In vitro 3D cancer spheroids generally exhibit a drug resistance profile similar to that found in solid tumors. Due to this property, these models are an appealing for anticancer compounds screening. Nevertheless, the techniques and methods aimed for drug discovery are mostly standardized for cells cultured in 2D. The development of 2D cell culture models displaying a drug resistant profile is required to mimic the in vivo tumors, while the equipment, techniques, and methodologies established for conventional 2D cell cultures can continue to be employed in compound screening. In this work, the response of 3D-derived MCF-7 cells subsequently cultured in 2D in medium supplemented with glutathione (GSH) (antioxidant agent found in high levels in breast cancer tissues and a promoter of cancer cells resistance) to Doxorubicin (DOX) is evaluated. These cells demonstrated a resistance toward DOX closer to that displayed by 3D spheroids, which is higher than that exhibited by standard 2D cell cultures. In fact, the 50% inhibitory concentration (IC50 ) of DOX in 3D-derived MCF-7 cell cultures supplemented with GSH is about eight-times higher than that obtained for conventional 2D cell cultures (cultured without GSH), and is only about two-times lower than that attained for 3D MCF-7 spheroids (cultured without GSH). Further investigation revealed that this improved resistance of 3D-derived MCF-7 cells may result from their increased P-glycoprotein (P-gp) activity and reduced production of intracellular reactive oxygen species (ROS).

Sertoli cells are crucial for the success of spermatogenesis, which is the biological process that ensures male fertility. These cells present high metabolic rates, being often subjected to high oxidative stress levels that, if uncontrolled, may compromise male fertility. Since the most abundant tea catechin, epigallocatechin-3-gallate (EGCG), has demonstrated a potent preventive activity against oxidative stress, we have evaluated its effect at concentrations of 5 and 50μM, on the metabolism, mitochondrial functionality and oxidative profile of human Sertoli cells (hSCs). While, the highest concentration of EGCG (50μM) increased glucose and pyruvate consumption, it decreased the conversion of pyruvate to alanine to sustain a regular lactate production. However, despite maintaining Krebs cycle functionality, EGCG (50μM) decreased mitochondrial membrane potential of hSCs, which could compromise the normal rates of ATP production. Interestingly, oxidative damages to proteins and lipids decreased in this experimental group, which may be valuable for the nutritional support of spermatogenesis.

Polyazamacrocycles are currently being studied and used in a variety of applications beyond their traditional place in supramolecular and co-ordination chemistry. This study suggests additional applications of these compounds with particular emphasis on their use as antiproliferative agents that could be potentially used to treat cancer. Four polyazamacrocycles were tested in human prostate cancer LNCaP and prostate epithelial PNTA1 cells to analyze changes in cell proliferation and cell death capabilities. Their intracellular localization was also evaluated by confocal microscopy. The results show a decrease in proliferation rate and cell viability of LNCaP and PNTA1, after treatment with these compounds. The decrease in the number of viable cells is similar for the majority of the compounds studied, and at higher concentration, the proliferation efficiency decreased significantly in the cell lines studied. Also, our results suggest that L and L2 induce early apoptosis in PNTA1 cells and late apoptosis/necrosis in LNCaP cells. The compounds did not induce a significant increase in necrosis of both cell types. Although the compounds did not localize in a unique organelle, all of them have as main target the Golgi apparatus and other localization profiles differed depending on the cell line.

The Notch-signaling ligand DLL1 has emerged as an important player and promising therapeutic target in breast cancer (BC). DLL1-induced Notch activation promotes tumor cell proliferation, survival, migration, angiogenesis and BC stem cell maintenance. In BC, DLL1 overexpression is associated with poor prognosis, particularly in estrogen receptor-positive (ER+) subtypes. Directed therapy in early and advanced BC has dramatically changed the natural course of ER+ BC; however, relapse is a major clinical issue, and new therapeutic strategies are needed. Here, we report the development and characterization of a novel monoclonal antibody specific to DLL1. Using phage display technology, we selected an anti-DLL1 antibody fragment, which was converted into a full human IgG1 (Dl1.72). The Dl1.72 antibody exhibited DLL1 specificity and affinity in the low nanomolar range and significantly impaired DLL1-Notch signaling and expression of Notch target genes in ER+ BC cells. Functionally, in vitro treatment with Dl1.72 reduced MCF-7 cell proliferation, migration, mammosphere formation and endothelial tube formation. In vivo, Dl1.72 significantly inhibited tumor growth, reducing both tumor cell proliferation and liver metastases in a xenograft mouse model, without apparent toxicity. These findings suggest that anti-DLL1 Dl1.72 could be an attractive agent against ER+ BC, warranting further preclinical investigation.

