uBibliorum
Repositório Digital da Universidade da Beira Interior
Entradas recentes
A Ansiedade na Era Digital: A Transformação da Angústia à Luz de Jean-Paul Sartre
Publication . Oliveira, Thaís de Sá; Nascimento, André Barata; Castro, Fernando José Gastal de
A era digital instaurou um novo regime de presença e ação, no qual a liberdade humana se exerce sob mediações técnicas e algorítmicas que antecipam, modulam e quantificam cada ato. Nesse contexto, o problema filosófico que orienta esta investigação consiste em compreender como a experiência originária da angústia – pela qual o ser humano se descobre condenado à liberdade – foi historicamente transfigurada em ansiedade digital, uma forma difusa de alienação e sofrimento que caracteriza a subjetividade contemporânea. Trata-se de interrogar o modo de ser do para-si em um mundo em que as mediações técnicas precedem e orientam a transcendência, convertendo a liberdade em cálculo e a escolha em probabilidade. O objeto da tese é examinar a relação entre liberdade, angústia e ansiedade no pensamento de Jean-Paul Sartre, em diálogo com o contexto técnico e cultural da era digital. A análise concentra-se nas obras de Sartre produzidas entre 1936 e 1960 – da psicologia fenomenológica à Crítica da Razão Dialética – período em que se estruturam as categorias centrais de sua fenomenologia existencial: intencionalidade, emoção, imaginação, liberdade, má-fé, temporalidade e o prático-inerte. Essas categorias são reinterpretadas à luz das mediações digitais contemporâneas, trazendo a filosofia de Sartre ao diálogo com teóricos da cibercultura e da sociologia do tempo, como Pierre Lévy, Manuel Castells, Jean Baudrillard, Sherry Turkle, Marc Augé, Hartmut Rosa, Byung-Chul Han, e André Barata. A hipótese central sustenta que a ansiedade digital constitui um desdobramento histórico e ontológico da má-fé – entendida como a tentativa da consciência de escapar à angústia inerente à liberdade. Argumenta-se que a ansiedade emerge como conduta psicofísica pela qual o ser humano busca atenuar o confronto com o nada que o fundamenta, ao mesmo tempo em que interioriza as coerções de um mundo tecnicamente mediado. Assim, a liberdade acaba por se traduzir em heteronomia técnica, reproduzida pelas estruturas do prático-inerte algorítmico. A pesquisa adota uma abordagem tripla: fenomenológica, para descrever a ansiedade digital como experiência intencional da consciência, evitando reduzi-la à patologia; hermenêutica, para reinterpretar as categorias centrais de Sartre no contexto contemporâneo da tecnologia e das redes digitais; e crítico-interdisciplinar, para dialogar com as teorias da aceleração, da atomização e da precariedade temporal, integrando-as à ontologia fenomenológica da liberdade. A trajetória argumentativa se desenvolve ao longo de seis capítulos interligados. O primeiro reconstrói a psicologia fenomenológica do jovem Sartre, examinando a intencionalidade, a emoção, a imaginação e a categoria do mágico como fundamentos ontológicos da liberdade. O segundo analisa a ontologia fenomenológica de O Ser e o Nada: Ensaio de Ontologia Fenomenológica, abordando liberdade, angústia, má-fé e intersubjetividade. O terceiro desenvolve a dialética entre liberdade e alienação por meio do método progressivo-regressivo e das mediações do prático-inerte. O quarto transpõe essa dialética para o contexto digital, interpretando o ciberespaço como manifestação contemporânea do prático-inerte e analisando seu papel na estruturação da experiência. O quinto investiga a crise da temporalidade na modernidade tardia, articulando a noção sartriana de ek-stasis com as teorias da aceleração, da atomização e da precariedade. Por fim, o sexto propõe uma interpretação fenomenológica da ansiedade digital como experiência psicofísica diante da angústia existencial, examinando manifestações paradigmáticas – como avatares, ghosting, doomscrolling e FOMO – e suas implicações ético-políticas. A tese demonstra que a ansiedade digital não é um sintoma clínico, mas um desdobramento histórico da má-fé, na qual a liberdade se oculta sob as mediações técnicas do algoritmo. Ao integrar a ontologia fenomenológica de Sartre às estruturas da cibercultura, o trabalho oferece uma interpretação original da subjetividade contemporânea como experiência de liberdade alienada – em que o sujeito busca escapar da indeterminação refugiando-se nas garantias do código. Sua principal contribuição consiste em oferecer a primeira sistematização filosófica da ansiedade digital como atualização histórica da fuga da angústia existencial pela má-fé, reposicionando a ontologia sartriana no centro dos debates contemporâneos sobre tecnologia, temporalidade e sofrimento na era digital.
