, the present study aimed to study autophagy in SCA2 and to understand whether its induction can become a future therapeutic strategy. The results demonstrated a dysfunction of the autophagic pathway in Neuro-2A cells transfected with mutant forms of ataxin-2, by verifying an increase in the levels of SQSTM1 and a decrease in the levels of LC3B-II, and in the post-mortem brain tissue of patients with SCA2, by observing an increase in ataxin-2 aggregates and an abnormal accumulation of autophagic markers. Furthermore, administration of cordycepin has been shown to lead to decreased neuronal loss and reduced aggregation of ataxin-2 protein. Thus, and despite the need for further complementary studies, this study demonstrated that the upregulation of autophagy may be a future therapeutic strategy for the treatment of SCA2 disease.

Le tiopurine, come la mercaptopurina e l'azatioprina, sono agenti leucotossici usati per trattare la leucemia linfoblastica acuta (LLA) e le malattie infiammatorie croniche intestinali (MICI). La tiopurina-metiltransferasi (TPMT) è un enzima coinvolto nell'inattivazione delle tiopurine; la sua funzione è influenzata principalmente dalle varianti geniche di TPMT e di Protein Kinase C And Casein Kinase Substrate In Neurons 2 (PACSIN2). La variante rs2413739 in PACSIN2 è considerata un expression quantitative trait locus (eQTL) associato a gravi effetti gastrointestinali da mercaptopurina in pazienti pediatrici con LLA infantile e all'efficacia dell'azatioprina in pazienti pediatrici affetti da MICI attraverso un meccanismo non caratterizzato, che potrebbe essere correlato all'autofagia. Il presente studio mira a chiarire il ruolo di PACSIN2 nell'autofagia e nella citotossicità delle tiopurine in modelli linfoidi ed intestinali. Inoltre, questo progetto mira a valutare il possibile effetto dell'esposizione a mercaptopurina e del silenziamento di PACSIN2 sia sull'autofagia che sull’apoptosi in un modello cellulare intestinale. I risultati ottenuti sulle linee cellulari immortalizzate linfoidi (NALM6) ed intestinali (LS180) con il silenziamento stabile (knock-down, KD) di PACSIN2 hanno identificato livelli autofagici maggiori, valutati tramite quantificazione dei livelli dei marcatori autofagici LC3-II e SQSTM1/P62, indicando il possibile ruolo di PACSIN2 come modulatore negativo delll’autofagia. È stata svolta un’analisi del flusso autofagico mediante trattamento con clorochina, che ha mostrato un aumento della quantità di LC3-II e SQSTM1/P62, suggerendo il coinvolgimento di PACSIN2 nella regolazione delle prime fasi dell'autofagia. Inoltre, la correlazione inversa tra la quantità di PACSIN2 ed i livelli autofagici è stata osservata anche in un gruppo di biopsie di tratti infiammati del colon rispetto a biopsie di colon non infiammato di pazienti pediatrici con MICI, confermando i risultati ottenuti in vitro. Non è stata rilevata alcuna associazione significativa tra la quantità di TPMT e i livelli dei marcatori autofagici, indicando che la modulazione di PACSIN2 sull'attività di TPMT possa coinvolgere meccanismi diversi dall’autofagia. Inoltre, analisi HPLC-UV hanno dimostrato che PACSIN2 non influenza la farmacocinetica della mercaptopurina sulle linee cellulari intestinali. È stata identificata un’interazione proteina-proteina tra PACSIN2 e LC3, mediata da un dominio di legame a LC3 presente nella sequenza amminoacidica di PACSIN2. Infine, è stato dimostrato che il silenziamento di PACSIN2 è correlato ad una maggior sensibilità alla mercaptopurina nelle cellule LS180 ma non nelle cellule NALM6, suggerendo un ruolo tessuto specifico di PACSIN2 nell'intestino dopo il trattamento con mercaptopurina. È interessante notare come l'esposizione alla mercaptopurina abbia ridotto i livelli autofagici delle cellule LS180 e questo effetto sia meno evidente nelle cellule con il PACSIN2 KD; inoltre, sia l'esposizione alla mercaptopurina che il PACSIN2 KD hanno aumentato i livelli apoptotici delle cellule LS180. I risultati di questo lavoro dimostrano per la prima volta il ruolo di PACSIN2 come inibitore delle prime fasi dell'autofagia, presumibilmente attraverso il legame del dominio LIR di PACSIN2 a LC3-II. Infine, è stato dimostrato il ruolo di PACSIN2 come modulatore della citotossicità alle tiopurine in modelli cellulari immortalizzati intestinali, suggerendo che i livelli di autofagia regolati da PACSIN2 potrebbero influenzare la sensibilità alle tiopurine. Thiopurines, such