Browsing by Author "Marques, Vanda"
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- Adiponectin, Leptin, and IGF-1 are useful diagnostic and stratification biomarkers of NAFLDPublication . Marques, Vanda; Afonso, Marta; Bierig, Nina; Duarte-Ramos, Filipa; Santos-Laso, Álvaro; Jimenez-Agüero, Raul; Eizaguirre, Emma; Bujanda, Luis; Pareja, Maria J.; Luís, Rita; Costa, Adília; Machado, Mariana; Alonso, Cristina; Arretxe, Enara; Alustiza, José M.; Krawczyk, Marcin; Lammert, Frank; Tiniakos, Dina G.; Flehmig, Bertram; Cortez-Pinto, Helena; Banales, Jesus M.; Castro, Rui E.; Normann, Andrea; Rodrigues, Cecília M. P.Background: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease where liver biopsy remains the gold standard for diagnosis. Here we aimed to evaluate the role of circulating adiponectin, leptin, and insulin-like growth factor 1 (IGF-1) levels as non-invasive NAFLD biomarkers and assess their correlation with the metabolome. Materials and Methods: Leptin, adiponectin, and IGF-1 serum levels were measured by ELISA in two independent cohorts of biopsy-proven obese NAFLD patients and healthy-liver controls (discovery: 38 NAFLD, 13 controls; validation: 194 NAFLD, 31 controls) and correlated with clinical data, histology, genetic parameters, and serum metabolomics. Results: In both cohorts, leptin increased in NAFLD vs. controls (discovery: AUROC 0.88; validation: AUROC 0.83; p < 0.0001). The leptin levels were similar between obese and non-obese healthy controls, suggesting that obesity is not a confounding factor. In the discovery cohort, adiponectin was lower in non-alcoholic steatohepatitis (NASH) vs. non-alcoholic fatty liver (AUROC 0.87; p < 0.0001). For the validation cohort, significance was attained for homozygous for PNPLA3 allele c.444C (AUROC 0.63; p < 0.05). Combining adiponectin with specific serum lipids improved the assay performance (AUROC 0.80; p < 0.0001). For the validation cohort, IGF-1 was lower with advanced fibrosis (AUROC 0.67, p < 0.05), but combination with international normalized ratio (INR) and ferritin increased the assay performance (AUROC 0.81; p < 0.01). Conclusion: Serum leptin discriminates NAFLD, and adiponectin combined with specific lipids stratifies NASH. IGF-1, INR, and ferritin distinguish advanced fibrosis.
- Genetic diagnosis of hypertrophic cardiomyopathy using mass spectrometry DNA arrays and high resolution meltingPublication . Santos, Susana; Lança, Vasco; Oliveira, Helena; Branco, Patrícia; Silveira, Leonor; Marques, Vanda; Brito, Dulce; Madeira, Hugo; Bicho, Manuel; Fernandes, Alexandra R.Hypertrophic cardiomyopathy (HCM), a complex myocardial disorder with an autosomal dominant genetic pattern and prevalence of 1:500, is the most frequent cause of sudden death in apparently healthy young people. The benefits of gene-based diagnosis of HCME for both basic research and clinical medicine are limited by the considerable costs of current genetic testing due to the large number of genes and mutations involved in this pathology. However, coupling two high-throughput techniques--mass spectrometry genotyping (MSG) and high resolution melting (HRM)--is an encouraging new strategy for HCM diagnosis. Our aim was to evaluate the diagnostic efficacy of both techniques in this pathology by studying 13 individuals with a clinical phenotype of HCM. Methods: Peripheral blood samples were collected from: (i) seven subjects with a clinical diagnosis of HCM, all bearing known mutations previously identified by dideoxy sequencing and thus being used as blinded samples (sample type 1); (ii) one individual with a clinical diagnosis of HCM negative for mutations after dideoxy sequencing of the five most common HCM genes, MYH7, MYBPC3, TNNI3, TNNT2 and MYL2 (sample type 2); and (iii) five individuals individual with a clinical diagnosis of HCM who had not previously been genetically studied (sample type 3). Results: The 13 samples were analyzed by MSG for 534 known mutations in 32 genes associated with HCM phenotypes and for all coding regions and exon-intron boundaries of the same HCM genes by HRM. The 32 studied genes include the most frequent HCM-associated sarcomere genes, as well as 27 genes with lower reported HCM phenotype association. This coupled genotyping strategy enabled us to identify a c.128delC (p.A43Vfs165) frame-shift mutation in the CSRP3 gene, a gene not usually studied in current HCM genetics. The heterozygous CSRP3 mutation was found in two patients (sample types 2 and 3) aged 50 and 52 years, respectively, both with diffuse left ventricular hypertrophy. Furthermore, this coupled strategy enabled us to find a novel mutation, c.817C>T (p.Arg273Cys), in MYBPC3 in an individual from sample type 3, subsequently confirmed by dideoxy sequencing. This novel mutation in MYBPC3, not present in 200 chromosomes from 200 healthy individuals, affects a codon known to harbor an HCM-causing mutation – p.Arg253His. Conclusion: In conclusion, in the cohort used in this work coupling two technologies, MSG and HRM, with high sensitivity and low false positive results, enabled rapid, innovative and lowcost genotyping of HCM patients, which may in the short term be suitable for accurate genetic diagnosis of HCM.