Potentially Inappropriate Prescribing in Elderly: Application of STOPP/START Criteria in Portuguese Patients
Publication . Candeias, Catarina Diogo; Alves, Gilberto Lourenço; Ferreira, Amílcar Celta Falcão Ramos; Rodrigues, Márcio José de Abreu Marques
The ageing of the population is a multifactorial phenomenon, primarily resulting from low birth rates and an increase in average life expectancy. While the increase in life expectancy reflects the success of public health measures, population ageing leads to greater morbidity and an increased incidence of chronic diseases. This raises concerns about the sustainability of patient management and health systems. Chronic diseases are more prevalent in older people (those aged 65 or over) and result in multimorbidity, which leads to the simultaneous use of several drugs. Polypharmacy — defined as the use of five or more drugs — is associated with an increased risk of adverse drug reactions, drug-related problems, and potentially inappropriate prescribing. Adverse drug reactions are responsible for hospital admissions, health costs, morbidity and mortality, even though more than half of them are preventable. The management of these patients with an increasingly complex pharmacotherapeutic profile requires a careful approach, particularly when transitioning care, requires the definition and monitoring of an individual intervention plan for each patient. In Portugal, where the average life expectancy is slightly higher than the European average, the National Network for Integrated Continuing Care (RNCCI) was created in 2006 by the Ministries of Labour and Social Solidarity, and Health. The RNCCI comprises a group of public and private institutions that provide long-term healthcare and social support to the patients leaving hospital or joining from the community who are dependent and require assistance with mental, social and/or physical limitations. Although this support is independent of age, a significant proportion of patients are elderly. The RNCCI has more than 10,000 inpatient beds, which are distributed across several Units for Integrated Continuous Care (UCCI) nationwide. However, research into the medication of these patients is still lacking, particularly on polypharmacy and potentially inappropriate prescribing. This is also associated with the occurrence of adverse drug reactions, the risk of hospitalisation and readmission, high costs, a lower quality of life and even higher mortality. Potentially inappropriate prescribing is a growing concern in terms of potentially inappropriate medication (PIM), overprescribing (prescribing medication without a clear clinical indication) and incorrect prescribing (prescribing an indicated medication when the risk outweighs the benefit, or when a safer or more effective alternative is available); it also refers to potential prescribing omissions (PPO), or underprescribing (failing to prescribe a beneficial medication for which there is a clear clinical indication). Various tools have been developed in the context of clinical practice to identify potentially inappropriate prescriptions. One of the first to be developed was the Beers list, which has been widely used. However, as many of the medicines included in this list are not marketed in most European countries, its applicability is limited. The STOPP/START criteria (Screening Tool of Older People’s Prescriptions/Screening Tool to Alert to Right Treatment) can identify PIM and PPO, both of which can have harmful consequences for patients. The updates of the criteria have been shown to cover PIM with greater clinical significance. Despite the relevance of these criteria, they are rarely applied in Portugal and are practically non-existent in specific contexts, such as UCCI. Therefore, this study aimed to address several gaps in Portuguese research, particularly regarding UCCI patients, by analysing the prescriptions of 180 patients from eight different UCCI. Firstly, the relationship between demographic and clinical characteristics and medication use patterns, including polypharmacy was analysed. On average, eight drugs were prescribed per patient, and around 90% of patients were polymedicated. Multivariate logistic regression revealed that polypharmacy was significantly associated