FCS - DCM | Dissertações de Mestrado e Teses de Doutoramento
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- Androgens and calcium-binding protein regucalcin in the control of prostate cells metabolism and survival: implications in carcinogenesis.Publication . Vaz, Cátia Alexandra Vicente; Socorro, Sílvia Cristina da Cruz Marques; Baptista, Cláudio Jorge MaiaProstate Cancer (PCa) is one of the most common cancers in men and continues to be a source of considerable morbidity and mortality worldwide. The progression of PCa from an early stage, confined to prostate, to a more aggressive phenotype is characterized by the loss of androgen-responsiveness, which means that tumor cells gain the ability to growth independently of the circulating androgens. Furthermore, the neoplastic transformation of prostate cells and tumor progression to more aggressive stages require several genetic and metabolic alterations. Warburg firstly demonstrated that tumor cells predominantly use glycolysis for obtaining energy in detriment of oxidative phosphorylation, with the production of high amounts of lactate. This metabolic adaptation is common to the majority of cancers and is now established as a hallmark of cancer. However, the alterations in the glycolytic metabolism that occur in the progression to the androgen-independent stage of PCa as well as the role of androgens in regulating the glycolytic flux are poorly studied. Regucalcin (RGN) is a calcium (Ca2+)-binding protein that plays a determinant role in the maintenance of intracellular Ca2+ homeostasis. RGN also has been suggested as a tumor suppressor protein, since it is involved in the regulation of proliferation, apoptosis, and diverse metabolic pathways, and its antioxidant properties also have been reported. In addition, androgens, Ca2+ and age, all well-known factors implicated in PCa development, regulate RGN expression in different tissues but their action in prostate remains to be elucidated. It was also demonstrated that RGN is under expressed in human PCa cases comparatively to normal tissues, which suggest that the loss of RGN may be an event favoring tumor development. Moreover, considering the biological processes under the influence of RGN it is also predictable that it may exert a crucial role in the maintenance of cell tissue homeostasis. Notwithstanding, little is known about the factors that regulate RGN expression in the prostate, and no studies exist regarding the role of RGN in the regulation of cell proliferation, apoptosis, Ca2+ homeostasis, and metabolism in this tissue. The present thesis firstly established the glycolytic profile of androgen-responsive (LNCaP) and non-responsive (PC3) PCa cells. We demonstrated that LNCaP and PC3 cells display a distinct glycolytic profile, with PC3 presenting higher production and export of lactate as a result of increased expression of target regulators of this metabolic pathway. The role of androgens in the regulation of the glycolytic flux in PCa cells was also studied. Treatment of LNCaP cells with 5α-dihydrotestosterone (DHT, 10nM) significantly increased glucose uptake, glycolysis rate and the export of lactate by upregulating the expression of important regulators of glycolysis, in a mechanism that seems to involve the androgen receptor (AR). These results highlighted the importance of controlling glucose and lactate metabolism as possible therapeutic approaches in PCa. The effect of androgens, Ca2+ and age on the expression of RGN in prostate cells in vitro and in vivo was investigated. The expression of RGN in rat prostate was downregulated by androgens and aging. Administration of extracellular Ca2+ to human prostate cells differentially regulated the expression of RGN in LNCaP and PNT1A cells in a time- and dose-dependent manner, with effects more pronounced in the up-regulation of RGN expression in PNT1A cells. The fact that androgens and Ca2+ regulate RGN levels in prostate cells, and the evidence of RGN down-regulation with aging, strongly points this protein as an important target in the regulation of prostatic physiology. Indeed, the work of this thesis also proved the role of RGN in controlling the crucial mechanisms involved in prostate cells homeostasis. RGN exerted a negative effect on the expression of Ca2+ sensing receptor, which was essential in the promotion of Ca2+-dependent proliferation, as demonstrated by in vitro and in vivo assays. In addition, overexpression of RGN suppressed cell proliferation, apoptosis and the glycolytic metabolism in rat prostate, as a result of the regulation of the expression and activity of several proteins involved in these pathways. RGN also decreased lipid peroxidation in rat prostate, which supports their cytoprotective function. Finally, we verified that augmented levels of RGN improved the antioxidant defenses, prevented the age-induced resistance to apoptosis, and the excessive proliferation usually found in the prostate of aged animals. The obtained results suggest that the manipulation of RGN levels may prevent the development of aged-associated diseases in rat prostate. In conclusion, the main findings of this dissertation pointed androgens as the “good friends” of PCa cells, since they increase the glycolytic flux in PCa cells, which is stimulated with the progression of disease. In contrast, loss of RGN expression with aging led to deregulation of different mechanisms crucial for the maintenance of prostate physiology, and thus it may contribute to the emergence of aged-associated pathophysiologic alterations, and to the neoplastic transformation. The results presented here also suggest future approaches for the management of PCa, which could passing by: i) the development of new therapeutic strategies based on the use of combined therapies, targeting AR and glycolytic metabolic regulators; and ii) the manipulation of RGN expression as a therapeutic option.
