Novos alvos terapêuticos na hipertensão pulmonar

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Utilização de murganhos no estudo de novos alvos da terapia oncológica anti-angiogénica

Tese de Doutoramento em Ciências Veterinárias na Especialidade de Ciências Biológicas e Biomédicas

Silenciamento da expressão da enzima óxido nítrico sintase neuronal (nNOS) em linhagem de neuroblastoma via siRNAs sintéticos em dupla fita

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, Programa de Pós-Graduação em Saúde Animal, 2011.

Leishmanicidal and cytotoxic activity of extracts and saponins from Ilex laurina (Aquifoliaceae)

Purpose: To evaluate the leishmanicidal and cytotoxic activity of alcohol and non-alcohol extracts and saponins from Ilex laurina . Methods: Extracts were obtained by percolation with solvents of different polarities: hexane, dichloromethane, ethyl acetate and ethanol. The ethyl acetate extract was subjected to silica gel column chromatography eluting with a step gradient of dichloromethane-methanol. All products were evaluated in vitro for leishmanicidal activity against amastigotes of leishmania panamensis and cytotoxicity on U- 937 cells. Results: Two saponins were isolated from the ethyl acetate extract. The ethyl acetate extract showed high leishmanicidal activity against intracellular amastigotes of L. panamensis (EC50, 7.5 ± 1.5 μg/mL) and low activity against axenic amastigotes (EC50, 52.8 ±1.6 μg/mL); this extract showed also high cytotoxicity (LC50, 57.7 ± 12.1 μg/mL). Saponin 2 exhibited high activity against intracellular amastigotes (EC50, 5.9 ± 0.5 μg/mL) but also showed high cytotoxicity on U-937 cells (EC50, 25.7 ± 6.1 μg/mL). This compound showed similar leishmanicidal activity and cytotoxicity to meglumine antimoniate and amphotericin B, respectively, drugs currently used for the treatment of leishmaniasis. Conclusions: Based on these results, Ilex laurina is a potential source of compounds that can lead to the development of new therapeutic alternatives against leishmaniasis.

Effect of Arginine and Oscillatory Ca2+ on Vascular Response Mediated Via Nitric Oxide Signaling in Normal and Salt Sensitive Hypertensive Rat Mesenteric Arterioles

Hypertension, a major risk factor in the cardiovascular system, is characterized by an increase in the arterial blood pressure. High dietary sodium is linked to multiple cardiovascular disorders including hypertension. Salt sensitivity, a measure of how the blood pressure responds to salt intake is observed in more than 50% of the hypertension cases. Nitric Oxide (NO), as an endogenous vasodilator serves many important biological roles in the cardiovascular physiology including blood pressure regulation. The physiological concentrations for NO bioactivity are reported to be in 0-500 nM range. Notably, the vascular response to NO is highly regulated within a small concentration spectrum. Hence, much uncertainty surrounds how NO modulates diverse signaling mechanisms to initiate vascular relaxation and alleviate hypertension. Regulating the availability of NO in the vasculature has demonstrated vasoprotective effects. In addition, modulating the NO release by different means has proved to restore endothelial function. In this study we addressed parameters that regulated NO release in the vasculature, in physiology and pathophysiology such as salt sensitive hypertension. We showed that, in the rat mesenteric arterioles, Ca2+ induced rapid relaxation (time constants 20.8 ± 2.2 sec) followed with a much slower constriction after subsequent removal of the stimulus (time constants 104.8 ± 10.0 sec). An interesting observation was that a fourfold increase in the Ca2+ frequency improved the efficacy of arteriolar relaxation by 61.1%. Our results suggested that, Ca2+ frequency-dependent transient release of NO from the endothelium carried encoded information; which could be translated into different steady state vascular tone. Further, Agmatine, a metabolite of L-arginine, as a ligand, was observed to relax the mesenteric arterioles. These relaxations were NO-dependent and occurred via α-2 receptor activity. The observed potency of agmatine (EC50, 138.7 ± 12.1 µM; n=22), was 40 fold higher than L-arginine itself (EC50, 18.3 ± 1.3 mM; n = 5). This suggested us to propose alternative parallel mechanism for L-arginine mediated vascular relaxation via arginine decarboxylase activity. In addition, the biomechanics of rat mesentery is important in regulation of vascular tone. We developed 2D finite element models that described the vascular mechanics of rat mesentery. With an inverse estimation approach, we identified the elasticity parameters characterizing alterations in normotensive and hypertensive Dahl rats. Our efforts were towards guiding current studies that optimized cardiovascular intervention and assisted in the development of new therapeutic strategies. These observations may have significant implications towards alternatives to present methods for NO delivery as a therapeutic target. Our work shall prove to be beneficial in assisting the delivery of NO in the vasculature thus minimizing the cardiovascular risk in handling abnormalities, such as hypertension.

