Application of ionic liquids and enzymes for the removal of proteinaceous layers from polychrome of works of art and evaluation of the cleaning effectiveness

Dissertação Apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Ciências da Conservação, especialização em PinturaA novel use of ionic liquids as alternative solvents for enzymes in cleaning treatments for the removal of proteinaceous materials from painted or gilded surfaces is presented. The ionic liquids are potentially green solvents to be applied in restoration treatments being also called designer solvents, because of their peculiar properties which can be adjusted by selecting different cationanion combinations. Two ionic liquids were selected: IL1)1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4])and IL2) 1-ethyl-3-methylimidazolium ethylsulfate ([EMIM][EtSO4]). Formulations were prepared with these ionic liquids and two different proteases: one acid (pepsin) and one alkaline (from Aspergillus sojae). Additionally aqueous gel formulations were prepared with these enzymes for reference purpose. A third enzyme provided by the Bromatology Department at the Faculty of Pharmacy from the Porto University was tested only in gel formulation in order to assess its potential use in cleaning treatments. To understand the enzyme activity of these formulations and predict their ability as cleaning agents, analyses were performed with ultraviolet–visible (UV-Vis) spectroscopy and highperformance liquid chromatography (HPLC) prior cleaning; and with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) after cleaning. These formulations were tested on mock-up samples prepared in accordance with documented and historical sources of artistic techniques of egg tempera and oil painting, and gilding. A non-invasive non-destructive multi-scale analytical protocol was carried out for cleaning effectiveness evaluation and surface characterization before and after treatment. Different surface analytical techniques were adopted to this purpose: stereomicroscopy (SM), optical microscopy (OM) with visible and fluorescence light, atomic force microscopy (AFM), scanning electron microscopy (SEM) and electron dispersive spectroscopy (EDS) and colorimetry (CIE L*a*b* system). The surface analytical protocol proved to be adequate, not only, for monitoring the cleaning process but also for complete characterization of the surface, before and after treatment, including information on the presence of residues and possible surface deterioration. It was also proved that the formulations of enzymes combined with ILs can be used successfully for the removal of proteinaceous material as alternatives to gel formulations. More studies should be conducted to determine the most suitable IL or group of ILs, the main concern should focus on improving aspects such as compatibility with other surface materials, and possible long-term effects of residues after cleaning

Nucleocytoplasmic Shuttling Activity of Ataxin-3

Spinocerebellar ataxia type-3, also known as Machado-Joseph Disease (MJD), is one of many inherited neurodegenerative disorders caused by polyglutamine-encoding CAG repeat expansions in otherwise unrelated genes. Disease protein misfolding and aggregation, often within the nucleus of affected neurons, characterize polyglutamine disorders. Several evidences have implicated the nucleus as the primary site of pathogenesis for MJD. However, the molecular determinants for the nucleocytoplasmic transport of human ataxin-3 (Atx3), the protein which is mutated in patients with MJD, are not characterized. In order to characterize the nuclear shuttling activity of Atx3, we performed yeast nuclear import assays and found that Atx3 is actively imported into the nucleus, by means of a classical nuclear localizing sequence formed by a cluster of lysine and arginine residues. On the other hand, when active nuclear export was inhibited using leptomycin B, a specific inhibitor of the nuclear export receptor CRM1, both endogenous Atx3 and transfected GFP-Atx3 accumulated inside the nucleus of a subpopulation of COS-7 cells, whereas both proteins are normally predominant in the cytoplasm. Additionally, using a Rev(1.4)-GFP nuclear export assay, we performed an extensive analysis of six putative aliphatic nuclear export motifs identified in Atx3 amino acid sequence. Although none of the tested peptide sequences were found to drive nuclear export when isolated, we have successfully mapped the region of Atx3 responsible for its CRM1-independent nuclear export activity. Curiously, the N-terminal Josephin domain alone is exported into the cytoplasm, but the nuclear export activity of Atx3 is significantly enhanced in a longer construct that is truncated after the two ubiquitin interaction motifs, upstream from the polyQ tract. Our data show that Atx3 is actively imported to and exported from the cell nucleus, and that its nuclear export activity is dependent on a motif located at its N-terminal region. Since pathological Atx3 aggregates in the nucleus of affected neurons in MJD, and there is in vivo evidence that nuclear localization of Atx3 is required for the manifestation of symptoms in MJD, defects in the nucleocytoplasmic shuttling activity of the protein may be involved in the nuclear accumulation and aggregation of expanded Atx3.This work was supported by a grant from the Portuguese Foundation for Science and Technology (POCI/SAU-MMO/60156/2004) and by Crioestaminal/ Associacao Viver a Ciencia. LC was a recipient of a post-doctoral fellowship from the Portuguese Foundation for Science and Technology (SFRH/BPD/20686/2004/ 22). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Neuropeptide Y inhibits interleukin-1β-induced phagocytosis by microglial cells

