Nitric oxide synthase and NAD(P)H oxidase modulate coronary endothelial cell growth

Reactive oxygen species (ROS) including nitric oxide (NO) and superoxide anion (O2-) are associated with cell migration, proliferation and many growth-related diseases. The objective of this study was to determine whether there was a reciprocal relationship between rat coronary microvascular endothelial cell (CMEC) growth and activity/expressions (mRNA and protein) of endothelial NO synthase (eNOS) and NAD(P)H oxidase enzymes. Proliferating namely, 50% confluent CMEC possessed approximately three-fold increased activity and expression of both enzymes compared to 100% confluent cells. Treatment of CMEC with an inhibitor of eNOS (L-NAME, 100M) increased cell proliferation as assessed via three independent methods i.e. cell counting, determination of total cellular protein levels and [3H]thymidine incorporation. Similarly, treatment of CMEC with pyrogallol (0.3-3 mM), a superoxide anion (O2-)- generator, also increased CMEC growth while spermine NONOate (SpNO), a NO donor, significantly reduced cell growth. Co-incubation of CMEC with a cell permeable superoxide dismutase mimetic (Mn-III-tetrakis-4-benzoic acid-porphyrin; MnTBAP) plus either pyrogallol or NO did not alter cell number and DNA synthesis thereby dismissing the involvement of peroxynitrite (OONO-) in CMEC proliferation. Specific inhibitors of NAD(P)H oxidase but not other ROS-generating enzymes including cyclooxygenase and xanthine oxidase, attenuated cell growth. Transfection of CMEC with antisense p22-phox cDNA, a membrane-bound component of NAD(P)H oxidase, resulted in substantial reduction in [3H]thymidine incorporation, total cellular protein levels and expression of p22-phox protein. These data demonstrate a cross-talk between CMEC growth and eNOS and NAD(P)H oxidase enzyme activity and expression, thus suggesting that the regulation of these enzymes may be critical in preventing the initiation and/or progression of coronary atherosclerosis.

Two novel COLVI long chains in zebrafish that are essential for muscle development

Collagen VI (COLVI), a protein ubiquitously expressed in connective tissues, is crucial for structural integrity, cellular adhesion, migration and survival. Six different genes are recognized in mammalians, encoding six COLVI-chains that assemble as two ‘short’ (α1, α2) and one ‘long’ chain (theoretically any one of α3–6). In humans, defects in the most widely expressed heterotrimer (α123), due to mutations in the COL6A1-3 genes, cause a heterogeneous group of neuromuscular disorders, collectively termed COLVI-related muscle disorders. Little is known about the function(s) of the recently described α4-6 chains and no mutations have been detected yet. In this study, we characterized two novel COLVI long chains in zebrafish that are most homologous to the mammalian α4 chain; therefore, we named the corresponding genes col6a4a and col6a4b. These orthologues represent ancestors of the mammalian Col6a4-6 genes. By in situ hybridization and RT-qPCR, we unveiled a distinctive expression kinetics for col6a4b, compared with the other col6a genes. Using morpholino antisense oligonucleotides targeting col6a4a, col6a4b and col6a2, we modelled partial and complete COLVI deficiency, respectively. All morphant embryos presented altered muscle structure and impaired motility. While apoptosis was not drastically increased, autophagy induction was defective in all morphants. Furthermore, motoneuron axon growth was abnormal in these morphants. Importantly, some phenotypical differences emerged between col6a4a and col6a4b morphants, suggesting only partial functional redundancy. Overall, our results further confirm the importance of COLVI in zebrafish muscle development and may provide important clues for potential human phenotypes associated with deficiency of the recently described COLVI-chains.

