Mechanisms of DNA Damage Response to Targeted Irradiation in Organotypic 3D Skin Cultures

DNA damage (caused by direct cellular exposure and bystander signaling) and the complex pathways involved in its repair are critical events underpinning cellular and tissue response following radiation exposures. There are limited data addressing the dynamics of DNA damage induction and repair in the skin particularly in areas not directly exposed. Here we investigate the mechanisms regulating DNA damage, repair, intracellular signalling and their impact on premature differentiation and development of inflammatory-like response in the irradiated and surrounding areas of a 3D organotypic skin model. Following localized low-LET irradiation (225 kVp X-rays), low levels of 53BP1 foci were observed in the 3D model (3.8±0.28 foci/Gy/cell) with foci persisting and increasing in size up to 48 h post irradiation. In contrast, in cell monolayers 14.2±0.6 foci/Gy/cell and biphasic repair kinetics with repair completed before 24 h was observed. These differences are linked to differences in cellular status with variable level of p21 driving apoptotic signalling in 2D and accelerated differentiation in both the directly irradiated and bystander areas of the 3D model. The signalling pathways utilized by irradiated keratinocytes to induce DNA damage in non-exposed areas of the skin involved the NF-κB transcription factor and its downstream target COX-2.

Discrete cyclic di-GMP-dependent control of bacterial predation versus axenic growth in Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus is a Delta-proteobacterium that oscillates between free-living growth and predation on Gram-negative bacteria including important pathogens of man, animals and plants. After entering the prey periplasm, killing the prey and replicating inside the prey bdelloplast, several motile B. bacteriovorus progeny cells emerge. The B. bacteriovorus HD100 genome encodes numerous proteins predicted to be involved in signalling via the secondary messenger cyclic di-GMP (c-di-GMP), which is known to affect bacterial lifestyle choices. We investigated the role of c-di-GMP signalling in B. bacteriovorus, focussing on the five GGDEF domain proteins that are predicted to function as diguanylyl cyclases initiating c-di-GMP signalling cascades. Inactivation of individual GGDEF domain genes resulted in remarkably distinct phenotypes. Deletion of dgcB (Bd0742) resulted in a predation impaired, obligately axenic mutant, while deletion of dgcC (Bd1434) resulted in the opposite, obligately predatory mutant. Deletion of dgcA (Bd0367) abolished gliding motility, producing bacteria capable of predatory invasion but unable to leave the exhausted prey. Complementation was achieved with wild type dgc genes, but not with GGAAF versions. Deletion of cdgA (Bd3125) substantially slowed predation; this was restored by wild type complementation. Deletion of dgcD (Bd3766) had no observable phenotype. In vitro assays showed that DgcA, DgcB, and DgcC were diguanylyl cyclases. CdgA lacks enzymatic activity but functions as a c-di-GMP receptor apparently in the DgcB pathway. Activity of DgcD was not detected. Deletion of DgcA strongly decreased the extractable c-di-GMP content of axenic Bdellovibrio cells. We show that c-di-GMP signalling pathways are essential for both the free-living and predatory lifestyles of B. bacteriovorus and that obligately predatory dgcC- can be made lacking a propensity to survive without predation of bacterial pathogens and thus possibly useful in anti-pathogen applications. In contrast to many studies in other bacteria, Bdellovibrio shows specificity and lack of overlap in c-di-GMP signalling pathways.

Primary airway epithelial cell culture and asthma in children-lessons learnt and yet to come

Until recently the airway epithelial cell (AEC) was considered a simple barrier that prevented entry of inhaled matter into the lung parenchyma. The AEC is now recognized as having an important role in the inflammatory response of the respiratory system to inhaled exposures, and abnormalities of these responses are thought to be important to asthma pathogenesis. This review first explores how the challenges of studying nasal and bronchial AECs in children have been addressed and then summarizes the results of studies of primary AEC function in children with and without asthma. There is good evidence that nasal AECs may be a suitable surrogate for the study of certain aspects of bronchial AEC function, although bronchial AECs remain the gold standard for asthma research. There are consistent differences between children with and without asthma for nasal and bronchial AEC mediator release following exposure to a range of pro-inflammatory stimulants including interleukins (IL)-1β, IL-4, and IL-13. However, there are inconsistencies between studies, e.g., release of IL-6, an important pro-inflammatory cytokine, is not increased in children with asthma relative to controls in all studies. Future work should expand current understanding of the "upstream" signalling pathways in AEC, study AEC from children before the onset of asthma symptoms and in vitro models should be developed that replicate the in vivo status more completely, e.g., co-culture with dendritic cells. AECs are difficult to obtain from children and collaboration between centers is expected to yield meaningful advances in asthma understanding and ultimately help deliver novel therapies. 