<p>Abstract</p> <p>Background</p> <p>Neuropeptide Y (NPY) is emerging as a modulator of communication between the brain and the immune system. However, in spite of increasing evidence that supports a role for NPY in the modulation of microglial cell responses to inflammatory conditions, there is no consistent information regarding the action of NPY on microglial phagocytic activity, a vital component of the inflammatory response in brain injury. Taking this into consideration, we sought to assess a potential new role for NPY as a modulator of phagocytosis by microglial cells.</p> <p>Methods</p> <p>The N9 murine microglial cell line was used to evaluate the role of NPY in phagocytosis. For that purpose, an IgG-opsonized latex bead assay was performed in the presence of lipopolysaccharide (LPS) and an interleukin-1β (IL-1β) challenge, and upon NPY treatment. A pharmacological approach using NPY receptor agonists and antagonists followed to uncover which NPY receptor was involved. Moreover, western blotting and immunocytochemical studies were performed to evaluate expression of p38 mitogen-activated protein kinase (MAPK) and heat shock protein 27 (HSP27), in an inflammatory context, upon NPY treatment.</p> <p>Results</p> <p>Here, we show that NPY inhibits phagocytosis of opsonized latex beads and inhibits actin cytoskeleton reorganization triggered by LPS stimulation. Co-stimulation of microglia with LPS and adenosine triphosphate also resulted in increased phagocytosis, an effect inhibited by an interleukin-1 receptor antagonist, suggesting involvement of IL-1β signaling. Furthermore, direct application of LPS or IL-1β activated downstream signaling molecules, including p38 MAPK and HSP27, and these effects were inhibited by NPY. Moreover, we also observed that the inhibitory effect of NPY on phagocytosis was mediated <it>via </it>Y₁receptor activation.</p> <p>Conclusions</p> <p>Altogether, we have identified a novel role for NPY in the regulation of microglial phagocytic properties, in an inflammatory context.</p

Neuropeptide Y Enhances Progerin Clearance and Ameliorates the Senescent Phenotype of Human Hutchinson-Gilford Progeria Syndrome Cells

Hutchinson-Gilford progeria syndrome (HGPS, or classical progeria) is a rare genetic disorder, characterized by premature aging, and caused by a de novo point mutation (C608G) within the lamin A/C gene (LMNA), producing an abnormal lamin A protein, termed progerin. Accumulation of progerin causes nuclear abnormalities and cell cycle arrest ultimately leading to cellular senescence. Autophagy impairment is a hallmark of cellular aging, and the rescue of this proteostasis mechanism delays aging progression in HGPS cells. We have previously shown that the endogenous Neuropeptide Y (NPY) increases autophagy in hypothalamus, a brain area already identified as a central regulator of whole-body aging. We also showed that NPY mediates caloric restriction-induced autophagy. These results are in accordance with other studies suggesting that NPY may act as a caloric restriction mimetic and plays a role as a lifespan and aging regulator. The aim of the present study was, therefore, to investigate if NPY could delay HGPS premature aging phenotype. Herein, we report that NPY increases autophagic flux and progerin clearance in primary cultures of human dermal fibroblasts from HGPS patients. NPY also rescues nuclear morphology and decreases the number of dysmorphic nuclei, a hallmark of HGPS cells. In addition, NPY decreases other hallmarks of aging as DNA damage and cellular senescence. Altogether, these results show that NPY rescues several hallmarks of cellular aging in HGPS cells, suggesting that NPY can be considered a promising strategy to delay or block the premature aging of HGPS

Towards a structural understanding of the fibrillization pathway in Machado-Joseph’s disease: trapping early oligomers of non-expanded ataxin-3