Caracterização funcional de dois cDNAs homólogos à ciclofilina e à proteína reprimida por auxina (ARP) identificados em bibliotecas subtrativas a para floração em tomateiro

Flowering is a fundamental process in the life cycle for plant. This process is marked by vegetative to reproductive apical meristem conversion, due to interactions between several factors, both internal and external to plant. Therefore, eight subtractive libraries were constructed using apical meristem induced or not induced for two contrasting species: Solanum lycopersicum cv. Micro-Tom and Solanum pimpinellifolium. Several cDNAs were identified and among these, were selected two cDNAs: one homologous cDNA to cyclophilin (LeCYP1) and the other to Auxin repressed protein (ARP). It has observed that LeCYP1 and ARP genes are important in the developmental process to plants. In silico analysis, were used several databases with the exclusion criterion E-value <1.0x10-15. As a result, conservation was observed for proteins analyzed by means of multiple alignments and the presence of functional domains. Then, overexpression cassettes were constructed for the ARP cDNA in sense and antisense orientations. For this step, it was used the CaMV35S promoter. The cDNA orientation (sense or antisense) in relation to the promoter was determined by restriction enzymes and sequencing. Then, this cassette was transferred to binary vector pZP211 and these cassettes were transferred into Agrobacterium tumefaciens LBA4404. S. lycopersicum cv. Micro-Tom (MT) and MT-Rg1 plants were transformed. In addition, seedlings were subjected to hormone treatments using a synthetic auxin (- naphthalene acetic acid) and cyclosporin A (cyclophilin inhibitor) treatments and it was found that the hormone treatment there were changes in development of lateral roots pattern, probably related to decreases in auxin signaling caused by reduction of LeCYP1 in MT-dgt plants while cyclosporin A treatments, there was a slight delay in flowering in cv. MT plants. Furthermore, assay with real-time PCR (RT-qPCR) were done for expression level analysis from LeCYP1 and ARP in order to functionally characterize these sequences in tomato plants.

Epigenetics and alternative splicing

Dissertação de Mestrado, Oncobiologia: Mecanismos Moleculares do Cancro, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

Investigation of H19/RsaI Polymorphism in Children With Low Birth Weight in Pernambuco, Brazil

Background: H19 is a strong candidate gene for influencing birth weight variation and is exclusively imprinted maternally. In an attempt to understand the relationship of this gene polymorphism with low birth weight children, we investigated association of H19/RsaI polymorphism with low birth weight and normal birth weight in children and their mothers. Objectives: The aim of our study was to establish the association between H19 gene polymorphism and LW in children born in Pernambuco, state of Brazil. Patients and Methods: It were selected 89 children, 40 low birth weight (LW) and 49 normal birth weight (NW) and 71 mothers (40 mothers of newborns NW and 31 mothers of newborns LW) attended at Dom Malan Hospital, Petrolina, Pernambuco - Brazil. Peripheral blood samples were collected from patients and genomic DNA was extracted and detected by electrophoresis agarose gel, stained by Blue Green Loading Dye. DNA PCR amplification was done using the primers H1 (sense) and H3 (antisense). PCR products were digested with RsaI and electrophoresed on agarose gel stained by ethidium bromide. Statistical analyses were performed using the program BioEstat version 5.0. Results: The RsaI polymorphism in the H19 gene showed that genotype frequencies did not differ statistically between low birth weight (AA = 12.5%, AB = 45%, BB = 42.5%) and control (AA = 8.6% AB = 36.73%, BB= 55.10% groups) and the allele frequencies were not significantly different (P = 0.2897). We also did not observe any association between maternal H19 allele polymorphism and low birth weight newborns (P =0.7799) or normal birth weight children (P = 0.8976). Conclusions: The small size of sample may be the explanation for these results; future studies with more patients are needed to confirm the effect of H19/RsaI polymorphism on birth weight of LW newborns.

La perturbation du locus Nr2f1-K12 entraine une différenciation gliale précoce dans un nouveau modèle murin de mégacôlon aganglionnaire