with UCCI when two institutions were compared, as well as with the Charlson Comorbidity Index (CCI). The CCI is an index resulting from the sum of age weighting (one point for each decade over the age of 50, up to a maximum of four points) and a weighting assigned to various diseases (from one to six). It predicts the 10-year survival rate of patients with multiple comorbidities: higher scores indicate more serious health conditions and, consequently, a worse prognosis. Then, the prevalence of PIM and PPO in elderly patients was determined by applying the STOPP/START criteria, after which potential predictors were investigated. PIM was found to have a prevalence of 85.1%, mainly involving drugs that act on the central nervous system and psychotropic drugs. PPO had a prevalence of 81.4%, mainly involving drugs that act on the musculoskeletal and cardiovascular systems. Logistic regression analysis showed that the predictors of PIM were female gender, hospital admission and the number of medications taken. In turn, PPO was significantly associated with CCI and a recent history of fractures. Finally, we explored differences in the prevalence and associated factors of PIM and PPO between the patients aged 75–84 years and the oldest-old patients (i.e., aged 85 or over). The oldest-old patients were more dependent on activities of daily living and had a higher CCI. However, patients aged 75–84 patients took more daily drugs and had higher oral doses; additionally, patients in this age group were more likely to have been hospitalized and to be obese. Although PIM was lower in older patients (≥85 years), the prevalence of PIM and PPO did not differ significantly between the two age groups. The most prevalent cases of PIM and PPO were the same, but older patients with a history of falls were more likely to have a PPO associated with an omitted vitamin D prescription. Logistic regression analysis showed that the number of medications was a common predictor of PIM. For PPO, male gender and risk of falls were predictors in the 75–84 age group, while number of comorbidities was a predictor in the group aged 85 or over. This study aims to improve the quality of healthcare provided to the elderly by highlighting the need to optimise drug therapy with a holistic but individualised approach. The dynamic interaction between ageing, multimorbidity, polypharmacy and inappropriate prescribing can result in significant harm, both economically and, more importantly, in terms of patients' lives. Adopting good prescribing practices alongside the early identification of PIM and PPO could reduce risks and improve patients' health outcomes. Furthermore, identifying predictors of inappropriate prescribing could provide valuable information for the development of future health policies and clinical practices tailored to Portugal's growing elderly population.
Characterization of Effector-Memory CD8+ T cells and their Association with Human Cognitive Function
Publication . Esgalhado, André João Gabriel ; Arosa, Fernando Aguilar; Uhrberg, Markus
Human effector-memory CD8+ T cells consist of highly differentiated cells that differ in the expression of the tyrosine phosphatase isoform CD45RA, being designated as CD8+ TEM and CD8+ TEMRA cells. These highly heterogeneous and polyfunctional cells possess cytotoxic, regulatory, and suppressive features, and are capable of migrating to non-lymphoid tissues and organs, including the brain under certain conditions. Expansions of CD8+ TEM and CD8+ TEMRA cells have been described in chronic inflammatory diseases, tumors and viral infections, as well as in healthy elderly individuals, including centenarians. Over the past few decades, the role of CD8+ T cells, particularly CD8+ TEMRA cells, has been the subject of several studies in the context of aging, cognition, and neurodegeneration, where they have been generally regarded as detrimental to the central nervous system (CNS), though recent investigations have challenged this view. These highly differentiated CD8+ T cells are known to arise through TCR-dependent and TCR-independent mechanisms, such as cytokine-driven proliferation via interleukin (IL)-15. Intriguingly, chronic antigenic stimulation has been shown to drive the generation of CD8+ T cells expressing