Increased expression of the RIα subunit of the cAMP-dependent protein kinase A is associated with advanced stage ovarian cancer

The primary element in the cAMP signal transduction pathway is the cAMP-dependent protein kinase (PKA). Expression of the RIα subunit of type I PKA is elevated in a variety of human tumours and cancer cell lines. The purpose of this study was to assess the prognostic importance of RIα expression in patients with ovarian cancer. We have evaluated the expression of RIα in a panel of human ovarian tumours (n = 40) and five human ovarian cancer cell lines using quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. The human ovarian cell lines OAW42 and OTN14 express high endogenous levels of RIα mRNA and protein (at significantly higher mRNA levels than high tissue expressors, P < 0.05). The ovarian cell line A2780 expresses low endogenous levels of RIα mRNA and protein (also at higher mRNA levels than low tissue expressors, P < 0.05). Quantitative RT-PCR revealed no significant difference in RIα mRNA expression between different ovarian histological subtypes in this study. No associations were found between RIα mRNA expression and differentiation state. RIα mRNA expression was significantly associated with tumour stage (P = 0.0036), and this remained significant in univariate analysis (P = 0.0002). A trend emerged between RIα mRNA expression levels and overall survival in univariate analysis (P = 0.051), however, by multivariate analysis, stage remained the major determinant of overall survival (P = 0.0001). This study indicates that in ovarian epithelial tumours high RIα mRNA expression is associated with advanced stage disease. RIα expression may be of predictive value in ovarian cancer and may be associated with dysfunctional signalling pathways in this cancer type.

The roles of Pellino E3 ubiquitin ligases in immunity

Pellino proteins were initially characterized as a family of E3 ubiquitin ligases that can catalyse the ubiquitylation of interleukin-1 receptor-associated kinase 1 (IRAK1) and regulate innate immune signalling pathways. More recently, physiological and molecular roles for members of the Pellino family have been described in the regulation of innate and adaptive immune responses by ubiquitylation. This Review describes the emerging roles of Pellino proteins in innate and adaptive immunity and discusses the mechanistic basis of these functions.

A BRCA1 deficient, NFκB driven immune signal predicts good outcome in triple negative breast cancer

Triple negative (TNBCs) and the closely related Basal-like (BLBCs) breast cancers are a loosely defined collection of cancers with poor clinical outcomes. Both show strong similarities with BRCA1-mutant breast cancers and BRCA1 dysfunction, or 'BRCAness', is observed in a large proportion of sporadic BLBCs. BRCA1 expression and function has been shown in vitro to modulate responses to radiation and chemotherapy. Exploitation of this knowledge in the treatment of BRCA1-mutant patients has had varying degrees of success. This reflects the significant problem of accurately detecting those patients with BRCA1 dysfunction. Moreover, not all BRCA1 mutations/loss of function result in the same histology/pathology or indeed have similar effects in modulating therapeutic responses. Given the poor clinical outcomes and lack of targeted therapy for these subtypes, a better understanding of the biology underlying these diseases is required in order to develop novel therapeutic strategies.We have discovered a consistent NFκB hyperactivity associated with BRCA1 dysfunction as a consequence of increased Reactive Oxygen Species (ROS). This biology is found in a subset of BRCA1-mutant and triple negative breast cancer cases and confers good outcome. The increased NFκB signalling results in an anti-tumour microenvironment which may allow CD8+ cytotoxic T cells to suppress tumour progression. However, tumours lacking this NFκB-driven biology have a more tumour-promoting environment and so are associated with poorer prognosis. Tumour-derived gene expression data and cell line models imply that these tumours may benefit from alternative treatment strategies such as reprogramming the microenvironment and targeting the IGF and AR signalling pathways.