as mercaptopurine and azathioprine, are leukotoxic agents used to treat acute lymphoblastic leukemia (ALL) and inflammatory bowel disease (IBD). Thiopurine-methyltransferase (TPMT) is an enzyme involved in thiopurines inactivation; its function is affected mainly by known TPMT polymorphisms and by Protein Kinase C And Casein Kinase Substrate In Neurons 2 (PACSIN2) through a less characterized molecular mechanism. The expression quantitative trait locus (eQTL) rs2413739 variant in PACSIN2 is associated with severe gastrointestinal effects of mercaptopurine in childhood ALL and with azathioprine efficacy in IBD pediatric patients through an uncharacterized mechanism that could be related to autophagy. The present study aims to clarify the role of PACSIN2 in autophagy and on thiopurine cytotoxicity in leukemic and intestinal models. Moreover, this project aims to evaluate the possible effect of both mercaptopurine exposure and PACSIN2 KD on both autophagy and apoptosis induction in an intestinal model. Leukemic (NALM6) and intestinal (LS180) cells with PACSIN2 knock-down (KD) showed higher autophagy, assessed as LC3-II and SQSTM1/P62 amount. Autophagy flux analysis by chloroquine treatment showed increased LC3-II and SQSTM1/P62 amount, suggesting that PACSIN2 affects the early autophagy phases. The inverse association between PACSIN2 amount and autophagy levels was observed also in inflamed compared to non-inflamed colon biopsies of IBD pediatric patients, confirming the in vitro results. No significant association between TPMT amount and autophagy levels was detected, indicating that modulation of autophagy is not the molecular mechanism at the basis of PACSIN2 impact on TPMT activity. Moreover, HPLC-UV analysis showed that PACSIN2 did not affect mercaptopurine pharmacokinetics on intestinal in vitro models. PACSIN2 has a LC3 interacting region (LIR) motif and co-immunoprecipitation analysis confirms that PACSIN2 binds LC3. Transient expression of PACSIN2 with a mutated LIR motif reduced the level of co-immunoprecipitated LC3, showing a role of the LIR motif of PACSIN2 in the interaction. PACSIN2 KD increased mercaptopurine sensitivity in LS180 cells but not in NALM6 cells, suggesting a tissue specific role of PACSIN2 in the intestine after mercaptopurine treatment. Interestingly, mercaptopurine exposure reduced autophagy levels, but PACSIN2 KD counteracted this effect; moreover, both mercaptopurine exposure and PACSIN2 KD are able to stimulate apoptosis. Taken together, these results demonstrate for the first time a clear role of PACSIN2 as an inhibitor of autophagy, putatively through inhibition of autophagosome formation by a protein-protein interaction with LC3-II mediated by a LIR motif present in the PACSIN2 sequence. Moreover, in vitro evidences of the role of PACSIN2 as a modulator of mercaptopurine-induced cytotoxicity in intestinal cells were provided, suggesting that PACSIN2-regulated autophagy levels might influence thiopurine sensitivity.

The non-toxic C-terminal fragment of the tetanus toxin (TTC) has been described as a neuroprotective molecule since it binds to Trk receptors and activates Trk-dependent signaling, activating neuronal survival pathways and inhibiting apoptosis. Previous in vivo studies have demonstrated the ability of this molecule to increase mice survival, inhibit apoptosis and regulate autophagy in murine models of neurodegenerative diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy. Prion diseases are fatal neurodegenerative disorders in which the main pathogenic event is the conversion of the cellular prion protein (PrPC) into an abnormal and misfolded isoform known as PrPSc. These diseases share different pathological features with other neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson’s disease or Alzheimer’s disease. Hitherto, there are no effective therapies to treat prion diseases. Here, we present a pilot study to test the therapeutic potential of TTC to treat prion diseases. C57BL6 wild-type mice and the transgenic mice Tg338, which overexpress PrPC, were intracerebrally inoculated with scrapie prions and then subjected to a treatment consisting of repeated intramuscular injections of TTC. Our results indicate that TTC displays neuroprotective effects in the murine models of prion disease reducing apoptosis, regulating autophagy and therefore increasing neuronal survival, although TTC did not increase survival time in these models.