- Avaliação In Vitro dos Potenciais Efeitos Terapêuticos e Toxicológicos Associados ao Consumo de AyahuascaPublication . Gonçalves, Joana Domingos; Duarte, Ana Paula Coelho; Alba, Maria Eugénia Gallardo; Luís, Ângelo Filipe SantosAyahuasca is a psychoactive beverage that has been consumed for centuries and was originally used by indigenous tribes in the northwest of the Amazon. This beverage consists of a decoction, thick, oily, and brownish in color, which is originally prepared from the leaves of Psychotria viridis Ruiz & Pav. and the scrapings of the stem of Banisteriopsis caapi (Spruce ex Griseb.) C.V.Morton, although over the years several modifications of this decoction have been developed. Currently, some natural or synthetic adulterants that can mimic the effects of the plants originally used are known. This decoction contains the hallucinogenic compound N,N-dimethyltryptamine (DMT) from Psychotria viridis, and β-carboline alkaloids such as harmine, tetrahydroharmine (THH) and harmaline, which come from Banisteriopsis caapi. DMT is the main psychoactive component of ayahuasca, since it acts as a serotonin receptor (5-HT1A/2A/2C) agonist and, when ingested alone, is metabolized by peripheral monoamine oxidase A (MAO-A), making it if harmless. However, together with the β-carboline alkaloids, it can reach the circulation and the central nervous system, by temporarily inhibiting MAO-A. Additionally, THH also inhibits serotonin reuptake enhancing the effects of DMT. This synergy between compounds is known by the indigenous peoples for about 3000 years. Thus, originally, the tribes resorted to this beverage for therapeutic and religious purposes. It was also used by native healers to treat psychological disorders, stimulate visual creativity and creative thinking. More recently, non-indigenous religious entities in various countries, especially in Latin America, also used this beverage for their ceremonies, namely Barquinha, União do Vegetal and Santo Daime. These last two, currently spread to other continents. In recent decades, the popularity of ayahuasca has increased worldwide, being seen as a natural remedy used for millennia to cure various diseases. Thus, currently this beverage, despite continuing to be used in a traditional way, is also consumed recreationally all over the world, as well as used in modern medicine. When consuming ayahuasca, vomiting, diarrhea and nausea often occur. In addition to these physical symptoms, changes in body temperature, pupil size and immunological, endocrine, and cardiovascular changes were observed. Psychological symptoms such as changes in perception (time, space, and the senses) and at the cognitive level have been also described. Additionally, connections with divine and mythical entities are also described, which is the aspect that motivates its consumption in a religious context. Despite all the effects described, over the years several beneficial properties have been pointed out to ayahuasca, even believing in its therapeutic potential in psychological illnesses, such as depression, anxiety, addiction, and psychological disorders. Additionally, beneficial properties in physiological treatments are also pointed out. In recent decades, the consumption of psychoactive substances has been increasing, namely substances of natural origin, such as ayahuasca. This consumption is often incited by cultural motivations or by spiritual and religious beliefs, making it difficult to measure its worldwide use. According to the literature, the ayahuasca consumption of in controlled environments is not dangerous and is not associated with psychotic episodes. However, the expansion of its consumption has raised some concerns. Although the consumption of DMT source plants is not controlled, it has become a controlled substance in some countries, due to the increase in its demand. However, the legislation regulating psychoactive substances is very variable and may even be ambiguous. Considering the potential of ayahuasca, the first objective of this thesis was to develop an analytical methodology for determine and quantify the main compounds present in ayahuasca decoctions, as well as to understand their behavior throughout the digestive process. The second objective was to carry out a phytochemical characterization of ayahuasca decoctions and to evaluate their potential antioxidant, anti-inflammatory, antimicrobial effects and their therapeutic properties in wound healing and cancer treatment. Thus, initially decoctions, in sufficient quantity to carry out the entire study, of four individual plants commonly used in ayahuasca preparations (P. viridis (leaves), Mimosa hostilis Benth. (root), B. caapi (stem) and Peganum harmala L. (seeds)), a decoction of a commercial mixture and four decoctions of mixtures of two of the above plants (P. viridis and B. caapi, P. viridis and Peganum harmala, Mimosa hostilis and B. caapi and M. hostilis and P. harmala) were prepared. The samples were submitted to three microextraction procedures and later, an analytical method was developed and validated for the determination and quantification of the main compounds of these samples. The Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) technique proved to be the most promising and, therefore, was chosen for application in the samples. The analytical method showed to be linear between 0.16 and 10 μg/mL for β-carbolines and between 0.016 and 1 μg/mL for DMT, with determination coefficients (R2) between 0.9968 and 0.9993. The limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.16 μg/mL (0.016 μg/mL for DMT) and extraction efficiencies ranged from 60 to 88%. The bioavailability and bioaccessibility of the same compounds were also evaluated in vitro. The bioaccessibility of the samples was assessed using an in vitro digestion process, and subsequently their bioavailability was evaluated using the colorectal adenocarcinoma cell line (Caco-2). After quantification, it was verified that the compounds (DMT, harmine, harmaline, harmol, harmalol and THH) were released from the matrix during the in vitro digestion process, becoming bioaccessible. Likewise, some of these compounds, after being incubated with the cell monolayer, were absorbed, becoming bioavailable, without presenting a risk to its integrity. The phytochemical profile of the samples was evaluated, and it was demonstrated that, in general, the samples have a high content of phenolic compounds and flavonoids. These results reflect the antioxidant and anti-inflammatory activities also determined. Additionally, the samples showed antimicrobial properties, with emphasis on the effect of B. caapi and P. harmala on the A. baumannii strain, which resulted in the inhibition of both biofilm formation and quorum sensing. Ayahuasca samples also demonstrated their therapeutic potential, particularly in terms of wound healing. Using normal human dermal fibroblasts (NHDF), a scratch assay was performed, and it was found that only one sample showed cytotoxicity and the others promoted the migration of skin fibroblasts, without cutaneous absorption of DMT and β-carboline alkaloids. Likewise, the ayahuasca samples showed a great anticancer potential in Caco-2 cells, having been verified that after incubation, the samples significantly induced apoptosis while cell proliferation decreased significantly. There was also a significant reduction in oxidative stress with some samples, with a significant increase in the activity of antioxidant enzymes. Similarly, in gastric adenocarcinoma (AGS) cells, an increase in the induction of apoptosis and a reduction in oxidative stress were observed after incubation with the ayahuasca samples. In conclusion, the main results of this thesis demonstrated that ayahuasca does indeed have a great therapeutic potential, with emphasis on its antimicrobial, antioxidant, anti-inflammatory, and healing actions, but above all its anticancer activity studied in two different cell lines. Also noteworthy was the development and validation of the analytical method where three miniaturized extraction techniques were compared and where the QuEChERS technique was applied to ayahuasca samples for the first time.
