Repositório Científico de Acesso Aberto da ULisboa
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Testing miRNA-engineered exosomes as regulators of inflammation in Alzheimer’s disease research models
No full textTese de mestrado, Bioquímica e Biomedicina , 2024, Universidade de Lisboa, Faculdade de CiênciasAlzheimer’s disease (AD) is a progressive neurodegenerative disorder marked by cognitive decline and
chronic inflammation, with no treatments able to halt disease progression. Several pro- and antiinflammatory microRNAs (miRNAs), such as miRNA(miR)-124-3p, have been found dysregulated in
AD. Previously, our laboratory identified elevated levels of miR-124-3p in SH-SY5Y neuroblastoma
cells with the Swedish mutation (APP695) and in PSEN1 mutant neurons. Interestingly, when
upregulated, miR-124 improved AD-associated features, causing a reduction in APP expression, Tau
phosphorylation and amyloid species. Moreover, soluble and exosomal miR-124 were able to protect
microglia activation by interferon-gamma stimulation.
To explore the NLRP3-autophagy axis in the context of AD, we assessed relevant markers in the
hippocampus and prefrontal cortices of 6- and 9-month-old female and male 5xFAD mice. Additionally,
glial activation and synaptic markers were assessed to identify the best conditions for testing our therapeutic
approach. We then assessed the effectiveness of the retro-orbital delivery of miR-124-transfected neural
exosomes (miR-124-EXOs) in wild-type mice. Lastly, 5xFAD mice were treated with mock/miR-124-
transfected EXOs, and cognitive performance was evaluated using behavioral tests. 5xFAD mice at 9-
months of age displayed the greatest alterations in messenger RNA (mRNA) levels of Nlrp3/Il1b/Becn1.
mRNA levels of Trem2/P2yr12/Cx3cr1/Gfap/Il6/Asc were most significantly increased in female mice.
Conversely, Dlg4/Syp mRNA levels were decreased. Western blots of TREM2/S100B supported the
activation of glial cells in females and downregulated levels of PSD95 in the hippocampus indicated
synaptic loss. Retro-orbital delivery of miR-124-EXOS successfully increased the expression of miR124 up to 14 days post injection. Delivery of mock/miR-124-transfected EXOs to 9-month-old female
5xFAD mice was able to prevent cognitive decline. This study contributes to the understanding of the
NLRP3-autophagy axis and inflammatory phenotype of 5xFAD mice. Moreover, it highlights miR-124-
EXOs delivery via retro-orbital route as a promising approach to slow AD progression and cognitive loss
Role of Short-Chain Fatty Acids and Ffar2 in Dendritic Cell Metabolism and Function
No full textTese de mestrado, Bioquímica e Biomedicina, 2024, Universidade de Lisboa, Faculdade de CiênciasDendritic Cells (DCs) play a pivotal role in the regulation of the immune system, acting as a conduit
between innate and adaptive immunity by presenting antigens to naïve T cells. Research has
demonstrated that Short Chain Fatty Acids (SCFAs), particularly butyrate, significantly regulate immune
responses. This project aims to examine the role of butyrate in DC activation and function, as well as its
potential in promoting immune tolerance, particularly through its interaction with regulatory T (Treg)
cells. Bone marrow-derived progenitors from mice were cultured with the growth factor FMS-like
Tyrosine Kinase (FLT3-L) alone or in combination with Granulocyte-Macrophage Colony-Stimulating
Factor (GM-CSF) and exposed to varying butyrate concentrations. Flow Cytometry (FACS) enabled the
acquisition of data on DC composition and activation, focusing on the expression of several surface
markers. Western Blot (WB) was conducted to assess butyrate’s impact on acetylation. RT-qPCR was
employed to examine the gene expression associated with anti-inflammatory cytokines, DC metabolism
enzymes, and SCFA receptors, all linked to immune tolerance. Co-culture experiments assessed the
influence of butyrate pre-treated DCs on Foxp3+ Treg cell differentiation from naïve CD4+ T cells.
