Abstract (Summary) Wood turtles ( Glyptemys insculpta ) are of conservation interest rangewide. Anecdotal accounts demonstrate that some populations have been decimated since 1850, and recent studies demonstrate that declines are still underway. From 2004-2008 I investigated the ecology of wood turtles in Massachusetts and New Hampshire. I obtained between one and five years of annual home range data for 150 turtles, and evaluated population structure at 31 sites in five major watersheds. Seasonal floods displaced 7% of wood turtles annually in one watershed, and accounted for elevated mortality. Twelve wood turtles were displaced < 16.8 km, and two were displaced over a 65-foot dam. Several turtles overwintered at their displacement site and two returned successfully, indicating that floods are a mechanism of population connectivity. Several homing turtles ended up in new areas. Turtles occupied stream segments with gradient < 1%, lower than generally available. Agricultural machinery accounted for most observed mortality, followed by automobiles and mammals. Female turtles exhibit smaller home ranges in agricultural areas. Older turtles move farther from the river than do young turtles, possibly reflecting their familiarity with a former landscape. Population density ranged from 0-40.4 turtles/river-kilometer. The highest densities occur in central New Hampshire and lower densities occur in the Housatonic watershed. Population density is negatively correlated with agriculture at both riparian and watershed scales, and responds unimodally to forest cover. Wood turtle populations in western Massachusetts are declining by 6.6-11.2% annually. I estimated ages of turtles by assessing shell-wear rates from photographs. Wood turtles regularly achieve ages over 80 years, and like related species, do not exhibit clear signs of senescence. Old wood turtles are reproductively dominant, and their survival rates are twice as high as young turtles. Carapace scutes appear to require 80 years to become worn. Population modeling indicates that wood turtle populations are declining in New England due to anthropogenic and natural factors. Conservation efforts must address the effects of agriculture on adult survival. Climate change may negatively affect northeastern wood turtles through increased flooding. Populations in mountainous areas may be likely candidates for conservation because they don't occupy prime agricultural land, but may be more susceptible to floods.

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[ES] La presente tesis doctoral titulada "La terapia senolítica como nuevo enfoque terapéutico para la prevención de la cardiotoxicidad inducida por la doxorrubicina" se centra en explorar la eliminación de células senescentes (senolisis) in vitro e in vivo mediante la administración del fármaco senolítico, navitoclax, como opción terapéutica para prevenir la cardiotoxicidad asociada al tratamiento antitumoral con doxorrubicina. La exposición a doxorrubicina afecta gravemente a la población de células cardíacas tanto en ratones como en corazones humanos al inducir senescencia prematura y navitoclax es uno de los senolíticos más potentes y ampliamente conocido en el campo de la senescencia celular pero su administración está asociada a la aparición de efectos adversos, principalmente trombocitopenia. Con el objetivo de mejorar el perfil terapéutico del fármaco, se han desarrollado dos estrategias específicamente dirigidas a células senescentes que incluyen la encapsulación y el desarrollo de un profármaco basado en navitoclax. En la primera sección experimental, abordamos la inducción de senescencia en un modelo in vitro de cardiomiocitos y evaluamos la terapia senolítica como estrategia terapéutica. Desarrollamos y caracterizamos, así mismo, las nanopartículas y la prodroga basada en navitoclax. Este nanodispositivo senolítico se basa en MSNs cargadas con navitoclax y funcionalizadas con un hexa-galacto-oligosacárido (galactán). Por su parte, la prodroga se obtiene tras la conjugación del navitoclax a una molécula de galactosa acetilada. En ambos casos, el mecanismo de la terapia dirigida radica en la presencia de la enzima lisosomal ß-galactosidasa, la cual presenta mayor expresión en las células senescentes. Al entrar en el lisosoma de células senescentes, los enlaces glicosídicos se hidrolizan por acción de la enzima, liberando el fármaco para su función. Los resultados muestran un aumento del efecto terapéutico del fármaco libre. A continuación, ponemos a punto un modelo murino de cardiotoxicidad donde demostramos que la administración sistémica de doxorrubicina induce la expresión de marcadores de cardiotoxicidad y senescencia en el corazón de ratones tratados y contribuye al deterioro de la función cardíaca de los animales seguido por ecografía. En este modelo preclínico, el tratamiento combinado de doxorrubicina con el senolítico navitoclax, en las diferentes formulaciones descritas, conduce a la disminución significativa de los marcadores de senescencia y cardiotoxicidad junto con el restablecimiento de la función cardíaca y, se observa, de manera similar con las tres estrategias mencionadas: fármaco libre, fármaco encapsulado y fármaco en formulación de prodroga. Los resultados científicos presentados en esta tesis resaltan el papel de la senescencia en la progresión de