PERSISTENT DNA DAMAGE AT TELOMERES, CAUSED BY TRF2-MEDIATED DNA REPAIR INHIBITION, TRIGGERS CELLULAR SENESCENCE AND IS ASSOCIATED WITH PRIMATES AGEING

The DNA damage response (DDR) coordinates DNA repair events and transiently arrests cell-cycle progression until DNA damage has been removed. If the damage is not resolved, cells can enter an irreversible cell cycle arrest called cellular senescence. In irradiation-induced senescent cells a large fraction of persistent DDR markers are associated with telomeric DNA, both in cultured cells and in in vivo tissues. The aim of my PhD project was to investigate the mechanism underlying this phenomenon. I showed that persistent DDR activation has a causative role for the senescence-associated cell cycle exit and that a double-strand break (DSB) within telomeric repeats is inducing a more protracted DDR activation compared with a non-telomeric one in human cells. The DDR persistency at telomeres is neither dependent on their heterochromatic state nor on TRF2 loss from telomeres during senescence establishment. Rather, TRF2 recruitment next to a DSB, in the absence of telomeric DNA, is sufficient to induce a more protracted site-specific DDR focus and to impair DSB repair in mouse cells. Ageing is associated with accumulation of markers of DDR activation. In terminally differentiated brain neurons from old primates, I observed DDR activation at telomeres that were not critically short. Taken together, these results strongly suggest that TRF2 inhibits DNA repair at broken telomeres, contributing to the accumulation of unrepaired, DDR-positive telomeres during ageing. This can in turn trigger cellular senescence and impair tissue homeostasis providing a mechanism for ageing also in non-proliferating tissues. Finally, I focused my attention on DICER and DROSHA-dependent DNA damage response RNAs (DDRNAs), novel components of the DDR machinery, which have been described to be necessary for DDR activation at DSBs. I showed that RNase A treatment as well as DICER or DROSHA down-regulation impair DDR activation at uncapped telomeres and that DICER and DROSHA may have a role in chromosomal fusions. Furthermore, in cells with dysfunctional telomeres, the inhibition of telomeric DDRNAs using inhibitory oligonucleotide molecules with a complementary sequence can prevent DDR activation and senescent-associated cell cycle arrest. These data indicate that at uncapped telomeres, DDRNAs with telomeric sequences are generated and that they are necessary for DDR activation and chromosomal fusions

Differential expression of cyclooxygenases in hypertrophic scar and keloid tissues

Hypertrophic scar (HS) and keloid (KL) are two forms of an abnormal cutaneous scarring process, mainly characterized by excessive extracellular matrix deposition and fibroblast proliferation. Despite the increased understanding of the molecular and cellular events leading to HS and KL, the pathogenesis of these lesions remains poorly understood. A pivotal role in the formation of abnormal scars has been ascribed to transforming growth factor-β, whose activity appears to be mediated through a link with pathways acting via cyclooxygenases (COX-1 and COX-2). To date, there is no report on the in vivo expression of COX-1 and COX-2 in human HS and KL tissues. Therefore, using immunohistochemistry and Western blot analysis, we investigated 36 cases of KL, 32 cases of HS, and 25 cases of normal skin in order to define the localization and distribution of COX-1 and COX-2 in the tissues of these scar lesions and the overlying epidermis. The results mainly show the following: (a) a significant overexpression of COX-1 in HS tissues and the overlying epidermis as compared with normal skin and KL tissues and (b) a significant overexpression of COX-2 in KL tissue and the overlying epidermis in contrast to normal skin and HS tissues. Our data support the hypothesis that both COXs are involved in the pathogenesis of scar lesions in different ways and, particularly, COX-1 in the formation of HS and COX-2 in the formation of KL. In addition, the overexpression of COX-1 and COX-2 in the epidermis overlying HS and KL tissues, respectively, underlines the importance of epithelial- mesenchymal interactions in the pathogenesis of scar lesion