- Biological properties of aromatic plants used in folk medicine: a focus on the essential oil of Thymus zygisPublication . Coimbra, Alexandra Teixeira; Duarte, Ana Paula Coelho; Ferreira, Susana Margarida ParaísoFor centuries, plants have been used for a wide variety of purposes, such as treating diseases, food flavouring and preservation and perfumery. The essential oils (EOs) are aromatic, oil-like volatile substances present in different plant materials that have a wide range of biological activities and can be used in different areas, such as chemical, cosmetic, food, perfumery, and pharmaceutical industries and have been used as therapeutic agents since ancient times. Thymus zygis is a widespread plant, mainly used as a culinary flavouring agent, which EO has demonstrated bioactive properties, such as antioxidant and antimicrobial, and that may even enhance the effect of certain antimicrobial agents. Furthermore, the potential application as a food preservative has been described on different matrixes mainly due to its antimicrobial activity against pathogenic and spoilage microorganisms in food. Considering the different applications of EOs, this work aimed to study the composition, antioxidant, and antimicrobial properties and also the cytotoxicity of the EOs of Foeniculum vulgare, Helichrysum stoechas, Mentha pulegium, Pinus pinaster, Ruta graveolens, and Thymus mastichina. The chemical composition, and the bioactive properties of the T. zygis EO were also evaluated with a focus on antimicrobial activity against Staphylococcus aureus and Listeria monocytogenes, and application of the EO in food. The F. vulgare, H. stoechas, M. pulegium, P. pinaster, R. graveolens, T. mastichina and T. zygis EOs showed antioxidant activity acting through inhibition of lipid peroxidation, while only the H. stoechas, M. pulegium and T. zygis EOs scavenged the free radicals of DPPH. M. pulegium and T. zygis EOs showed the strongest antimicrobial activity and only volatiles compounds from these EOs demonstrated inhibitory activity. Regarding the EO cytotoxicity on a fibroblasts cell line, it was observed that the effect was directly proportional to the EOs concentration, in which the highest cytotoxic effect was obtained with the R. graveolens EO. Considering the antimicrobial potential of the EO from T. zygis and since it showed the greater activity among the tested EOs, it was decided to proceed with further evaluation of this EO, thus deepening its bioactive activities, mainly the antibacterial activity against S. aureus and L. monocytogenes. Taking into account that S. aureus is a Gram-positive bacterium that causes a wide variety of clinical infections, from less severe to serious and life-threatening infections and because these are aggravated by antibiotic resistance, the antibacterial activity of T. zygis EO against strains of S. aureus was evaluated. The T. zygis EO demonstrated antimicrobial activity against S. aureus strains with bactericidal effect. The EO of T. zygis also revealed a synergistic or additive effect in combination with the antibiotic’s ampicillin, ciprofloxacin, or vancomycin against S. aureus strains and, in some cases, changed the antibiotic-resistance phenotype from resistant to susceptible. The T. zygis EO inhibit the formation of biofilms by S. aureus and partially eliminate the preformed biofilms even at subinhibitory concentrations. The EO reduced the haemolytic activity of S. aureus SA 03/10 (the only strain tested that causes haemolysis) as well the quorum-sensing in Chromobacterium violaceum biosensor. Therefore, these results demonstrate the good bioactive properties of the T. zygis EO, mainly the antimicrobial activity against S. aureus, revealing its potential to be used as an antibacterial agent and/or as an enhancer of the effect of antibiotics. Since L. monocytogenes is a foodborne Gram-positive bacterium with a high mortality rate and has the ability to survive and replicate in adverse conditions, allowing the wide distribution of this bacterium in different environments and matrices, such as water, soil, and food products, the antimicrobial activity of T. zygis EO against strains of L. monocytogenes was evaluated. The T. zygis EO presented good antibacterial activity against L. monocytogenes with MIC value of 0.05% while showing a bactericidal effect. The EO significantly reduced the biofilm formation with inhibition percentages from 16.85 to 89.86% and also the motility, while not inducing cross-resistance to antibiotics. The concentration of 2× MIC of T. zygis EO reduced the L. monocytogenes counts (initial inoculum of ~106 CFU/mL) in the chicken juice (1.53 log CFU/mL) and the lettuce model (to below the detection limit) after two days of storage. Regarding the sanitizing of fresh vegetables, the use of 0.2% (v/v) of EO for 5 min of immersion, reduce L. monocytogenes and natural microbiota counts for values below the detection limit of the method for iceberg lettuce. For the spinach leaves, L. monocytogenes and the natural microbiota counts were reduced to 4.35 log CFU/mL and in a range of 4.47 to 5.94 log CFU/mL, respectively, when compared with the washing with water. Thus, the T. zygis EO has demonstrated promising antibacterial activity against L. monocytogenes and these results point to the potential use of the T. zygis EO as a natural food preservative or sanitiser for controlling L. monocytogenes and the natural microbiota in food products. Overall, the present study revealed significant bioactive properties of different EOs, highlighting T. zygis EO to be considered in further studies for potential use in food preservation, due to their antioxidant and antimicrobial properties as well as in the control and reduction of the pathogenicity of the L. monocytogenes and S. aureus.
