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New signaling pathways associated with mitochondrial dysfunction induced by obesogens in Sertoli cells
Publication . Bastos, Ana Luísa Marques de; Rato, Luís Pedro Ferreira; Sousa, Ana Catarina; Silva, Branca Maria Cardoso Monteiro da
In recent years, obesity has emerged as a significant public health concern, particularly with regard to its effects on male fertility. Of particular interest is the role of obesogens, a class of environmental chemicals implicated in the development of obesity. These compounds are increasingly recognized as major contributors to excessive weight gain, primarily due to their function as endocrine-disrupting chemicals (EDCs), which interfere with hormonal regulation and metabolic processes. Tributyltin (TBT), a welldocumented obesogen, is recognized as one of the most toxic compounds introduced into ecosystems. Owing to its lipophilic properties, TBT exhibits a strong tendency to bioaccumulate in lipid-dense tissues, including the testes. Upon accumulation in these organs, TBT can interfere with critical physiological and metabolic processes necessary for spermatogenesis and steroidogenesis. This disruption may lead to detrimental reproductive outcomes, including elevated oxidative stress (OS) within the testes and the manifestation of sperm abnormalities. Sertoli cells (SCs), which play a crucial role in providing nutritional and structural support to developing and maturing germ cells, are especially susceptible to these disruptions. SCs have been identified as primary targets of environmental toxicants, which can significantly impair their structural integrity and functional capacity. Furthermore, the hypothalamic-pituitary-testicular (HPT) axis, commonly known as the reproductive axis, exhibits marked sensitivity to metabolic disruptions induced by environmental toxicants, potentially resulting in compromised reproductive function. This study sought to elucidate the relationship between TBT exposure and male infertility by investigating the deleterious effects of this obesogen on SCs mitochondrial function and its broader implications for male reproductive health. To accomplish this objective, an animal model was employed to assess the expression of key proteins involved in the regulation of mitochondrial biogenesis and the activation of the antioxidant defense system. Two concentrations of TBT were evaluated: 0,1 nM, a sub-toxic dose known to activate the retinoid X receptor/peroxisome proliferatoractivated receptor gamma (RXR/PPAR?) heterodimer, and 10 nM, a concentration comparable to the levels detected in the serum and tissues of certain individuals. The results demonstrated that exposure to the highest concentration of TBT (10 nM) elicited the most pronounced effects on SCs. Nevertheless, significant alterations were also observed at the sub-toxic concentration of 0,1 nM, indicating that even low-level exposure to TBT can induce substantial cellular and functional changes. The antioxidant capacity of Sertoli cells (SCs) was assessed, revealing a reduction following exposure to both concentrations of TBT (10 nM and 0,1 nM). Additionally, oxidative stress-induced damage was evaluated, encompassing measurements of protein carbonylation and lipid peroxidation. Lipid peroxidation was found to decrease following exposure to the highest concentration of TBT (10 nM), whereas a slight increase was observed at the lower concentration (0,1 nM). Regarding protein oxidation, levels of carbonylation decreased in response to both concentrations of TBT. Several molecular regulators were also affected by TBT exposure, with increased expression observed for sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1a) at both concentrations. In contrast, levels of sirtuin 3 (SIRT3) were found to be diminished following TBT exposure. Additionally, other regulators of PGC-1a were also impacted, including a decrease in CREB-binding protein (CBP) at both concentrations. Notably, acetylated CREB-binding protein (A-CBP) and homolog 2 of the general control of amino acid synthesis in yeast (GCN2) exhibited an increase at 10 nM TBT, while levels decreased at the lower concentration of 0,1 nM. Conversely, the p300/CBP-associated factor (PCAF) demonstrated an inverse pattern, with levels increasing at 0.1 nM TBT and decreasing at 10 nM. The results indicate that exposure to TBT induces significant alterations in the expression of key regulators of mitochondrial biogenesis within reproductive organs, thereby disrupting the molecular pathways essential for mitochondrial function. This dysregulation compromises the integrity of the antioxidant defense system, which is crucial for cellular homeostasis. Expanding our understanding of mitochondrial bioenergetics is therefore essential for devising strategies to combat the increasing prevalence of male infertility, particularly in developed countries, where metabolic disorders are increasingly acknowledged as significant public health concerns.
