Open Access Theses - Health Science - 2011-2020
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PublicationStress Reaction in Outer Segments of Photoreceptors after Blue Light Irradiation( 2016)"The retina is prone to oxidative stress from many factors which are also involved in the pathogenesis of degenerative diseases. In this study, we used the application of blue light as a physiological stress factor. The aim of this study was to identify the major source of intracellular ROS that mediates blue light-induced detrimental effects on cells which may lead to cytotoxicity. We hypothesized that outer segments are the major source of blue light induced ROS generation. In photoreceptors, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) enzymes and the recently found respiratory chain complexes may represent a major source for reactive oxygen species (ROS), beside mitochondria and chromophores. Therefore, we investigated this hypothesis and analysed the exact localization of the ROS source in photoreceptors in an organotypic culture system for mouse retinas. Whole eyeball cultures were irradiated with visible blue light (405 nm) with an output power of 1 mW/cm2. Blue light impingement lead to an increase of ROS production (detected by H2DCFDA in live retinal explants), which was particularly strong in the photoreceptor outer segments. Nox-2 and Nox-4 proteins are sources of ROS in blue light irradiated photoreceptors; the Nox inhibitor apocynin decreased ROS stimulated by blue light. Concomitantly, enzyme SOD-1, a member of the antioxidant defense system, indicator molecules of protein oxidation (CML) and lipid oxidation (MDA and 4-HNE) were also increased in the outer segments. Interestingly, outer segments showed a mitochondrial-like membrane potential which was demonstrated using two dyes (JC-1 and TMRE) normally exclusively associated with mitochondria. As in mitochondria, these dyes indicated a decrease of the membrane potential in hypoxic states or cell stress situations. The present study demonstrates that ROS generation and oxidative stress occurs directly in the outer segments of photoreceptors after blue light irradiation."
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PublicationEpigenetic signature in persons with Down Syndrome( 2015)"Persons affected by Down Syndrome show a heterogeneous phenotype that includes developmental defects and cognitive and haematological disorders. Premature accelerated aging and the consequent development of age associated diseases like Alzheimer Disease (AD) seem to be the cause of higher mortality late in life of DS persons. Down Syndrome is caused by the complete or partial trisomy of chromosome 21, but it is not clear if the molecular alterations of the disease are triggered by the specific functions of a limited number of genes on chromosome 21 or by the disruption of genetic homeostasis due the presence of a trisomic chromosome. As epigenomic studies can help to shed light on this issue, here we used the Infinium HumanMethilation450 BeadChip to analyse blood DNA methylation patterns of 29 persons affected by Down syndrome (DSP), using their healthy siblings (DSS) and mothers (DSM) as controls. In this way we obtained a family-based model that allowed us to monitor possible confounding effects on DNA methylation patterns deriving from genetic and environmental factors. We showed that defects in DNA methylation map in genes involved in developmental, neurological and haematological pathways. These genes are enriched on chromosome 21 but localize also in the rest of the genome, suggesting that the trisomy of specific genes on chromosome 21 induces a cascade of events that engages many genes on other chromosomes and results in a global alteration of genomic function. We also analysed the methylation status of three target regions localized at the promoter (Ribo) and at the 5’ sequences of 18S and 28S regions of the rDNA, identifying differently methylated CpG sites. In conclusion, we identified an epigenetic signature of Down Syndrome in blood cells that sustains a link between developmental defects and disease phenotype, including segmental premature aging."
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PublicationData management and data analysis in the large European projects GEHA (GEnetics of Healthy Aging) and NU-AGE (NUtrition and AGEing): a bioinformatic approach( 2015)The aging process is characterized by the progressive fitness decline experienced at all the levels of physiological organization, from single molecules up to the whole organism. Studies confirmed inflammaging, a chronic low-level inflammation, as a deeply intertwined partner of the aging process, which may provide the “common soil” upon which age-related diseases develop and flourish. Thus, albeit inflammation per se represents a physiological process, it can rapidly become detrimental if it goes out of control causing an excess of local and systemic inflammatory response, a striking risk factor for the elderly population. Developing interventions to counteract the establishment of this state is thus a top priority. Diet, among other factors, represents a good candidate to regulate inflammation. Building on top of this consideration, the EU project NU-AGE is now trying to assess if a Mediterranean diet, fortified for the elderly population needs, may help in modulating inflammaging. To do so, NU-AGE enrolled a total of 1250 subjects, half of which followed a 1-year long diet, and characterized them by mean of the most advanced –omics and non –omics analyses. The aim of this thesis was the development of a solid data management pipeline able to efficiently cope with the results of these assays, which are now flowing inside a centralized database, ready to be used to test the most disparate scientific hypotheses. At the same time, the work hereby described encompasses the data analysis of the GEHA project, which was focused on identifying the genetic determinants of longevity, with a particular focus on developing and applying a method for detecting epistatic interactions in human mtDNA. Eventually, in an effort to propel the adoption of NGS technologies in everyday pipeline, we developed a NGS variant calling pipeline devoted to solve all the sequencing-related issues of the mtDNA.