- High resolution melting : improvements in the genetic diagnosis of hypertrophic cardiomyopathy in a Portuguese cohortPublication . Santos, Susana; Marques, Vanda; Pires, Marina; Silveira, Leonor; Oliveira, Helena; Lança, Vasco; Brito, Dulce; Madeira, Hugo; Esteves, J. Fonseca; Freitas, António; Carreira, Isabel M.; Gaspar, Isabel M.; Monteiro, Carolino; Fernandes, Alexandra R.Background: Hypertrophic Cardiomyopathy (HCM) is a complex myocardial disorder with a recognized genetic heterogeneity. The elevated number of genes and mutations involved in HCM limits a gene-based diagnosis that should be considered of most importance for basic research and clinical medicine. Methodology: In this report, we evaluated High Resolution Melting (HRM) robustness, regarding HCM genetic testing, by means of analyzing 28 HCM-associated genes, including the most frequent 4 HCM-associated sarcomere genes, as well as 24 genes with lower reported HCM-phenotype association. We analyzed 80 Portuguese individuals with clinical phenotype of HCM allowing simultaneously a better characterization of this disease in the Portuguese population. Results: HRM technology allowed us to identify 60 mutated alleles in 72 HCM patients: 49 missense mutations, 3 nonsense mutations, one 1-bp deletion, one 5-bp deletion, one in frame 3-bp deletion, one insertion/deletion, 3 splice mutations, one 5’UTR mutation in MYH7, MYBPC3, TNNT2, TNNI3, CSRP3, MYH6 and MYL2 genes. Significantly 22 are novel gene mutations. Conclusions: HRM was proven to be a technique with high sensitivity and a low false positive ratio allowing a rapid, innovative and low cost genotyping of HCM. In a short return, HRM as a gene scanning technique could be a cost-effective gene-based diagnosis for an accurate HCM genetic diagnosis and hopefully providing new insights into genotype/phenotype correlations.
- Metabolic cues in cancer diagnosis and therapeuticsPublication . Marques, Vanda; Rodrigues, Cecília Maria Pereira; Afonso, Marta Bento; Saraiva, Lucília Helena AtaídeThe worldwide social and economic impact of cancer keeps increasing and proper disease management is a critical step to reducing the cancer burden on society, where appropriate diagnosis, staging, and treatment are key factors. Concomitantly, the increasing prevalence of obesity brings forward its importance as a risk factor for cancer development but also allows to explore associated metabolites and signaling pathways as tools for cancer research. As such, the studies presented in this thesis aim to tackle two pillars in cancer research: early diagnosis and therapy resistance, by exploring metabolism-related hormones as disease biomarkers and their impact on resistance to chemoradiotherapy, as well as unraveling novel molecules targeting cancer stem cells as major culprits in refractory cancer. In our first study, we focused on nonalcoholic fatty liver disease (NAFLD) as a predisposing risk factor for the development of hepatocellular carcinoma. Our goal was to evaluate serum levels of metabolism hormones as non-invasive biomarkers aiming to produce diagnostic options alternative to liver biopsy. Using two independent cohorts of biopsy-proven obese NAFLD patients and healthy-liver controls, we measured adiponectin, leptin, and insulin-like growth factor 1 (IGF-1) serum levels. Leptin serum levels were able to discriminate NAFLD with obesity posing as a potential confounding factor. Adiponectin serum levels combined with specific serum lipids composed a biomarker panel with good assay performance for the stratification of nonalcoholic steatohepatitis versus nonalcoholic fatty liver, however, it can be influenced by the presence of patatin-like phosphate domain-containing protein 3 (PNPLA3) genetic polymorphisms. In turn, IGF-1 levels combined with INR and ferritin were able to distinguish advanced liver fibrosis outranking other fibrosis evaluation scores used in the clinical practice. Next, we followed to evaluate how adiponectin and leptin modulated cell stemness and influenced resistance to chemoradiotherapy in rectal cancer patients, and ultimately providing novel biomarker candidates to identify patients not responding to neoadjuvant therapy. By evaluating serum and biopsy tissue from a cohort of locally advanced rectal cancer, we observed that leptin and adiponectin serum levels were not able to identify non-responder patients to chemoradiotherapy. Yet, high leptin and low adiponectin levels were associated with worsened clinical outcomes and unfavorable patient survival. At a molecular level, leptin serum profile was associated with increased expression of Yamanaka factors mRNA, particularly of octamer-binding transcription factor 4 (OCT4) and Kruppel-like factor 4 (KLF4), as well as signal transducer and activator of transcription 3 (STAT3) activation in the biopsy tissues, and all three