Although the generation of new neurons in the adult nervous system ('adult neurogenesis') has been studied intensively in recent years, little is known about this phenomenon in non-mammalian vertebrates. Here, we examined the generation, migration, and differentiation of new neurons and glial cells in the Mozambique tilapia (Oreochromis mossambicus), a representative of one of the largest vertebrate taxonomic orders, the perciform fish. The vast majority of new cells in the brain are born in specific proliferation zones of the olfactory bulb; the dorsal and ventral telencephalon; the periventricular nucleus of the posterior tuberculum, optic tectum, and nucleus recessi lateralis of the diencephalon; and the valvula cerebelli, corpus cerebelli, and lobus caudalis of the cerebellum. As shown in the olfactory bulb and the lateral part of the valvula cerebelli, some of the young cells migrate from their site of origin to specific target areas. Labeling of mitotic cells with the thymidine analog 5-bromo-2'-deoxyuridine, combined with immunostaining against the neuron-specific marker protein Hu or against the astroglial marker glial fibrillary acidic protein demonstrated differentiation of the adult-born cells into both neurons and glia. Taken together, the present investigation supports the hypothesis that adult neurogenesis is an evolutionarily conserved vertebrate trait.

Novel additive manufacturing processes are increasingly recognized as ideal techniques to produce 3D biodegradable structures with optimal pore size and spatial distribution, providing an adequate mechanical support for tissue regeneration while shaping in-growing tissues. With regard to the mechanical and biological performances of 3D scaffolds, pore size and geometry play a crucial role. In this study, a novel integrated automated system for the production and in vitro culture of 3D constructs, known as BioCell Printing, was used only to manufacture poly(ε-caprolactone) scaffolds for tissue engineering; the influence of pore size and shape on their mechanical and biological performances was investigated. Imposing a single lay-down pattern of 0°/90° and varying the filament distance, it was possible to produce scaffolds with square interconnected pores with channel sizes falling in the range of 245–433 µm, porosity 49–57% and a constant road width. Three different lay-down patterns were also adopted (0°/90°, 0°/60/120° and 0°/45°/90°/135°), thus resulting in scaffolds with quadrangular, triangular and complex internal geometries, respectively. Mechanical compression tests revealed a decrease of scaffold stiffness with the increasing porosity and number of deposition angles (from 0°/90° to 0°/45°/90°/135°). Results from biological analysis, carried out using human mesenchymal stem cells, suggest a strong influence of pore size and geometry on cell viability. On the other hand, after 21 days of in vitro static culture, it was not possible to detect any significant variation in terms of cell morphology promoted by scaffold topology. As a first systematic analysis, the obtained results clearly demonstrate the potential of the BioCell Printing process to produce 3D scaffolds with reproducible well organized architectures and tailored mechanical properties.

Glial cells are master regulators of synaptic plasticity and essential controllers of synapse formation and maintenance, and neuronal function. Gliomas are intrinsic brain tumors originating from the brain's and spinal cord's neuroglial progenitor cells. They constitute the majority of primary brain tumors and 80% of cancers of the central nervous system (CNS). This type of cancer can be classified into four categories according to the current World Health Organization (WHO) guidelines published in 2021: grades I and II represent low-grade gliomas, while grades III and IV represent high-grade gliomas (HGG). This latter carries an incidence rate of 3.2 per 100,000 individuals and makes up around 25% of all brain tumors. Recovery with minimal aggression on the patient's health is essential. Furthermore, the neurogenesis ability in the adult brain is extremely limited and highly restricted to a few regions. Cell reprogramming is a new promising, cell-specific therapy that induces cell fate conversion through the forced expression of transcription factors (TFs) and can be used to regain neuronal functional features from other cell types In this work, we studied different candidates to reprogram glioma cells into induced neurons in vitro and their capability of impeding tumor cell proliferation. Here we aimed to induce a targeted expression using a functional Cre-flex system that can infect U87-MG-Luc2 cells and lead to the overexpression of Cre and the selected TFs. Remarkably, we detected an increased tendency of expression of neuronal markers under these conditions and a decreased migration postreprogramming. In summary, this thesis highlights the promising role of transcription factors in converting glioma cells into neurons by which target reprogramming may impede tumor proliferation. This approach deepens our insight into glioma pathophysiology and enhances our understanding of cell reprogramming-based therapeutic strategies for brain tumors.