Development and production of 3D-printed composite scaffolds enriched with Beeswax for bone tissue regeneration
Publication . Francisco, Martinho Jorge ; Moreira, André Ferreira; Correia, Ilídio Joaquim Sobreira; Cabral, Cátia Solange Duarte
The regeneration of bone defects caused by trauma or disease is a significant challenge in modern medicine. Conventional therapeutic approaches like bone grafting (including autologous, allogeneic, and xenogeneic grafts), are widely used but have limitations such as donor scarcity, development of immune reactions, and risks of disease transmission, such as hepatitis, acquired immunodeficiency syndrome, bacterial infections, and bovine spongiform encephalopathy commonly known as “mad cow disease”. Bone tissue engineering has emerged as a promising alternative, focusing on developing bioactive scaffolds that mimic the native structure of bone. To this end, various therapeutic solutions under development aim to replicate the characteristics of native bone, namely the inorganic phase (i.e., calcium phosphate in the form of hydroxyapatite) and the organic phase (e.g., collagen type I, bone cells, and non-collagenous proteins), to support the natural bone healing process. This study aimed to attain the production of 3D scaffolds displaying suitable properties for bone tissue engineering, using a novel composite mixture comprising tricalcium phosphate (TCP), hydroxyapatite (HAp), sodium alginate (SA), beeswax (BW), and thymol (TM). The scaffolds were fabricated through a rapid prototyping technique, using the Fab@Home 3D-Plotter extruder via a top-down approach. The results demonstrated that the 3D-printed scaffolds exhibited well-defined structural and morphological features, with a porosity suitable for cell attachment and growth. Furthermore, scaffolds containing BW and TM showed rougher surfaces, which are more favorable for cell adhesion. The mechanical properties of the scaffolds, including compressive strength and Young’s modulus, were within the range required for bone regeneration. Otherwise, biological assays showed that the scaffolds were cytocompatible and were able to support human osteoblast cell adhesion and proliferation. In addition, the scaffolds containing TM demonstrated significant antibacterial activity, against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria, making them an attractive option for preventing infections in bone healing applications. In conclusion, this study allowed the successful production of novel 3D-printed scaffolds composed of TCP, HAp, SA, BW, and TM. Compared to previous work, the addition of BW resulted in structures with improved mechanical properties, especially in humid conditions. In addition, BW also allowed the optimization of various physicochemical properties such as hydrophobicity and surface roughness, favoring cell growth. On the other hand, the incorporation of TM demonstrates the potential of this phenolic compound for application in bone regeneration, as a viable alternative to conventional antibiotic-based methods to prevent the establishment of bacterial infections and biofilm development. In this way, the results show that the scaffolds produced, incorporating materials of natural origin, could be highly effective for future clinical applications in bone tissue regeneration.
Evaluation of the Permeability of Brain Barriers to Compounds with Anticancer Properties
Publication . Carreira, Maria Carolina Lareiro; Duarte, Ana Catarina Abreu; Santos, Cecília Reis Alves dos
Brain tumors remain one of the most aggressive and lethal forms of cancer, posing significant challenges to treatment due to their critical location, infiltrative nature, and resistance to conventional therapies. One of the main factors limiting the effectiveness of treatment in brain tumors is the difficulty of chemotherapeutic agents reaching the central nervous system in adequate concentrations. Brain barriers, despite being essential for central nervous system protection and homeostasis, have a high selective nature that restricts the ability of chemotherapeutic agents to reach tumors, reducing the available therapeutic options. This restricted permeability is mostly mediated by the ABC transporter superfamily, which is expressed in both the blood-brain barrier and the blood–cerebrospinal fluid barrier and