Carlos Frederico Martins Menck.

Projeto desenvolvido como pesquisador visitante senior, no Erasmus Medical Center, Erasmus University, Rotterdam, the Netherlands, durante o período de fevereiro a julho de 2023.

This project is supported under the International Collaboration Research Funding from Sao Paulo Research Foundation (FAPESP, SP, Brazil) and The Netherlands Organization for Scientific Research (NWO, The Netherlands), Grant # 2019/19435-3. 

The world population is getting older and older. Unfortunately, this increased life expectancy is generally associated by so-called age-related vascular diseases, such as dementia, heart infarct, and stroke, which profoundly compromise the quality of life of the elderly. Age-related disorders are generally viewed as necessary evil. Our project challenges this dogma, as we propose that the aforementioned age-related disorders, despite their very diverse symptom profiles, share vascular ageing as common denominator. Moreover vascular ageing is caused by cumulative by accumulation of unrepaired DNA damage and mitochondrial dysfunction in the vessel wall which both increase with age. These events interplay and induce progressive senescence, inflammation and function loss of the vessel wall, which eventually will manifest as cardiovascular dysfunction or neurodegeneration, two very important hallmarks of ageing. Several of the predominant human diseases, such as obesity, diabetes and cardiovascular diseases, are directly related to this chain of cellular and physiological events that are part of and accelerate the process of ageing. This project joins experts on DNA damage repair, mitochondrial dysfunction, inflammation and cardiovascular disease to deploy and share knowledge and knowhow to unravel this common disease axis. To do so they will combine cutting edge technologies (e.g. scRNASEq, MitoNGS. CRISPR-Cas, organoids, MacroScreen functionomics platform, (intravital or multispectral) imaging and process reporters) with unique cellular and mouse models, deficient in processes that lead to accelerated ageing phenotypes. The insights gained in this project will be harnessed to the design of new (metabolic) biomarkers of vascular ageing and for novel therapeutic measures to revert this deleterious axis. By targeting a unique common mechanism in vascular ageing we expect that this project will help to reduce these age-related diseases thus improving quality of life for the elderly, and bringing the vista of healthy ageing within reach.
Among the hypothesis raised to explain the ageing processes, the accumulation of unrepaired DNA damage during life is one of the main explanations and supported by many evidence. In a second part of the project we will investigate several aspects of DNA repair mechanisms and how unrepaired DNA damage may cause aging and cancer. In most of our studies we will use human cells with deficiencies in DNA repair processes, mainly derived from xeroderma pigmentosum (XP) and Cockayne syndrome (CS) patients. Cells from these patients will be employed in order to investigate mutagenesis by DNA damaging agents, including UVA and UVB. Mutations in XP Brazilian patients will also be identified, aiming to know the distribution of these mutations in the country. Also, the DNA damaging action of certain chemotherapeutic drugs drive us to investigate mechanisms of tumor cell resistance to treatments. A pioneer work to investigate how intracellular parasites affect host cells? genome metabolism, including DNA repair, will focus the parasite Trypanosoma cruzi and the human respiratory syncytial virus. Finally, DNA repair processes are extremely conserved, existing in virtually all life forms. The biological processes of defense against DNA damage in prokaryotes will also be studied in model bacteria. DNA damage responses will also be studied in samples from the Antartic continent, understand responses in extreme environments. We hope these more general studies will help us to contribute to the understanding of the aging process, investigated in first part of this project.