low levels of the CD8β chain, though antigen-independent mechanisms remain poorly understood. Herein, we have performed a comprehensive characterization of peripheral blood mononuclear cells (PBMC) as well as of human leukocyte antigen (HLA) molecules in a cohort of elderly volunteers differing in their cognitive status. A detailed analysis of the level of expression of CD45RA in the CD8+ TEMRA compartment revealed the presence of two distinct populations: CD8+ TEMRAlow and CD8+ TEMRAhigh cells. Notably, CD8+ TEMRAhigh cells formed a well-defined and sharply delineated population that was significantly expanded in cognitively impaired volunteers, whereas cognitively unimpaired volunteers were enriched in CD8+ TEMRAlow cells. Further analysis of CD8α and CD8β expression also identified the existence of two distinct CD8+ T cells subsets based on the expression of CD8β: CD8αβlow and CD8αβhigh T cells, with the former being more prevalent among cognitively unimpaired individuals. Moreover, stimulation with PMA and Ionomycin revealed significantly increased IFN-γ production by CD4+ T cells from cognitively impaired elderly. Noteworthy, all but one of the volunteers studied were cytomegalovirus (CMV) seropositive. Finally, a higher prevalence of the HLA-B8 serotype, which belongs to the ancestral haplotype HLA-A1, Cw7, B8, DR3, DQ2, was found among the cognitively impaired elderly. Additionally, we assessed the impact of IL-15 on the cell surface expression of CD8β using CFSE-labeled purified human naïve CD8+ T cells cultured for 12 days. IL-15 induced a robust proliferation and differentiation, resulting in a cell cycle-dependent down-modulation of CD8β from the cell surface, while CD8α expression remained stable or increased slightly. This led to the generation of CD8αβlow and CD8αβ– (i.e., CD8αα) T cells. In contrast, IL-2 and IL-7 alone were unable to replicate this effect. Determination of mRNA levels for CD8α and CD8β isoforms by qPCR revealed that IL-15 promoted a significant decrease in mRNA levels of the CD8β M-4 isoform, while increasing the levels of the M-1 and M-2 isoforms alongside with CD8α. Remarkably, analysis of the level of the tyrosine kinase Lck showed a significant increase in CD8+ T cell blasts after culture of CD8+ T cells with IL-15, when compared to CD8+ T cells at the beginning of the culture. Our findings show an association with certain CD8+ T cell subsets that is compatible with a protective role in cognition and neurodegenerative diseases by identifying novel markers that define discrete subsets of highly differentiated CD8+ T cells expanded in cognitively unimpaired elderly individuals and identify IL-15 as a factor involved in the generation of these subsets. In-depth phenotypic, functional, and transcriptomic characterization of ex vivo and in vitro obtained CD8+ T cell subset is warranted to further elucidate their unique functional properties.
Portuguese Natural Resources as Modulators of Acne Vulgaris Hallmarks
Publication . Oliveira, Ana Sofia Domingues ; Oliveira, José António Martinez Souto de; Oliveira, Ana Cristina Palmeira de; Teixeira, João Paulo Fernandes
Acne vulgaris, commonly known as acne, is a chronic skin disease of the pilosebaceous unit, that affects millions worldwide, particularly adolescents and young adults. Clinically, acne is characterized by non-inflammatory and inflammatory lesions, predominantly on the face, but also affecting the neck, chest, and upper back. Beyond its signs and symptoms, acne is also associated with a substantial psychological and emotional burden. The pathogenesis of the disease is multifactorial and involves a complex interplay of several biological events or hallmarks. Traditionally, four mechanisms have been associated with its development, namely: increased sebum production, follicular hyperproliferation/hyperkeratinization, colonization by Cutibacterium acnes bacterium (formerly Propionibacterium acnes), and the release of inflammatory mediators. However, recent research has further complicated the disease’s pathophysiology, with new factors such as changes in sebum composition, differential involvement of innate and adaptive immunity, and dysbiosis of the follicular microbiota, being increasingly described by the scientific community.