actively extrudes a wide variety of substances out of the brain, including many anticancer compounds. Taste receptors, traditionally associated with oral cavities, are also expressed in extraoral tissues, including the brain and many tumors. Lactisole, a well-known inhibitor of sweet taste receptors, affects cancer cell proliferation, possibly due to the high glucose dependency of these cells. Quercetin, a bitter compound that is a known ligand of TAS2R14, has demonstrated potential therapeutic effects by overcoming chemoresistance mechanisms and inducing apoptosis in cancer cells. This study aimed to investigate the permeability of the blood-brain and blood– cerebrospinal fluid barriers to lactisole and quercetin, and whether quercetin's interaction with TAS2R14 affects ABC transporter expression. For that a human choroid plexus papilloma cell line (HIBCPP) and a human cerebral microvascular endothelial cell line (hCMEC/D3) were used to establish in vitro models that mimics the in vivo phenotypic characteristics of the blood–cerebrospinal fluid barrier and the blood-brain barrier, respectively. It was verified that lactisole did not reduce viability in the HIBCPP cell line but in hCMEC/D3 cells, a decrease in viability was observed. Through transepithelial electrical resistance measurements and immunocytochemistry, it was possible to optimize the blood-brain barrier model. The 6th day of culture proved to be the most appropriate for the permeability assays, presenting the highest integrity of the cell monolayer and the highest levels of TAS2R14 expression. Additionally, RT-PCR was performed to confirm TAS2R14 expression in both cell lines. The permeability of the barriers to quercetin was not determined, due to completely degradation after 3 hours. Nevertheless, the expression levels of ABCC1 and ABCC4 were assessed in the blood–cerebrospinal fluid barrier cell line following TAS2R14 knockout and subsequent exposure to quercetin. However, no alterations in ABCC1 and ABCC4 expression in mock or TAS2R14 silenced conditions were detected. Lastly, lactisole permeability was assessed in the blood–cerebrospinal fluid barrier model, and a threefold higher concentration was observed in the apical compartment after 4 hours. Thus, it was concluded that lactisole crosses the blood–cerebrospinal fluid barrier in a timedependent manner.
Híbridos de Biginelli com potencial ação para o melanoma
Publication . Mateia, Ana Carolina Passos; Matias, Mariana Ruivo; Silvestre, Samuel Martins
O melanoma é a principal causa de morte relacionada com o cancro de pele e apresenta taxas de incidência e mortalidade crescentes. O tratamento do melanoma beneficia de várias opções terapêuticas como cirurgia, radioterapia, quimioterapia, terapia direcionada e imunoterapia. No entanto, devido ao facto do melanoma apresentar uma elevada resistência aos tratamentos atualmente utilizados, é imperativo descobrir e desenvolver novas abordagens terapêuticas que se revelem mais eficientes e seguras. Neste contexto, a hibridação molecular tem-se revelado uma abordagem encorajadora no desenvolvimento de novos fármacos com potencial ação anticancerígena. Esta estratégia consiste na fusão covalente de dois ou mais farmacóforos para criar uma única molécula que apresenta múltiplos mecanismos de ação. Deste modo, neste estudo, é proposta a síntese de diversos híbridos, tendo como base a reação de Biginelli. Esta reação multicomponente baseia-se na ciclocondensação entre um aldeído, um ßcetoéster/acetilacetona e ureia/tioureia, da qual se podem obter diversos híbridos de Biginelli com uma ampla diversidade de atividades biológicas, nomeadamente anticancerígena. Assim, com o intuito de desenvolver novos compostos antitumorais, procedeu-se com sucesso à síntese de doze híbridos de Biginelli, combinando o farmacóforo 3,4-dihidropirimidin-2-(1H)-ona/tiona, com o núcleo tiofeno ou furano. A atividade antiproliferativa, em diferentes linhas celulares, bem como a atividade inibitória da xantina oxidase e atividade antioxidante, foram avaliadas de forma a elucidar a potencial ação de cada molécula. Na sua generalidade, os compostos revelaram ser seletivos, tendo-se verificado que a presença de átomos de cloro nas moléculas pode ser importante para a sua atividade anti-proliferativa nas células humanas de melanoma (SK-MEL-28), sem