- Biomarkers in Barrett’s EsophagusPublication . Mesquita, Marta Catarina da Piedade Sirgado; Pascoal, Maria Paula Guerreiro Chaves; Dias, Mónica BettencourtBarrett’s esophagus (BE) is a recognized premalignant condition of the distal esophagus that constitutes the major risk factor for the development of esophageal adenocarcinoma (EA). Despite the known low rates of BE progression to EA, the incidence of both has increased profoundly over the last decades and esophageal malignancy remains to be a deadly cancer with high morbidity and mortality unless diagnosed at early stages. Current BE clinical management has revealed unsuccessful in reverting this worrisome epidemiological picture and a major hurdle has been the incapacity to discriminate among BE patients those who have a higher risk of malignant progression. In fact, to this date, besides dysplasia none of the existing clinical and histologic criteria could anticipate malignant progression. It is therefore imperative to find reliable molecular biomarkers to guide medical practice and improve the standard of care for BE patients. The global aim of this thesis was to better understand the pathways underlying BE malignant progression and thereby identify reliable biomarkers relevant for the diagnosis, prognosis and management of BE patients thus contributing to an improved understanding of BE biology and an optimized support for clinical decisions. To accomplish these challenging goals an unbiased and a hypothesis-driven strategies were followed. Through the unbiased approach, a meta-analysis of transcriptome datasets and subsequent experimental validation in a cohort BE patients in follow-up was used to define a gene set associated with BE cancer development and, therefore, identify early biomarkers predictive of BE malignant progression. In silico analysis singled out two genes, CYR61 and TAZ as candidate predictive markers for BE malignant progression and experimental validation using quantitative PCR and immunohistochemistry revealed that both genes are upregulated and overexpressed in non-dysplastic BE index biopsies from progressors years before cancer development when compared with index biopsies from BE patients that did not progressed. We also found that EMT and stemness-related genes were also significantly over represented in BE associated with progression. Together, these results support that CYR61 and TAZ are promising early biomarkers to stratify BE patients according to their cancer risk and suggest a novel mechanist route for BE neoplastic progression. Using an hypothesis-driven approach, we explored when and how centrosome abnormalities arise along BE malignant pathway, from the early premalignant condition stage to metastatic disease, by establishing an accurate method to identify and score centrosomes, at the single-cell level, in patient samples and cell lines. We found that centrosome amplification arises as early as the premalignant condition of patients that progress to malignancy and significantly expands at dysplasia stage, which is dependent of p53 loss of function, being then present along cancer progression, namely in EA and metastasis. So, these finding suggest that centrosome amplification could contribute to BE initiation and malignant progression. Considering that centrosome amplification is specific of patients that progress to cancer, this could be further explored to be translated into useful tools to be used in the clinical setting and potentially improve its diagnosis, prognosis and treatment. Moreover, given widespread occurrence of both p53 mutations and centrosome abnormalities in human tumors, our findings are likely to be extended to other cancers. Collectively, both research avenues suggest the existence of different cellular and molecular abnormalities dictating different pathological propensity for malignant progression in BE, right from the beginning, and this could be further explored to trace a cancer risk profile for every patient and guide medical decisions and improve patient care.
- Characterization of bitter taste receptors expression and function in the human blood-cerebrospinal fluid barrierPublication . Duarte, Ana Catarina Abreu; Santos, Cecília Reis Alves dos; Gonçalves, Isabel Maria Theriaga Mendes VarandaBitter taste receptors (TR2) expression and functionality was recently reported in the rat choroid plexus (CP). CP epithelial cells establish a major brain barrier, the blood-cerebrospinal fluid barrier (BCSFB). Given their capacity to bind a large array of chemical compounds, we hypothesised that TR2 might be involved in monitoring the composition of blood and cerebrospinal fluid. Brain barriers play a critical role in the protection of the central nervous system (CNS) by hindering the access of toxic substances to the brain. Consequently, many drugs targeting neurological disorders are impaired to cross these barriers. This is explained through the expression of several membrane transporters in brain barriers cells that efflux drugs, thus impairing drug cell accumulation in the brain. A wide range of compounds that bind to TR2 show neuroprotective and anti-tumoral properties. However, their low bioavailability in the CNS restrains its therapeutic application. Additionally, bitter compounds might interact with transporters that are also found in brain barriers. Therefore, bitter compounds might be effluxed which explains their low bioavailability but can also regulate the action of these transporters in order to increase their or other drugs’ intracellular accumulation. Considering that bitter compounds are TR2 agonists it is possible that TR2 play an important role on the bioactive effects of bitter compounds in the CNS, as reported in other tissues. The main goal of this doctoral thesis was to analyse the expression and function of the bitter signalling pathway in the human BCSFB. Additionally, the role of human TR2 (TAS2Rs) as modulator of specific neuroactive bitter compounds on ABC transporters function and activity at the BCSFB was also evaluated. The first research work presented showed the expression of 13 TAS2Rs as well as of downstream effector proteins of the taste signalling pathway in the human BCSFB. Moreover, we demonstrated that TAS2R14 and TAS2R39 are functional in a human cell model of the BCSFB and respond to bitter compounds quercetin and chloramphenicol, respectively. The second research work evaluated resveratrol transport across the BCSFB and the involvement of TAS2Rs. Results showed that resveratrol is able to cross the BCSFB from blood to cerebrospinal fluid in a dependent manner of TAS2R14 expression at CP epithelial cells. Further, efflux transporters ABCC1, ABCC4 and ABCG2, which are expressed at CP epithelial cells, transport resveratrol. Additionally, resveratrol upregulated ABCG2 expression and regulated ABCC4 and ABCG2 efflux activity in TAS2R14 dependent way. In conclusion, the results obtained during this project demonstrate that TAS2Rs are expressed and functional at the human BCSFB and support their participation in the monitorization of chemical composition of the surrounding fluids. Furthermore, the major achievements of this thesis strongly support the role of BCSFB in the regulation of the transport of molecules into the brain. In the future, it is necessary to further exploit the role of other TAS2Rs as mediators of the effects of bitter compounds in the brain, as well as in the regulation of transport and detoxifying systems at the BCSFB. The knowledge hereby created has far-reaching potential for improving the challenging task of delivering therapeutic drugs into the CNS.
