Senescent cells play an important role in normal biological processes such as wound healing and cancer prevention. If not effectively cleared by the immune system however, senescent cells can accumulate and have been linked to negative events; most notably fibrosis and ageing. To understand more about the molecular mechanisms underpinning senescence as well as the effects senescent cells have on surrounding cells, the organism as a whole and why some cells evade the immune system, it is necessary to study and manipulate senescent cells in vitro and in vivo. In order to do this, accurate identification of senescent cells is required, something which can currently only be achieved by staining for multiple markers, a process that requires invasive tissue sampling when using in vivo models, and is a limitation in human studies in particular. A secreted biomarker that is detectable in bio-fluids such as blood would facilitate more informative human and animal studies. In this thesis un-targeted metabolomic screens were performed using fibroblasts from multiple tissue types and two well characterised models of senescence: replicative senescence and irreparable DNA double strand break induced senescence. Controls for transient growth arrest and repairable DNA damage were also included and all groups were compared to young dividing cells. These investigations not only give insight into the metabolic changes occurring in senescence but also provided candidate biomarkers that were then more closely studied using targeted techniques. Extracellular citrate was identified as the most robust candidate, and its regulation was investigated at the molecular level. The work presented in this thesis represents a novel contribution to the field of senescence both in terms of the metabolic profiles of senescent and quiescent fibroblasts and the strong candidate biomarker, citrate, which has the potential to broaden studies of senescence in humans in vivo.

Efficient removal of apoptotic cells is critical for development, tissue remodelling, maintenance of homeostasis, and response to injury. Phagocytosis of apoptotic cells is mediated by many phagocytic receptors, soluble bridging molecules, and pro-phagocytic ligands on the surface of apoptotic cells. Macrophage phagocytosis in general is controlled by stimulatory and inhibitory mechanisms. An example of the latter mechanism is that mediated by the cell surface glycoprotein CD47, which by binding to the inhibitory receptor Signal Regulatory Protein alpha (SIRPα) on macrophages, is known to inhibit phagocytosis of viable host cells. The studies of the present thesis aimed at investigating possible changes to CD47 on apoptotic cells, which could influence their elimination by macrophages. The endoplasmatic protein calreticulin (CRT), in conjunction with Low density lipoprotein Receptorrelated Protein 1 (LRP1) on the phagocyte, can act as a receptor for collectin family members and mediate uptake of apoptotic cells. However, CRT itself was found to also be expressed on the surface of many viable cell types, and the CRT expression increased on apoptotic cells. By using antibodies to LRP1 or receptor‐associated protein (RAP), an antagonist blocking LRP1 ligand binding, we found that CRT on target cells could interact in trans with LRP1 on a phagocyte and stimulate phagocytosis. CD47 on the target cell inhibited LRP1‐mediated phagocytosis of viable cells (e.g. lymphocytes or erythtocytes), but not that of apoptotic cells. The inability of CD47 on apoptotic cells to inhibit LRP1‐ mediated phagocytosis could be explained in two ways: 1) Some apoptotic cell types (fibroblasts and neutrophils, but not Jurkat T cells) lost CD47 from the cell surface, or 2) CD47 is evenly distributed on the surface of viable cells, while it was redistributed into patches on apoptotic cells, segregated away from areas of the plasma membrane where the pro‐phagocytic ligands CRT and phoaphatidylserine (PS) were concentrated. Apoptotic murine thymocytes also showed a patched distribution of CD47, but no significant loss of the receptor. However, both PS‐independent and PS‐dependent macrophage phagocytosis of apoptotic CD47‐/‐ thymocytes was less efficient than uptake of apoptotic wild‐type (wt) thymocytes. This contradictory finding was explained by the fact that CD47 on apoptotic thymocytes did no longer inhibit phagocytosis, but rather mediated binding of the apoptotic cell to the macrophage. These effects could in part be dependent on the apoptotic cell type, since uptake of experimentally senescent PS+ wt or CD47‐/‐ erythrocytes by macrophage in vitro, or by dendritic cells (DC) in vivo, were the same. In vivo, PS+ erythrocytes were predominantly trapped by marginal zone macrophages and by CD8+ CD207+ DCs in the splenic marginal zone. DCs which had taken up PS+ erythrocytes showed a slight increase in expression levels of CD40, CD86 and MHC class II. These findings suggest that PS+ erythrocytes may be recognized by splenic macrophages and DCs in ways similar to that reported for apoptotic T cells. Uptake of senescent erythrocytes by DCs may serve as an important mechanism to maintain self‐tolerance to erythrocyte antigens, and defects in this function may facilitate development of AIHA. Glucocorticoids are used to treat inflammatory conditions and can enhance macrophage uptake of apoptotic cells. We found that the glucocorticoid dexamethasone time‐ and dose‐dependently stimulated macrophage cell surface LRP1 expression. Dexamethasone‐stimulated macrophages also showed enhanced phagocytosis of apoptotic thymocytes and unopsonized viable CD47‐/‐ erythrocytes. In summary, LRP1 can mediate phagocytosis of both viable and apoptotic cells by binding CRT on the target cell. Macrophage expression of LRP1 is increased by glucocorticoids, which could be one explanation for the anti‐inflammatory role of glucocorticoids. While CD47 on viable cells efficiently inhibits phagocytosis in macrophages, CD47 on apoptotic cells does not and can sometimes even promote their removal.