La maladie de Hirschsprung est une affection congénitale de la motilité intestinale caractérisée par un segment aganglionnaire dans le côlon terminal. Un criblage génétique par mutation insertionnelle aléatoire chez la souris nous a permis d’identifier la lignée transgénique Spot dont les homozygotes souffrent de mégacôlon aganglionnaire. L’analyse d’intestins d’embryons mutants a révélé une baisse de prolifération et un délai de migration des cellules de la crête neurale entériques (CCNe) progénitrices dus à leur différenciation gliale précoce, entrainant un défaut de colonisation de l’intestin et une aganglionose du côlon. Le séquençage du génome Spot indique que le transgène s’est inséré à l’intérieur du locus K12-Nr2f1 sur le chromosome 13, une région dépourvue de gènes préalablement associés à la maladie, perturbant également une séquence non-codante très conservée dans l’évolution. K12 est un gène d’ARN long non codant (ARNlnc) et antisens du gène Nr2f1, lui-même impliqué dans la gliogénèse du système nerveux central. Le séquençage du transcriptome des CCN a montré une surexpression de Nr2f1 et des formes courtes de K12 chez Spot et des essais luciférase ont révélé l’activité répressive de l’élément conservé. Nous avons observé l’expression de K12 dans les CCNe et sa localisation subcellulaire dans des zones transcriptionnellement actives du noyau. Avec l’émergence des ARNlnc régulateurs, ces données nous permettent de pointer deux nouveaux gènes candidats associés à une différenciation gliale prématurée du SNE menant au mégacôlon aganglionnaire, en supposant que la régulation de Nr2f1 se fait par son antisens, K12.

La perturbation du locus Nr2f1-K12 entraine une différenciation gliale précoce dans un nouveau modèle murin de mégacôlon aganglionnaire

La maladie de Hirschsprung est une affection congénitale de la motilité intestinale caractérisée par un segment aganglionnaire dans le côlon terminal. Un criblage génétique par mutation insertionnelle aléatoire chez la souris nous a permis d’identifier la lignée transgénique Spot dont les homozygotes souffrent de mégacôlon aganglionnaire. L’analyse d’intestins d’embryons mutants a révélé une baisse de prolifération et un délai de migration des cellules de la crête neurale entériques (CCNe) progénitrices dus à leur différenciation gliale précoce, entrainant un défaut de colonisation de l’intestin et une aganglionose du côlon. Le séquençage du génome Spot indique que le transgène s’est inséré à l’intérieur du locus K12-Nr2f1 sur le chromosome 13, une région dépourvue de gènes préalablement associés à la maladie, perturbant également une séquence non-codante très conservée dans l’évolution. K12 est un gène d’ARN long non codant (ARNlnc) et antisens du gène Nr2f1, lui-même impliqué dans la gliogénèse du système nerveux central. Le séquençage du transcriptome des CCN a montré une surexpression de Nr2f1 et des formes courtes de K12 chez Spot et des essais luciférase ont révélé l’activité répressive de l’élément conservé. Nous avons observé l’expression de K12 dans les CCNe et sa localisation subcellulaire dans des zones transcriptionnellement actives du noyau. Avec l’émergence des ARNlnc régulateurs, ces données nous permettent de pointer deux nouveaux gènes candidats associés à une différenciation gliale prématurée du SNE menant au mégacôlon aganglionnaire, en supposant que la régulation de Nr2f1 se fait par son antisens, K12.

Registro de pacientes con distrofinopatías en Colombia

INTRODUCCIÓN. La distrofia muscular de Duchenne es una enfermedad neuromuscular con una herencia recesiva ligada al X que afecta a 1 de cada 3500 niños nacidos vivos. Se produce por mutaciones en el gen DMD que codifica para la distrofina. Se caracteriza por manifestaciones clínicas variables típicas de una distrofia muscular proximal progresiva. OBJETIVO. Realizar el primer registro en Colombia de los pacientes identificados con distrofinopatías, teniendo en cuenta características clínicas y paraclínicas, así como las mutaciones causales de esta patología. METODOLOGÍA Es un estudio descriptivo, transversal, de la revisión de historias clínicas de los pacientes con diagnóstico de DMD atendidos en la consulta de Genética de la Universidad del Rosario durante los años 2006 a 2015. RESULTADOS Se identificaron 99 pacientes, de los cuales 56 (56,56%) corresponden al fenotipo Duchenne y 12 (12,12%) al Becker. No fue posible clasificar a 31 pacientes (31,3%) por falta de datos clínicos. La edad de inicio de los síntomas fue en promedio de 4,41 años. Las mutaciones más frecuentes fueron las deleciones (69%), seguidas por las mutaciones puntuales(14%), las duplicaciones (11%) y por otras mutaciones (4%). CONCLUSIONES Este registro de distrofinopatías es el primero reportado en Colombia y el punto de partida para conocer la incidencia de la enfermedad, caracterización clínica y molecular de los pacientes, garantizando así el acceso oportuno a los nuevos tratamientos de medicina de precisión que permitan mejorar la calidad de vida de los pacientes y sus familias.