parameters tended to increase in non-responders. Bringing together STAT3 activation, OCT4, and KLF4 mRNA expression with carcinoembryonic antigen levels – the only serological parameter that distinguished responders from non-responders – we were able to establish a high-performance biomarker panel to distinguish rectal cancer patient responders versus non-responders to chemoradiotherapy. In our third study, we focused on cancer stem cells as a cause of cancer relapse and resistance to therapy. Taking advantage of in vitro tumorsphere forming assays, we identified a curcumin analog that was able to reduce cellular viability of several colorectal cancer cell lines, by modulating stemness traits and inducing apoptosis. Importantly, this compound significantly reduced tumor burden in an in vivo xenograft mouse model following the same mechanisms of action. Moreover, in silico analysis identified nuclear factor kappa B (NF-ĸB) and STAT3 as putative molecular targets that were later confirmed both in vitro and in vivo. In conclusion, our studies highlight how hormones associated with obesity may pose as biomarkers in chronic disease diagnosis and valuable tools in cancer prognosis. Moreover, our results support the influence of obesity on cancer development and clinical outcome, and how its associated signaling pathways provide valuable targets for an innovative approach developed to treat cancer. Finally, we discovered a novel molecule that by targeting those signaling pathways could be a promising drug candidate for therapy insensitive tumors.
- The Role of Rosmarinic Acid on the Bioproduction of Gold Nanoparticles as Part of a Photothermal Approach for Breast Cancer TreatmentPublication . Ferreira-Gonçalves, Tânia; Gaspar, Maria Manuela; Coelho, João M. P.; Marques, Vanda; Viana, Ana S.; Ascensão, Lia; Carvalho, Lina; Rodrigues, Cecília M. P.; Ferreira, Hugo Alexandre; Ferreira, David; Reis, Catarina PintoBreast cancer is a high-burden malignancy for society, whose impact boosts a continuous search for novel diagnostic and therapeutic tools. Among the recent therapeutic approaches, photothermal therapy (PTT), which causes tumor cell death by hyperthermia after being irradiated with a light source, represents a high-potential strategy. Furthermore, the effectiveness of PTT can be improved by combining near infrared (NIR) irradiation with gold nanoparticles (AuNPs) as photothermal enhancers. Herein, an alternative synthetic method using rosmarinic acid (RA) for synthesizing AuNPs is reported. The RA concentration was varied and its impact on the AuNPs physicochemical and optical features was assessed. Results showed that RA concentration plays an active role on AuNPs features, allowing the optimization of mean size and maximum absorbance peak. Moreover, the synthetic method explored here allowed us to obtain negatively charged AuNPs with sizes favoring the local particle accumulation at tumor site and maximum absorbance peaks within the NIR region. In addition, AuNPs were safe both in vitro and in vivo. In conclusion, the synthesized AuNPs present favorable properties to be applied as part of a PTT system combining AuNPs with a NIR laser for the treatment of breast cancer.
- The Role of Rosmarinic Acid on the Bioproduction of Gold Nanoparticles as Part of a Photothermal Approach for Breast Cancer TreatmentPublication . Ferreira-Gonçalves, Tânia; Gaspar, Maria Manuela; Coelho, João M. P.; Marques, Vanda; Viana, Ana S.; Ascensão, Lia; Carvalho, Lina; Rodrigues, Cecília M. P.; Ferreira, Hugo Alexandre; Alves Ferreira, David; Reis, Catarina PintoBreast cancer is a high-burden malignancy for society, whose impact boosts a continuous search for novel diagnostic and therapeutic tools. Among the recent therapeutic approaches, photothermal therapy (PTT), which causes tumor cell death by hyperthermia after being irradiated with a light source, represents a high-potential strategy. Furthermore, the effectiveness of PTT can be improved by combining near infrared (NIR) irradiation with gold nanoparticles (AuNPs) as photothermal enhancers. Herein, an alternative synthetic method using rosmarinic acid (RA) for synthesizing AuNPs is reported. The RA concentration was varied and its impact on the AuNPs physicochemical and optical features was assessed. Results showed that RA concentration plays an active role on AuNPs features, allowing the optimization of mean size and maximum absorbance peak. Moreover, the synthetic method explored here allowed us to obtain negatively charged AuNPs with sizes favoring the local particle accumulation at tumor site and maximum absorbance peaks within the NIR region. In addition, AuNPs were safe both in vitro and in vivo. In conclusion, the synthesized AuNPs present favorable properties to be applied as part of a PTT system combining AuNPs with a NIR laser for the treatment of breast cancer.