Immunological analogues of band 3, the anion transporter of the human erythrocyte, have been identified in all cells, including both isolated neurons and neurons of the central nervous system. We hypothesized that the anion channel is altered in neurological disease associated with choreiform movements because γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in mammalian brain, binds to its receptor and opens an integral membrane chloride channel. In order to examine this hypothesis, we studied a family with a serious, progressive, genetic neurologic disorder with acanthocytosis (choreoacanthocytosis) that resembles Huntington's chorea. We selected choreoacanthocytosis because erythrocytes, which are readily obtained, are affected in this disease as well as the central nervous system. Biochemical studies of erythrocytes from the proposita, mother, and brother revealed that sulfate transport Vmax was increased, and glucose efflux was decreased. Erythrocytes exhibited immunological changes indicative of cellular aging/transporter damage. In addition, transporter reactive antibodies were present. This is the first evidence for abnormalities of membrane transport in this neurologic disorder.

This series of studies began with an examination of the effects of training status (Tr vs UTr) and sex on the resting levels and redistribution of senescent (CD28-CD57+) and naïve (CD28+CD57-) T-lymphocytes (CD4+, CD8+) following a treadmill test to volitional exhaustion. In this first study exercise elicited a redistribution of senescent CD4+, CD8+ and naïve CD4+,CD8+ T-lymphocytes. UTr had a higher proportion of senescent and a lower proportion of naïve CD8+ T-lymphocytes than Tr. Males had a higher proportion of senescent and lower proportion of naïve T-lymphocytes than females with the highest percentage of senescent and lowest percentage of naïve T-lymphocytes observed in UTr males. CMV was a covariate in the senescent and naïve CD8+ T-lymphocytes. This study highlighted important sex and training status differences in the senescent and naïve T-lymphocyte redistribution in response to exercise. These findings led on to an investigation of the T-lymphocyte (CD4+, CD8+, γδ+) response to a period of 2 weeks increased volume training (39% increase in volume) in trained females (Tr, n=13) compared to a period of 2 weeks habitual activity in female controls (UTr, n=13). This second study observed no difference in the resting T-lymphocyte profile from the pre to post increased volume training period. The resting number of CD3+ and proportion of γδ+ T-lymphocytes was greater in the Tr compared to the UTr. The resting proportion of CD4+T-lymphocytes and the CD4+:CD8+ ratio was greater in the UTr compared to the Tr. CMV was a covariate in the analysis of CD8+, CD28+ CD8+, and naïve CD8+ T-lymphocyte cell numbers but not in the analysis of T-lymphocyte proportions. The increased volume training period had no effect on resting T-lymphocyte populations in Tr females, and T-lymphocyte populations also did not change with 2 weeks of habitual exercise in UTr. The total energy, carbohydrate and protein intake was greater in Tr compared to the UTr during the increased volume training period and was greater than normal in the Tr group. These dietary influences may partly explain the absence of any change in T-lymphocyte proportions pre to post training period in Tr. Differences in the proportions of γδ+, CD4+ and the ratio of CD4+:CD8+ T-lymphocytes at rest between the Tr and UTr warrants further investigation. The final study of this series is presented in two parts. The first part focused on the influence of 4 weeks supplementation at 0.1g/kg body mass/day with n-3 polyunsaturated fatty acids (PUFA) as fish oil (FO, n=10), or short-chain saturated fatty acids (SFA) as coconut oil (CO, n=10) on T-lymphocyte (CD4+,CD8+, γδ+) differentiated populations at rest and in response to exercise in trained males. Changes were examined by Day (Baseline to pre supplementation, Pre Sup (4 week control period), and pre supplementation to post supplementation, Post Sup (4 week supplementation period)). During a 4 week baseline control period no changes were observed in the blood lipid profile in both FO and CO groups. During the control period a main effect of exercise was observed in all the CD3+ and γδ+ T-lymphocytes subsets. During the control period an interaction of group-by-day was observed in the senescent CD8+ T-lymphocytes from BL to Pre Sup the