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PublicationThe elderly health status and its correlation with ageing biomarkers: the European Project Mark-Age( 1971)According to the latest statistics projections formulated by Eurostat, the proportion of elderly EU-27’s population aged over 65 years old is predicted to increase from 17.5 % in 2011 to 29.5 % by 2060. This "population explosion" makes extremely important to identify the different genetic and molecular mechanisms which underpin the morbidity and mortality along with new strategies able to counteract or slow down its progress. In this scenario fits the European Project MARK-AGE whose aim was to identify a robust set of biomarkers of human ageing able to discriminate between chronological and biological ageing and to derive a model for healthy ageing through the analysis of three populations from different European countries, supposed to be characterized by different ageing rate: 1. Subjects representing the “Normal” or “Physiological” aging. 2. Subjects representing the “successful” or “decelerate” aging 3. Subjects representing the “accelerated” aging. The aim of this work was to recruit and characterize volunteers, to perform an accurate analysis of the health status of elderly recruited subjects (60-79 years) verifying any possible dissimilarity in their aging trajectories, to identify a set of robust ageing biomarkers and investigate possible correlations between ageing biomarkers and health status of recruited volunteers. The model proposed by MARK-AGE Project regarding different ageing trajectories has been confirmed and several ageing biomarkers have been identified.
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PublicationNutrient Transporter Inhibition Disrupts Mammary and Intestinal Polarized Epithelial Function( 2016)"The transporters primarily responsible for transporting important nutrients involved in energy metabolism have a wide substrate specificity setting up the potential for drug-nutrient transporter interactions. Pharmacological inhibition of nutrient transport across the lactating mammary and neonatal intestinal epithelial barrier can directly and indirectly affect growth and maturation of the developing neonate by either reducing the uptake of important nutrients by the neonate or by disrupting epithelial barrier integrity. My thesis focused on two transporters, OCTN2 and MCT1, expressed in immortalized intestinal and mammary epithelial cell cultures to assess the effects of their pharmacological inhibition on L-carnitine and butyrate flux, respectively, and polarized epithelial barrier integrity. Human colorectal adenocarcinoma (Caco-2) and bovine mammary (BME-UV) cell lines were grown into monolayers on 12-well tissue culture plates and subsequently exposed to the presence or absence of OCTN2 and MCT1 inhibitors for 6, 12, and 24 hours as well as 7 days. Failure to obtain a polarized mammary monolayer prevented the analysis of the direct effects of nutrient transport inhibition on nutrient flux forcing the focus on the indirect effects. To assess polarized epithelial barrier integrity, transepithelial electrical resistance and Lucifer yellow rejection rates were measured at each time point. No trend was noted between control and treated groups. To assess the acute and chronic effects of pharmacological exposure on polarized epithelial function, a limited appraisal of nutrient transporter expression and cellular homeostasis parameters was conducted. Following exposure at each time point, mRNA expression of OCTN1, OCTN2, MCT1, MCT2 and GADPH were measured using qPCR. Low mRNA yields resulted in an inability to assess transporter expression levels in the epithelial systems. Cellular homeostasis parameters were analyzed using the CellTiter-Glo Luminescent Cell Viability Assay, pH-Xtra Glycolysis Assay and MitoXpress Xtra Oxygen Consumption Assay. These assays measured ATP synthesis, glycolytic flux and cellular respiration, respectively. No significant trend was noted in ATP synthesis between control and treated groups. An upward trend in both glycolytic flux and cellular respiration was noted in treatment with both inhibitors in both cell lines. Complications in obtaining polarized monolayer forced the focus on the indirect affects, therefore, obtaining and utilizing a more accurate portrayal of the lactating mammary and neonatal intestinal epithelium is critical in answering this research question as both of these systems are highly synthetic and complex. By doing so, a more accurate representation of the effects of pharmacological inhibition of nutrient transporters essential for energy metabolism can be identified."