Results demonstrate that butyrate influences DC activation and functionality. Increased CD86
expression in iCD103-DC cultures and CD80 expression in FLT3-L DC cultures suggest that butyrate
modulates different activation pathways depending on the cell type. Butyrate induced a tolerogenic
profile in LPS-induced DCs, resulting in reduced IL-12 and TNF-α at gene and protein levels. WB
confirmed butyrate inhibits histone deacetylases (HDACs), resulting in increased acetylation of histone
3 (H3) and other proteins. This may be associated with changes in gene expression that favour a
tolerogenic environment. In the co-culture experiments, DCs treated with butyrate exhibited enhanced
efficacy in inducing Treg cells, indicating that butyrate conditions DCs to promote immune tolerance.
These findings suggest that butyrate profoundly influences immune responses
Synthetically-defined glycoconjugate vaccine candidates against cancer
No full textCancer is a major cause of death worldwide and despite the several treatments currently available (such as chemotherapy and hormonal treatments), these are often associated with severe side effects and often have poor results. Thus, more effective strategies are needed.
In the las years anti-cancer vaccines have gained a lot of interest, as these can act as preventive or therapeutic treatments, by triggering the immune system to attack the cancer cells, hence being a promising approach to fight cancer.
Importantly, as a result of its overexpression and shorter glycans in cancer cells, MUC1 is currently a drug target for many cancers, including its use as an antigen for the development of anticancer vaccines. However, therapeutic vaccines based on MUC1 face several challenges, as the natural tumor-associated MUC1 (taMUC1) is poorly immunogenic, and these derivatives can act as self-antigens, which can lead to immunosuppression.
Additionally, polysaccharide-based vaccines fail to generate memory, as polysaccharide antigens only interact with specific B-cells promoting low-affinity humoral responses. Hence, when conjugated, glycoconjugate vaccines have some advantages over traditional polysaccharide-based vaccines, such as triggering T cell dependent responses leading to the establishment of B-cell memory and longerlasting immune responses.
To overcome these issues, an artificial MUC1 was synthesized and conjugated to gold nanoparticles to be used as anti-cancer vaccine. The construct was tested for its ability to promote an immune response against human tumor associated MUC1 in mice.
With ELISA assays, we found that the synthetic MUC1 was able to trigger the production of specific antibodies against human tumor-associated MUC1. Furthermore, the anti-sera from the immunized mice were able to bind to the natural MUC1 antigen in human breast cancer cell lines and in tumor samples from breast cancer patients, and also were able to elicit antibody-dependent cell mediated cytotoxicity towards breast cancer cells.
Having obtained these promising results with the unnatural MUC1, another vaccine candidate was prepared using the same artificial MUC1 antigen conjugated with the highly immunogenic protein carrier CRM197, as the protein carrier was expected to trigger T-cell responses on its own, hence leading to a stronger immune response. Once again, specific antibodies against human MUC1 were detected, and these were able to specifically bind to the MUC1 expressed in human breast cancer cells and to promote cytotoxic T Lymphocyte responses. As predicted, antibodies against the protein carrier were also detected in high levels. Additionally, when tumors were induced in mice immunized with this second vaccine candidate, a delay in tumor growth was observed.
These encouraging results will lead to further studies on the potential therapeutic effect of these vaccine candidates
Cianobactérias e Legionella: sinergia ou antagonismo?
No full textTese de mestrado, Biologia Humana e Ambiente, 2024, Universidade de Lisboa, Faculdade de CiênciasAs cianobactérias são procariotas fotossintéticos, constituintes do fitoplâncton, que em condições
favoráveis crescem massivamente formando blooms frequentemente associados a toxicidade. A
Legionella é uma bactéria oportunista que ocorre em ambientes naturais ou artificiais e que causa
legionelose através da inalação de gotículas de água contaminada. As cianobactérias e a
Legionella são omnipresentes no ambiente aquático, mas a sua relação ainda é pouco conhecida.
Este trabalho teve como objetivos avaliar o papel dos blooms cianobacterianos na ocorrência de
Legionella em albufeiras e estudar a atividade antibacteriana de cianobactérias contra estirpes
clínicas (2), ambientais (4) e de referência (2) de Legionella pneumophila.