proportion and number decreased in the FO group and increased in the CO group. Inclusion of CMV as a covariate introduced a main effect of group on the CD4+ naïve proportions and cell counts and the group-by-day interaction observed on the CD8+ senescent T-lymphocyte proportions and cell counts disappeared. During the 4 week supplementation period this study observed an increase in the n-3 PUFAs, EPA (20:5n-3), DHA (22:6n-3) and DPA (22:5n-3) in the FO group but not in the CO group (with no changes in blood lipid profile on CO). During the supplementation period a main effect of exercise was observed in all the CD3+ and γδ+ T-lymphocyte subsets except for the proportion of CD8+ naïve T-lymphocytes. The proportion of CD8+ naïve T-lymphocytes was lower at rest and in response to exercise in FO and CO groups after supplementation. CMV was a significant covariate in senescent CD4+ T-lymphocyte cell counts. At the post exercise time point the γδ+ T-lymphocyte count increased in the FO group but decreased in the CO group, following the supplementation period. However, this observation did not quite reach statistical significance. Although a difference between the groups was evident for γδ+ T-lymphocyte count and proportion there was insufficient evidence to conclude whether the difference was supplement related. It would appear that dose, duration and type of fatty acids ingested could all be important in the overall response but these require further study. The second part of this final study investigated the influence of 4 week supplementation at 0.1g/kg body mass/day with n-3 polyunsaturated fatty acids (PUFA) as fish oil (FO, n=10) or short-chain saturated fatty acids (SFA) as coconut oil (CO, n=10) on plasma Th1 cytokine: IL-2, TNF- α and IFN-γ, and Th2 cytokine IL-4, IL-6 and IL-10 concentrations, and expression of the T-lymphocyte activation marker CD69 at rest and in response to exercise in trained males. Changes were examined by Day (Baseline to pre supplementation (4 week control period), and pre supplementation to post supplementation (4 week supplementation period)). This study observed an increase in n-3 PUFAs, EPA (20:5n-3), DHA (22:6n-3) and DPA (22:5n-3) in the FO group but not in the CO group. There was a significant mobilisation of activated CD4+ CD69+ and CD8+ CD69+ (P<0.05) T-lymphocyte numbers in response to exercise in both FO and CO groups. CMV infection was a significant covariate on the number and proportion of CD4+CD69+ T-lymphocytes (P<0.05) but not on the number or proportion of CD8+CD69+ T-lymphocytes. During the supplementation period there was a significant effect of Day on TNF-α, IL-6, IL-4 and IL-2 with IFN-γ and IL-10 trending towards a difference. The plasma cytokine concentration was greater at post supplementation compared to pre supplementation for both FO and CO groups. Latent CMV infection was a significant covariate for TNF-α, IL-6, IL-4, IL-2, IFN-γ and IL-10. In the current study we observed no evidence of a difference between the CO and FO groups for early T-lymphocyte activation marker or plasma cytokine concentrations despite the membrane lipid composition change over the 4 week supplementation period. It would appear that the plasma Th1 and Th2 cytokine concentration increased from pre supplementation to post supplementation on both PUFA and SFA, highlighting a potential link between fatty acid incorporation and cytokine expression that needs closer examination. The results of this series of studies highlight that sex and training status impact upon the T-lymphocyte pool at rest and in response to exercise. Increasing the volume of training for 2 weeks without dietary restriction does not alter the resting T-lymphocyte pool in trained females. Alterations to the T-lymphocyte pool at rest and in response to exercise are not related to FO or CO supplementation. Furthermore, the response of Th1, Th2 plasma cytokines, and the early activation marker CD69 at rest and in response to exercise does not differ between a group supplemented with FO compared to a group supplemented with CO it would appear that Th1 and Th2 plasma cytokines increase post supplementation in both groups. Particular avenues of interest for future research would be, to explore the sex differences in T-lymphocyte subsets at rest and in response to exercise, to determine whether these sex differences are key in susceptibility to disease/infection and to determine the tissue targets of lymphocytes mobilised during exercise.