Machado-Joseph’s disease is caused by a CAG trinucleotide repeat expansion that is translated into an abnormally long polyglutamine tract in the protein ataxin-3. Except for the polyglutamine region, proteins associated with polyglutamine diseases are unrelated, and for all of these diseases aggregates containing these proteins are the major components of the nuclear proteinaceous deposits found in the brain. Aggregates of the expanded proteins display amyloid-like morphological and biophysical properties. Human ataxin-3 containing a non-pathological number of glutamine residues (14Q), as well as its Caenorhabditis elegans (1Q) orthologue, showed a high tendency towards self-interaction and aggregation, under nearphysiological conditions. In order to understand the discrete steps in the assembly process leading to ataxin-3 oligomerization, we have separated chromatographically high molecular mass oligomers as well as medium mass multimers of non-expanded ataxin-3. We show that: (a) oligomerization occurs independently of the poly(Q)-repeat and it is accompanied by an increase in b-structure; and (b) the first intermediate in the oligomerization pathway is a Josephin domain-mediated dimer of ataxin- 3. Furthermore, non-expanded ataxin-3 oligomers are recognized by a specific antibody that targets a conformational epitope present in soluble cytotoxic species found in the fibrillization pathway of expanded polyglutamine proteins and other amyloid-forming proteins. Imaging of the oligomeric forms of the non-pathological protein using electron microscopy reveals globular particles, as well as short chains of such particles that likely mimic the initial stages in the fibrillogenesis pathway occurring in the polyglutamine-expanded protein. Thus, they constitute potential targets for therapeutic approaches in Machado-Joseph’s disease, as well as valuable diagnostic markers in disease settings

CuMV VLPs Containing the RBM from SARS-CoV-2 Spike Protein Drive Dendritic Cell Activation and Th1 Polarization.

Dendritic cells (DCs) are the most specialized and proficient antigen-presenting cells. They bridge innate and adaptive immunity and display a powerful capacity to prime antigen-specific T cells. The interaction of DCs with the receptor-binding domain of the spike (S) protein from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a pivotal step to induce effective immunity against the S protein-based vaccination protocols, as well as the SARS-CoV-2 virus. Herein, we describe the cellular and molecular events triggered by virus-like particles (VLPs) containing the receptor-binding motif from the SARS-CoV-2 spike protein in human monocyte-derived dendritic cells, or, as controls, in the presence of the Toll-like receptors (TLR)3 and TLR7/8 agonists, comprehending the events of dendritic cell maturation and their crosstalk with T cells. The results demonstrated that VLPs boosted the expression of major histocompatibility complex molecules and co-stimulatory receptors of DCs, indicating their maturation. Furthermore, DCs' interaction with VLPs promoted the activation of the NF-kB pathway, a very important intracellular signalling pathway responsible for triggering the expression and secretion of proinflammatory cytokines. Additionally, co-culture of DCs with T cells triggered CD4+ (mainly CD4+Tbet+) and CD8+ T cell proliferation. Our results suggested that VLPs increase cellular immunity, involving DC maturation and T cell polarization towards a type 1 T cells profile. By providing deeper insight into the mechanisms of activation and regulation of the immune system by DCs, these findings will enable the design of effective vaccines against SARS-CoV-2

Unlocking the potential of snake venom-based molecules against the malaria, Chagas disease, and leishmaniasis triad

Funding Information: This work received financial support from PT national funds ( FCT/MCTES , Fundação para a Ciência e Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) through the project CIRCNA/BRB/0281/2019 . Funding Information: This work received financial support from PT national funds (FCT/MCTES, Fundação para a Ciência e Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) through the project CIRCNA/BRB/0281/2019.The authors further thank FCT/MCTES for supporting Research Units LAQV-REQUIMTE (UIDB/50006/2020), GHTM (UID/Multi/04413/2020). Publisher Copyright: © 2023 The AuthorsMalaria, leishmaniasis and Chagas disease are vector-borne protozoal infections with a disproportionately high impact on the most fragile societies in the world, and despite malaria-focused research gained momentum in the past two decades, both trypanosomiases and leishmaniases remain neglected tropical diseases. Affordable effective drugs remain the mainstay of tackling this burden, but toxicicty, inneficiency against later stage disease, and drug resistance issues are serious shortcomings. One strategy to overcome these hurdles is to get new therapeutics or inspiration in nature. Indeed, snake venoms have been recognized as valuable sources of biomacromolecules, like peptides and proteins, with antiprotozoal activity. This review highlights major snake venom components active against at least one of the three aforementioned diseases, which include phospholipases A2, metalloproteases, L-amino acid oxidases, lectins, and oligopeptides. The relevance of this repertoire of biomacromolecules and the bottlenecks in their clinical translation are discussed considering approaches that should increase the success rate in this arduous task. Overall, this review underlines how venom-derived biomacromolecules could lead to pioneering antiprotozoal treatments and how the drug landscape for neglected diseases may be revolutionized by a closer look at venoms. Further investigations on poorly studied venoms is needed and could add new therapeutics to the pipeline.publishersversionepub_ahead_of_prin

Verdade e vaidade : técnicas de retoque em negativos de gelatina e vidro de coleções portuguesas da primeira metade do século xx