Molecular Subtypes and Personalized Therapy in Metastatic Colorectal Cancer

Development of colorectal cancer occurs via a number of key pathways, with the clinicopathological features of specific subgroups being driven by underlying molecular changes. Mutations in key genes within the network of signalling pathways have been identified; however, therapeutic strategies to target these aberrations remain limited. As understanding of the biology of colorectal cancer has improved, this has led to a move toward broader genomic testing, collaborative research and innovative, adaptive clinical trial design. Recent developments in therapy include the routine adoption of wider mutational spectrum testing prior to use of targeted therapies and the first promise of effective immunotherapy for colorectal cancer patients. This review details current biomarkers in colorectal cancer for molecular stratification and for treatment allocation purposes, including open and planned precision medicine trials. Advances in our understanding, therapeutic strategy and technology will also be outlined.

Role of insulin and insulin-like peptides in bone formation: identification of bone-specific target genes and regulatory mechanisms, and characterization of the insulin-mimetic effect of vanadium

Tese de Doutoramento em Biologia, Especialidade em Biologia Molecular, Universidade do Algarve, 2008

Study the function of the newly discovered TrkB receptor fragment (TrkB-ICD) formed by calpain cleavage

Tese de mestrado, Neurociências, Faculdade de Medicina, Universidade de Lisboa, 2016

HES1 in immunity and cancer

Hairy and enhancer of split homolog-1 (HES1) is a part of an extensive family of basic helix-loop-helix (bHLH) proteins and plays a crucial role in the control and regulation of cell cycle, proliferation, cell differentiation, survival and apoptosis in neuronal, endocrine, T-lymphocyte progenitors as well as various cancers. HES1 is a transcription factor which is regulated by the NOTCH, Hedgehog and Wnt signalling pathways. Aberrant expression of these pathways is a common feature of cancerous cells. There appears to be a fine and complicated crosstalk at the molecular level between the various signalling pathways and HES1, which contributes to its effects on the immune response and cancers such as leukaemia. Several mechanisms have been proposed, including an enhanced invasiveness and metastasis by inducing epithelial mesenchymal transition (EMT), in addition to its strict requirement for tumour cell survival. In this review, we summarize the current biology and molecular mechanisms as well as its use as a clinical target in cancer therapeutics.

Urocortin – From Parkinson’s disease to the skeleton

Urocortin (Ucn 1), a 40 amino acid long peptide related to corticotropin releasing factor (CRF) was discovered 19 years ago, based on its sequence homology to the parent molecule. Its existence was inferred in the CNS because of anatomical and pharmacological discrepancies between CRF and its two receptor subtypes. Although originally found in the brain, where it has opposing actions to CRF and therefore confers stress-coping mechanisms, Ucn 1 has subsequently been found throughout the periphery including heart, lung, skin, and immune cells. It is now well established that this small peptide is involved in a multitude of physiological and pathophysiological processes, due to its receptor subtype distribution and promiscuity in second messenger signalling pathways. As a result of extensive studies in this field, there are now well over one thousand peer reviewed publications involving Ucn 1. In this review, we intend to highlight some of the less well known actions of Ucn 1 and in particular its role in neuronal cell protection and maintenance of the skeletal system, both by conventional methods of reviewing the literature and using bioinformatics, to highlight further associations between Ucn 1 and disease conditions. Understanding how Ucn 1 works in these tissues, will help to unravel its role in normal and pathophysiological processes. This would ultimately allow the generation of putative medical interventions for the alleviation of important diseases such as Parkinson's disease, arthritis, and osteoporosis.

Characterization of the effects of antiepileptic drugs on bone metabolism: in vitro studies with human osteoblastic and osteoclastic cells