cardiotoxicidad por la administración de doxorrubicina y se concluye que los sistemas senolíticos aquí evaluados podrían ser una herramienta importante para el desarrollo de nuevas estrategias terapéuticas en el campo de la prevención de efectos secundarios asociados a terapia. / [CA] La present tesi doctoral titulada "La teràpia senolítica com a nou enfocament terapèutic per a la prevenció de la cardiotoxicitat induïda per la doxorrubicina" se centra en explorar l'eliminació de cèl·lules senescents (senolisis) in vitro i in vivo mitjançant l'administració del fàrmac senolític, navitoclax, com a opció terapèutica per a previndre la cardiotoxicitat associada al tractament antitumoral amb doxorrubicina. L'exposició a doxorrubicina afecta greument a la població de cèl·lules cardíaques tant en ratolins com en cors humans en induir senescència prematura y navitoclax és un dels senolítics més potents i àmpliament conegut en el camp de la senescència cel·lular però la seua administració està associada a l'aparició d'efectes adversos, principalment trombocitopenia. Amb l'objectiu de millorar el perfil terapèutic del fàrmac, s'han desenvolupat dues estratègies específicament dirigides a cèl·lules senescents que inclouen l'encapsulació i el desenvolupament d'un profàrmac basat en navitoclax. En la primera secció experimental, abordem la inducció de senescència en un model in vitro de cardiomiòcits i avaluem la teràpia senolítica com a estratègia terapèutica. Desenvolupem i caracteritzem, així mateix, les nanopartícules i la prodroga basada en navitoclax. Aquest nanodispositiu senolític es basa en MSNs carregades amb navitoclax i modificades amb un hexa-galacto-oligosacàrid (galactán). Per part seua, la prodroga s'obté després de la conjugació del navitoclax a una molècula de galactosa acetilada. En tots dos casos, el mecanisme de la teràpia dirigida radica en la presència de l'enzim lisosòmic ß-galactosidasa, la qual presenta major expressió en les cèl·lules senescents. En entrar en el lisosoma de cèl·lules senescents, els enllaços glicosídics s'hidrolitzen per acció de l'enzim, alliberant el fàrmac per a la seua funció. Els resultats mostren un augment de l'efecte terapèutic del fàrmac lliure. A continuació, posem a punt un model de ratolí on vam demostrar que l'administració sistèmica de doxorrubicina indueix l'expressió de marcadors de cardiotoxicitat i senescència en el cor de ratolins tractats i contribueix a la deterioració de la funció cardíaca dels animals seguit per ecografia. En aquest model preclínic, el tractament combinat de doxorrubicina amb el senolític navitoclax, en les diferents formulacions descrites, condueix a la disminució significativa dels marcadors de senescència i cardiotoxicitat juntament amb el restabliment de la funció cardíaca i, s'observa, de manera similar amb les tres estratègies esmentades: fàrmac lliure, fàrmac encapsulat i fàrmac en formulació de prodroga. Els resultats científics presentats en aquesta tesi ressalten el paper de la senescència en la progressió de cardiotoxicitat per l'administració de doxorrubicina i es conclou que els sistemes senolítics ací avaluats podrien ser una eina important per al desenvolupament de noves estratègies terapèutiques en el camp de la prevenció d'efectes secundaris associats a teràpia. / [EN] The present PhD thesis entitled " Pharmacological senolysis as a new therapeutic approach for the prevention of doxorubicin-induced cardiotoxicity" focuses on exploring the elimination of senescent cells (senolysis) in vitro and in vivo by administration of the senolytic drug, navitoclax, as a therapeutic option to prevent cardiotoxicity associated with antitumor treatment with doxorubicin. Doxorubicin exposure severely affects the cardiac cell population in both mouse and human hearts by inducing premature senescence and navitoclax is one of the most potent and widely known senolytics in the field of cellular senescence, but its administration is associated with the occurrence of adverse effects, mainly thrombocytopenia. In the first experimental section, we addressed the induction of senescence in an in vitro cardiomyocyte model and evaluated senolytic therapy as a therapeutic strategy. We also developed and characterized the nanoparticles targeting senescent cells and the navitoclax-based prodrug. The senolytic nanodevice is based on MSNs loaded with navitoclax and functionalized with a hexa-galacto-oligosaccharide (galactan). The prodrug is obtained after conjugation of navitoclax to an acetylated galactose molecule. In both cases, the mechanism of targeted therapy lies in the presence of the lysosomal enzyme ß-galactosidase, which is highly expressed in senescent cells. Upon entering the lysosome of senescent cells, the glycosidic bonds are hydrolyzed by the action of the enzyme, releasing the drug. The results show an increase in the therapeutic effect of the free drug. Next, we developed a murine model of cardiotoxicity in which we demonstrated that systemic administration of doxorubicin induces the expression of markers of cardiotoxicity and senescence in the heart of treated mice and contributes to the deterioration of the cardiac function of the animals followed by echocardiography. In this preclinical model, the combined treatment of doxorubicin with the senolytic navitoclax, in the different formulations described