Caracterisation fonctionelle d'un facteur d'elongation mitochondrial leEF-Tsmt chez la tomate: approaches par transgenese et proteomique.

Esta tese é relacionada ao estudo funcional de um gene que codifica um fator de elongação LeEF-Tsmt em tomate. Este gene participa no processo de síntese de proteína em mitocôndrias e apresenta uma forte expressão durante o processo de maturação quando comparado a outros órgãos. Nós demonstramos que o mesmo se exprime fortemente durante as primeiras fases do processo maturação em paralelo com a crise respiratória climatérica e que sua expressão é estimulada pelo etileno, ferimento e altas temperaturas. Porém, os mutantes de tomate insensíveis ao etileno, exibem uma expressão normal. Frutos transgênicos foram gerados, nos quais o LeEF-Tsmt foi aumentado ou inibido de uma forma constitutiva. Porém, a alteração da expressão do gene através da transformação genética com construções sentido e antisense do gene LeEF-Tsmt não afeta o padrão de respiração e produção de etileno durante a maturação e após o ferimento. Além disso, a expressão do gene da alternativa oxidase, que é conhecida por apresentar um papel importante no climatério respiratório, não foi afetada. Todos estes dados indicam que apesar de sua forte regulação, o LeEF-Tsmt não é limitante da atividade respiratória mitocondrial. A expressão do gene de LeEF-Tsmt é estimulada pelo efeito do estresse oxidativo induzido nas partes vegetativas da planta pela seca e o paraquat. A sensibilidade ao estresse oxidativo avaliado em folhas pela presença de necrose e em calos através de crescimento celular, foi reduzido em plantas antisentido. Entre as enzimas conhecidas por apresentar um papel na detoxificação de espécies reativas de oxigênio, superóxido dismutase (SOD), catalases (CAT), peroxidase (PX) e glutation redutase (GR), nós demostramos que a GR e PX exibem atividade mais alta em linhas antisentido, explicando assim, pelo menos em parte, sua melhor tolerância ao estresse. O papel da proteína de LeEF-Tsmt na síntese de proteínas mitocondriais foi estudado pela análise do proteôma mitocondrial em linhas antisentido e sentido do gene LeEF-Tsmt. A comparação dos proteômas de linhas transformadas e selvagem foi tratado com a ajuda de uma técnica de dupla marcagem 14N/15N aplicadas à tecidos de tomate cultivados in vitro. A linha sentido super expressa fortemente a proteína, enquanto que as linhas antisentidos diminuem ligeiramente. Uma proteína do tipo ?heat-shock? segue as variações da proteína LeEF-Tsmt, sugerindo um possível papel chaperona. Uma análise global do proteôma mitocondrial foi executada, fornecendo novas informações sobre um conjunto de ao redor 500 proteínas mitocondriais de tomate.

The mitochondrial elongation factor LeEF-Tsmt is regulated during tomato fruit ripening and upon wounding and ethylene treatment.

A gene encoding an elongation factor LeEF-Tsmt that participates in the protein synthesis process in mitochondria shows strong expression in ripening fruit as compared to other organs. It is strongly up-regulated during the first stages of the ripening process in parallel with the climacteric rise in respiration. LeEF-Tsmt expression is stimulated by ethylene, wounding and high temperature but ethylene-insensitive mutants exhibit normal expression. Transgenic fruit have been generated in which LeEF-Tsmt has been constitutively up- and down-regulated. Surprisingly, altering the expression of the gene by genetic transformation with antisense and sense LeEF-Tsmt constructs did not affect the pattern of respiration and ethylene production during ripening and upon wounding. In addition, expression of the alternative oxidase gene which is known to play an important role in respiratory climacteric was not affected. Possible reasons for the absence of effect on respiration of variations of LeEF-Tsmt gene expression are discussed.