Nesta pesquisa, foi avaliado o hábito alimentar de pacientes senescentes totalmente desdentados, antes e após a reabilitação por próteses totais bimaxilares convencionais. A avaliação foi realizada por meio de exame clínico, entrevista e a aplicação de questionários pela própria pesquisadora. O trabalho foi conduzido em três fases distintas: a primeira, desenvolvida antes do início de qualquer procedimento relacionado com a confecção das próteses e consistiu no preenchimento de ficha clínica, avaliação protética e da realização de uma anamnese alimentar, sendo essa última elaborada com a orientação de uma nutricionista. Já a segunda e a terceira fases foram executadas em períodos compreendidos entre aproximadamente dois a três meses e cinco a seis meses após a reabilitação protética respectivamente, quando novos exames clínicos eram realizados. Os dados coletados nesses três períodos distintos foram processados por um computador utilizando-se do software BioEstat 3.0,sendo o nível de significância estabelecido para p< 0,05, em uma prova bilateral. Os resultados demonstraram que após a instalação de uma nova prótese total convencional confeccionada no rigor da técnica tivemos, não só melhora funcional do aparelho protético, como alteração positiva na habilidade mastigatória dos indivíduos, além de alteração da consistência da dieta acompanhada da introdução de novos alimentos. Os resultados demonstraram ainda, que quando o aparelho protético apresentava retenção e estabilidade consideradas satisfatórias, aquele acabava influenciando positivamente o hábito intestinal do paciente.Contudo, através desse estudo, não foi possível identificar se houve uma real modificação da dieta dos pacientes com inserção de nutrientes, uma vez que para obtermos esses dados seriam necessários exames complementares. / In this research, the alimentary attitude of senescent edentulous patients was evaluated, before and after the rehabilitation by new complete dentures. A clinical exam, an interview and a survey were performed. The work was accomplished in three different phases: the first was developed before the beginning of any procedure related to the construction of the new dentures and consisted of a clinical record, a prosthetic evaluation and an alimentary anamnesis. The second and the third phases took place after the prosthetic rehabilitation and were done in periods between two to three months and five to six months respectively, together with new clinical exams. The data collected during those three different periods were processed using software BioEstat 3.0 ; the level of significant was p <0,05, in a bilateral proof. The results demonstrated that after the installation of new complete dentures made according to the technique there was a functional improvement of the dentures noticed as a better performance in the individuals' masticatory ability. It was reported also changes in the consistence of the diet associated to the increasing of different nutrients to the diet. It was also possible to verify that when the prostheses presented satisfactory retention and stability, intestinal habit was influenced positively. However, through this study only, it was not possible to identify if there was a real modification in patients’ alimentary habit or if different nutrients were inserted to it. To make sure those modifications had happened complementary exams should have been performed as well.

Nesta pesquisa, foi avaliado o hábito alimentar de pacientes senescentes totalmente desdentados, antes e após a reabilitação por próteses totais bimaxilares convencionais. A avaliação foi realizada por meio de exame clínico, entrevista e a aplicação de questionários pela própria pesquisadora. O trabalho foi conduzido em três fases distintas: a primeira, desenvolvida antes do início de qualquer procedimento relacionado com a confecção das próteses e consistiu no preenchimento de ficha clínica, avaliação protética e da realização de uma anamnese alimentar, sendo essa última elaborada com a orientação de uma nutricionista. Já a segunda e a terceira fases foram executadas em períodos compreendidos entre aproximadamente dois a três meses e cinco a seis meses após a reabilitação protética respectivamente, quando novos exames clínicos eram realizados. Os dados coletados nesses três períodos distintos foram processados por um computador utilizando-se do software BioEstat 3.0,sendo o nível de significância estabelecido para p< 0,05, em uma prova bilateral. Os resultados demonstraram que após a instalação de uma nova prótese total convencional confeccionada no rigor da técnica tivemos, não só melhora funcional do aparelho protético, como alteração positiva na habilidade mastigatória dos indivíduos, além de alteração da consistência da dieta acompanhada da introdução de novos alimentos. Os resultados demonstraram ainda, que quando o aparelho protético apresentava retenção e estabilidade consideradas satisfatórias, aquele acabava influenciando positivamente o hábito intestinal do paciente.Contudo, através desse estudo, não foi possível identificar se houve uma real modificação da dieta dos pacientes com inserção de nutrientes, uma vez que para obtermos esses dados seriam necessários exames complementares. / In this research, the alimentary attitude of senescent edentulous patients was evaluated, before and after the rehabilitation by new complete dentures. A clinical exam, an interview and a survey were performed. The work was accomplished in three different phases: the first was developed before the beginning of any procedure related to the construction of the new dentures and consisted of a clinical record, a prosthetic evaluation and an alimentary anamnesis. The second and the third phases took place after the prosthetic rehabilitation and were done in periods between two to three months and five to six months respectively, together with new clinical exams. The data collected during those three different periods were processed using software BioEstat 3.0 ; the level of significant was p <0,05, in a bilateral proof. The results demonstrated that after the installation of new complete dentures made according to the technique there was a functional improvement of the dentures noticed as a better performance in the individuals' masticatory ability. It was reported also changes in the consistence of the diet associated to the increasing of different nutrients to the diet. It was also possible to verify that when the prostheses presented satisfactory retention and stability, intestinal habit was influenced positively. However, through this study only, it was not possible to identify if there was a real modification in patients’ alimentary habit or if different nutrients were inserted to it. To make sure those modifications had happened complementary exams should have been performed as well.

