Convergent Sets of Data from In Vivo and In Vitro Methods Point to an Active Role of Hsp60 in Chronic Obstructive Pulmonary Disease Pathogenesis

BACKGROUND: It is increasingly clear that some heat shock proteins (Hsps) play a role in inflammation. Here, we report results showing participation of Hsp60 in the pathogenesis of chronic obstructive pulmonary diseases (COPD), as indicated by data from both in vivo and in vitro analyses. METHODS AND RESULTS: Bronchial biopsies from patients with stable COPD, smoker controls with normal lung function, and non-smoker controls were studied. We quantified by immunohistochemistry levels of Hsp10, Hsp27, Hsp40, Hsp60, Hsp70, Hsp90, and HSF-1, along with levels of inflammatory markers. Hsp10, Hsp40, and Hsp60 were increased during progression of disease. We found also a positive correlation between the number of neutrophils and Hsp60 levels. Double-immunostaining showed that Hsp60-positive neutrophils were significantly increased in COPD patients. We then investigated in vitro the effect on Hsp60 expression in bronchial epithelial cells (16HBE) caused by oxidative stress, a hallmark of COPD mucosa, which we induced with H\u2082O\u2082. This stressor determined increased levels of Hsp60 through a gene up-regulation mechanism involving NFkB-p65. Release of Hsp60 in the extracellular medium by the bronchial epithelial cells was also increased after H\u2082O\u2082 treatment in the absence of cell death. CONCLUSIONS: This is the first report clearly pointing to participation of Hsps, particularly Hsp60, in COPD pathogenesis. Hsp60 induction by NFkB-p65 and its release by epithelial cells after oxidative stress can have a role in maintaining inflammation, e.g., by stimulating neutrophils activity. The data open new scenarios that might help in designing efficacious anti-inflammatory therapies centered on Hsp60 and applicable to COP

Characterisation of the HSP70-HSP90 organising protein gene and its link to cancer

HOP (Heat shock protein 70/ Heat shock protein 90 organising protein) is a co-chaperone essential for client protein transfer from HSP70 to HSP90 within the HSP90 chaperone machine and has been found to be up-regulated in various cancers. However, minimal in vitro information can be found on the regulation of HOP expression. The aim of this study was to analyse the HOP gene structure across known orthologues, identify and characterise the HOP promoter, and identify the regulatory mechanisms influencing the expression of HOP in cancer. We hypothesized that the expression of HOP in cancer cells is likely regulated by oncogenic signalling pathways linked to cis-elements within the HOP promoter. An initial study of the evolution of the HOP gene speciation was performed across identified orthologues using Mega5.2. The evolutionary pathway of the HOP gene was traced from the unicellular organisms to fish, to amphibian and then to land mammal. The synteny across the orthologues was identified and the co-expression profile of HOP analysed. We identified the putative promoter region for HOP in silico and in vitro. Luciferase reporter assays were utilized to demonstrate promoter activity of the upstream region in vitro. Bioinformatic analysis of the active promoter region identified a large CpG island and a range of putative cis-elements. Many of the cis-elements interact with transcription factors which are activated by oncogenic pathways. We therefore tested the regulation of HOP levels by rat sarcoma viral oncogene homologue (RAS). Cancer cell lines were transfected with mutated RAS to observe the effect of constitutively active RAS expression on the production of HOP using qRT-PCR and Western Blot analyses. Additionally, inhibitors of the RAS signalling pathway were utilised to confirm the regulatory effect of mutated RAS on HOP expression. In cancer cell lines containing mutated RAS (Hs578T), HOP was up-regulated via a mechanism involving the MAPK signalling pathway and the ETS-1 and C/EBPβ cis-elements within the HOP promoter. These findings suggest for the first time that Hop expression in cancer may be regulated by RAS activation of the HOP promoter. Additionally, this study allowed us to determine the murine system to be the most suited genetic model organism with which to study the function of human HOP