Given its multifactorial nature, different therapeutic strategies are adopted depending on the severity of the disease. Topical treatments, when used alone, are typically reserved for comedonal or non-inflammatory acne, while in combination topical therapy is considered the first-line approach for mild to moderate acne. In more severe cases, systemic treatments are used as first-line options, with isotretinoin being the standard choice despite its severe risks and side effects. These adverse effects associated with classical acne therapies, together with a behavioral shift among consumers who increasingly prefer more “natural” and/or “green” solutions, have driven both the cosmetic and pharmaceutical industries to invest in the research and development of alternatives or adjuvants to conventional treatments that meet these expectations. Plant-derived ingredients have gained particular attention and, among them, essential oils (EOs), which are secondary metabolites from aromatic plants, stand out for their spectrum of bioactive properties and are extensively used across various health fields, including dermatology.
Based on this premise, the aim of this doctoral project was to investigate the bioactive potential of preparations from aromatic and medicinal plants produced in Portugal, with a particular focus on their ability to modulate the main hallmarks involved in acne pathophysiology. Two autochthonous Portuguese species, namely Thymus mastichina (L.) L. (Thymus mastichina; TM) and Cistus ladanifer L. (Cistus ladanifer; CL), and the allochthonous hybrid Thymus × citriodorus (Pers.) Schreb. (Thymus × citriodorus; TC) were investigated for their anti-acne potential, through the study of their EOs in addition to their hydrolates, by-products of EO production, thus aligning this work with the principles of circular economy.
Initial screenings of the autochthonous taxa revealed that CL EO, rich in α-pinene, exhibited the highest anti-inflammatory potential (EC₅₀ = 0.002% v/v), although with associated cytotoxicity in murine macrophage cell model (RAW 264.7; IC₅₀ = 0.012% v/v). TM preparations, mainly composed by 1,8-cineole, showed balanced anti-inflammatory effects with superior biocompatibility in both murine macrophages and fibroblasts (L929). CL EO showed the highest antimicrobial activity, with minimum inhibitory concentrations (MICs) ranging from 0.06% (v/v) to 2% (v/v) against the tested microorganisms, which included those used in the Challenge test for cosmetics and representatives of skin microbiota.
Regarding the preparations from the allochthonous hybrid, TC EO, mainly composed of geraniol, stood out for its antimicrobial activity against acne-associated bacteria (C. acnes, Staphylococcus epidermidis and S. aureus) and for its ability to prevent and disrupt C. acnes biofilms, even at sub-inhibitory concentrations. TC hydrolate retained some bioactive potential, particularly in modulating C. acnes biofilms, still being deprived of relevant antimicrobial activity.
Considering the higher anti-acne potential of TC EO, and to address variability concerns relevant to large-scale application of EOs, four TC EOs from different Portuguese regions were compared. Although all shared geraniol as a major compound, significant differences in yield, efficacy, and cytotoxicity were observed, underscoring the need for standardization protocols. Still, all EOs exhibited antimicrobial activity, with particular effectiveness against C. acnes, as evidenced by MICs ranging from 0.016% (v/v) to 0.031% (v/v), corroborating the anti-acne potential of this hybrid.
Building upon previous results and considering the anti-acne potential of the studied preparations, their ability to modulate key acne-related events was further explored using more complex in vitro models that better mimic in vivo conditions. These closer-to-disease models further explored the preparations’ ability to modulate sebum production in human sebocytes (SZ95 cell line), C. acnes-induced inflammation and oxidative stress, keratinocyte proliferation, and bacterial adhesion. Additionally, potential antimicrobial interactions between the different preparations along with their cytotoxic and mutagenic potential were also investigated.
Regarding anti-acne efficacy, comprising the above-described models, EOs exhibited overall greater bioactivity compared to their corresponding hydrolates. The EOs of TC and CL stood out for their ability to modulate all the studied hallmarks, demonstrating a multitarget potential. Although less potent, the hydrolates still showed relevant activity, particularly the one from CL, displaying multi-target potential, failing only in inhibiting lipase activity. Regarding the combined antimicrobial activity of the preparations against C. acnes, EO–EO combinations showed consistent synergistic effects (FICIs between 0.250 and 0.375), while the presence of additive interactions depended on the specific combinations. As for safety, TC and CL EOs were the least biocompatible upon human keratinocytes and sebocytes, and all preparations were considered non-mutagenic, except for CL EO, which showed a “likely-positive” result in one of the Salmonella typhimurium strains included in the Ames test.