Investigation of the enhanced antimicrobial activity of combination dry powder inhaler formulations of lactoferrin

The airways of most people with cystic fibrosis are colonized with biofilms of the Gram-negative, opportunistic pathogen Pseudomonas aeruginosa. Delivery of antibiotics directly to the lung in the form of dry powder aerosols offers the potential to achieve high local concentrations directly to the biofilms. Unfortunately, current aerosolised antibiotic regimes are unable to efficiently eradicate these biofilms from the airways. We investigated the ability of the innate antimicrobial, lactoferrin, to enhance the activity of two aminoglycoside antibiotics (tobramycin and gentamicin) against biofilms of P. aeruginosa strain PAO1. Biofilms were prepared in 96 well polystyrene plates. Combinations of the antibiotics and various lactoferrin preparations were spray dried. The bacterial cell viability of the various spray dried combinations was determined. Iron-free lactoferrin (apo lactoferrin) induced a 3-log reduction in the killing of planktonic cell by the aminoglycoside antibiotics (p < 0.01) and also reduced both the formation and persistence of P. aeruginosa biofilms (p < 0.01). Combinations of lactoferrin and an aminoglycoside displays potential as an effective new therapeutic strategy in the treatment of P. aeruginosa biofilms infections such as those typical of the CF lungs.

The microbiome and the pathophysiology of asthma

Asthma is a chronic respiratory disease whose prevalence is increasing in the western world. Recently research has begun to focus on the role the microbiome plays in asthma pathogenesis in the hope of further understanding this respiratory disorder. Considered sterile until recently, the lungs have revealed themselves to contain a unique microbiota. A shift towards molecular methods for the quantification and sequencing of microbial DNA has revealed that the airways harbour a unique microbiota with apparent, reproducible differences present between healthy and diseased lungs. There is a hope that in classifying the microbial load of the asthmatic airway an insight may be afforded as to the possible role pulmonary microbes may have in propagating an asthmatic airway response. This could potentially pave the way for new therapeutic strategies for the treatment of chronic lung conditions such as asthma.

Imunoterapia

O cancro oral é uma neoplasia maligna relativamente frequente, sendo por isso responsável por uma taxa de mortalidade elevada. Em particular, o carcinoma espinocelular é o tipo histológico mais frequente das neoplasias malignas da cavidade oral, estando claramente associada a factores de risco como o tabaco, o consumo de álcool e a infecção pelo vírus do papiloma humano (HPV). Actualmente, no mundo ocidental, observa-se um aumento na incidência do cancro da língua que parece estar relacionado com infecções pelos vírus HPV. Tendo em conta os fenómenos associados à cancerização da mucosa oral e a progressão do mesmo, este trabalho tem como função a pesquisa de possíveis alternativas de tratamentos, nomeadamente a imunoterapia, com a utilização de anticorpos monoclonais, terapia de vacinas, terapia de transferência adoptiva de células T, entre outras, uma vez que nem sempre os tratamentos convencionais como a quimioterapia, radioterapia, ou tratamento cirúrgico se revelam completamente eficazes. Contudo, existe uma carência de protocolos definidos, sendo a imunoterapia ainda uma terapêutica a evoluir, por isso esta monografia pretende fazer uma revisão sobre o ‘’estado da arte’’ deste tema tão complexo, com base em literatura de vários autores ao longo desta última década. Este trabalho pretende mencionar novos alvos terapêuticos que permitem desenhar terapêuticas mais dirigidas e, eventualmente, com menos efeitos adversos. A utilização por exemplo do cetuximab (anti-EGFR), que na prática clínica é já uma realidade.

Communication du risque et clarification des valeurs, deux éléments essentiels de la décision partagée : étude descriptive dans cinq unités de médecine familiale du Québec

Cette étude descriptive transversale visait à estimer la proportion de consultations en médecine familiale dans lesquelles sont observées une communication des risques et une clarification des valeurs et des préférences. Au sein de 238 dyades de cliniciens-patients (238 patients et 71 cliniciens), nous avons observé que 63 % (intervalle de confiance [IC] à 95 % : 54 % - 70 %) des consultations comportaient ces deux éléments. Aussi, nous avons observé que six facteurs étaient associés à la présence de ces deux éléments lors de la consultation : 1) les nouvelles options thérapeutiques (rapport de cotes [RC] = 3,54; IC à 95 % : 1,32 - 9,48); 2) les options de traitement (RC = 3,56, IC à 95 % : 1,52 - 8,36); 3) la présence de cinq décisions et plus (RC = 5,00; IC à 95 % : 1,5 - 16,9); 4) report de la décision (RC = 4,92; IC à 95 % : 1,35 - 17,87); 5) la durée de la consultation (RC = 1,03; IC à 95 % : 1,002 - 1,07) et 6) le style de prise de décision collaboratif du clinicien (RC = 8,78; IC à 95% : 1,62 - 47,71). Des interventions ciblant directement les facteurs modifiables sont à considérer en vue d’augmenter la présence de ces deux éléments durant la consultation en médecine familiale.