Introdução: Lesão dos podócitos e subsequente nefropatia crônica progressiva são características proeminentes da Doença de Fabry (DF), desordem de depósito lisossômico com padrão de herança ligada ao X provocada por mutações do gene GLA levando à atividade insuficiente da enzima α-GAL (α-galactosidase A). Embora as consequências clínicas tardias da nefropatia da DF sejam bem conhecidas, os eventos moleculares que deflagram a lesão podocitária carecem de estudos. O objetivo do presente estudo foi investigar a expressão diferencial de proteínas em podócitos humanos imortalizados com características fenotípicas da DF obtidas pela edição prévia do gene GLA através da tecnologia CRISPR/Cas9. Métodos: Os podócitos foram avaliados comparativamente: (i) quanto ao padrão proteico e processos biológicos alterados, através da abordagem proteômica e gene ontology (GO), (ii) quanto às vias de sinalização afetadas, por protocolo de western blot (WB) e RT-PCR e (iii) quanto à indução de apoptose, por meio de microscopia de fluorescência utilizando o marcador iodeto de propídio (IP). Resultados: A proteômica exploratória mostrou que as proteínas UCHL1 (1,4x) e p62 (>1,7x), biomarcadores da autofagia, revelando inibição do fluxo autofágico. (2) TGF- (>1,5x), VEGF (>1,5x) e Vimentina (>2,3x), marcadores das vias pró-fibróticas, resposta vascular e transição epitélio mesenquimal, respectivamente; (3) PI3K 110 (>2,5x) e PI3K 110 (>1,45x), vias associadas com hipertrofia e apoptose. Paralelamente, os podócitos editados, em relação ao grupo controle, apresentaram significativa menor expressão de mRNA das proteínas estruturais podocalixina (1.4x) and p62 (>1.7x), biomarkers of autophagy, revealing inhibition of autophagic flux. (2) TGF-β (>1.5x), VEGF (>1.5x) and Vimentin (>2.3x), markers of profibrotic pathways, vascular response and mesenchymal epithelial transition, respectively; (3) PI3K 110 (>2.5x) and PI3K 110 (>1.45x) associated with hypertrophy and apoptosis. In parallel, significantly lower mRNA expression of the structural proteins podocalyxin (

[cat] Actualment, el tractament de la malaltia d’Alzheimer (MA) és una necessitat mèdica no coberta, ja que només existeixen tres grups de fàrmacs aprovats que no són suficientment eficaços pel control de la malaltia. La naturalesa complexa de la fisiopatologia de la MA i la manca de biomarcadors són els principals obstacles per al desenvolupament de nous fàrmacs pel tractament de la MA. Tenint en compte l’alta taxa de fracàs, l’optimització dels fàrmacs ja en el mercat representa una eina interessant ja que es reduirien els costos en comparació amb l’assaig de nous compostos dirigits a noves dianes. La memantina, un antagonista dels receptors N-metil-D-aspartat (NMDA), ha demostrat beneficis clínics en relació tant en aspectes cognitius i no cognitius, com en vies moleculars implicades en el procés neurodegeneratiu. Malgrat tot, la seva efectivitat és limitada. Per aquest motiu, aquesta tesi doctoral s’ha centrat en caracteritzar farmacològicament en models in vivo dos nous compostos anàlegs a la memantina (l’RL-208 i l’UB-ALT-EV) que s’han desenvolupat amb l’objectiu de millorar l’eficàcia terapèutica de la memantina. Durant la tesi doctoral, es van dur a terme estudis cognitius i de comportament en el nematode C.elegans, en un model de ratolí amb senescència accelerada (SAMP8) i en un model de ratolí transgènic de la MA, el 5XFAD, després del tractament amb els nous antagonistes. A més, es va avaluar molecularment vies associades a la MA per poder avaluar l’efecte modulador dels antagonistes dels receptors NMDA sobre elles. Els resultats obtinguts en el models animals de ratolí demostren com el tractament crònic per via oral a una dosi baixa amb els nous compostos és capaç de millorar les alteracions de la memòria, així com regular les alteracions de la conducta social i el comportament de tipus ansiós. Per altra banda, el compost UB-ALT-EV va demostrar efectes positius sobre els dèficits locomotors i