Foram recolhidas amostras de água em 5 albufeiras do Alentejo nas 4 estações do ano, para
identificar, quantificar e isolar cianobactérias (microscopia ótica), Legionella (métodos culturais
e qPCR) e para determinar cianotoxinas (ELISA). Os blooms de cianobactérias e as cianotoxinas
foram frequentes em todas as albufeiras, sobretudo no verão e primavera. A Legionella spp. foi
detetada em todas as amostragens. Porém, não foi observada uma relação entre a ocorrência de
cianobactérias/ cianotoxinas e L. pneumophila, que apenas foi detetada no outono, em 2
albufeiras.
A atividade anti-Legionella de extratos de 15 estirpes de cianobactérias da coleção ESSACC,
preparados com diferentes solventes [n-Hexano, DCM:MetOH (2:1), MetOH 70% e H2O, por
ordem de polaridade crescente (A) e decrescente (B)] foi avaliada pelo método de Difusão em
Disco. Identificou-se atividade inibitória do crescimento das 8 estirpes de L. pneumophila nos
extratos LMECYA 182 (Dolichospermum spp.) preparados com MetOH 70% B e DCM:MetOH
A. Adicionalmente, ambos os extratos potenciaram o efeito dos antibióticos levofloxaciana e
ciprofloxacina nas 8 estirpes de L. pneumophila.
A ocorrência de blooms cianobacterianos não influencia, aparentemente, a ocorrência de L.
pneumophila nas albufeiras, mas foi identificada uma relação de antagonismo entre a
cianobactéria LMECYA 182 e estirpes de L. pneumophila, com potencial interesse
biotecnológico.Cyanobacteria are photosynthetic prokaryotes, constituents of phytoplankton, which under
favorable conditions grow massively, forming blooms often associated with toxicity. Legionella
is an opportunistic bacterium that occurs in natural or artificial environments and causes
legionellosis through inhalation of droplets of contaminated water. Cyanobacteria and Legionella
are ubiquitous in the aquatic environment, but their relationship is still poorly understood.
This work aimed to evaluate the role of cyanobacterial blooms in the occurrence of Legionella in
reservoirs and to study the antibacterial activity of cyanobacteria against clinical (2),
environmental (4) and reference (2) strains of Legionella pneumophila.
Water samples were collected in 5 reservoirs in Alentejo in the 4 seasons of the year, to identify,
quantify and isolate cyanobacteria (optical microscopy), Legionella (cultural methods and qPCR)
and to determine cyanotoxins (ELISA). Cyanobacterial blooms and cyanotoxins were frequent in
all reservoirs, especially in summer and spring. Legionella spp. was detected in all samples.
However, a relationship was not observed between the occurrence of cyanobacteria/cyanotoxins
and L. pneumophila, which was only detected in autumn, in 2 reservoirs.
The anti-Legionella activity of extracts from 15 cyanobacteria strains from the ESSACC
collection, prepared with different solvents [n-Hexane, DCM:MetOH (2:1), MetOH 70% and
H2O, in order of increasing (A) and decreasing polarity (B)] was evaluated by the Disk Diffusion
method. Growth inhibitory activity of the 8 strains of L. pneumophila was identified in LMECYA
182 (Dolichospermum spp.) extracts prepared with MetOH 70% B and DCM:MetOH A.
Additionally, both extracts potentiated the effect of the antibiotics levofloxacin and ciprofloxacin
on the 8 strains of L. pneumophila.
The occurrence of cyanobacterial blooms does not apparently influence the occurrence of L.
pneumophila in reservoirs, but an antagonistic relationship was identified between the
cyanobacterium LMECYA 182 and strains of L. pneumophila, with potential biotechnological
interest
Atividades ao ar livre em família: equipamento lúdico em parques urbanos
No full textO presente projeto debruça-se sobre a atividade de brincar ao ar livre e a sua importância no
desenvolvimento infantil, bem como aquisição de capacidades e valores necessários à interação com
outras crianças, adultos e na sociedade em geral.
Com a pandemia instalaram-se novos hábitos e novas rotinas que ainda perduram. Crianças e famílias
refugiaram-se no conforto e na segurança do lar e, neste contexto específico, passaram a dedicar ainda
mais tempo aos ecrãs digitais promovendo um estilo de vida mais sedentário e intensificando a
dependência destes gadgets, democratizando-se o acesso e a sua banalização em todas as faixas etárias.