Bone is constantly being molded and shaped by the action of osteoclasts and osteoblasts. A proper equilibrium between both cell types metabolic activities is required to ensure an adequate skeletal tissue structure, and it involves resorption of old bone and formation of new bone tissue. It is reported that treatment with antiepileptic drugs (AEDs) can elicit alterations in skeletal structure, in particular in bone mineral density. Nevertheless, the knowledge regarding the effects of AEDs on bone cells are still scarce. In this context, the aim of this study was to investigate the effects of five different AEDs on human osteoclastic, osteoblastic and co-cultured cells. Osteoclastic cell cultures were established from precursor cells isolated from human peripheral blood and were characterized for tartrate-resistant acid phosphatase (TRAP) activity, number of TRAP+ multinucleated cells, presence of cells with actin rings and expressing vitronectin and calcitonin receptors and apoptosis rate. Also, the involvement of several signaling pathways on the cellular response was addressed. Osteoblastic cell cultures were obtained from femur heads of patients (25-45 years old) undergoing orthopaedic surgery procedures and were then studied for cellular proliferation/viability, ALP activity, histochemical staining of ALP and apoptosis rate. Also the expression of osteoblast-related genes and the involvement of some osteoblastogenesis-related signalling pathways on cellular response were addressed. For co-cultured cells, osteoblastic cells were firstly seeded and cultured. After that, PBMC were added to the osteoblastic cells and co-cultures were evaluated using the same osteoclast and osteoblast parameters mentioned above for the corresponding isolated cell. Cell-cultures were maintained in the absence (control) or in the presence of different AEDs (carbamazepine, gabapentin, lamotrigine, topiramate and valproic acid). All the tested drugs were able to affect osteoclastic and osteoblastic cells development, although with different profiles on their osteoclastogenic and osteoblastogenic modulation properties. Globally, the tendency was to inhibit the process. Furthermore, the signaling pathways involved in the process also seemed to be differently affected by the AEDs, suggesting that the different drugs may affect osteoclastogenesis and/or osteoblastogenesis through different mechanisms. In conclusion, the present study showed that the different AEDs had the ability to directly and indirectly modulate bone cells differentiation, shedding new light towards a better understanding of how these drugs can affect bone tissue.

Insights into the role of almond CBF transcription factors in the environmental control of cold acclimation and dormancy break

Dissertation presented to obtain the Ph.D degree in Biology

Expression analysis of the AtPHO1 gene family in Arabidopsis thaliana and the involvement of AtPHO1 in the regulation of stomatal movements