Unraveling the pathophysiological mechanisms of visceral pain in the murine model of Fabry disease

Fabry disease (FD) is an X‐linked inherited, lysosomal storage disorder characterized by a deficient activity of the enzyme α-Galactosidase A (α-Gal A). This deficiency causes an accumulation of globotriaosylceramide 3 (Gb3), in nearly all organs. Gastrointestinal (GI) symptoms are among the earliest and most frequent symptoms of FD. It has been hypothesized that Gb3 accumulation is the leading cause of these, but their pathophysiology is complex and still poorly understood. Here, we aim at understanding the molecular mechanisms underpinning the GI symptoms of FD. For this purpose, we used the α‐Gal A (-/0) male mouse, a murine model of FD, to characterize morphological and molecular features of the colon tract. Our results show that α‐Gal A (-/0) mice display a thickening of the muscular layer due to a hypertrophic state of myenteric plexus ganglia, caused by an accumulation of Gb3 in neurons. Also, α-Gal A (-/0) mice present a decreased density of mucosal nerve fibres. Furthermore, α-Gal A (-/0) mice presented visceral hyperalgesia, by showing greater visceromotor response (VMR) values and obtaining higher abdominal withdrawal reflex (AWR) scores, following colorectal distension (CRD). Subsequently, the immunoreactivity of the pain-related ion channels TRPV1, TRPV4, TRPA1 and TRPM8 was detected at level of myenteric and submucosal plexus ganglia of both the genotypes. Further studies are required to assess differences of expression between α-Gal A (-/0) and control mice. Finally, we optimized the protocols to obtain three types of primary cultures from mouse intestine to be tested electrophysiologically: a mixed culture containing neurons and glia, an enriched culture of neurons, and one of glia. In summary, we revealed alterations that are likely to be part of the pathophysiological causes of FD GI symptoms. Therefore, together with further studies, this work could help identify new therapeutic targets for the treatment of visceral pain in FD.

Design and Synthesis of Novel Kinase Inhibitors for the Treatment of Chronic Respiratory Diseases

In 2017, Chronic Respiratory Diseases accounted for almost four million deaths worldwide. Unfortunately, current treatments are not definitive for such diseases. This unmet medical need forces the scientific community to increase efforts in the identification of new therapeutic solutions. PI3K delta plays a key role in mechanisms that promote airway chronic inflammation underlying Asthma and COPD. The first part of this project was dedicated to the identification of novel PI3K delta inhibitors. A first SAR expansion of a Hit, previously identified by a HTS campaign, was carried out. A library of 43 analogues was synthesised taking advantage of an efficient synthetic approach. This allowed the identification of an improved Hit of nanomolar enzymatic potency and moderate selectivity for PI3K delta over other PI3K isoforms. However, this compound exhibited low potency in cell-based assays. Low cellular potency was related to sub optimal phys-chem and ADME properties. The analysis of the X-ray crystal structure of this compound in human PI3K delta guided a second tailored SAR expansion that led to improved cellular potency and solubility. The second part of the thesis was focused on the rational design and synthesis of new macrocyclic Rho-associated protein kinases (ROCKs) inhibitors. Inhibition of these kinases has been associated with vasodilating effects. Therefore, ROCKs could represent attractive targets for the treatment of pulmonary arterial hypertension (PAH). Known ROCK inhibitors suffer from low selectivity across the kinome. The design of macrocyclic inhibitors was considered a promising strategy to obtain improved selectivity. Known inhibitors from literature were evaluated for opportunities of macrocyclization using a knowledge-based approach supported by Computer Aided Drug Design (CADD). The identification of a macrocyclic ROCK inhibitor with enzymatic activity in the low micro molar range against ROCK II represented a promising result that validated this innovative approach in the design of new ROCKs inhibitors.