Genetic and functional analysis of the PDB6 locus in Paget's disease of bone

Tese de doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015Paget’s disease of bone (PDB) is the most frequent metabolic bone disease after osteoporosis, and despite having a strong genetic component, Sequestosome 1 is the only gene directly linked, so far, to this disease. Genome wide linkage and association studies have suggested an association of PDB6 locus with PDB, however no functional studies were performed to try to explain that association. The main goal of this work was to assess the relative contribution of each significant variant identified in the PDB6 locus to the functionality of the candidate genes and their involvement in PDB pathophysiology. In this regard we analyzed the entire PDB6 locus to search for the best candidate genes likely to be involved in bone metabolism. OPTN, CCDC3, UCMA/GRP, CAMK1D, PHYH and SEPHS1 were selected and screened for genetic variants. Together with the variants found in a previous work from our group (Michou et al 2012) we studied 82 genetic variants, from which we selected rs2234968 and rs3829923 for an association study. The results showed that only rs2234968 was significantly associated with PDB (p-value = 6 x 10-3). In addition, we assessed the effect of a rare variant (RV) found in one PDB patient, RV -9906, and rs1561570, a SNP in OPTN strongly associated with PDB. Our functional studies showed that (i) rs3829923 was responsible for an increase of OPTN promoter activity due to the appearance of new binding sites for activator nuclear factors E47 and E2F1, while (ii) RV -9906 was responsible for an increase of OPTN promoter activity but due to the loss of SP1 binding and consequent loss of its inhibitory effect in gene transcription. Rs2234968 was found to be in linkage disequilibrium with two SNPs that changed OPTN splicing, thus creating a premature stop codon, and probably resulting in a nonfunctional protein. In this work we showed that rs1561570, the SNP most associated to PDB, was responsible for the loss of a methylation site that subsequent increase in OPTN expression, which in turn increases NF-κB translocation into the nucleus and activation of NF-κB target genes. This will explain the increase in number and activity of the osteoclasts of PDB patients carrying of the rs1561570. In this work we provide new insights about OPTN regulation and its role in bone metabolism, with emphasis in osteoclastogenesis, and more importantly the contribution of OPTN variants to PDB pathophysiology.A doença óssea de Paget (DOP) é a doença óssea metabólica mais frequente a seguir à osteoporose, afectando 3% da população Caucasiana a partir dos 55 anos. Esta doença é caracterizada por um aumento focal e desorganizado do metabolismo ósseo devido ao aumento do número, tamanho e atividade dos osteoclastos, as células responsáveis pela reabsorção óssea. No entanto, posteriormente existe também um aumento no número e atividade dos osteoblastos que, na tentativa de recuperar o osso que foi reabsorvido, dão origem a um osso que não possui uma estrutura normal, tem fraca qualidade e é mais susceptível de sofrer deformações e fraturas. Este tecido ósseo desorganizado é caracterizado por apresentar hipertrofia, fragilidade e hipervascularização, podendo diminuir drasticamente a qualidade de vida dos pacientes. No entanto, a maioria dos casos são assintomáticos. A sua prevalência aumenta com a idade e é mais frequente em países da Europa, como a Inglaterra. As causas da DOP ainda não foram clarificadas. Pensa-se que possa ter uma componente ambiental e que a exposição a agentes como o fumo do tabaco ou arsénio, ou a factores relacionados com uma vida rural como o consumo de leite não pasteurizado e o contacto com animais de quinta, pode contribuir para um aumento da susceptibilidade para desenvolver DOP. A existência de uma infeção viral pelo vírus do sarampo também foi considerada um forte factor de risco, mas essa relação continua ainda por comprovar. A DOP possui também uma componente genética, com um padrão de hereditariedade autossómico dominante com penetrância incompleta, que vai desde os 17% antes dos 50 anos de idade até aos 80% depois dos 60 anos de idade. Cerca de 15 a 40% dos pacientes apresentam uma história familiar positiva, no entanto pensa-se que este número será muito maior devido ao facto desta ser uma patologia assintomática. O único gene associado à DOP até ao momento é o Sequestosoma 1 (SQSTM1/p62), que codifica para a proteína p62 que, por sua vez, está envolvida na iniciação da via do NF-κB, regulando a ubiquitinação da TRAF6 e a consequente degradação do inibidor do NF-κB (IκB), o que resulta na libertação do NF-κB para o núcleo. No entanto mais