The genetic material is under constant assault from the environment or by-products of cell metabolism, inducing damage in the DNA molecule. Cells exhibit several responses, which protect from these lesions, including removal by DNA repair mechanisms, or DNA damage tolerance, where the genetic material can be replicated or transcribed, despite the damage. Our group has been working on scientific questions that try to understand how these mechanisms and cellular responses operate maintaining genomic stability, as well as what are the consequences of unrepaired DNA damage for cells and organisms. The importance of these systems for cell function is attested by the high degree of evolutionary conservation and, in humans, dramatically illustrated by genetic syndromes related to defects in DNA repair mechanisms or impairment of responses to DNA injuries. These defects result in clinical phenotypes such as high frequency of carcinogenesis, development problems, neurodegeneration and premature aging. Thus, an important part of this project focus studies in human cells, especially those derived from patients with deficiencies in DNA repair mechanisms, such as xeroderma pigmentosum (XP), Cockayne syndrome (CS), trichothiodystrophy (TTD), and other syndromes identified more recently. In one of the subprojects, we propose to investigate the damaging effects of the UV components of sunlight. The mechanism of DNA damage formation by UVA irradiation, for example, is not fully understood, nor the cellular responses to these lesions, especially in relation to death or mutagenesis. Given the importance of oxidative processes in the generation of cellular endogenous damage, the different types of cell death induced by oxidative stress will be investigated in cells with different defects in DNA repair, searching cellular different responses, that may mimic the different clinical phenotypes observed in patients with neurodegeneration/premature aging. In this project we also intend to investigate the cellular processes that allow the replication of damaged genome. Approaches of next-generation sequencing (NGS) are proposed for studies of mutagenesis and also the identification of mutations and molecular diagnosis of Brazilian patients with XP or other DNA repair related diseases. These studies can provide interesting data on the causes of certain clinical phenotypes, and help patients and their families, with the recognition of the disease, made possible by the molecular diagnosis. XP patients include those from the region of Goiás, due to the high frequency of XP patients observed in Faina, where the incidence is the highest in the world due to geographical isolation and consanguineous marriage. Yet, this project also intends to study how human cells respond to anti-tumor chemotherapeutic agents, aiming to understand the repair mechanisms involved in the repair of DNA damage induced by them and seeking alternative therapies that may enhance the fight against cancer. Finally, we also plan to study genes related to DNA repair in bacteria of basic (Caulobacter crescentus) or medical (Pseudomonas aeruginosa) interest. In the first case, it is an excellent model for studies of bacterial genetics, and we will continue to identify the function of new genes involved in the response to DNA damage. With the model of P. aeruginosa, we aim to investigate the involvement of DNA repair and damage tolerance genes in the responses to antimicrobial compounds, as well as mutagenesis that leads to the emergence of antibiotic resistance.

O material genético celular está constantemente sujeito a agressões, gerando lesões na molécula de DNA. Estas afetam diretamente a replicação do DNA e transcrição do RNA, desencadeando respostas que interferem em vários processos metabólicos, inclusive ciclo celular. Para as células, lesões não removidas no DNA podem resultar em mutagênese ou morte celular e, para organismos multicelulares, as conseqüências são carcinogênese e envelhecimento. Este projeto busca estudar as respostas celulares após a indução de lesões no genoma, visando compreender os processos genéticos envolvidos na remoção (reparo de DNA), ou na tolerância aos danos. Empregamos vários modelos biológicos para esse estudo, eucariontes e procariontes, graças ao alto grau de conservação evolutiva dessas respostas, e que, por outro lado, nos dá uma visão abrangente do tema.

Este projeto visa o estudo de sistemas de proteção ao material genético celular, assim como a evolução, com a busca de genes novos relacionados a esses sistemas. Quatro subprojetos são apresentados. Nos dois primeiros propomos o estudo mecanismos de reparo de DNA em células animais com prioridade para estudos com células humanas primárias. Pretendemos empregar vetores genéticos, sobretudo derivados de adenovírus, para transduzir genes de reparo de DNA em células humanas, sobretudo relacionados a síndromes humanas com deficiência nesse processo (síndromes conhecidas como xeroderma pigmentosum). Esses vetores estão sendo previstos para serem usados em processos de diagnóstico que identificam o defeito celular e no estudo da dinâmica dessas proteínas dentro das células. Pretendemos também continuar a decifrar os sinais apoptóticos que são induzidos pela presença de lesões no genoma. Em outro subprojeto, buscamos compreender os sistemas pelos quais os vegetais protegem seu genoma de lesões no seu genoma. Como alvo, pretendemos estudar a função e a expressão de dois genes clonados por nós (AtXPB e Thi1). Finalmente, pretendemos realizar uma investigação de genômica funcional, para identificar genes envolvidos em reparo de DNA na bactéria modelo Caulobacter crescentus. Este subprojeto é exploratório e pretende a construção e o estudo de mutantes bacterianos. A facilidade de sincronização do ciclo celular nessas bactérias também pode ser útil na determinação efeito do ciclo em funções de reparo nessa célula.