Abstract Chronic, non-healing wounds constitute a massive financial burden on health care system. The healing processes of these wounds and their underlying pathology are only partly understood. In this study, important biological functions performed by Retinoic acid with its regulatory protein cellular retinoic acid binding protein 2 (CRABP2) were discussed. Possibly, these biological func-tions might be linked with chronic wound therapeutic by inducing antiproliferative activity of cells which leads to reduction in migration and growth rate of fibroblast during skin regeneration pro-cess in chronic wound healing. The aim of this study was to comparatively analyze the expression pattern of CRABP2 and P16 cyclic dependent kinase inhibitor in dermal fibroblasts at mRNA levels along with their morphological pattern, migration and growth rate. Fibroblasts were cultured and their morphology were observed by phase-contrast imaging. Difference in viability, migratory capacity was examined by Cell titer blue and scratch assay respectively and expression were meas-ured by polymerase chain reaction. Interestingly, the date revealed that morphology was altered and growth rate and migration velocity was significantly lower in chronic wound fibroblasts and senescent fibroblasts when compared with their control. Expression pattern revealed that CRABP2 was highly up-regulated only by senescent cells but not in chronic wound fibroblasts which point novel function for this protein in term of replicative senescence. However, P16 was not signifi-cantly altered among all fibroblasts which demands supplementary studies to conform the role of CRABP2 in fibroblast dysfunction and cellular senescence in chronic wounds.

La presente tesis doctoral titulada ¿Supramolecular and heterosupramolecular chemistry in controlled release and molecular recognition processes¿ está centrada en los dos aspectos principales de la química supramolecular que han experimentado un gran auge en los últimos años: el reconocimiento molecular y los procesos de liberación controlada. En particular la primera parte de la tesis se focaliza en el diseño y síntesis de moléculas orgánicas que pueden ser empleados cómo sensores para especies aniónicas y neutras. El paradigma seleccionado para los procesos de reconocimiento molecular fue la aproximación del dosimetro químico. Esta aproximación presenta ventajas con respecto a los otros dos métodos de determinación de aniones (desplazamiento y unidad coordinanteunidad indicadora), cómo, por ejemplo, la posibilidad de determinar los analitos en disolución acuosa. Así se sintetizaron dos sensores selectivos, uno para el anión fluoruro (F-) y el otro para glutatión (GSH). El sensor selectivo para la determinación de F- está basado en un colorante azoico funcionalizado, en su ¿OH fenólico, cómo silileter. Esta molécula presenta una banda de absroción muy intensa centrada a 350 nm que, después de la adición de F- , sufre un efecto hipocrómico significativo y un desplazamiento batocromico ligero (de ca. 10 nm), mientras aparece una nueva banda a 470 nm, determinando un cambio de incoloro a amarillorojo. Para obtener un sensor selectivo para GSH se sintetizó una sonda químico basado en una sal de 2,6-difenilpirilio. Sucesivamente se preparó una disolución de este compuesto en agua/CTAB, que se caracterizaba por un intenso color azul. En este caso, la adición de GSH produce una disminución significativa de la banda del visible, acompañada por la consecuente decoloración. Además la adicón de GSH induce la aparición de Resumen vi una intensa banda de emisión centrada a 485 nm (después de la irradiación a 350 nm). La segunda parte de esta tesis doctoral se basa en el diseño y síntesis de nuevos sistemas híbridos orgánicos-inorgánicos para procesos de liberación controlada en ambiente celular. Estos materiales híbridos se componen en general, de dos unidades: una matriz inorgánica mesoporosa de base silícea, capaz de almacenar moléculas orgánicas (colorantes, farmacos...) y un compuesto orgánico anclado covalentemente a la superficie externa del soporte inorgánico mesoporoso, que actúa cómo puerta molecular. La aplicación de un estímulo externo puede modificar la conformación de la puerta molecular permitiendo o bien impidiendo la difusión de la carga almacenada en los mesoporos hacía el exterior (disolución o citoplasma). El primer sistema sintetizado y estudiado se compone de una matriz inorgánica mesoporosa (MCM-41), cargada con el colorante Ru(bipy)3 2+ y funcionalizada en la superficie con un oligoetilen glicol mediante un grupo ester. La adición de la enzima esterasa determinaba la hidrólisis del grupo ester y la consecuente reducción del tamaño de la puerta molecular, acompañada por la liberación del colorante previamente cargado. Otro sistema de liberación preparado consiste en el uso de la misma matriz MCM-41 nanoscópica y el mismo colorante Ru(bipy)3 2+, pero se funcionalizó la superficie con una puerta molecular fotolabil. La irradiación en el maximo de absorción de la puerta molecular inducía la fotodegradación de la misma y la consecuente liberación del colorante. Un tercer ejemplo de sistema de liberación consiste en una puerta molecular caracterizada por la presencia de dos grupos funcionales hidrolizables con enzimas diferentes: grupos urea y amida. vii El material final, caracterizado por la presencia del mismo esqueleto inorgánico, y cargado con Ru(bipy)3 2+, era capaz de liberar selectivamente cantidades distintas de colorante, dependiendo del enzima empleado. Así se podían conseguir dos tipos de perfiles de liberación: uno muy rápido y poco intenso y otro más lento pero mucho mas intenso. Finalmente se sintetizó un material híbrido siempre basado en la misma matriz de MCM- 41, cargado con rodamina-B y funcionalizado en la superficie con galactooligosacáridos. Con este material se podía conseguir una liberación controlada del colorante selectivamente en células senescentes, debido a que estas sobreexpresan el enzima ß-galactosidasa que es capaz de hidrolizar los galactooligosacáridos. / Agostini, A. (2013). Supramolecular and heterosupramolecar chemistry in controlled release and molecular recognition processes [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/29397