Inhibition of DNA damage response at telomeres improves the detrimental phenotypes of Hutchinson–Gilford Progeria Syndrome

Hutchinson–Gilford progeria syndrome (HGPS) is a genetic disorder characterized by premature aging features. Cells from HGPS patients express progerin, a truncated form of Lamin A, which perturbs cellular homeostasis leading to nuclear shape alterations, genome instability, heterochromatin loss, telomere dysfunction and premature entry into cellular senescence. Recently, we reported that telomere dysfunction induces the transcription of telomeric non-coding RNAs (tncRNAs) which control the DNA damage response (DDR) at dysfunctional telomeres. Here we show that progerin-induced telomere dysfunction induces the transcription of tncRNAs. Their functional inhibition by sequence-specific telomeric antisense oligonucleotides (tASOs) prevents full DDR activation and premature cellular senescence in various HGPS cell systems, including HGPS patient fibroblasts. We also show in vivo that tASO treatment significantly enhances skin homeostasis and lifespan in a transgenic HGPS mouse model. In summary, our results demonstrate an important role for telomeric DDR activation in HGPS progeroid detrimental phenotypes in vitro and in vivo

Rooting Pattern Distribution and Spatial Variability of Italian Ryegrass (Lolium Multiflorum Lam) in a Mediterranean Region

It is estimated that less than 10% of the studies on pastures and forages have evaluated the subterranean biomass production. The objective of this study was to evaluate for a Mediterranean region the rooting characteristics and spatial variability of Italian ryegrass (Lolium multiflorum Lam) under two different soil water status conditions

DNA damage response at telomeres boosts the transcription of SARS-CoV-2 receptor ACE2 during aging

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19), known to be more common in the elderly, who also show more severe symptoms and are at higher risk of hospitalization and death. Here, we show that the expression of the angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 cell receptor, increases during aging in mouse and human lungs. ACE2 expression increases upon telomere shortening or dysfunction in both cultured mammalian cells and in vivo in mice. This increase is controlled at the transcriptional level, and Ace2 promoter activity is DNA damage response (DDR)-dependent. Both pharmacological global DDR inhibition of ATM kinase activity and selective telomeric DDR inhibition by the use of antisense oligonucleotides prevent Ace2 upregulation following telomere damage in cultured cells and in mice. We propose that during aging telomere dysfunction due to telomeric shortening or damage triggers DDR activation and this causes the upregulation of ACE2, the SARS-CoV-2 cell receptor, thus contributing to make the elderly more susceptible to the infection

Aspectos climáticos do noroeste fluminense, RJ.

bitstream/CNPS/11829/1/doc_43_2003_noroeste_fluminense.pd

Uso de clorofilometro na avaliação da adubação nitrogenada e potássica no capim Tifton 85.