- Comparative analysis of metallated phthalocyanines for photodynamic therapy of solid tumorsPublication . Dias, Lionel Mendes; Heger, Michal; Cavaco, José Eduardo Brites; Ding, BaoyuePhotodynamic therapy (PDT) comprises the administration of a photosensitizer (PS) and its accumulation within the tumor site, followed by irradiation with light of a specific wavelength. Consequently, singlet oxygen and other reactive oxygen/nitrogen species (ROS/RNS) are produced from bioavailable oxygen at the tumor’s microenvironment and are responsible for the tumor’s eradication. PDT works effectively in certain types of cancer, but poorly in tumors that reside in internal organs and organ structures such as the pancreas and biliary tree. Moreover, adverse effect such as skin phototoxicity is a major obstacle to more widespread clinical applicability. To that end, we encapsulated a second-generation of PS into liposomal carriers that are targeted to the tumor interstitium after intravenous administration. Therefore, this doctoral thesis describes essentially the evaluation and comparison between lipophilic metallated-phthalocyanines (ZnPC and AlPC) encapsulated in interstitially-targeted liposomes (ITLs), and their corresponding hydrophilic derivatives (ZnPCS4 and AlPCS4). To do so, we initiated the doctoral research through our first study by performing an attritional assessment in vitro using A431 cells as a template for tumor cells with a dysfunctional P53 tumor suppressor gene and epidermal growth factor receptor (EGFR) overexpression. As a methodology for our investigations, we have first assessed the dark toxicity as a function of PS concentration using the water-soluble tetrazolium salt (WST-1) and sulforhodamine B dyes as an indication of the cell viability. Using the same principle, we then drew the LC50 values for each PS through PDT at 671 nm and a light exposure of 15 J/cm2 following 1 hour of PS exposure. We continued our research looking into a time-dependent uptake and intracellular distribution of the PS, and we finalized our first study with the assessment of the mode of cell death as well as the cell cycle arrest at 24 hours after PDT. Through this research we observed that, in the absence of illumination, AlPC and ZnPC in ITLs were not toxic to cells up to a 1.5 μM PS concentration and exposure for up to 72 h, but for AlPCS4 and ZnPCS4, the dark toxicity was at 5 μM and at 2.5 μM, respectively. However, PDT of cells photosensitized with ZnPC, AlPC, and AlPCS4 yielded LC50 values of 0.13 μM, 0.04 μM, and 0.81 μM, respectively (24 hours post-PDT based on sulforhodamine B assay). The uptake of all PSs was observed as early as 1 min after PS addition to cells and increased in amplitude during a 2-h incubation period. ZnPCS4 did not induce notable phototoxicity, which was echoed in the mode of cell death and cell cycle arrest data. However, AlPCS4 induced considerable necrosis in addition to apoptosis, whereby most of the cell death had already manifested as early as 2 h after PDT. Cell death signaling coincided with a reduction in cells in the G0/G1 phase (ZnPC, AlPC, AlPCS4) and cell cycle arrest in the S-phase (ZnPC, AlPC, AlPCS4) and G2 phase (ZnPC and AlPC). With the intention of validating our previous research, we have moved forward with the investigations through a second study by using our comparative model with the four metallated-phthalocyanines in a human cholangiocarcinoma cell line and tumor-comprising cells (endothelial cells, fibroblasts, and macrophages), as a representation of the tumor’s microenvironment. In addition to all parameters assessed in the previous study, we went one step further and evaluated the systemic toxicity of each PS in zebrafish and in chicken embryos, and while using BALB/c nude mice as our in vivo model, we researched for signs of skin phototoxicity. A pilot study on PDT efficacy was also performed in BALB/c nude mice bearing human triple-negative breast cancer (MDA-MB-231) xenografts. The key findings were that photodynamically active PSs (all except ZnPCS4) were able to effectively photosensitize cancer cells and non-cancerous cells. In addition, PSs in study did not induced any notable systemic toxicity in zebrafish and chicken embryos. However, ITL-delivered ZnPC and ZnPCS4 were associated with skin phototoxicity, while the aluminum containing PSs did not exert any detectable sign of cutaneous phototoxicity. Last but not least, ITL-delivered ZnPC and AlPC are equally effective in their tumor-killing capacity in human tumor breast cancer xenografts, and superior to other non-phthalocyanine PSs when appraised on a per mole administered dose basis. In summary, the research on the applications of ZnPCS4 for oncological PDT will be discontinued in our group as it failed the attrition step regarding phototoxicity and it showed alarming signs of cutaneous phototoxicity. It is therefore concluded that AlPC and its derivative AlPCS4 are the least toxic and most effective PSs to employ with respect to ITLs as part of the comprehensive tumor targeting and PS delivery platform.