No sentido de contrariar esta tendência, propõe-se o conceito de um parque urbano intergeracional,
onde crianças e adultos poderão partilhar o tempo e as brincadeiras, ao ar livre. Em família, pretende-se
que os novos equipamentos sejam apelativos e capazes de influenciar positivamente as crianças e
envolver os pais, avós e cuidadores nas dinâmicas do brincar, enquanto realizam uma supervisão proativa
e integrada.
Para concretizar estas ideias utilizaram-se diversas metodologias. Num primeiro momento realizou-se a
revisão da literatura e a recolha de informação sobre os equipamentos existentes no mercado. Ainda na
fase exploratória fez-se a análise de casos de estudo e a investigação ativa com a visita de quatro escolas
na região da Grande Lisboa e as entrevistas aos especialistas. Utilizou-se também o método Fly on the
Wall para realizar o trabalho de campo e a observação do público alvo, em contexto escolar. O
desenvolvimento projetual resultou na elaboração de uma proposta de conceito e respetivas maquetes e
protótipos de estudo que possibilitaram a validação dos especialistas e, consequentemente, a integração
das suas recomendações.
O presente projeto confirma a pertinência do tema e a capacidade que as crianças têm de criar novas
dinâmicas nas famílias, sendo que a brincadeira passa a incluir também os adultos. Este novo conceito
para a fruição dos parques urbanos procura reconquistar o tempo e o gosto pelas brincadeiras
despendidos ao ar livre, em detrimento dos espaços fechados e sedentários, em prol da saúde física e
mental das crianças e das famílias.This project focuses on the activity of playing outdoors and its importance for children's development, as
well as acquiring the skills and values needed to interact with other children, adults and society in general.
With the pandemic, new habits and routines were established that still persist. Children and families took
refuge in the comfort and safety of home and, in this specific context, began to spend even more time on
digital screens, promoting a more sedentary lifestyle and intensifying dependence on these gadgets,
democratizing access and making it commonplace for all age groups.
In order to counteract this trend, we propose the concept of an intergenerational urban park, where
children and adults can share time and play outdoors. As a family, the aim is for the new facilities to be
appealing and capable of positively influencing children and involving parents, grandparents and
caregivers in the dynamics of play, while providing proactive and integrated supervision.
Several methodologies were used to make these ideas a reality. Firstly, a literature review was carried out
and information was gathered on the equipment on the market. Still in the exploratory phase, case studies
were analyzed and active research was carried out by visiting four schools in the Lisbon area and
interviewing specialists. The Fly on the Wall method was also used to carry out fieldwork and observe the
target audience in a school context. The design development resulted in the preparation of a concept
proposal and respective mock-ups and study prototypes that enabled the experts to validate and,
consequently, integrate their recommendations.
This project confirms the relevance of the theme and the ability of children to create new dynamics in
families, with play now also including adults. This new concept for the enjoyment of urban parks seeks to
recapture the time and taste for play spent outdoors, to the detriment of closed and sedentary spaces, in
favor of the physical and mental health of children and families.N/
Edifício principal revertido em hotel
No full textO presente Projeto Final de Mestrado desenvolve uma proposta de reabilitação no Quartel do Regimento de Lanceiros 2, na freguesia de Belém.
O quartel está desativado e insere-se numa área com valor patrimonial e histórico, atraindo várias pessoas a visitar os polos turísticos existentes.
Assim, propõe-se programas que revitalizem o edificado, salvaguardando a sua memória e identidade, integrando-o na envolvente.
Na relação do edificado com a comunidade e o meio urbano, pretende-se dar uma resposta às necessidades da população local e nacional, promovendo o desenvolvimento cultural e económico.
Em suma, procura-se resolver esta problemática de desuso através de uma reflexão urbana e populacional, suportada em princípios que permitam a reutilização deste edifício respeitando a sua história e identidade.This Master's Final Project develops a rehabilitation proposal in the 2nd Lancers Regiment Barracks, in the parish of Belém.
