Comprometimento da homeostase telomérica e indução do fenótipo senescente no contexto da obesidade

O envelhecimento é um processo biológico caracterizado pela deterioração progressiva das funções fisiológicas do organismo. A perda de função e o acúmulo de dano tecidual estão relacionados a uma série de marcadores celulares, dentre eles, o encurtamento acelerado de telômeros, acúmulo de células senescentes e o comprometimento da função mitocondrial. O estabelecimento do fenótipo senescente pode ser caracterizado pelo aumento na transcrição do gene CDKN1A (P21) e pela atividade da enzima β-galactosidase (β-gal), além de um conjunto de citocinas que constituem um secretoma capaz de contribuir para um estado de inflamação crônico, observado em doenças relacionadas à aceleração do envelhecimento. A obesidade é uma condição fisiológica caracterizada pela alteração na composição e a estrutura do tecido adiposo e uma desregulação metabólica. É uma doença associada a um estado de inflamação crônica, compartilhando aspectos relacionados ao envelhecimento e ao aparecimento de características de senescência de forma antecipada. Dessa maneira, o objetivo principal desta tese foi avaliar parâmetros relacionados à homeostasia do complexo telomérico e o impacto do ambiente oxidativo, característico da obesidade, na indução do fenótipo senescente. Para isso, relacionamos a homeostase do complexo telomérico de células mononucleares de sangue periférico (PBMC) com a indução do fenótipo senescente relacionado à disfunção mitocondrial em células tronco adipo-derivadas (ADSC) expostas ao plasma de indivíduos portadores de obesidade após 10 dias de tratamento. Nossos resultados demonstraram que indivíduos portadores de obesidade possuem telômeros mais curtos e uma desregulação na expressão dos componentes do complexo shelterin em PBMC, independente da ocorrência de comorbidades associadas à doença. Além disso, observamos um aumento do dano oxidativo relacionado ao aumento da lipoperoxidação e o conteúdo de proteínas carboniladas no plasma de indivíduos portadores de obesidade. A Análise de Componentes Principais (PCA) ainda sugere que o comprometimento da homeostase telomérica pode ser parcialmente explicado pela desregulação de TRF1, o qual não pôde ser revertido pelo aumento na resposta antioxidante não enzimática, decorrente de uma possível resposta celular adaptativa. Os efeitos crônicos do plasma de indivíduos portadores de obesidade sugeriram o gatilho da indução do fenótipo senescente em ADSC pelo aumento da atividade da enzima β-gal e um aumento na expressão de CDKN1A. Observamos um acúmulo de mitocôndrias danificadas e indícios de inibição da autofagia celular, características que podem contribuir na indução do fenótipo senescente no contexto da obesidade. Complementando os achados obtidos no sistema periférico (PBMC), nossos resultados ainda demonstram um aumento na expressão de TRF1 nas ADSC no início do fenótipo senescente, sugerindo novamente que TRF1 tem um papel importante na progressão do envelhecimento, tanto a nível de tecido adiposo quando a nível sistêmico. Os dados obtidos nessa tese sugerem que a obesidade é uma doença que está associada à aceleração do fenótipo senescente em diferentes contextos celulares, induzindo a ativação de respostas moleculares antecipadas, similares aos mecanismos observados no envelhecimento.Aging is a biological process characterized by the progressive deterioration of the physiological functions of the organism. Loss of function and accumulation of tissue damage are related to several cell hallmarks, such as accelerated telomere shortening, senescent cell accumulation, and mitochondrial dysfunction. The establishment of the senescent phenotype can be characterized by the increase in CDKN1A (P21) gene transcription and the enzyme β-galactosidase (β-gal) activity, as well as a set of cytokines that constitute a secretome that contributes to a chronic inflammation state, observed in age-related diseases. Obesity is a multifactorial condition characterized by changes in the composition and structure of adipose tissue and excessive metabolic dysregulation. It is associated with a chronic low-grade inflammatory condition that accompanies metabolic dysfunction, sharing aspects related to anticipation of aging characteristics. Thus, the aim of this thesis was to evaluate parameters related to homeostasis of the telomeric complex and the impact of the obesity-related oxidative environment on the induction of senescent phenotype. To this end, we evaluated the telomere homeostasis of the peripheral blood mononuclear cell (PBMC) and the senescence induction on adipose-derived stem cells (ADSC) by chronic exposure to an obesogenic environment. We demonstrated that obese individuals have shorter telomeres and dysregulated gene expression of shelterin components in PBMC, regardless of the occurrence of disease-associated comorbidities. In addition, we observed increased oxidative damage related to augmented lipoperoxidation and carbonyl protein content in plasma of obese individuals. Also, Principal Component Analysis (PCA) suggests that the impairment of telomeric homeostasis may be partially explained by the positive regulation of TRF1 and could not be reversed by the increase in non-enzymatic antioxidant response, observed as an adaptive cellular response. The chronic effects of obesogenic environment were observed after plasma incubation for 10 days. Our data sugested that the onset of senescent phenotype induction in ADSC was observed by increased β-gal enzyme activity and an increase in CDKN1A expression. After plasma incubation for 10 days. Furthermore, the accumulation of damaged mitochondria may be a response due to autophagy machinery impairment, so that it may contribute to the induction of senescent phenotype in the context of obesity. Our data further demonstrated an increase in TRF1 expression in ADSC at the onset of the senescent phenotype, suggesting that TRF1 plays an important role in the progression of aging, both at adipose and systemic levels. The data obtained in this thesis suggest that obesity is a disease that is associated with acceleration of the senescent phenotype in different cellular contexts, contributing to the activation of molecular responses that trigger characteristics observed in aging

Retinoic acid downregulates thiol antioxidant defences and homologous recombination while promotes A549 cells sensitization to cisplatin