- Computational Analysis of Fundus Images: Rule-Based and Scale-Space ModelsPublication . Soares, Ivo Miguel da Fonseca Gravito; Pinheiro, António Manuel Gonçalves; Sousa, Miguel Castelo Branco Craveiro deFundus images are one of the most important imaging examinations in modern ophthalmology because they are simple, inexpensive and, above all, noninvasive. Nowadays, the acquisition and storage of highresolution fundus images is relatively easy and fast. Therefore, fundus imaging has become a fundamental investigation in retinal lesion detection, ocular health monitoring and screening programmes. Given the large volume and clinical complexity associated with these images, their analysis and interpretation by trained clinicians becomes a timeconsuming task and is prone to human error. Therefore, there is a growing interest in developing automated approaches that are affordable and have high sensitivity and specificity. These automated approaches need to be robust if they are to be used in the general population to diagnose and track retinal diseases. To be effective, the automated systems must be able to recognize normal structures and distinguish them from pathological clinical manifestations. The main objective of the research leading to this thesis was to develop automated systems capable of recognizing and segmenting retinal anatomical structures and retinal pathological clinical manifestations associated with the most common retinal diseases. In particular, these automated algorithms were developed on the premise of robustness and efficiency to deal with the difficulties and complexity inherent in these images. Four objectives were considered in the analysis of fundus images. Segmentation of exudates, localization of the optic disc, detection of the midline of blood vessels, segmentation of the vascular network and detection of microaneurysms. In addition, we also evaluated the detection of diabetic retinopathy on fundus images using the microaneurysm detection method. An overview of the state of the art is presented to compare the performance of the developed approaches with the main methods described in the literature for each of the previously described objectives. To facilitate the comparison of methods, the state of the art has been divided into rulebased methods and machine learningbased methods. In the research reported in this paper, rulebased methods based on image processing methods were preferred over machine learningbased methods. In particular, scalespace methods proved to be effective in achieving the set goals. Two different approaches to exudate segmentation were developed. The first approach is based on scalespace curvature in combination with the local maximum of a scalespace blob detector and dynamic thresholds. The second approach is based on the analysis of the distribution function of the maximum values of the noise map in combination with morphological operators and adaptive thresholds. Both approaches perform a correct segmentation of the exudates and cope well with the uneven illumination and contrast variations in the fundus images. Optic disc localization was achieved using a new technique called cumulative sum fields, which was combined with a vascular enhancement method. The algorithm proved to be reliable and efficient, especially for pathological images. The robustness of the method was tested on 8 datasets. The detection of the midline of the blood vessels was achieved using a modified corner detector in combination with binary philtres and dynamic thresholding. Segmentation of the vascular network was achieved using a new scalespace blood vessels enhancement method. The developed methods have proven effective in detecting the midline of blood vessels and segmenting vascular networks. The microaneurysm detection method relies on a scalespace microaneurysm detection and labelling system. A new approach based on the neighbourhood of the microaneurysms was used for labelling. Microaneurysm detection enabled the assessment of diabetic retinopathy detection. The microaneurysm detection method proved to be competitive with other methods, especially with highresolution images. Diabetic retinopathy detection with the developed microaneurysm detection method showed similar performance to other methods and human experts. The results of this work show that it is possible to develop reliable and robust scalespace methods that can detect various anatomical structures and pathological features of the retina. Furthermore, the results obtained in this work show that although recent research has focused on machine learning methods, scalespace methods can achieve very competitive results and typically have greater independence from image acquisition. The methods developed in this work may also be relevant for the future definition of new descriptors and features that can significantly improve the results of automated methods.
- Contribution to the evaluation of clinical prognostic factors in canine lymphomaPublication . Henriques, Joaquim José Garcia Pereira; Pascoal, Maria Paula Guerreiro Chaves; Almeida, José Manuel Pereira de; Cabeçadas, José Manuel Valente SequeiraNon-Hodgkin Lymphoma (NHL) is the most common hematopoietic cancer in dogs, from which up to 50% of the cases are diffuse large B-cell lymphomas (DLBCL). The etiology, like in humans, is believed to be multifactorial. To the date, the best response rate and best survival times are offered by a combination therapy including cyclophosphamide, vincristine, doxorubicin and prednisolone, known by the acronym of CHOP. Infectious agents, namely vector-borne agents (VBA), can induce chronic B cell stimulation and immune deregulation permitting lymphomagenesis. Also, there are several reports in literature associating NHL with VBA, namely from the genus Borrelia and Leishmania mimicking or co-existing with hematopoietic malignancies either in humans or dogs. Vector-borne agents can induce haematological and clinical changes in hosts that, when existing in cancer patients, can either mislead the interpretation of clinical signs or interfere with recognized prognostic markers, namely blood cell populations. Prognosis, after quality of life, is determinant in veterinary oncology to further proceed with a treatment. Short survival times and therapy response rates determine the option for a non standard-of-care treatment or, lately, animal euthanasia without treatment. Consequently, easy to perform, “bench-to-bedside” widely available and cost-effective prognostic markers are fundamental to obtain client financial compliance and support treatment planning and disease outcome. Obtaining serological results on vector-borne agents or haematological information trough a total blood cell count and biochemical parameters are widely available and cost effective either by an “in-house” laboratory or trough commercial laboratories. Having in mind the existing published literature on human medicine regarding haematological parameters as prognostic indicators in lymphoma and the scarse information on the veterinary field, we aimed to: 1) investigate the prevalence of infection by four vector-borne agents (Leishmania infantum, Ehrlichia