de 60% dos pacientes não têm qualquer mutação no SQSTM1, o que sugere que outros genes estarão envolvidos na DOP. Em 2010 estudos de ligação e de associação sugeriram que existia uma relação entre o locus PDB6, nomeadamente o polimorfismo rs1561570 localizado no gene da Optineurina (OPTN), e a DOP, mas não existem estudos funcionais que expliquem esta relação. Assim, o objectivo principal deste trabalho foi analisar o locus PDB6 e tentar explicar a contribuição das variações genéticas encontradas para a DOP. Para isso analisámos toda a região do locus PDB6 e procurámos genes que pudessem ter um papel no metabolismo ósseo. Assim, selecionámos os genes OPTN, CCDC3, UCMA/GRP, CAMK1D, PHYH e SEPHS1 que foram sequenciados utilizando uma amostra de 30 casos familiares e 5 controlos saudáveis. Juntamente com um trabalho anterior elaborado pelo nosso grupo foram encontradas no total 82 variantes genéticas nos seis genes analisados. Destas, duas foram selecionadas devido à sua frequência no grupo de pacientes e devido à sua localização genómica – o rs2234968 (G>A), localizado no primeiro exão codante da OPTN, e o rs3829923 (C>T), localizado no promotor partilhado entre os genes OPTN e CCDC3. Posteriormente, foi feito um estudo de associação com 225 pacientes e 298 controlos saudáveis que mostrou existir uma associação à DOP estatisticamente significativa (p-value = 6 x 10-3) unicamente para o polimorfismo rs2234968. Os estudos funcionais foram feitos com o objectivo de explicar o efeito (i) do polimorfismo rs1561570 - posteriormente associado à DOP -, (ii) do polimorfismo rs2234968 - descrito como associado à DOP neste trabalho -, e (iii) de duas variantes localizadas no promotor da OPTN, o polimorfismo rs3829923 - encontrada neste trabalho - e a variante rara RV -9906 - encontrada num único paciente, num trabalho anterior do nosso grupo. Os resultados obtidos mostraram que o polimorfismo rs3829923 era responsável por um aumento da atividade basal do promotor da OPTN devido à criação de um novo local de ligação dos factores de transcrição E47 e E2F1. Também foi demonstrado que a variante rara RV -9906 é responsável por um aumento da atividade do promotor da OPTN, mas neste caso devido à perda de ligação e consequente efeito inibitório do factor de transcrição SP1. Em relação ao polimorfismo rs2234968, os resultados mostram que este se encontra em desequilíbrio de ligação com outros dois polimorfismos - o rs10906303 e o rs79529484 - que são responsáveis por uma alteração no padrão de splicing da OPTN, alterando o quadro de leitura e originando um codão stop prematuro. A proteína resultante perde a maioria dos seus domínios proteicos e por isso é provável que seja degradada. Em relação ao polimorfismo rs1561570 (C>T), através do tratamento do DNA de pacientes e controlos saudáveis com bissulfito, e de técnicas como o qPCR ou western blot, verificámos que o alelo mais comum nos pacientes - o alelo T - era responsável pela perda de um local de metilação e pelo aumento da expressão de OPTN. Também foi possível verificar que a presença do alelo T aumenta a translocação do NF-κB para o núcleo e a expressão dos seus genes alvo envolvidos na osteoclastogénese (tal como o NFATc1). Além disso a presença do alelo T também está relacionada com um aumento da taxa de diferenciação de osteoclastos, assim como um aumento no número de núcleos e na atividade destes osteoclastos, explicando assim o fenótipo pagético. Por fim, foi feita uma análise bioinformática em que se comparou a conservação, ao longo da evolução, dos genes mais relevantes para a DOP - o SQSTM1 e a OPTN -, nomeadamente entre o humano e o peixe-zebra. Através deste estudo verificou-se que ambos os genes são conservados entre humano e peixe-zebra e que aparentam possuir mecanismos de regulação semelhantes, com base na presença de locais putativos de ligação de factores de transcrição em comum nas duas espécies. É de destacar a conservação do resíduo P392 no SQSTM1 (frequentemente mutado em pacientes com DOP) nas duas espécies, o que mostra que o peixe-zebra poderá ser um bom modelo para estudar o efeito desta mutação no metabolismo ósseo. Em relação ao gene da OPTN é de realçar o facto deste gene ser expresso nos mesmos tecidos nas duas espécies, entre eles o tecido ósseo, o que indicia que o papel da OPTN possa ser semelhante nestas duas espécies. Este trabalho permite concluir que o locus PDB6 é relevante no estudo da DOP, dado que possui vários genes com um possível papel no osso, nomeadamente o gene da OPTN. Foi possível decifrar novos mecanismos de regulação do gene OPTN desconhecidos até à data e propor um mecanismo que relacione este gene com a osteoclastogénese e com a DOP.Fundação para a Ciência e Tecnologia (FCT) - SFRH/BD/77227/201