Ruolo del gene umano CYYR1: dallo studio funzionale dell'ortologo in Danio rerio alla potenziale implicazione in processi tumorigenici

Il locus CYYR1 identificato e clonato sul cromosoma 21 umano è stato caratterizzato dal punto di vista molecolare come un sistema multitrascritto, esclusivo dei vertebrati che ad oggi è orfano di una funzione specifica. Dati presenti in lettura e rintracciati mostrano una possibile relazione tra il gene CYYR1 e il pathway di Sonic Hedgehog (SHH). In questo progetto di tesi è stato utilizzato il modello animale Danio rerio per indagare il ruolo funzionale dell’ortologo (cyyr1), attraverso esperimenti di gain e loss of function che hanno permesso di dimostrare un suo coinvolgimento nello sviluppo del sistema nervoso centrale, del cuore e del tessuto muscolare. Lo studio dell’ortologo in zebrafish è stato associato all’utilizzo di linee cellulari di rabdomiosarcoma umano. I risultati ottenuti dall’induzione al differenziamento miogenico di queste linee, insieme ai dati ottenuti in Danio rerio, confermano il possibile coinvolgimento del gene CYYR1 nella miogenesi. Lo studio delle relazione tra il pathway di SHH e l’espressione del gene CYYR1 è stato condotto in entrambi i modelli con l’utilizzo di differenti inibitori della via di segnalazione. I risultati ottenuti mostrano che sistemi inibitori agenti direttamente sul recettore SMO riducono l’espressione del gene. Un dato inaspettato in Danio rerio ottenuto durante questi esperimenti di inibizione, ha aperto una nuova linea di ricerca in collaborazione con l’Università di Warwick tesa a verificare la relazione tra il gene cyyr1 e il gene lefty1. Gli esperimenti condotti presso il laboratorio della Prof.ssa Sampath hanno dimostrato la localizzazione del prodotto proteico cyyr1 in Danio rerio e indagato co-localizzazioni con la proteina lefty1. Infine, in collaborazione con Dr. Deflorian e della Prof.ssa Pistocchi, è stato generato un mutante di Danio rerio deleto per il gene cyyr1 con la tecnica CRISPR/Cas9. La caratterizzazione del mutante cyyr1 -/- ha confermato alcuni dei dati ottenuti attraverso esperimenti di loss of function.

TAZ role in lung cancer: resistance to BET inhibitors and functional interaction with lncRNA

Despite extensive research and introduction of innovative therapy, lung cancer prognosis remains poor, with a five years survival of only 17%. The success of pharmacological treatment is often impaired by drug resistance. Thus, the characterization of response mechanisms to anti-cancer compounds and of the molecular mechanisms supporting lung cancer aggressiveness are crucial for patient’s management. In the first part of this thesis, we characterized the molecular mechanism behind resistance of lung cancer cells to the Inhibitors of the Bromodomain and Extraterminal domain containing Proteins (BETi). Through a CRISPR/Cas9 screening we identified three Hippo Pathway members, LATS2, TAOK1 and NF2 as genes implicated in susceptibility to BETi. These genes confer sensitivity to BETi inhibiting TAZ activity. Conversely, TAZ overexpression increases resistance to BETi. We also displayed that BETi downregulate both YAP, TAZ and TEADs expression in several cancer cell lines, implying a novel BETi-dependent cytotoxic mechanism. In the second part of this work, we attempted to characterize the crosstalk between the TAZ gene and its cognate antisense long-non coding RNA (lncRNA) TAZ-AS202 in lung tumorigenesis. As for TAZ downregulation, TAZ-AS202 silencing impairs NSCLC cells proliferation, migration and invasion, suggesting a pro-tumorigenic function for this lncRNA during lung tumorigenesis. TAZ-AS202 regulates TAZ target genes without altering TAZ expression or localization. This finding implies an uncovered functional cooperation between TAZ and TAZ-AS202. Moreover, we found that the EPH-ephrin signaling receptor EPHB2 is a downstream effector affected by both TAZ and TAZ-AS202 silencing. EPHB2 downregulation significantly attenuates cells proliferation, migration and invasion, suggesting that, at least in part, TAZ-AS202 and TAZ pro-oncogenic activity depends on EPH-ephrin signaling final deregulation. Finally, we started to dissect the mechanism underlying the TAZ-AS202 regulatory activity on EPHB2 in lung cancer, which may involve the existence of an intermediate transcription factor and is the object of our ongoing research.