comprometer, de forma significativa, a viabilidade dos fibroblastos normais da derme humana (NHDF). De entre os compostos testados (incluindo aqueles que foram sintetizados neste projeto e outros análogos que já tinham demonstrado atividade noutras linhas celulares), o CM 8 e CM 11, ambos derivados de tiofeno, e MM 55, derivado diclorado de benzeno, revelaram ser os mais promissores, tendo demonstrado citotoxicidade acentuada nas células SK-MEL-28 (IC50=1,28 e 1,71 µM, respetivamente para CM8 e CM 11 e IC50<0,01 µM para MM 55). Para além disso, estes compostos não apresentaram atividade anti-proliferativa relevante nas células NHDF (IC50=35,14, 55,89 e 100,4 µM, respetivamente), o que se traduz num índice de seletividade bastante elevado. De seguida, um estudo preliminar do potencial mecanismo de ação anti-proliferativa demonstrou que a atividade inibitória da xantina oxidase foi inferior a 20%, com destaque para os derivados com uma cadeia lateral maior, quer de tiofeno (ex, MM96 e MM 106, com inibição de 18,77 e 18,71%), quer de furano (ex, MM 93 e MM 99 com inibição de 18,14 e 17,62%, respetivamente). Adicionalmente, através da avaliação da ação antioxidante dos compostos, verificou-se que eles podem desempenhar a sua atividade anti-proliferativa, pelo menos em parte, através da sua ação oxidante. Foi ainda possível verificar que os derivados diclorados (MM 54, MM 55, MM 56, MM 57 e MM 81) foram aqueles que exibiram menor atividade oxidante. No geral, este estudo demonstrou que os híbridos de Biginelli testados exibem potencial anti-melanoma e que poderão servir de base para investigações futuras. De facto, os híbridos poderão ser otimizados com base nos resultados obtidos, tendo em consideração o potencial demonstrado pelos derivados diclorados e tiofeno. Para além disso, será também relevante realizar ensaios que clarifiquem o(s) mecanismo(s) de ação destes candidatos a fármacos.
Caracterização de leveduras colonizadoras da vulva e avaliação de fatores de risco para a infeção
Publication . Silva, Maria Margarida Santos; Mateus, Joana Rita Gonçalves Araújo Rolo
Os casos de infeções fúngicas, com particularidade em infeções causadas por espécies do género Candida spp., nas últimas décadas, têm aumentado a sua incidência no mundo. A Candidose Vulvovaginal é uma infeção vaginal, causada por leveduras do género Candida, prevalente em mulheres em idade fértil, que afeta milhões de mulheres no mundo. Caracteriza-se pela ocorrência de sintomas na vagina e na vulva, como prurido, inchaço, irritação da vulva e uma secreção espessa e irregular. Classicamente, os sintomas na vulva estão associados à disseminação do fluido vaginal infetado. No entanto, estudos mais recentes descrevem a ocorrência de sintomas vulvares associados à recuperação da levedura deste nicho sem envolvimento vaginal. Assim, de modo a compreender melhor o papel da vulva nessa dinâmica, neste estudo, objetivamos identificar e caracterizar isolados de leveduras vulvares, através da suscetibilidade aos antifúngicos, da formação de biofilmes e produção de fosfolipases e hemolisinas, assim como avaliar a adesão, in-vitro, às células HeLa de isolados clínicos. Os resultados indicaram a presença de uma diversidade de espécies de leveduras Candida não - albicans; entre os 46 isolados vulvares, foram identificadas 11 espécies diferentes. A espécie mais frequentemente recuperada entre as amostras vulvares foi Candida parapsilosis (25%), seguida por Nakaseomyces glabratus (15%) e Meyerozyma guilliermondii (15%). No geral, verificou-se que 45% dos isolados vulvares eram resistentes ao fluconazol. A espécie Clavispora lusitanae foi a que produziu uma maior quantidade de biomassa de biofilme. Já a Pichia kudriavzevii foi a única espécie que produziu fosfolipase e detetou-se a produção de hemolisinas em isolados pertencentes a Nakaseomyces glabratus, Candida parapsilosis, Candida intermedia, Meyerozyma guilliermondii, Candidozyma haemulid e Clavispora lusitanae. Finalmente, verificou-se que Pichia kudriavzevii e Clavispora lusitanae aderiram às células HeLa. A grande diversidade de espécies encontrada, aliada à elevada taxa de resistência ao fluconazol e ao perfil virulento, leva a concluir que se considera necessária uma vigilância epidemiológica, para auxiliar o desenvolvimento de estratégias terapêuticas mais eficazes.