Exploring Genetic Susceptibility: Using a combined systems biology, in vitro and ex vivo approach to understand the pathology of ulcerative colitis

The overall aim of this PhD is to use a multidisciplinary approach to determine the function of Ulcerative Colitis (UC) associated SNPs, to help understand the role of SNPs in the pathogenesis of UC in general and in a patient specific context. UC is a chronic, relapsing inflammatory disease of the large bowel for which the aetiology is thought to be a trifecta of 1) dysregulation of the immune system in response to 2) an environmental trigger in a 3) genetically susceptible host. Genetic susceptibility or susceptibility loci for UC have been identified by Genome Wide Associations Scanning and subsequent fine mapping and deep sequencing. This work intended to further characterise these susceptibility loci at a global level and a patient specific level using both a systems biology approach and experimental validation of the in-silico work. Using publicly available datasets non exonic UC associated SNPs were functionally annotated to regulatory regions within the genome. Exonic SNPs were also analysed looking at impacts in protein linear motifs and splice enhancement motifs. Bioinformatics was used to identify interacting proteins and create a UC-interactome network. This suggested that UC was a disease of fine regulators as opposed to a disease of specific target proteins. Analysis of the UC-interactome identified the focal adhesion complex (FAC) that is involved in regulating wound healing as major component of the network. One member of the FAC, Leupaxin (LPXN), was identified as a potential target for validation. Using CRISPR-Cas9 technology, LPXN overexpressing cell lines and knock out cell lines were created. Wound healing assays and cytokine analysis identified that overexpression of LPXN impaired wound healing and reduced the secretion of MCP-1. In addition, using genotyped colonic biopsies from UC patients and control patients in a polarised in vitro organ culture (pIVOC) system we show that the LPXN risk allele may impact on cytokine production. Finally, UKIBD genetics consortium data was used to access a pilot dataset of 58 patients’ SNP profiles from Immunochip data who were patients at the Norfolk and Norwich University Hospital to create patient-specific UC-interactomes. Analysis of these footprints identified convergent interacting proteins affected by multiple SNPs and novel pathogenic pathways

A transcriptomic approach for studying the activation of dendritic cells in response to mycobacterial infections

Mycobacterium tuberculosis (M. tuberculosis) is responsible for 2 million deaths annually. In recent years co-infection with HIV and drug-resistant strains has led to the increase in clinical cases of tuberculosis. BCG is the only vaccine currently available but is ineffective against adult pulmonary forms of the disease. Understanding the principle components of the host immune response against the pathogen will aid in the design of a better vaccine. Dendritic cells are potent antigen presenting cells that play a key role in priming naïve T cells. Effective T cell priming is necessary for a successful protective immune response in the host against the pathogen. I have been investigating the interaction of dendritic cells with M. tuberculosis, with a view to better understanding the signalling pathways affecting priming of anti-mycobacterial T cells by dendritic cells. Using DNA microarray and bio-informatics, I have been able to study early transcriptional signatures of bone marrow-derived dendritic cells (BMDCs) in response to M. tuberculois infection. Interferon Regulatory Factors (IRFs) and NFkB (Nuclear Factor kappa B) appear to be the principal transcription factors involved in regulating the cellular responses in M. tuberculosis infected BMDCs. It has been found that IRFs can function independent of the adaptor MyD88 (Myeloid Differentiation Factor 88) in M. tuberculosis infected BMDCs, which is surprising considering the important role played by MyD88 in the Toll mediated signalling pathway. Exvivo experiments also show that MyD88 may not be absolutely essential for mounting anti-mycobacterial T cell responses. Hence immune responses generated by M. tuberculosis infected BMDCs appear to be mediated via the MyD88 dependent and independent pathways

Modelling the molecular mechanisms of ageing

This document is the Accepted Manuscript version of a published work that appeared in final form in Bioscience reports. To access the final edited and published work see http://www.bioscirep.org/content/37/1/BSR20160177.The ageing process is driven at the cellular level by random molecular damage which slowly accumulates with age. Although cells possess mechanisms to repair or remove damage, they are not 100% efficient and their efficiency declines with age. There are many molecular mechanisms involved and exogenous factors such as stress also contribute to the ageing process. The complexity of the ageing process has stimulated the use of computational modelling in order to increase our understanding of the system, test hypotheses and make testable predictions. As many different mechanisms are involved, a wide range of models have been developed. This paper gives an overview of the types of models that have been developed, the range of tools used, modelling standards, and discusses many specific examples of models which have been grouped according to the main mechanisms that they address. We conclude by discussing the opportunities and challenges for future modelling in this field

Translational and clinical research applications of exome sequencing to neurodevelopmental disorders of childhood