Insight into the molecular and functional determinants of HP1043, the essential master regulator of Helicobacter pylori.

Helicobacter pylori is one of the most widespread and successful human pathogens, colonizing half of the population stomach mucosa and causing gastric malignancies in 1% of carriers. Due to the increasing number of antimicrobial-resistant strains, in 2017 the WHO included H. pylori among pathogens that pose a major threat for humankind. In this study, we propose as a molecular target for novel antimicrobial strategies HP1043, an orphan response regulator essential for the viability of H. pylori as it orchestrates all the most important cellular processes. Amino acids most relevant for HP1043 dimerization and target DNA recognition were identified and used to guide an in-silico protein-DNA docking and generate a high-resolution structural model of the interacting HP1043 dimer and its target DNA. The model was experimentally validated and exploited to carry out a virtual screening of small molecule libraries, identifying 8 compounds potentially able to interfere with HP1043 function and likely block H. pylori infection. A second line of research aimed at the characterization of the regulatory function of HP1043 and the tight mechanisms of regulation of hp1043 gene expression. In particular, we proved a direct interaction between HP1043 and the housekeeping sigma80 factor of the RNA polymerase. A conditional mutant H. pylori strain overexpressing a synthetic copy of the hp1043 gene altered in nucleotide sequence yet encoding the wild-type protein was generated, achieving increased intracellular levels of HP1043. However, overexpression of HP1043 did not result in an upregulation of target genes transcription nor modulation of hp1043 transcript levels, pinpointing the existence of multiple overlayed mechanisms of regulation that affect both protein levels and functionality as well as maintain steady the amount of hp1043 transcript. Finally, we proposed that a mechanism of post-transcriptional regulation could depend on an antisense transcript to the hp1043 gene which was validated in two different strains.

TIDES unveiled: pioneering green peptides synthesis and oligonucleotides innovations in science

My PhD research focused on the development of environmentally sustainable methods for peptide synthesis. The traditional and toxic solvents and bases used in solid-phase peptide synthesis (SPPS) were replaced with eco-friendly alternatives to reduce the environmental impact. In particular, N-octylpyrrolidone was found to be an effective green solvent in combination with dimethyl carbonate, resulting in a 63-66% reduction in process mass intensity (PMI). In addition, a green base, DEAPA, was identified for Fmoc removal, which showed comparable results to piperidine, while being less regulated and toxic, and able to better control aspartimide-related side reactions. The study extended beyond SPPS to explore liquid-phase peptide synthesis (LPPS) and solution-phase peptide synthesis (SolPPS) using propylphosphonic anhydride (T3P®) as a coupling reagent. The developed green SolPPS using Cbz amino acids achieved exceptional efficiency, minimal racemisation and a PMI of 30 to introduce a single amino acid in the iterative process. This PMI value is the lowest ever reported for an oligopeptide synthesis protocol. This technique was extended to N-Boc amino acids in DCM, requiring aqueous workups and achieving 95% purity of Leu-Enkephalin. Finally, T3P® was found to be suitable for LPPS. An anchor, mimicking a resin, was used to allow precipitation or solubilisation of the growing anchored-peptide, depending on the polarity of the solvent used. Anisole and DCM resulted in a pentapeptide purity of over 95%. While at Oxford University, I synthesized a cleavable fragment that is sensitive to cathepsin B (CatB) and incorporated it into a cyclic antisense oligonucleotide (ASO) targeting the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). ASO demonstrated good stability in a simulated in vivo environment using human serum and high affinity with complementary RNA. The Cyclic-ASO was opened by CatB in optimal conditions. Experiments highlight therapeutic potential and a novel method for controlling cyclic oligonucleotide activity, potentially enhancing cellular uptake.