Analysis of mortality development in Russia using various decomposition methods Abstract The aim of this thesis is to analyze and evaluate mortality development in Russia in last 50 years by using various decomposition methods. The first part presents the method of decomposition of the difference between two demographic indicators (E. Kitagawa's method or methods from E. Arriaga, R. Pressat and J. Pollard). In the second part mortality development in Russia is analyzed by using methods that decomposed the value of demographic indicators in a given year. Mortality is divided into senescent and background component by using the Gompertz-Makeham formula and by using the logistic model. Afterwards, avoidable and unavoidable mortality and mortality due to endogenous and exogenous causes of death is analyzed. Hypotheses, set out in the introductory chapter, are verified by using different decomposition methods. Using multiple methods of decomposition enable to obtain a more complex view of the evolution of mortality in the observed period so that could be viewed from multiple perspectives and identify specific population trends in Russia. Keywords: mortality, Russia, decomposition, senescent and background mortality, avoidable mortality, endogenous, exogenous, causes of death, mortality crisis

A. Background<p><p>Skin wound healing (WH) is a dynamic and extremely determinate process of cellular, humoral and molecular mechanisms which begins directly after wounding and can last for years. WH is described as is an intricate process in which the skin (or another organ-tissue) repairs itself after injury. The process of skin WH occurs through the actions of an interplay of cells, growth factors and cytokines leading to wound closure.<p><p>WH occurs in three precisely and highly programmed phases: the inflammatory phase (day 0 to day 7) followed by the proliferative phase or vasculogenic phase (day 7 to day 21) and finally the remodeling phase (2 days - up to 2 years). For a successful healing, all three phases must occur in the proper sequence and time frame.<p><p>Many factors can interfere with one or more phases of the WH process, thus causing improper or impaired healing. The proliferation phase, in particular, requires the participation of various cells types such as fibroblasts, endothelial cells (ECs) and endothelial progenitor cells (EPCs), to produce a healthy well-vascularized granulation tissue for epithelization and wound closure.<p><p>A.1 Wound Healing And Obesity<p><p>In 2008, over 1.4 billion adults, 20 and older, were overweight. Of these, obesity has been shown to affect over 500 million people (OMS website). Moreover, the prevalence of obesity continues to rise, and by 2018, it is estimated that obesity will cost $ 347 billion annually.<p><p>Each year, in the US, approximately 33 million overweight and obese patients undergo surgery. Obesity causes a number of known health problems and increased post-surgical complications such as wound infection, dehiscence, hematoma and seroma. Surgeons anecdotally report WH complications among obese patients; however, little research has been conducted to investigate the mechanisms mediating impaired obesity-related WH. <p><p>Some previous work on diabetic patients and diabetic mice showed an imbalance between pro-oxydant and anti-oxydant genes as well as impaired EPCs proliferation and tube formation during the WH process. More then a hundred cytologic factors have been found to impair WH in the type 2 diabetic patient. It is a very complex and multifactorial problem involving decreased growth factors secretion, impaired keratinocyte and fibroblast functions, impaired EPs function, alteration of the macrophage function and granulation tissue synthesis, etc. <p><p>Based on these findings and because obesity is associated with the development of type 2 diabetes, we hypothetize that, impaired balance between pro-apoptotic/anti-apoptotic and pro- oxydant /anti-oxydant genes is involved in impaired WH. Furthermore, we hypothetize that impaired EPCs function leads to the perturbation of the proliferation phase of obesity impaired WH.<p><p>A.2. Wound Healing and Age<p><p>The world population is aging; by 2030, nearly 20% of Americans, (± 72 million people), will be 65 years old and older. In 2010, 17% of the European population was over the age of 65. By 2060, it is projected that the share of those aged 65 and over will rise to 30%, accounting for more then 150 million people. (ec.europa.eu) These aging subjects undergo an increasing number of surgical procedures: in the past two decades, the percentage of surgeries in patients over 65 has doubled to nearly 40%.<p>As a corollary, it is well established knowledge that elderly WH is impaired. However, little is known about the underlying mechanisms of age-related impaired WH.