Neurodevelopmental disabilities (NDDs) are a group of chronic clinically distinct disorders sharing a documented disturbance, quantitative, qualitative, or both, in developmental progress in one or more developmental domains compared with established norms. These domains are not mutually independent or exclusive and include: (1) motor (gross or fine), (2) speech and language, (3) cognition, (4) personal-social, and (5) activities of daily living (Shevell et al., 2008). Whole Exome Sequencing (WES) has provided a huge contribution to the discovery of disease-causing variants for rare diseases, especially neurodevelopmental disorders. Furthemore, the use of WES in clinical practice has improved the diagnostic rate in several rare genetic conditions, including neurodevelopmental disorders, which previously remained unexplained (Xue et al., 2014). This has led to a relevant improvement in patient management in selected disorders. Indeed, the better understanding of the pathophysiology underlying a specific condition has helped clinicians in developing a disease-specific approach in patient care. Furthermore, the identification of several new possible therapeutic targets has promoted the development of new therapeutic strategies or specific drugs. In this study, we investigated the use of exome sequencing in three different genomic research approaches: genotype-phenotype correlations; pathophysiological mechanisms; gene discovery. Our findings show that NGS techniques play a pivotal role in the NDDs research

Unravelling the mechanisms of resistance to imatinib mesylate in chronic myeloid leukemia: a proteomic approach

Imatinib mesylate is a potent inhibitor of the Bcr-Abl tyrosine kinase, an oncoprotein that plays a key role in the development of chronic myeloid leukemia. Consequently, imatinib is used as front-line therapy for this disease. A major concern in imatinib treatment is the emergence of resistance to the drug. The aim of this study was to obtain further insights into the Bcr-Abl activity-independent mechanisms underlying imatinib resistance, in chronic myeloid leukemia. The imatinib-resistant KCL22R and sensitive KCL22S cells were used as experimental model. None of the already described resistance mechanisms has been detected so far in KCL22R cells; therefore additional mechanisms independent of Bcr-Abl kinase activity could be envisaged. Moreover, KCL22S cells exhibited typical features of the quiescent hematopoietic Ph+ stem cells, thereby representing a good experimental model to investigate imatinib resistance. To this aim differentially expressed proteins between KCL22S and KCL22R cells were characterized using a proteomic approach: two-dimensional differential gel electrophoresis (2D-DIGE) coupled with Tandem Mass Spectrometry. 51 proteins were identified: 27 over-expressed and 24 under-expressed in KCL22R cells versus KCL22S cells. Bioinformatic analysis with GeneSpring and Ingenuity Pathway Analysis (IPA) softwares showed that several of these proteins were involved in the modulation of redox balance and activation of anti-apoptotic pathways mediated by NF-kB and Ras-MAPK signaling. Since the Erk pathway has been shown to influence chemotherapeutic drug resistance of hematopoietic cells, the level of activation of Erk in KCL22R and KCL22S cells was investigated. This analysis demonstrated that continuous activation of Erk occurred in KCL22R cells as compared to sensitive cells. Interestingly, examination of the most statistically significant protein network showed that several differentially expressed proteins, between KCL22R and KCL22S cells, were directly or indirectly connected with Erk. In particular, among them, this study focused on two SH2-containing, non-receptor protein tyrosine phosphatases: Shp1 (PTPN6) and Shp2 (PTPN11). It has been shown that Shp2 positively regulates the Ras-Erk pathway and is activated by phosphorylation. This study demonstrated that the level of phosphorylation and hence of activation of Shp2 in KCL22R cells was higher than in KCL22S cells. In addition the knock-down of Shp2 expression, in combination with imatinib treatment, significantly reduced the activation of Erk 1/2 in KCL22R cells and produced a reversion of the KCL22R phenotype, suggesting that Shp2 plays a role in the Bcr-Abl activity-independent mechanisms of imatinib resistance. Interestingly this study also demonstrated that Shp1, that was found down-regulated in KCL22R cells, interacted with Shp2 and that Shp1 played a negative role in the Shp2 activation in KCL22S cells. Moreover Annexin A1 and Hsp70, belonging to the same protein network, were found down-regulated in KCL22R cells. They could also play a role in imatinib resistance by the direct or indirect interaction with Shp2. Taken together these results suggest that a reduced Shp1 expression in KCL22R cells could contribute to a continuous Shp2 activation, sustaining a Bcr-Abl activity-independent pathway of proliferation and survival to imatinib treatment. These two proteins could be used as putative biomarkers to evaluate the efficacy of imatinib treatment and to develop new combinatorial therapeutic approaches