Abstract of Hospital Pharmacy Internship Report:This report, carried out within the scope of the "Curricular Internship" unit, inserted in the “Mestrado Integrado em Ciências Farmacêuticas” (MICF), consists of a reflection about the period of about 8 weeks that allowed the knowledge of the pharmacist's work as a health professional in the area of Pharmacy Hospital, more precisely in the public health agency that is the “Administração Regional de Saúde do Centro” (ARSC, I.P.). During this time, I was given the opportunity to join the team of Pharmacists, Diagnostic and Therapeutic Technicians and Operational Assistants, at the Central Pharmacy of the Office of Pharmacy and Pharmaceutical Services of ARSC, IP. The choice of this stage stems from a desire to integrate a team of hospital pharmacists and realize how important their work is in the mission to bring health care to the best possible and equal way to the patient, giving priority to the correct use of the medicine. This report is based on a SWOT analysis (Strenghts, Weaknesses, Opportunities, Threats), allowing the reader to clearly and objectively understand the way the institution operates, the work performed by the trainee and the importance was retained during this period and could be used in future work situations in the pharmaceutical area. Each topic in this analysis is duly identified by the initials S, W, O, and T for the acronym SWOT. Summary of Internship Reports in Community Pharmacy: In the final stretch of all these years of learning, students are put to the test in the real environment of exercising the pharmaceutical profession, in what is their place par excellence: Community Pharmacy (CF). It is in this way that we prepare ourselves for a future as a pharmacists, acquiring an active vision of how is the day to day contact with the user and what is required for the execution of the various tasks that aim at the proper functioning of a CF. This report presents a critical reflection of the activity carried out as a trainee in CF within the scope of the curricular unit in the curriculum of this Masters Degree. This stage was presented as the first opportunity for direct contact with the work performed in a CF in all its aspects, from the backoffice tasks to the pharmacotherapeutic advice. Thus, during a period of 4 months between March and June 2018, I joined the work team of the Pharmacy Sitália in Coimbra, and had the opportunity, with experienced professionals, to acquire new skills and experience common situations in CF practice , under the guidance of Dr. Susana Sousa. This moment in my training constituted, then, a form of reconciliation of the theoretical knowledge learned over the last years with practical effect on the diversity of services provided by the Pharmacy Sitália, as well as at the organizational and resource management level in CF; besides this I was allowed to verify the social character inherent to the profession as well as the importance of communication with the user for a health gain of the community that enjoys the exclusive services of the Pharmacy. This report is also presented in the form of a SWOT analysis, showing internally the strengths (Strenghts) and weaknesses (Weaknesses), and at the external level, Opportunities and threats (Threats) relating to my experience in CF and the method of work implemented in the Pharmacy Sitália. Each topic in this analysis is duly identified by the initials S, W, O, and T for the acronym SWOT.Abstract of the thesis titled “H4R as a new target on cancer therapy”: Cancer continues to be a major health problem, accounting for 1 in 7 deaths worldwide. According to the World Health Organization, in 2012, approximately 14.1 million new cases were diagnosed and 8.2 million cancer-related deaths ocurred, with current estimates pointing to 21.6 million of new cases in 2030.The discovery of the histamine H4 receptor and its presence in different tissue types and malignant cell lines has led to the assumption of its involvement in the processes of cell proliferation, angiogenesis, immunosuppression and metastasis. Thus, numerous research institutes have been struggling to evaluate the pharmacological activity of H4R ligands in experimental models, in order to find new approaches in the therapy of this disease.

In this paper we propose a mathematical model to describe the evolution of glioma cells taking into account the viscoelastic properties of brain tissue. The mathematical model is established considering that the glioma cells are of two phenotypes: migratory and proliferative. The evolution of the migratory cells is described by a diffusion-reaction equation of non Fickian type deduced considering a mass conservation law with a non Fickian migratory mass flux. The evolution of the proliferative cells is described by a reaction equation. A stability analysis that leads to the design of efficient protocols is presented. Numerical simulations that illustrate the behavior of the mathematical model are included.

Human mesenchymal stem cells (hMSCs) have been proposed as possible therapeutic agents for central nervous system (CNS) disorders. Recently, it has been suggested that their effects are mostly mediated through their secretome, which contains a number of neuroregulatory molecules capable of increasing cell proliferation, differentiation, and survival in different physiological conditions. Here, we present an overview of the hMSC secretome as a possible candidate in the creation of new cell-free therapies, demonstrating the process of its collection and route of administration, focusing our attention on their effects in CNS regenerative medicine.