Based on the higher overall efficacy of the tested EOs, the final experimental work focused on potential phylotype-selective efficacy of EOs against clinical C. acnes strains, with different virulence patterns. Phylotype IA1 strains (typically associated with acne lesions), exhibited higher lipase activity and biofilm formation than phylotype II strains (more associated with healthy skin). However, not all virulence factors were phylotype-dependent, which supports the multifactorial nature of this disease. TC EO showed the lowest MICs across all strains (MICs between 0.03% v/v and 0.06% v/v) when compared to the other EOs, demonstrating less variation in MIC values between phylotypes. It was also able to reduce both biofilm biomass and metabolic activity from IA1 phylotype strains, being effective at lower concentrations. These results further support the relevance of TC EO for this application, despite its lack of phylotype-dependent selectivity.
In conclusion, this work provides evidence for the anti-acne potential of Portuguese plant preparations, particularly TC and CL EOs, supported by their capacity to modulate different pathophysiological mechanisms associated with disease development. This research also contributes to the scientific valorization of Portuguese natural resources and supports their integration into sustainable dermocosmetic formulations aligned with the principles of circular economy.
The Bridge Between Nanotechnology and Chronobiology: Circadian Control of Cancer Therapy by Gene Delivery Systems
Publication . Neves, Ana Raquel Bastos; Costa, Diana Rita Barata; Paixão, Telma Alexandra Quintela; Fan, Donglei Emma
Cancer is nowadays the second leading cause of death worldwide, making it a significant public health concern. Europe, following Asia, is, according to the Global Cancer Observatory (GLOBOCAN), the second continent where the incidence, mortality, and prevalence are highest. Statistics indicate that the number of cancer-related deaths in Europe is projected to increase 36.4% by 2050 compared to 2022. In particular, glioblastoma (GB), designated by the World Health Organization (WHO) as a grade 4 astrocytoma, represents the most prevalent (more than 50% of the total cases) and highly aggressive type of primary brain cancer in adults. This cancer is characterized by a high inter- and intratumourally genetic heterogeneity, aggressiveness, angiogenesis, invasiveness, resistance to current standard treatment protocols, poor patient prognosis (12-15 months), and low survival rate. Molecularly, several altered signalling pathways (p53 tumour suppressor pathway inclusive), gene mutations (tumour suppressor p53 gene (TP53), epidermal growth factor receptor (EGFR), isocitrate dehydrogenase (IDH)-1/2, etc.), and methylation of O6-methylguanine-DNA methyltransferase (MGMT) promoter contribute to its hyperproliferation, growth, and chemotherapeutic resistance. For these reasons, actual care treatments, as the classical Stupp protocol, face several challenges. This protocol focuses on first removing the tumour by surgical resection, without compromising the normal neurological function, followed by radiotherapy and chemotherapy with Temozolomide (TMZ), whose effectiveness depends on the MGMT gene promotor methylation status. However, after being subjected to this, patients prognosis remains very poor, and 90% of them have a tumour recurrence within two years. Thus, the development of new and more effective therapeutic approaches is essential to fight this disease. Innovative therapeutic strategies as nanotechnology, gene therapy, and chronotherapy, have been developed and explored to improve the treatment efficacy of GB. Gene therapy consists of delivering exogenous nucleic acids to cells to activate, silence, modify, or edit certain genes and correct genetic defects that may contribute to disease progression. In this case, correcting abnormal gene expression, since several mutations have been associated with glioblastoma, seems a promising approach to treat it. However, delivering nucleic acids to cells is difficult unless delivery systems are used. Nanotechnology, with the use of non-viral vectors (cell-penetrating peptides (CPPs), liposomes, polymers, exosomes, dendrimers, etc.), leads to ground-breaking possibilities for a precise delivery of anticancer drugs and nucleic acids to specific sites, overcoming several physiological barriers, as the blood-brain barrier (BBB). Targeting tumour-specific receptors using ligands at the delivery systems surface can minimize side effects and improve therapeutic responses. Additionally, in the past few years, the study of cancer cells circadian rhythms, normally disrupted, has shown that the circadian clock as a significant role in cancer development and therapeutic efficacy. Circadian rhythm is defined as the approximately 24 h oscillation of physiological and metabolic processes, which are synchronized with the Earth's diurnal cycle. Several studies indicated that rhythms could modulate drug’s effectiveness and their side effects, and delivery systems cellular uptake and efficacy. Studies have shown that some chemotherapy drugs are better tolerated or more effective at certain times of the day. Accordingly, the main object of this doctoral thesis was to design and develop a delivery system, functionalized to target glioblastoma cells, and to evaluate the influence of circadian rhythms on the efficacy of targeting, internalization, cargo release, and, ultimately, therapeutic effect for a precise cancer therapy strategy.