This thesis aims to understand better the retouchings on dry plate negatives and contribute to their preservation, updating established conservation practices. Archives of old photography studios, and photographers, are kept for their recognized historical and cultural value. These are mostly negative collections, which are frequently retouched. The research focused on several Portuguese negative collections from the first half of the twentieth century. It was divided into a three-way approach: 1, establish a historical framework to explain the retouching practices, their acceptance, criticisms, and how they relate with contemporary aesthetics, social-cultural dimensions and contribute to a better knowledge of the visual culture of the time; 2, review the practice of retouching to identify motivations, identification of materials and techniques and understand intended effects; 3, revise the established conservation procedures. Frequently not valued in the past, retouched photography exists since the birth of photography itself. Furthermore, although often criticized, retouching photography is an accepted part of the photographic process. However, it is not only relevant to explain and characterize a photographer's technique. While a hidden practice, retouching has a significant impact on the visual culture of its time and even how images and aesthetics of that time help evolve and influence photography today. Many authors identified the aproximation to the truth and the photographer's eye as the primary motivation. When photography was still debating its role in the arts, retouching was central to the discussion and used by each side as an argument both in favor and against. However, for the professional photographer, retouching negatives was necessary to please the client, assure flawless prints, and even a measure of pride in their work's quality. Fourier-transform infrared spectroscopy (FT-IR) was used to complement the identification of retouching materials. The result is a compilation of spectra that can be used as references in future studies. Some flaws in current conservation procedures have been identified, with consequences in the value and preservation of retouched negative collections. Conservation of archive collections can no longer be dissociated from digital dissemination. The conservator needs to take a more active role in advocating for collections' materiality in digital environments to ensure their preservation and truthful representation of the photographic cultural heritage. The retouching practices were kept forgotten for a long time. This work now shines a light on the importance of preserving retouched collections and open directions for future research in the fields of Conservation, Art History, and other Social Sciences.Este trabalho visa compreender melhor o retoque em negativos de gelatina e vidro e contribuir para a sua preservação, atualizando as práticas de conservação estabelecidas. Os arquivos de antigos estúdios fotográficos, , são preservados pelo seu reconhecido valor hitórico e enltura Tratasse cobremo colecoes de negativos, frequentemente retocados. A investigação focou-se em várias coleções de negativos portuguesas da primeira metade do século XX. Dividiu-se numa abordagem tripla: 1, estabelecer um contexto histórico que explique as práticas de retoque, a sua aceitação e críticas, e como se relacionam com a estética contemporánea, as suas dimensões social-culturais. e como contribuem para a cultura visual da época; 2, rever a prática de retoque, identificar motivações, materiais e técnicas e compreender os efeitos pretendidos; 3, rever procedimentos de conservação estabelecidos. Muitas vezes não valorizado no passado, o retoque existe desde o nascimento da própria fotografia. Além disso, embora muitas vezes criticado, o retoque de fotografia é aceite como parte do processo fotográfico. No entanto, não é apenas relevante para explicar e caracterizar a técnica de um fotógrafo. Embora seja uma prática escondida, o retoque tem impacto significativo na cultura visual do seu tempo e até na forma como as imagens e a estética desse tempo influenciam a cultura visual até à atualidade. Muitos autores identificaram a aproximação à verdade e ao olho do fotógrafo como as principais motivações para o retoque. Quando a fotografia ainda debatia o seu papel nas artes, o retoque foi fulcral para a discussão e usado por ambos lados como argumento tanto a favor como contra. No entanto, para o fotógrafo profissional, retocar os negativos era uma necessidade, para agradar ao cliente, para agradar o cliente, garantir a obtenção de provas perfeitas e até um motivo de orgulho na qualidade do seu trabalho. A Espectroscopia no infravermelho com transformada de Fourier (FT-IR) foi usada para complementar a identificação de materiais retoques. O resultado é a compilação de espectros que poderão ser usados como referências em estudos futuros. Foram identificadas algumas falhas nos atuais procedimentos de conservação, com consequências na valorização e preservação de coleções de negativos retocados. A conservação das coleções de arquivos já não pode ser dissociada da disseminação digital O conservador precisa de assumir um papel mais ativo na defesa e promoção da materialidade das coleções em ambientes digitais para garantir a preservação e verdadeira representação do património cultural fotográfico. As práticas de retoque foram mantidas esquecidas por muito tempo. Este trabalho pretende trazer a luz a importância de preservar coleções retocadas e abre caminhos para futuras investigações nas áreas da Conservação, História e Outras Ciências Sociais