Résumé Le transfert du phosphate des racines vers les feuilles s'effectue par la voie du xylème. Il a été précédemment démontré que la protéine AtPHO1 était indispensable au transfert du phosphate dans les vaisseaux du xylème des racines chez la plante modèle Arabidopsis thaliana. Le séquençage et l'annotation du génome d'Arabidopsis ont permis d'identifier dix séquences présentant un niveau de similarité significatif avec le gène AtPHO1 et constituant une nouvelle famille de gène appelé la famille de AtPHO1. Basée sur une étude moléculaire et génétique, cette thèse apporte des éléments de réponse pour déterminer le rôle des membres de ia famille de AtPHO1 chez Arabidopsis, inconnue à ce jour. Dans un premier temps, une analyse bioinformatique des séquences protéiques des membres de la famille de AtPHO1 a révélé la présence dans leur région N-terminale d'un domaine nommé SPX. Ce dernier est conservé parmi de nombreuses protéines impliquées dans l'homéostasie du phosphate chez la levure, renforçant ainsi l'hypothèse que les membres de la famille de AtPHO1 auraient comme AtPHO1 un rôle dans l'équilibre du phosphate dans la plante. En parallèle, la localisation tissulaire de l'expression des gènes AtPHO dans Arabidopsis a été identifiée par l'analyse de plantes transgéniques exprimant le gène rapporteur uidA sous le contrôle des promoteurs respectifs des gènes AtPHO. Un profil d'expression de chaque gène AtPHO au cours du développement de la plante a été obtenu. Une expression prédominante au niveau des tissus vasculaires des racines, des feuilles, des tiges et des fleurs a été observée, suggérant que les gènes AtPHO pourraient avoir des fonctions redondantes au niveau du transfert de phosphate dans le cylindre vasculaire de ces différents organes. Toutefois, plusieurs régions promotrices des gènes AtPHO contrôlent également un profil d'expression GUS non-vasculaire, indiquant un rôle putatif des gènes AtPHO dans l'acquisition ou le recyclage de phosphate dans la plante. Dans un deuxième temps, l'analyse de l'expression des gènes AtPHO durant une carence en phosphate a établi que seule l'expression des gènes AtPHO1, AtPHO1; H1 et AtPHO1; H10 est régulée par cette carence. Une étude approfondie de leur expression en réponse à des traitements affectant l'homéostasie du phosphate dans la plante a ensuite démontré leur régulation par différentes voies de signalisation. Ensuite, une analyse détaillée de la régulation de l'expression du gène AtPHO1; H1O dans des feuilles d'Arabidopsis blessées ou déshydratées a révélé que ce gène constitue le premìer gène marqueur d'une nouvelle voie de signalisation induite par l'OPDA, pas par le JA et dépendante de la protéine COI1. Ces résultats démontrent pour la première fois que l'OPDA et le JA peuvent activer différents gènes via des voies de signalisation dépendantes de COI1. Enfin, cette thèse révèle l'identification d'un nouveau rôle de la protéine AtPHO1 dans la régulation de l'action de l'ABA au cours des processus de fermeture stomatique et de germination des graines chez Arabidopsis. Bien que les fonctions exactes des protéines AtPHO restent à être déterminées, ce travail de thèse suggère leur implication dans la propagation de différents signaux dans la plante via la modulation du potentiel membranaire et/ou l'affectation de la composition en ions des cellules comme le font de nombreux transporteurs ou régulateur du transport d'ions. Summary Phosphate is transferred from the roots to the shoot via the xylem. The requirement for AtPHO1 protein to transfer phosphate to the xylem vessels of the root has been previously demonstrated in Arabidopsis thaliana. The sequencing and the annotation of the Arabidopsis genome had allowed the identification of ten sequences that show a significant level of similarity with the AtPHO1 gene. These 10 genes, of unknown functions, constitute a new gene family called the AtPHO1 gene family. Based on a molecular and genetics study, this thesis reveals some information needed to understand the role of the AtPHO1 family members in the plant Arabidopsis. First, a bioinformatics study revealed that the AtPHO sequences contained, in the N-terminal hydrophilic region, a motif called SPX and conserved among multiple proteins involved in phosphate homeostasis in yeast. This finding reinforces the hypothesis that all AtPHO1 family members have, as AtPHO1, a role in phosphate homeostasis. In parallel, we identified the pattern of expression of AtPHO genes in Arabidopsis via analysis of transgenic plants expressing the uidA reporter gene under the control of respective AtPHO promoter regions. The results exhibit a predominant expression of AtPHO genes in vascular tissues of all organs of the plant, implying that these AtPHO genes could have redundant functions in the transfer of phosphate to the vascular cylinder of various organs. The GUS expression pattern for several AtPHO promoter regions was also detected in non-vascular tissue indicating a broad role of AtPHO genes in the acquisition or in the recycling of phosphate in the plant. In a second step, the analysis of the expression of AtPHO genes during phosphate starvation established that only the expression of the AtPHO1, AtPHO1; H1 and AtPHO1; H10 genes were regulated by Pi starvation. Interestingly, different signalling pathways appeared to regulate these three genes during various treatments affecting Pi homeostasis in the plant. The third chapter presents a detailed analysis of the signalling pathways regulating the expression of the AtPHO1; H10 gene in Arabidopsis leaves during wound and dehydrated stresses. Surprisingly, the expression of AtPHO1; H10 was found to be regulated by OPDA (the precursor of JA) but not by JA itself and via the COI1 protein (the central regulator of the JA signalling pathway). These results demonstrated for the first time that OPDA and JA could activate distinct genes via COI1-dependent pathways. Finally, this thesis presents the identification of a novel role of the AtPHO1 protein in the regulation of ABA action in Arabidopsis guard cells and during seed germination. Although the exact role and function of AtPHO1 still need to be determined, these last findings suggest that AtPHO1 and by extension other AtPHO proteins could mediate the propagation of various signals in the plant by modulating the membrane potential and/or by affecting cellular ion composition, as it is the case for many ion transporters or regulators of ion transport.

Development and preclinical assessment of a bioartificial pancreas.

Transplantation of insulin secreting cells is regarded as a possible treatment for type 1 diabetes. One major difficulty in this approach is, however, that the transplanted cells are exposed to the patient's inflammatory and autoimmune environment, which originally destroyed their own beta-cells. Therefore, even if a good source of insulin-secreting cells can be identified for transplantation therapy, these cells need to be protected against these destructive influences. The aim of this project was to evaluate, using a clonal mouse beta-cell line, whether genetic engineering of protective genes could be a viable option to allow these cells to survive when transplanted into autoimmune diabetic mice. We demonstrated that transfer of the Bcl-2 anti-apoptotic gene and of several genes specifically interfering with cytokines intracellular signalling pathways, greatly improved resistance of the cells to inflammatory stresses in vitro. We further showed that these modifications did not interfere with the capacity of these cells to correct hyperglycaemia for several months in syngeneic or allogeneic streptozocin-diabetic mice. However, these cells were not protected against autoimmune destruction when transplanted into type 1 diabetic NOD mice. This suggests that in addition to inflammatory attacks by cytokines, autoimmunity very efficiently kills the transplanted cells, indicating that multiple protective mechanisms are required for efficient transplantation of insulin-secreting cells to treat type 1 diabetes.