Recent studies have investigated the use of retinoic acid (RA) molecule in combined chemotherapies to cancer cells as an attempt to increase treatment efficiency and circumvent cell resistance. Positive results were obtained in clinical trials from lung cancer patients treated with RA and cisplatin. Meanwhile, the signalling process that results from the interaction of both molecules remains unclear. One of the pathways that RA is able to modulate is the activity of NRF2 transcription factor, which is highly associated with tumour progression and resistance. Therefore, the aim of this work was to investigate molecular mechanism of RA and cisplatin co-treatment in A549 cells, focusing in NRF2 pathway. To this end, we investigated NRF2 and NRF2-target genes expression, cellular redox status, cisplatin-induced apoptosis, autophagy and DNA repair through homologous recombination. RA demonstrated to have an inhibitory effect over NRF2 activation, which regulates the expression of thiol antioxidants enzymes. Moreover, RA increased reactive species production associated with increased oxidation of thiol groups within the cells. The expression of proteins associated with DNA repair through homologous recombination was also suppressed by RA pre-treatment. All combined, these effects appear to create a more sensitive cellular environment to cisplatin treatment, increasing apoptosis frequency. Interestingly, autophagy was also increased by combination therapy, suggesting a resistance mechanism by A549 cells. In conclusion, these results provided new information about molecular mechanisms of RA and cisplatin treatment contributing to chemotherapy optimization

Shorter telomeres in children with severe asthma, an indicative of accelerated aging

Severe therapy-resistant asthma (STRA) is closely associated with distinct clinical and inflammatory pheno-endotypes, which may contribute to the development of age-related comorbidities. Evidence has demonstrated a contribution of accelerated telomere shortening on the poor prognosis of respiratory diseases in adults. Eotaxin-1 (CCL11) is an important chemokine for eosinophilic recruitment and the progression of asthma. In the last years has also been proposed as an age-promoting factor. This study aimed to investigate the association of relative telomere length (rTL) and eotaxin-1 in asthmatic children. Children aged 8-14 years (n=267) were classified as healthy control (HC, n=126), mild asthma (MA, n=124) or severe therapy-resistant asthma (STRA, n=17). rTL was performed by qPCR from peripheral blood. Eotaxin-1 was quantified by ELISA from fresh-frozen plasma. STRA had shorter telomeres compared to HC (p=0.02) and MA (p=0.006). Eotaxin-1 levels were up-regulated in STRA [median; IQR25-75)] [(1,190 pg/mL; 108–2,510)] compared to MA [(638 pg/mL; 134–1,460)] (p=0.03) or HC [(627 pg/mL; 108–1,750)] (p<0.01). Additionally, shorter telomeres were inversely correlated with eotaxin-1 levels in STRA (r=-0.6, p=0.013). Our results suggest that short telomeres and up-regulated eotaxin-1, features of accelerated aging, could prematurely contribute to a senescent phenotype increasing the risk for early development of age-related diseases in asthma

Telomere length and epigenetic age acceleration in adolescents with anxiety disorders

Evidence on the relationship between genetics and mental health are flourishing. However, few studies are evaluating early biomarkers that might link genes, environment, and psychopathology. We aimed to study telomere length (TL) and epigenetic age acceleration (AA) in a cohort of adolescents with and without anxiety disorders (N = 234). We evaluated a representative subsample of participants at baseline and after 5 years (n = 76) and categorized them according to their anxiety disorder diagnosis at both time points: (1) control group (no anxiety disorder, n = 18), (2) variable group (anxiety disorder in one evaluation, n = 38), and (3) persistent group (anxiety disorder at both time points, n = 20). We assessed relative mean TL by real-time quantitative PCR and DNA methylation by Infinium HumanMethylation450 BeadChip. We calculated AA using the Horvath age estimation algorithm and analyzed differences among groups using generalized linear mixed models. The persistent group of anxiety disorder did not change TL over time (p = 0.495). The variable group had higher baseline TL (p = 0.003) but no accelerated TL erosion in comparison to the non-anxiety control group (p = 0.053). Furthermore, there were no differences in AA among groups over time. Our findings suggest that adolescents with chronic anxiety did not change telomere length over time, which could be related to a delay in neuronal development in this period of life

Effects of Achyrocline satureioides inflorescence extracts against pathogenic intestinal bacteria : chemical characterization, in vitro tests, and in vivo evaluation

Three Achyrocline satureioides (AS) inflorescences extracts were characterized: (i) a freeze-dried extract prepared fromthe aqueous extractive solution and (ii) a freeze-dried and (iii) a spray-dried extract prepared from hydroethanol extractive solution (80% ethanol).The chemical profile, antioxidant potential, and antimicrobial activity against intestinal pathogenic bacteria of AS extracts were evaluated. In vitro antioxidant activity was determined by the total reactive antioxidant potential (TRAP) assay. In vivo analysis and characterization of intestinal microbiota were performed in male Wistar rats (saline versus treated animals with AS dried extracts) by high-throughput sequencing analysis: metabarcoding. Antimicrobial activity was tested in vitro by the disc diffusion tests. Moisture content of the extracts ranged from 10 to 15% and 5.7 to 17mg kg−1 of fluorine. AS exhibited antioxidant activity, especially in its freeze-dried form which also exhibited a wide spectrum of antimicrobial activity against intestinal pathogenic bacteria greater than those observed by the antibiotic, amoxicillin, when tested against Bacillus cereus and Staphylococcus aureus. Antioxidant and antimicrobial activities of AS extracts seemed to be positively correlated with the present amount of flavonoids. These findings suggest a potential use of AS as a coadjuvant agent for treating bacterial-induced intestinal diseases with high rates of antibiotic resistance