This approach focused on the use of a non-viral CPP, namely the WRAP5 peptide, for the construction of a delivery system bearing a transferrin (Tf) receptor ligand sequence (TfR) to co-deliver the anticancer TMZ drug and a plasmid DNA (pDNA) coding for p53 to glioblastoma cells. This system was designed to present an improved brain cell targeting ability and cellular uptake and to penetrate the brain barriers easily. The first step consisted of acquiring its physicochemical properties (size, polydisperse index, surface charge, and complexation capacity), morphology, and biocompatibility. Results revealed these properties to be under the influence of the N/P ratio, which can be optimized to improve complexes desired characteristics. These formulations demonstrated appropriate physicochemical characteristics for in vitro applications, and confocal microscopy using U87 glioblastoma cells confirmed their ability to internalize into cells and deliver the pDNA into the nucleus. Following nuclear localization, successful transcription and translation of the TP53 were observed. The resulting complexes significantly reduced the viability of glioblastoma cells. In a three-dimensional (3D) 9-day U87 spheroid model, generated by two different protocols, complexes were shown to have some effect on the spheroids morphology and size after a single dose treatment. Moreover, complexes have been revealed to be biocompatible with several non-cancerous cells and with zebrafish Danio rerio embryos. Experiments with other two glioma cell lines (SNB19 and U373) also highlighted the complexes ability for internalization and p53 expression levels increase. In these cell lines, apoptosis activation by the intrinsic pathway was implicated. This thesis also aimed to elucidate the influence of circadian core clock components in the performance of developed peptide nanocomplexes, namely, TfR expression, complexes internalization, and p53 expression promotion. The obtained results demonstrated that, at specific time-points, the highest circadian activity of Period circadian regulator 2 (PER2) and TfR led to higher cellular uptake of complexes, TP53 expression induction, and consequently p53 expression.
In summary, our comprehensive dataset provides strong evidence supporting the high potential of TMZ/TP53 co-delivery WRAP5 complexes for targeted cellular transfection, p53 expression, and for effectively triggering apoptotic pathways, holding promising therapeutic value toward glioblastoma. Moreover, aligning the timing of complexes administration with the circadian rhythms of GB cells may significantly enhance cellular uptake and gene/protein expression. This chronobiologically optimized approach offers a promising possibility for developing more precise and impactful treatment strategies against glioblastoma. Future preclinical research should prioritize the study of delivery systems bioavailability, tumour targeting, and pharmacokinetics at specific times of day in patient-derived 3D models and in vivo xenograft models. Additionally, it will be relevant to study the temporal expression of glioblastoma biomarkers and the effects of their knockout on survival and therapeutic outcomes. Understanding how brain barrier dynamics, such as permeability, for example, oscillate with circadian changes and impact delivery systems uptake, will bring noticeable improvements to this strategy. Ultimately, this research should instigate complexes clinical translation toward more effective glioblastoma therapy.