The barracks is deactivated and is located in an area with heritage and historical value, attracting several people to visit the existing tourist centers.
Therefore, programs are proposed that revitalize the building, safeguarding its memory and identity, integrating it into the surroundings.
In the relationship between buildings and the community and the urban environment, the aim is to respond to the needs of the local and national population, promoting cultural and economic development.
In short, we seek to resolve this problem of disuse through urban and population reflection, supported by principles that allow the reuse of this building while respecting its history and identity.N/
Artificial intelligence in personalised cosmetics
No full textTrabalho Final de Mestrado Integrado, Ciências Farmacêuticas, 2024, Universidade de Lisboa, Faculdade de Farmácia.A história da cosmética tem sido marcada pelo progresso e, atualmente, tem seguido um caminho que anda de mãos dadas com a indústria tecnológica. Esta evolução criou uma indústria caracterizada pela tecnologia da beleza onde técnicas avançadas de inteligência artificial e de aprendizagem digital são utilizadas para analisar e compreender diferentes elementos de beleza e cosméticos com vista a fornecer a melhor resposta.
As formulações personalizadas de cuidados da pele com o uso de Inteligência Artificial têm vindo a ganhar popularidade na indústria da beleza e dos cuidados da pele, em consequência do interesse dos consumidores pelos seus produtos de cuidados da pele e pela sua própria saúde. O segmento de mercado de inteligência artificial em beleza e cosméticos apresentou um crescimento exponencial, com receitas de US 7,79 bilhões em 2028, crescendo a uma taxa composta de crescimento anual de 19,6%. O crescimento do sector do comércio eletrónico, a crescente adoção da realidade aumentada para fazer compras, o envelhecimento da população e o aumento dos rendimentos disponíveis; são os principais fatores que se prevê que impulsionem o crescimento, enquanto as preocupações com a privacidade são vistas como uma potencial barreira ao crescimento.
A aplicação de inteligência artificial abrange, atualmente, desde o fabrico e desenvolvimento ao marketing pós-venda. As principais empresas que já assumiram a liderança neste sector estão a utilizar inteligência artificial para o aconselhamento de produtos ao consumidor, ensaios/amostragem de produtos e descoberta de novos ingredientes. Deste modo, é possível obter uma melhor compreensão sobre os hábitos dos consumidores, o desenvolvimento de estratégias de mercado, a concetualização de produtos, a otimização de formulações e muito mais para a investigação e desenvolvimento de cosméticos.
Com os avanços e a descoberta de novos caminhos, surgem desafios. Um deles é o facto de o Regulamento Europeu de Cosméticos n.º 123/2009 da União Europeia não se pronunciar sobre a personalização, levantando muitas questões relativamente à notificação destes produtos e criando uma zona cinzenta, abrindo uma porta para futuros desenvolvimentos.The history of cosmetics has been marked by progress, and today it has followed a path that
goes hand in hand with the technology industry. This evolution created an industry characterised by beauty tech where advanced artificial intelligence and machine learning techniques are used to analyse and comprehend different elements of beauty and cosmetics to provide the best response.
Personalised skin care formulations using artificial intelligence have been growing in popularity in the beauty and skin care industry driven by consumers' growing interest in their skin care products and health. The artificial intelligence in beauty and cosmetics market segment has witnessed exponential growth, with revenues surging from 7.79 billion by 2028, growing at a compound annual growth rate of 19.6%. The growth of the e commerce sector, the increasing adoption of augmented reality for shopping, the growing aging population, and rising disposable i ncomes are the key factors predicted to drive growth, while privacy concerns are seen as a potential barrier to growth.