above, leads to significant decrease in senescence and cardiotoxicity markers along with restoration of cardiac function. Similar results were observed with the three strategies mentioned: free drug, encapsulated drug, and prodrug formulation. The scientific results presented in this thesis highlight the role of senescence in the progression of cardiotoxicity by doxorubicin administration and it is concluded that the senolytic systems evaluated here could be an important tool for the development of new therapeutic strategies in the field of prevention of therapy-associated side effects and represent an alternative to the limitations of current treatments. / Lérida Viso, A. (2023). Pharmacological Senolysis as a New Therapeutic Approach for the Prevention of Doxorubicin-Induced Cardiotoxicity [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/194709

Despite the advent of stents, intimal hyperplasia subsequent to vascular interventional procedures remains a major obstacle. Vascular smooth muscle cells (VSMC) play a critical role in the pathogenesis of intimal hyperplasia; therefore, regulation of VSMC gene expression is a logical intervention point. FXR1 is a muscle-enhanced RNA binding protein and its expression is increased in injured arteries. We have shown that modulation of FXR1 levels affects stability and abundance of inflammatory transcripts in VSMC, suggesting that FXR1 is a negative regulator of inflammation. RNA-sequencing analysis in FXR1-depleted human VSMC (hVSMC) identified a number of transcripts with decreased abundance, the overwhelming majority of which were associated with proliferation and cell division. This drives our hypothesis that FXR1 is involved in mitigating vascular disease by regulating inflammatory and proliferative mRNA in VSMC. The mRNA abundance and stability of a number of these transcripts was decreased in FXR1 depleted hVSMC, and RIP-sequencing demonstrated that FXR1 interacts with transcripts involved in cell cycle control, and stability of these transcripts is decreased with FXR1 depletion. FXR1-depleted cells showed decreased proliferation (p<0.05), however, an increase in β-galactosidase (p<0.05) and γH2AX (p<0.01), indicative of senescence was noted. Senescent cells exhibit a senescence associated secretory phenotype (SASP) with characteristic gene expression leading to increased inflammation in the tissue microenvironment. HVSMC depleted of FXR1 had increased transcripts abundance of many SASP genes, as well as an increase of both mRNA and protein expression of canonical senescence markers p53 and p21. We developed a novel SMC-specific conditional knockout mouse (FXR1SMC/SMC) to further study these results in a more translational context. In a carotid artery ligation model of intimal hyperplasia, FXR1SMC/SMC mice have significantly reduced neointima formation (p<0.001) post-ligation compared to controls. qPCR analysis from FXR1 conditional knockout mouse VSMC (mVSMC) show increased transcripts associated with senescence (p21, p16, p53) as well as increased SASP-associated mRNA, a decrease in proliferation, and an increase in β-galactosidase staining. Our results are the first to suggest that in addition to destabilization of inflammatory transcripts, FXR1 may stabilize cell cycle related genes in VSMC, and absence of FXR1 leads to induction of a senescent phenotype, an increase in SASP genes, and reduction of intimal hyperplasia. / Organ Systems & Translational Medicine

Background: Ageing is associated with age-related disease and it has been divided into 12 hallmarks and cellular senescence is one of them. Cellular senescence increases with age and has different pathological inducing aspects in the tissue. Hyperbaric oxygen therapy is used in the clinic to treat different pathological conditions and has emerged as a possible intervention for the reduction of senescent cells. Reducing senescent cells could be a way to reduce the effects of ageing and therefore possibly in the future treat age-related diseases. Aim: This systematic literature review aims to investigate articles researching the effect of HBO exposure on senescent cells and the properties of senescent cells. Methods: PubMed database was used in this systematic literature review. Exclusion and inclusion criteria were specified using the PECO format. A search plan was created using both MeSH words and free text words. An article search was performed twice on 11 th September 2023 and 20 th November 2023. The exclusion and inclusion criteria were used to filter the search results and for the full-text review. Result: 6 articles in total were included in this study. 2 of those included articles were conducted on humans while the rest were conducted on animals or animal cells. Two studies showed that HBOT decreases the number of senescent cells, one study showed that the senescent cell markers and SASP were decreased post-HBOT, two studies showed that the properties of senescent cells were decreased post-HBOT while one study showed the contrary with an increased senescent cell properties. Conclusion: The result of this systematic literature review suggests that there is an association between HBOT and a decrease in senescent cells or its properties. More research is needed though to better understand the relationship between HBOT and the effect it imposes on senescent cells.