Dissecting the role of zyxin as a mediator of aggressiveness in Ewing sarcoma

Zyxin is a phosphoprotein localized at the focal adhesions and on the actin stress fibres, where it regulates the cytoskeleton organization. In addition, zyxin can shift into the nucleus and modulates the gene expression, affecting key cellular processes. Consequently, zyxin is as a crucial factor in the malignancy of several cancers, like Ewing sarcoma (EWS). EWS is a rare tumour of the bones, affecting children and adolescents. The main features of EWS are the presence of a chimeric transcriptional factor, EWS-FLI1 and the high expression of CD99, a glycoprotein necessary for the maintenance of the malignant phenotype. Triggering of CD99 with specific antibodies causes massive cell death, an effect that requires zyxin presence. In EWS zyxin is repressed by EWS-FLI1 and its forced re-expression counteracts the malignant phenotype. In this work we decided to deepen our knowledge on how zyxin affects EWS malignancy. We proved that zyxin is a negative regulator of cell migration, survival and growth in anchorage-independent conditions, confirming the tumour suppressor role of zyxin. Then we focused on the relation between CD99 and zyxin. Loss of function of CD99, by engagement with specific antibodies or use of shRNA, increases zyxin levels and promotes its nuclear translocation. Here, we observed that zyxin impairs the transcriptional activity of the Glioma associated oncogene 1 (Gli1), a member of the Hedgehog signalling pathway, which has a relevant oncogenic function in EWS. To support these evidences, we also reported that the loss of function of CD99 inhibits, trough zyxin mediation, the expression of Gli1 up-regulated target genes, such as NKX2-2, PTCH1 and cyclins, whilst enhances the expression of its down-regulated target GAS1. In conclusion, we presented a more accurate depiction of zyxin role in EWS, which in the future could be further developed in hope to offer new therapeutic approaches.

The role of Phospholipases as novel potential modulators of aggressiveness in Glioblastoma

Glioblastoma is the most malignant brain tumor in adults. The standard care of treatment is tumor resection, radiotherapy, and chemotherapy. Despite these invasive therapeutic approaches, glioblastoma prognosis remains unchanged. Therefore, a better understanding of the molecular mechanisms driving tumor transformation is needed to uncover novel therapeutic strategies. Several studies have shown the significance of lipid signaling and phospholipases (PLCs) in the regulation of different mechanisms in the central nervous system as well as in glioblastoma pathogenesis. This work suggests a potential role of PLCβ1 in the maintenance of a less aggressive phenotype of the tumor. Indeed, it was demonstrated that PLCβ1 gene was relatively less expressed in glioblastoma patients compared to their healthy/low-grade counterparts. Moreover, PLCβ1 silencing, in both immortalized and primary cell lines, led to increased cell migration, invasion, proliferation, cell survival and induced the upregulation of mesenchymal markers and metalloproteinases. Moreover, PLCγ1, another abundant PLC isoform in the brain, has been identified as a key element for the aggressiveness of glioblastoma. Data collected on patients’ biopsies and engineered cell models, suggested a strong correlation between PLCγ1 expression level and the acquisition of a more aggressive tumor phenotype. Finally, this trend was further probed using patient-derived glioblastoma stem cells (GSCs), which are a specific tumor population that drives aggressiveness, resistance, and recurrence in glioblastoma. GSCs analysis on the transcriptomic profiles confirmed that PLCγ1 downregulation modulated positively the activation of pathways that negatively regulate cell motility and migration and led to a decreased expression of genes involved in cancer development and progression. Taken together, these data highlight the importance of further investigating phospholipases as potential prognostic biomarkers and targets in the development of new therapeutic strategies for glioblastoma.

New frontiers in therapeutic resistance in cancer.

Despite advances in personalized medicine and targeted therapies, therapeutic resistance remains a persistent dilemma encountered by clinicians, scientists and patients. In this article we summarize the highlights of the third Quebec Conference on Therapeutic Resistance in Cancer. This unique meeting provided researchers and clinicians with insights into: intrinsic and acquired resistance; tumor heterogeneity; complexities of biomarker-driven trials; challenges of 'omics data analysis; and models of clinical applications of personalized medicine. Emphasized throughout the conference was the importance of collaborations - between industry and academia, and between basic researchers and clinicians - so that therapeutic resistance can be studied where it matters most, in patients.

Development of new methodologies for entry to targets of therapeutic interest

This Account provides an overview of strategies that have been reported from our laboratories for the synthesis of targets of therapeutic interest, namely carbohydrates, and prodrugs for the treatment of melanoma. These programmes have involved the development of new synthetic methodologies including the regio- and stereoselective synthesis of specific carbohydrate isomers, and new protecting group methodologies. This review provides an insight into the progress of these research themes, and suggests some applications for the targets that are currently being explored.