The application of artificial intelligence ranges from manufacturing and development to marketing after sales. The major companies that have already taken the lead in this sector are using artificial intelligence from consumer facing product advice and product trials/sampling to ingredient discovery, better understanding of consumer habits, market strategy development, product conceptualisation , formulation optimisation and more for cosmetics research and development
With these advances and the discovery of new paths come challenges. The European Union Regulation No 123/2009 is silent on personalisation, raising questions regarding the notification of these products and creating a grey area in the regulation and ethical considerations, opening a door for future developments
Alginate rubber mask as a triple friend: cosmetic, medicine and skin barrier protector
No full textTrabalho Final de Mestrado Integrado, Ciências Farmacêuticas, 2024, Universidade de Lisboa, Faculdade de Farmácia.O alginato, um polissacarídeo naturalmente abundante, é reconhecido por sua excepcional capacidade de retenção de água e pelas suas funções como agente gelificante, viscosante e estabilizador, tornando-o valioso em diversos setores, incluindo o farmacêutico e cosmético. Este estudo explora o desenvolvimento de uma “máscara de borracha” à base de alginato, desenvolvida como uma solução de duplo propósito: um dispositivo médico para proteção da pele e um medicamento de aplicação tópica para tratar patologias cutâneas, como as causadas pelo uso prolongado de equipamentos de proteção individual (EPI). A máscara incorpora ingredientes ativos, especificamente ácido salicílico e metronidazol, conhecidos pelas suas propriedades anti-inflamatórias, queratolíticas e antimicrobianas.
A “máscara de borracha” à base de alginato foi desenvolvida e reconstituída com quatro águas potáveis com diferentes propriedades físico-químicas (pH, turbidez, dureza total e perfil mineral). Usando o analisador de Textura TA.XT plus, foram avaliadas características-chave como espalhabilidade, firmeza, força de ruptura e adesão, enquanto as medições de pH foram realizadas com um medidor de pH digital. Estudos de libertação in vitro foram realizados usando células de difusão de Franz, demonstrando que a formulação otimizada proporcionou uma libertação lenta e sustentada dos ingredientes ativos. O estudo não demonstrou diferença significativa no tempo de secagem entre as formulações. Testes reológicos provaram que a formação da máscara não depende da temperatura, mas sim do tempo, e que o tempo de secagem foi cerca de 10 minutos para todas as águas testadas. A formulação reconstituída com água muito dura resultou na maior viscoelasticidade, indicando uma estrutura de gel mais forte (G'=6390 Pa). Todas as formulações apresentaram boa espalhabilidade e, quando secas, a água muito dura apresentou uma alta força de ruptura (0,626 ± 0,041 N), seguida pela água dura (0,542 ± 0,013 N). As diferenças entre os tipos de água podem ser devido ao seu conteúdo em níveis de pH e dureza.
Os resultados sugerem que esta máscara de borracha à base de alginato uma abordagem inovadora é promissora para proteger e tratar a pele, particularmente em condições associadas ao uso de EPI.Alginate, a naturally abundant polysaccharide, is recognized for its exceptional water-retention capacity and its roles as a gelling, viscosifying, and stabilizing agent, making it valuable in various sectors, including pharmaceuticals and cosmetics.
This study explores the development of an alginate-based rubber mask designed as a dual-purpose solution: a medical device for skin protection and a therapeutic tool for treating skin conditions such as those caused by prolonged use of personal protective equipment (PPE). The mask incorporates active ingredients, specifically salicylic acid and metronidazole, known for their anti-inflammatory, keratolytic, and antimicrobial properties.
The alginate-based rubber mask was developed and reconstituted with four drinking waters with different physicochemical properties (pH, turbidity, total hardness and mineral profile). Using the TA.XT plus Texture Analyzer, key characteristics such as spreadability, firmness, burst strength, and adhesion were assessed, while pH measurements were conducted using a digital pH meter. In vitro release studies were performed using Franz diffusion cells), revealing that the optimized formulation provided a slow and sustained release of active ingredients. The study demonstrated no significant difference in drying time between formulations. Rheological tests proved that the mask is not temperature-dependent for its formation to occur, but time-dependent and that the drying time was around 10 minutes in all tested waters. The formulation reconstituted with very hard water resulted in the highest viscoelasticity, indicating a stronger gel structure (G’=6390 Pa.s). All formulations presented good spreadability, and when dry, very hard water presented a high rupture force (0.626 ± 0.041N), followed by hard water (0.542 ± 0.013 N). Differences between water types may be due to their content in pH levels and hardness.
The findings suggest that this alginate-based rubber mask holds promise as an innovative approach to both protect and treat the skin, particularly under conditions associated with PPE use