del comportament els cucs C.elegans causats per l’acumulació del pèptid β-amiloide. Així mateix, els antagonistes del receptor NMDA avaluats van reduir els marcadors neuropatològics típics de la MA com la formació i l’acumulació del pèptid Aβ i la fosforilació de la proteïna tau. De la mateixa manera es va demostrar l’efectivitat dels nous compostos en la modulació de vies de senyalització molecular implicades en la neurodegeneració, com la desregulació dels nivells de calci intracel·lular, l’estrès oxidatiu, el flux autofàgic i la neuroinflamació. De manera global, els estudis duts a terme en aquesta tesi permeten demostrar l’efecte neuroprotector dels dos antagonistes del receptor NMDA a més de suggerir que l’optimització de fàrmacs aprovats suposa una estratègia de desenvolupament de nous fàrmacs que permetria l’abordatge de la MA de manera més eficaç. Particularment, donada la capacitat dels nous compostos de regular no només aspectes cognitius i no cognitius sinó també vies moleculars implicades en el procés neurodegeneratiu, aquestes noves teràpies podrien acabar sent considerades com teràpies modificadores de la malaltia i no només tractaments simptomatològics., [eng] Nowadays, the treatment of Alzheimer’s Disease (AD) is an unmet medical need, as there are only three groups of approved drugs that are not sufficiently effective for the control of the disease. The complex nature of the pathophysiology of AD and the lack of biomarkers constitute major obstacles for the development of new drugs for the treatment of AD. Given the high rate of failures, optimisation of already available drugs represents an interesting tool, as the cost would be reduced in comparison to the testing of new compounds against new targets. Memantine, an N-methyl-D-aspartate (NMDA) receptor antagonist, has shown clinical benefits regarding cognitive and non-cognitive aspects and molecular pathways involved in the neurodegenerative process. However, its efficacy is limited. Therefore, this thesis dissertation has focused on the pharmacological characterization of two new memantine-like compounds (RL-208 and UB-ALT-EV) that have been developed with the aim of improving the therapeutic efficacy of memantine. During the research, cognitive and behavioural studies have been performed in the nematode C.elegans, in a mouse model with accelerated senescence (SAMP8) and in a transgenic mouse model of MA, 5XFAD, after the treatment with the new antagonists. Furthermore, the molecular pathways associated to AD were evaluated to evaluate the modulating effect of NMDA receptor antagonists on these pathways. The results obtained with animal mice models demonstrate that the chronic oral treatment at low doses with the new compounds is able to improve memory impairment, as well as to regulate social and anxiety-like behavioural disturbances. Moreover, the compound UB-ALT-EV showed positive effects on locomotor and behavioural deficits in C.elegans worms caused by accumulation of amyloid-β peptide. Additionally, the NMDA receptor antagonists tested reduced typical neuropathological markers of AD, such as Amyloid-β formation and accumulation and tau protein hyperphosphorylation. The efficacy of the new compounds in modulating molecular signaling pathways involved in neurodegeneration, such as dysregulation of intracellular calcium levels, oxidative stress, autophagic flux and neuroinflammation, was also demonstrated. Overall, the studies conducted in this thesis demonstrate the neuroprotective effect of the two NMDA receptor antagonists and suggest that the optimization of approved drugs is a strategy for the development of new drugs that would address AD more effectively. In particular, given the ability of the new compounds to regulate not only cognitive and non-cognitive aspects, but also the molecular pathways involved in the neurodegenerative process, these new therapies could eventually be considered as disease-modifying therapies and not only as symptomatic treatments.