canis, Anaplasma phagocytophilum and Bartonella henselae), its potential role in lymphomagenesis and its possible association with the tumour subtype and with the haematological alterations present in dogs with lymphoma and, 2) determine the prognostic value of dogs’ sex, neutered status, clinical stage, presence of anaemia, presence of neutrophilia, presence of thrombocytopenia and the ratios lymphocyte-to-monocyte (LMR), neutrophil-tolymphocyte (NLR), platelet-to-lymphocyte (PLR) and platelet-to-neutrophil (PNR) in canine DLBCL that were naïve for treatment, fully staged and received chemotherapy with a 19 week-CHOP protocol. All dogs tested negative for B. henselae, A. phagocytophilum and E. canis by both serology and molecular detection. Regarding L. infantum, 8,2% of the dogs had a positive serologic result. Leishmania infantum DNA was detected in two samples of diffuse large B-cell lymphoma (DLBCL). These results show an increased, but not significant, seropositivity (8,2%, p=0,201) and molecular detection (3,3%, p=0,166), for L. infantum in dogs with lymphoma, when compared to matched historic controls in the same geographical area. In the second study, PNR showed to be an independent prognostic marker (p≤0,001) for TTPR at 180 and 365 days. Dogs with a PNR above 0,032 were more likely to progress before 180 days (sensitivity 46,5%, specificity 87,5%, p=0,004). On univariate analysis, NLR showed a prognostic significance for LSSR at 180 (p=0,006) and 365 days (p=0,009). A baseline NLR value below 7,45 was positively associated with survival at 180 days (sensitivity of 52%, specificity of 85.3%, p=0.025). The presence of substage b, was associated with early lymphoma progression and decreased survival at 180 days (p =0.031). Anaemia significantly reduced LSSR at 365 days (p=0,028). Although it was not possible to identify, in the first study, any significant association between canine lymphoma and the studied VBA, it was of extreme importance for the discussion of the second study on the possible effects on peripheral blood cell dynamics caused by the CBVD studied. Further studies, following dogs trough their CVBD disease evolution, are worthwhile and may help clarify a possible role of these agents in lymphomagenesis. This is the first study evaluating PLR and PNR in canine DLBCL and demonstrates that PNR could be a predictor of early lymphoma progression. Since peripheral blood cell composition can be affected by several non-oncological causes, the development of larger multicenter studies with homogeneous inclusion criteria could help to better determine the true predictive values of blood cell ratios in dogs suffering from DLBCL treated with CHOP chemotherapy.
- Desenvolvimento de novos biomateriais para aplicação como substitutos de pelePublication . Ribeiro, Maximiano José Prata; Correia, Ilídio Joaquim Sobreira; Borges, Paula Isabel Teixeira Gonçalves CoutinhoThe skin is the largest organ in mammals and acts as a barrier between the human body and the surrounding environment. It protects the underlying organs and defends the body against nocive agents. After an injury, skin integrity is compromised and the organism triggers the wound healing process for restoring the structure and functions of this organ. Wound healing is an extremely dynamic and interactive biological process that involves extracellular matrix molecules, soluble mediators, various resident cells (fibroblasts and keratinocytes) and infiltrating leukocyte subtypes which, together, act to re-establish the integrity of the damaged tissue and replace the lost one. To improve patients odds of survival and to minimize the loss of skin vital functions, this tissue has to be covered immediately after being damaged. Such highlights the importance of developing new wound dressings that improve the healing process, making it less painful and, simultaneously, contributing for the re-establishment of skin structure and functions in a shorter period of time. In recent years, various dressings have been developed, using natural or synthetic materials, for restoring skin native properties and structure. Although, none of them is capable of fully accomplish this objective. Taking this into account, the main objective of this work was to develop and characterize skin substitutes to be used in the treatment of skin disorders. Natural polymers such as agarose, chitosan and dextran were used for dressings production. Initially, we evaluated the applicability of a chitosan hydrogel as a wound dressing. The morphology of the developed system was characterized by scanning electron microscopy while its cytotoxic profile and degradation by-products were evaluated through in vitro assays. In vivo experiments were also performed to evaluate the potential of the chitosan hydrogel for the treatment of skin burns. The results obtained revealed that the hydrogel developed has the required properties for biomedical application intended, once the in vitro and in vivo assays revealed that chitosan hydrogel and its degradation by-products are biocompatible and possess the ability to promote the healing of skin wounds. In the second study, a dextran hydrogel was loaded with chitosan microparticles containing epidermal and vascular endothelial growth factors for the improvement of wound healing process. The hydrogel morphology and cytotoxicity profile and degradation by-products were characterized by scanning electron microscopy and in vitro assays. Furthermore, in vivo experiments were also performed to evaluate the applicability of the hydrogel for wound healing. The results obtained, revealed that the animals treated with this hydrogel showed a faster wound healing with no signs of local or systemic inflammatory response. Moreover, a unique application per week of this skin substitute allowed a faster healing than that obtained when growth factors were topically applied in the wound every two days. Dextran hydrogel proved that it can be used as a wound dressing and also as a carrier of microparticles containing growth factors involved in wound healing. In the third study, a thermoresponsive chitosan/agarose hydrogel was produced for being used in the wound healing process. The porosity, wettability, hydrophilicity, biocompatibility, and bactericidal activity of the hydrogel were characterized by scanning electron microscopy, studies of water uptake, determination of contact angle, confocal microscopy, cytotoxic assays, determination of minimum inhibitory concentration and biofilm deposition, respectively. The performance of this polymeric matrix in the wound healing process was evaluated through in vitro and in vivo assays. The attained results revealed that the hydrogel promotes cellular adhesion and proliferation and also its bactericidal activity. The in vivo studies showed also an improved healing and the lack of a reactive or a granulomatous inflammatory reaction in the skin lesions treated with this hydrogel. During this PhD, various natural polymers were used to produce three different hydrogels, aimed for wound healing. The in vitro and in vivo assays revealed promising results that may allow their use as skin substitutes in a near future.