RESUMO - Objetivou-se com este trabalho aferir o comportamento de um clorofilometro na gramínea forrageira Tifton 85 (Cynodon spp. cv. Tifton 85) e sua relação com a produção de massa seca em resposta à adubação nitrogenada e potássica. No primeiro ensaio de campo realizado foi feita a aplicação de nitrogênio e potássio separados em três tratamentos: 0,75 e 150kg/ha/corte, segundo um delineamento em blocos casualizados, com quatro repetições. Os teores de clorofila total, extraídos da primeira lâmina foliar totalmente expandida, correlacionaram-se bem com as leituras do clorofilometro e foram afetados pelas doses de N e número de cortes. No segundo corte, os teores de clorofila total foram significativamente maiores que no primeiro, enquanto a massa verde seca total da parte aérea respondeu apenas às doses de N, o que sugere a existência de uma produção ?de luxo? de clorofilas. No segundo ensaio, os tratamentos consistiram de cinco doses de N e K: 0; 150; 300; 450 e 600 kg/ha, dispostos na forma de blocos inteiramente casualizados, com quatro repetições e ao final de um período de 28 dias, os teores de clorofila total, as leituras do clorofilometro e a matéria seca de folhas variaram, segundo um modelo quadrático, em resposta às doses de N (entre 0 e 600kg N/ha) sem evidencias de produção de luxo de clorofilas. Embora esta última situação seja provavelmente a situação normal, a melhoria no desempenho do clorofilometro a altos níveis de disponibilidade de N demandará novos estudos com relação às frações nitrogenadas que se acumulam na planta nessas condições. ABSTRACT - The aim of this study was to assess the performance of a chlorophyll meter and its relation to dry matter production of Tifton 85 (Cynodon spp. cv. Tifton 85), in response to nitrogen fertilization. In the first field trial, the total chlorophyll contents extracted from the uppermost fully expanded leaf blade correlated well with chlorophyll meter readings and were affected by N rates and cut number. In the second cut in the first field trial, total chlorophyll contents were higher than the first cut while total dry mass of green shoots responded only to N rates, suggesting that there was a ?luxury? production of chlorophylls. In the second field trial, at the end of a single regrowth period of 28 days, total chlorophyll contents, chlorophyll meter readings and dry leaf biomass varied harmonically, according to a quadratic model of response to N dose (0 - 600kg N/ha), without evidence of a supraoptimal production of chlorophylls. Although this last it is probably the normal situation, the refinement of the chlorophyll meter sensitivity to high levels of soil N availability, will demand news studies in order to clarify the status of the nitrogen fractions accumulated in the plant in these conditions

YY1 overexpression is associated with poor prognosis and metastasis-free survival in patients suffering osteosarcoma

<p>Abstract</p> <p>Background</p> <p>The polycomb transcription factor Yin Yang 1 (YY1) overexpression can be causally implicated in experimental tumor growth and metastasization. To date, there is no clinical evidence of YY1 involvement in outcome of patients with osteosarcoma. Prognosis of osteosarcoma is still severe and only few patients survive beyond five years. We performed a prospective immunohistochemistry analysis to correlate YY1 immunostaining with metastatic development and survival in a selected homogeneous group of patients with osteosarcoma.</p> <p>Methods</p> <p>We studied 41 patients suffering from osteosarcoma (stage II-IVa). Multivariate analysis was performed using Cox proportional hazard regression to evaluate the correlation between YY1 expression and both metastasis development and mortality.</p> <p>Results</p> <p>YY1 protein is not usually present in normal bone; in contrast, a high number of patients (61%) showed a high score of YY1 positive cells (51-100%) and 39% had a low score (10-50% positive cells). No statistical difference was found in histology, anatomic sites, or response to chemotherapy between the two degrees of YY1 expression. Cox regression analysis demonstrated that the highest score of YY1 expression was predictive of both low metastasis-free survival (HR = 4.690, 95%CI = 1.079-20.396; p = 0.039) and poor overall survival (HR = 8.353, 95%CI = 1.863-37.451 p = 0.006) regardless of the effects of covariates such as age, gender, histology and chemonecrosis.</p> <p>Conclusion</p> <p>Overexpression of YY1 in primary site of osteosarcoma is associated with the occurrence of metastasis and poor clinical outcome.</p

Drosha drives the formation of DNA:RNA hybrids around DNA break sites to facilitate DNA repair

The error-free and efficient repair of DNA double-stranded breaks (DSBs) is extremely important for cell survival. RNA has been implicated in the resolution of DNA damage but the mechanism remains poorly understood. Here, we show that miRNA biogenesis enzymes, Drosha and Dicer, control the recruitment of repair factors from multiple pathways to sites of damage. Depletion of Drosha significantly reduces DNA repair by both homologous recombination (HR) and non-homologous end joining (NHEJ). Drosha is required within minutes of break induction, suggesting a central and early role for RNA processing in DNA repair. Sequencing of DNA:RNA hybrids reveals RNA invasion around DNA break sites in a Drosha-dependent manner. Removal of the RNA component of these structures results in impaired repair. These results show how RNA can be a direct and critical mediator of DNA damage repair in human cells