<p><p>As previously mentioned, adult BM-derived EPCs contribute to peripheral tissue repair and regeneration. In light of the abundant literature suggesting that neovascularization is impaired in the elderly, we characterize a novel model of senile cutaneous WH and investigate the role that vasculogenesis plays in the pathogenesis of age related impaired WH.<p>Aged mice colonies have traditionally been the model for aged small mammalian research, however, the ability to use a readily-available transgenic mouse model with features of accelerated aging would aid in the exploration of targeted therapies and a great number of age-related investigations.<p><p>We hypothesize that the Hutchinson-Gilford Progeria Syndrome (HGPS) Zmpste24 deficient (Zmpste24-/-) mouse mimics physiological ageing and can be used as a novel model for the study of senescent WH. We further hypothetized that impaired balance between pro-apoptotic/anti-apoptotic and pro-oxydant /anti-oxydant genes as well as impaired EPCs function are responsible for the impairment of the proliferative phase, leading to overall impaired WH.<p><p>A.3 Aims<p><p>Recently, a great deal of research has been directed at understanding the critical factors inducing poorly healing wounds. However, a lot remains unclear.<p><p>It is now well accepted that new blood vessel formation occurs not only by angiogenesis (blood vessels formation from a preexisting network of capillaries), but also by vasculogenesis (blood vessels formation from BM SCs recruitment) and that EPCs contribute to as much as 25% of new blood vessels formed in healing tissues4. They are mobilized from the BM in response to injury and production of local cytokines, are incorporate into wounds and play an integral role in systemic tissue repair. <p><p>Based on this finding, we hypothesized that co-morbidities related impaired WH may be due, in part, to decreased EPCs number, migration/homing, and/or function resulting in impaired vasculogenesis. Because age and/or obesity have been shown to be one of the most common predictors of altered WH, we decided to focus on these two parameters.<p><p>Following a bedside to bench approach the purpose of this work was to 1) develop coherent and translatable models of co-morbidity digging in the physiologic/pathologic mechanisms underlying altered healing in obese and senile mice; 2) develop targeted therapeutics to improve impaired WH.<p><p>B. Material and Methods<p><p>B.1 Human Model<p><p>Since obesity impairs WH and BM EPCs are important for tissue repair, we hypothesize that obesity- impaired WH is due, in part, to impaired EPCs mobilization, trafficking, and function. Peripheral blood was obtained from non diabetic, obese (BMI > 30, n = 25), and non obese (BMI < 30, n = 17) subjects. Peripheral blood human EPCs were isolated, quantified, and functionally assessed.<p>As for aged impaired WH, EPCs of aged subjects have already been found to have decreased adhesion, migration and proliferative properties as well as being decreased in number in elderly patients undergoing surgery compared to younger patients.<p><p>B.2. Mice Models<p><p>Two models of WH were developed and characterized.<p>In order to isolate the effect of obesity on EPCs and WH, OB non-diabetic female TallyHo/JngJ mouse were selected (Female mice don’t express hyperglycemia and hyperinsulinemia). Female SWR/J non-OB mice were used as control mice. In order to limit variables, TallyHO/JngJ obese mice were selected over other OB mice that exhibit a polygenic type of obesity (Jackson Laboratory Website). By selecting this mouse model, we have excluded in our selection of the ideal model common confounding factors such as hyperglycemia, hyperinsulinemia, immune disorders.<p><p>Zmpste24 is a metalloproteinase involved in the maturation of lamin A (LmnA), an essential component of the nuclear envelope. When Zmpste24 or LmnA are knocked-out, mice exhibit profound nuclear architectural abnormalities and histopathological defects that phenocopy an accelerated aging process. Of crucial importance, the lamin-A dependent nuclear alterations seen in Zmpste24-deficient mice have also been found in human physiological aging. We defined the utilization of the Hutchinson-Gilford Progeria Syndrome (HGPS) Zmpste24 deficient (Zmpste24- /-) mouse as a novel model for the study of senescent WH (controls used were C57BL/6J mice).<p><p>B.3. Wounding Model and Data Collection<p><p>All mice group underwent wounding using a stented wound model developed in our laboratory and previously published. Briefly, paired 6-mm circular, full-thickness wounds extending through the panniculus carnosus were made on the dorsal skin of the mouse. An O-ring, 12-mm splint made of silicone sheeting was then sutured to the skin around the wound. To minimize wound contraction and reliably recapitulated the granulation and re-epithelialization seen in human WH by secondary intention. Time to wound closure was measured using standardized digital photographs taken on days 0, 7, 14, and 21. Wound closure was calculated as a percentage of the original wound.<p><p>For each model, EPCs were harvested, quantified by flow-cytometry and their function tested. Wounds were harvested at various time points and RNA, DNA and protein analysis were conducted. Finally immunohistochemistry to assess epidermal thickness, vascularity and WH were also realized.<p><p>In a second step, after characterization of the models, local (using targeted siRNA gel) and systemic therapies (using AMD3100, a PC mobilizer) were applied on the wounds and compared to controls. WH was monitored. We conducted the previously mentioned analysis (RT-PCR, ELISA and DNA analysis) on the harvested samples.