- Development of IR780 based nanoparticles for photothermal therapy of breast cancerPublication . Alves, Cátia Gomes; Correia, Ilídio Joaquim Sobreira; Diogo, Duarte Miguel de MeloDespite all the efforts that have been done, cancer remains as one of the most common and deadliest diseases in the whole world. Surgery can be an effective strategy to treat this disease if the cancer is diagnosed at an early stage. In a more advanced stage of this disease, chemotherapy and radiotherapy are the most applied therapeutic strategies. However, these conventional approaches lack efficacy and selectivity towards cancer cells, causing several side effects in the patients such as hair loss, nausea, and severe weakness. These drawbacks of the clinically available therapeutics highlight the urgent need for developing new anticancer approaches with greater efficacy and safety. To face this challenge, the nanomaterials’ mediated phototherapy is one of the most promising strategies. Regarding this therapeutic modality, it is important to consider two main aspects: i) the physicochemical properties of the nanomaterials (e.g., size, corona composition) and ii) the optical properties of the laser (e.g., wavelength, intensity) and of the therapeutic agent (e.g., molar extinction coefficient, photothermal and photodynamic efficiencies). In what concerns the first point, the nanomaterials’ size, charge, shape, and corona composition play an important role in their ability to reach the tumor site. Classically, the optimization of the nanomaterials’ size has been intensively pursued to allow these nanostructures to accumulate at the tumor site by exploiting the tumor’s leaky vasculature (i.e., to take advantage from the so-called Enhanced Permeability and Retention effect). However, recently it was unveiled that dynamic vents (also known as eruptions) occur spontaneously at the tumor vasculature, facilitating nanostructures extravasation into the tumor site. This new phenomenon led to a paradigm shift, and thus, currently, researchers are focused on the optimization of the nanoparticles’ corona for improving their tumor uptake (i.e., to increase their blood circulation time and hence their likelihood to benefit from these dynamic vents). To improve the nanostructures’ blood circulation time and favor their tumor uptake, these nano-systems have been mainly coated with poly(ethylene glycol) (PEG). However, recently it was uncovered that PEGylated nanomaterials suffer from the Accelerated Blood Clearance phenomenon. Therefore, at the time of the first administration of PEGylated nanomaterials, anti-PEG antibodies are created. Then, these anti-PEG antibodies mediate the rapid clearance of the PEGylated nanoparticles in subsequent administrations. Due to the immunogenicity displayed by PEG-based coatings, it is crucial to develop and validate novel materials capable of improving the nanostructures’ biological properties. The efficacy of nanomaterials-based phototherapies also depends on the optical properties of the laser and of the photoresponsive agent. In this regard, the use of near infrared (NIR; 750-1000 nm) light is of utmost importance since it does not interact significantly with major body components (e.g., water, melanin, collagen) and achieves a high penetration depth (up to about 2 cm). Moreover, the laser power density and irradiation time can also affect the therapeutic outcome. In this way, phototherapies based on NIR light-responsive nanomaterials have been showing promising results. In this type of therapy, after the nanomaterials’ tumor uptake, this zone is irradiated with NIR light. Upon interaction with this radiation, the nanomaterials can produce a temperature increase (photothermal therapy) and/or reactive oxygen species (photodynamic therapy), which cause damages on cancer cells. Among the several NIR light responsive agents, IR780 stands out due to its high versatility. This prototypic heptamethine cyanine has multimodal properties since it can be used for both photothermal therapy and photodynamic therapy as well as for imaging applications (IR780 emits fluorescence in the NIR). However, this small molecule presents acute toxicity and low solubility, hindering its direct application in cancer therapy. In this way, encapsulating IR780 in nanomaterials can be pursued to surpass these disadvantages. However, most of the IR780-based nanomaterials have been produced using PEG in their corona. Considering the recent studies demonstrating the immunogenicity of PEGylated nanostructures, it is of utmost importance to develop new IR780-based nanoparticles that are functionalized with PEG alternatives. In this way, the main goal of the workplan developed during this Doctoral thesis was to validate the potential of novel materials, based on sulfobetaine methacrylate (SBMA) and Poly(2-ethyl-2-oxazoline) (PEtOx), in the coating of nanostructures incorporating IR780 for application in cancer photothermal therapy. This thesis includes two chapters presenting research work. In the first research work (Chapter 3), Bovine Serum Albumin (BSA) was grafted with SBMA, for the first time, being this polymer (SBMA-BSA) employed to encapsulate IR780 through the nanoprecipitation technique (IR/SBMA-BSA NPs). The produced nanoparticles presented an ideal size (≈ 96 nm) and surface charge (≈ -9 mV) for cancer-related applications. As importantly, the SBMA functionalization improved the colloidal stability of the nanostructures in different media as well as their uptake by breast cancer cells. In the phototherapeutic assays, the IR/SBMA-BSA NPs in combination with NIR light could decrease the cancer cells’ viability to just 12 %. In the second research work (Chapter 4), a novel amphiphilic PEtOx-IR780 conjugate was produced. For that, the cyclohexenyl ring of IR780 was chemically attached to thiol-terminated PEtOx (PEtOx-IR conjugate). Afterwards, PEtOx-IR and D-α-tocopheryl succinate (TOS) were combined through the nanoprecipitation technique, yielding PEtOx-IR/TOS NPs. These nanoparticles presented a size (≈ 190 nm) and surface charge (≈ -8 mV) compatible with anticancer applications. As importantly, the PEtOx-IR/TOS NPs also presented an optimal colloidal stability. The PEtOx-IR/TOS NPs could be successfully internalized by cancer cells. In the phototherapeutic assays, the combination of PEtOx-IR/TOS NPs with NIR light could decrease the viability of breast cancer cells (2D in vitro cancer models) to 9 %, and the heterotypic breast cancer spheroids’ (3D in vitro cancer models) viability was also reduced to just 15 %. Overall, the results obtained in this thesis validate the potential of SBMA-brushes and PEtOx in the coating of IR780-based nanomaterials. Moreover, these novel IR780-based nanomaterials also displayed a good in vitro performance, highlighting their potential for cancer photothermal therapy.