Lipid droplets (LDs) are organelles with many functions in cells and numerous protein interactors facilitate their biogenesis, maintenance, and turnover. The mammalian lipase responsible for LD turnover during lipophagy, LipA, has two candidate homologs in Arabidopsis: MPL1 and LIP1. One or both of these plant homologs may function in a similar manner to mammalian LipA, providing an LD breakdown pathway. To test this hypothesis, wild type (WT) Arabidopsis plants, MPL1 over-expressing (OE) mutants, and T-DNA insertion mutants of MPL1 (mpl1) and LIP1 (lip1) were examined for LD phenotypes in normal conditions and in environments where LD numbers are known to fluctuate. Plants to be imaged by confocal microscopy were exposed to heat stress and wounding to increase LD accumulation, senescence was induced in leaves to deplete lipids, and LDs were imaged throughout the day/night period to observe their diurnal regulation. The mutation of both MPL1 and LIP1 lead to an increase in LDs within the leaf mesophyll cells, although the spatial distribution of the LDs differed between the two mutants. mpl1 mutants had disrupted diurnal regulation of their LDs, but lip1 mutants did not. Alternately, lip1 mutants retained LDs during dark-induced senescence, and mpl1 mutants did not. Together these results suggest that MPL1 and LIP1 are likely both important for LD dynamics; however they appear have roles in different aspects of LD accumulation and turnover.

Aging of the brain is the leading risk factor for neurodegenerative diseases and brain cancers and has deleterious effects on brain functions. It follows that attempts to reverse the aging process may be therapeutically valuable. Neural stem cells (NSC) have been shown to play a critical role in maintaining brain functions, and their number is severely decreased with age. The development of senescence-like characteristics and declining functions in NSCs have been proposed to be responsible for brain aging and tumorigenesis. MS-818 is a pyrrolopyrimidine that has been shown to increase the NSC population and reverse the decline of behavioral function in aged rodent models. While MS-818 has demonstrated such benefits, the mechanism by which it affects particular pathways of biological age in NSCs is not yet known. Understanding how MS-818 relates to the molecular mechanisms underlying cellular aging may help accelerate the development of anti-aging therapies for neurodegenerative diseases and cancer. This study attempts to elucidate the mechanism of action of MS-818 on NSCs using an in vitro accelerated-aging model produced by Hydroxyurea (HU) treatment. Our analysis of NSC population size post-MS-818 exposure supports the idea that MS-818 treatment can increase NSC proliferation. qPCR analysis of aging-related genes revealed HU treatment produced a trend of increased p16 and Il-6 and decreased Lamin B1 relative expression, supporting the notion that HU treatment can induce senescence in NSCs. MS-818 treatment alone also produced notable trends for targets including BRCA1. In addition, MS-818 treatment post-HU exposure appeared to influence the relative expression of targets, including PGC1a and Lamin B1. Such MS-818 treatment produced similarly noteworthy trends for the expression of genes including PGC1a, Lamin B1, BRCA1, RPTOR, and Il-6, whether in media containing 2.5% or 7.5% serum. These results indicate that MS-818 may have influenced some aging-related pathways.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第23744号 / 理博第4834号 / 新制||理||1691(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 工藤 洋, 准教授 本庄 三恵, 教授 松下 智直 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

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