<p><p>All values are expressed as a mean ± standard error of mean (SEM). The number of mice per treatment group was determined using G*Power (G*Power©, Melbourne, Australia) to provide a power greater than 0.80. Student T test was realized to compare two groups among each other.<p><p>C. Results<p><p>C.1. Human EPCs Have Impaired Function<p><p>There was no difference in the number of baseline circulating human EPCs in non-diabetic OB and non-OB<p>subjects, but EPCs from OB subjects had impaired adhesion (p<0.05), migration (p<0.01), and proliferation (p<0.001).<p><p>C.2. Obesity and Wound Healing<p><p>TallyHo/JgnJ OB mice demonstrated significantly impaired healing when compared to SWR/J control mice. They healed at an average of 28 ± 2 days (p<0.05). Post-wounding circulating EPCs were quantified and wounds were analyzed. Circulating EPCs recruitment is impaired in wounded TallyHo/JngJ mice and their wounds shown significantly decreased new blood vessel formation through decreased HIF-1α/SDF-1α signaling (p<0.05). Their wounds are characterized by increased apoptosis, increased DNA damage and impaired pro-/anti-oxydant balance. Immunonistochemistry and histology showed decreased vascular vessels in TallyHo/JngJ wounds and thinner epidermal thickness.<p><p>In the local treatment phase, local p53 silencing consistently improved WH to a nearly normal healing time (wounds healed in 18 ± 2 days, p<0.05). sip53 treatment showed a significant decrease in pro-apoptotic markers (p53, Bax, PUMA p<0.05) and a significant increase in angiogenic markers (VEGF, SDF-1α, HIF-1α) with increased blood vessel formation and decreased DNA damage.<p><p>C.3. Age and Wound Healing<p><p>In these experiments, we show that not only is Zmpste24-/- WH impaired when compared to C57BL/6J mice (Zmpste24-/- mice healed at average 40 days ± 2 days p<0.05) at all time points but that they also showed decreased vascularity and proliferation in the wound bed (p<0.05).<p><p>Histological analysis was performed utilizing hematoxylin and eosin staining to assess epidermal thickness, CD31 immunofluorescence to assess vascular density, p53 and caspase 3 to assess apoptosis, 8’OHdG staining to assess DNA damage and PCNA to assess proliferation. Epidermal thickness was significantly decreased in Zmpste24-/- animals compared to WT as well as vascular density, and proliferation in Zmpste24-/- wound tissue (p<0.05). <p><p>Circulating vasculogenic EPCs recruitment was impaired in Zmpste24-/- mice and their wounds showed significantly decreased new blood vessel formation through decreased HIF-1α/SDF-1α signaling (p<0.05). Zmpste24-/- wounds are characterized by increased apoptosis and an abnormal rise in ROS.<p>In the treatment phase, local p53 silencing consistently improved healing by more then a two fold (18 ± 2 days). VEGF production was significantly increased and pro-apoptotic factors were significantly downregulated in siRNA-treated Zmpste24-/- mice (p<0.05). DNA damage due to ROS production was also shown to be significantly decreased following treatment. Our results suggest a vasculogenic dysfunction in wound closure and showed that the specific knock down of p53 significantly improves WH.<p><p>Because EPCs showed impaired function, lower peripheric blood counts and impaired SDF-1α/HIF-1α signaling, we hypothesized that improving their mobilization by using a progenitor cell mobilizer, AMD3100, known to mobilize SCs from the BM, in a systemic treatment phase will improve WH. Peripheral blood counts were significantly increased and time to wound closure significantly decreased (20 days ± 2, p<0.05). Vasculogenic markers and anti- apoptotic molecules were upregulated compare to non-treated animals.<p><p>D. Conclusions<p><p>Obesity impaired wound closure is a complex problem with many contributory factors. Our results suggest that obesity impairs the BM-derived EPCs response to peripheral injury and this, in turn, impairs wound closure. This impairment is associated with decreased new blood vessel formation and increased DNA damage leading to an increase in the p53 pathway. We also demonstrate that targeted siRNA therapy can partially rescue impaired WH due to obesity. Based on these results we support the encouraging argument that, WH and closure has the potential be improved through specific local and systemic therapies in vivo in our rodent model and that further studies are needed to support this in a clinical environment.<p><p>Impaired WH due to ageing is a complex phenomenon that is partially understood. We demonstrate that the Zmpste24-/- transgenic knockout mouse provides a model for age-related WH investigation. Zmpste24-/- animals heals their wounds with significant delays, showed impaired EPCs mobilization following wounding through an impaired HIF-1α/SDF-1α pathway and increased apoptosis. Furthermore, WH can be improved through specific local siRNA therapy and systemic stem cell mobilization therapies.<p><p>Our results suggest strong similar patterns between obesity and ageing in the way they mediate WH impairments trough (premature) ageing. Our encouraging endeavor to bring WH back to baseline in these diseased models underlines the possibility to reverse the microenvironment alterations and improves EPCs contribution to the WH process. Because EPCs are involved in virtually every tissue repair process happening in the human body, we hope that this work will lead the way for new research in various fields in medicine to improve wound care and quality of life of patients. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished