<p>Complications from cardiovascular disease, such as a heart attack or a stroke, represent the leading cause of death in the United States and many developed and developing countries. Atherosclerosis is the primary pathology underlying cardiovascular disease. It is caused by an increase in endothelial cell (EC) permeability, leading to the buildup of cholesterol and lipids which form the atherosclerotic plaque. Accelerated aging in regions of atherosclerosis contribute to the development and progression of the disease. The global hypothesis that motivated this research was that activation of deacetylase antioxidant regulator, Sirtuin1 (SIRT1), improved function in senescent endothelial cells ECs by increasing the integrity of cell-cell junctions. In turn, this led to elevated EC permeability, a decline in the response to shear stress, and elevated traction forces.</p><p>Aging of hCB-ECs significantly increased permeability due to changes in tight junction protein localization and phosphorylation. Activation of SIRT1 significantly reduced permeability in aged hCB-ECs and restored junction integrity. ECs under flow also exhibited changes in cell junctions with increasing age. Aged hCB-ECs were less responsive to shear stress, exhibiting lower levels of atheroprotective genes, KLF2 and eNOS. Activation of SIRT1 in aged hCB-ECs restored the response to shear stress by altering localization and phosphorylation of adherens junction protein, VE-cadherin. The endothelial glycocalyx is a layer of proteoglycans and glycoproteins on the surface of ECs that is important in maintaining EC barrier function. In aging ECs, the glycocalyx was thinner and less dense. However, activation of SIRT1 restored the structure of the glycocalyx, suggesting that the positive effect of SIRT1 on elevated permeability in aged hCB-ECs may also be due to restoration of the glycocalyx. Aged hCB-ECs also exhibited elevated traction forces for measurements done with single cells, cell clusters (2 to 3 cells), and cell monolayers (20 to 30 cells). The elevated traction forces correlated with altered actin localization and increased actin filament thickness. Activation of SIRT1 reduced traction forces and decreased actin stress fiber thickness in aged hCB-ECs, suggesting that the effects of SIRT1 on cell-cell junctions alters binding of junction proteins to the actin cytoskeleton and generation of cell traction forces. Together, these results implicate an important role for SIRT1 in regulating permeability and mechanotransduction in aging endothelium.</p> / Dissertation

Introduction: Mesenchymal stem cells (MSC) offer a novel cell therapy within tissue engineering and regenerative medicine (TERM)-based strategies, and the prospect of MSC therapies are widening since the discovery of MSCs within multiple locations of the body including bone marrow (BM-MSCs) and adipose tissue, (AD-MSCs). It is highly recognised that an organisms reparative and regenerative potential declines with advancing age, therefore aged patients are one of the primary target populations for TERM applications. Although information is available regarding the effects of patient age on the quality of human BM-MSCs, little and conflicting information currently exists for AD-MSCs. In addition, few studies have compared the quality of freshly isolated and expanded donor-matched BM and AD-MSCs to elucidate the more appropriate cell source. This study investigated the effect of donor age and in vitro ageing on functional behaviour (i.e. senescence state, population kinetics and differentiation potential) of donor-matched BM and AD-MSCs. Methods: The influence of donor age and in vitro ageing on mature (28-55 years) and elderly (75-86 years) donor-matched BM and AD-MSCs was assessed upon isolation (early life-span) and during extended (mid and late lifespan) timepoints through culture. During culture MSCs were characterised for cumulative population doublings (CPDs) and the expression of senescence associated marker genes, p16INK4A, p21 and p53, and transcription factor NANOG. At each lifespan telomere length was assessed along with differentiation efficiency along the osteogenic, adipogenic and chondrogenic lineages through lineage specific marker genes and histological staining. Results: Elderly BM and AD-MSCs displayed similar characteristics in terms of initial CPD number, p21, p53 and NANOG expression, telomere length and differentiation along osteogenic and adipogenic lineages. With increasing donor age there was a significant decline in p16INK4A expression within BM-MSCs, whilst expression of all chondrogenic markers significantly decreased within AD-MSCs. BM and AD-MSCs were comparable for the majority of outcome measures with the exception of chondrogenic differentiation which was superior with BM-MSCs in terms of COL2A1 expression and histological staining for proteoglycans. Donor age had a negative effect on BM-MSCs with long-term culture leading to a significantly longer PD time and decreased telomere lengths. Similar population kinetics was displayed between BM and AD-MSCs during long-term culture. Increasing culture time had effects on differentiation potential for both MSC sources with complete loss of osteogenic capacity and decreased adipogenic capacity; however chondrogenic capacity was only decreased within AD-MSCs. Differentiation potential after long-term culture between BM and AD-MSCs showed similar osteogenic and adipogenic ability yet superior chondrogenic ability was apparent within mature BM-MSCs compared to AD-MSCs, in terms of ECM deposition. Conclusions: In conclusion the source of MSCs for TERM will need to be considered depending upon the type of tissue regeneration required. The clinical outcome would be greater using MSCs during early stages of culture, as culture expansion has detrimental effects on functional properties of both BM and AD-MSCs.

O telômero é uma sequência repetitiva da dupla cadeia do DNA que protege as pontas dos cromossomos. Seu comprimento é mantido pela telomerase, cuja expressão ocorre em células de câncer, mas não em células somáticas. A célula apresenta um número definido de divisões antes do telômero sofrer erosão. A quebra do DNA ativa a p53, supressor tumoral que induz senescência e respostas de pontos de checagem. O desenvolvimento de inibidores de telomerase tem importância clínica para o câncer. Estudamos os efeitos dos inibidores de telomerase. Duas linhagens celulares LC-HK2 (NSCLC) e hTERT RPE-1 foram tratadas com os inibidores TMPyP4 (5µM) e Thymoquinone (10 e 40 µM) durante 72 e 120 h. TMPyP4 aumentou a porcentagem de células com dano na membrana, induziu mudança na morfologia da célula e diminuiu a expressão do mRNA da vimentina e vinculina. Thymoquinone aumentou a frequência de células senescentes, células com dano na membrana e induziu morte celular. Ambos os inibidores diminuíram a atividade da telomerase, afetando a proliferação e induzindo morte celular. / The telomere is a repetitive double-strand sequence of DNA that protects the chromosomes ends. Its length is maintained by telomerase, whose expression occurs in cancer cells, but not in somatic cells. The cell has a defined number of divisions before the telomere undergoes erosion. DNA break activates p53, tumor suppressor and induces senescence and checkpoint responses. The development of telomerase inhibitors is of clinical importance for cancer. We studied the effects of telomerase inhibitors. Two cell lines LC-HK2 (NSCLC) and hTERT RPE-1 were treated with inhibitors TMPyP4 (5 M) and Thymoquinone (10 and 40 M) for 72 and 120 h. TMPyP4 increased the percentage of cells with membrane damage, induced change in cell morphology, and decreased mRNA expression of vimentin and vinculin. Thymoquinone increased the frequency of senescent cells, cells with membrane damage and induced cell death. Both inhibitors decreased telomerase activity, affecting proliferation and inducing cell death.

Oxidative stress caused by excess reactive oxygen species (ROS) accelerates telomere erosion and mitochondrial injury, leading to impaired cellular functions and cell death. Whether oxidative stress-mediated telomere erosion induces mitochondrial injury, or vice versa, in human T cells—the major effectors of host adaptive immunity against infection and malignancy—is poorly understood due to the pleiotropic effects of ROS. Here we employed a novel chemoptogenetic tool that selectively produces a single oxygen (1O2) only at telomeres or mitochondria in Jurkat T cells. We found that targeted 1O2 production at telomeres triggered not only telomeric DNA damage but also mitochondrial dysfunction, resulting in T cell apoptotic death. Conversely, targeted 1O2 formation at mitochondria induced not only mitochondrial injury but also telomeric DNA damage, leading to cellular crisis and apoptosis. Targeted oxidative stress at either telomeres or mitochondria increased ROS production, whereas blocking ROS formation during oxidative stress reversed the telomeric injury, mitochondrial dysfunction, and cellular apoptosis. Notably, the X-ray repair cross-complementing protein 1 (XRCC1) in the base excision repair (BER) pathway and multiple mitochondrial proteins in other cellular pathways were dysregulated by the targeted oxidative stress. By confining singlet 1O2 formation to a single organelle, this study suggests that oxidative stress induces dual injury in T cells via crosstalk between telomeres and mitochondria. Further identification of these oxidation pathways may offer a novel approach to preserve mitochondrial functions, protect telomere integrity, and maintain T cell survival, which can be exploited to combat various immune aging-associated diseases.

T cell dysfunction has a crucial role in establishing and maintaining viral persistence. We have previously shown a decline in miR-181a, which regulates CD4+ T cell responses via DUSP6 overexpression, in individuals with hepatitis C virus (HCV) infection. Here, we describe accelerated T cell senescence in HCV-infected individuals compared with age-and sex-matched healthy subjects. Mechanistic studies revealed that up-regulation of transcription factor ∆Np63 led to the decline of miR-181a expression, resulting in an overexpression of the antiaging protein Sirt1, in CD4+ T cells from HCV-infected individuals. Either reconstituting miR-181a or silencing ∆Np63 or Sirt1 expression in CD4+ T cells led to accelerated T cell senescence, as evidenced by an increased senescence-associated b-galactosidase (SA-β-gal) expression, shortened telomere length, and decreased EdU incorporation; this suggests that HCV-induced T cell senescence is counterregulated by the ∆Np63-miR-181a-Sirt1 pathway. An increase of IL-2 production was observed in these senescent CD4+ T cells and was driven by a markedly reduced frequency of Foxp3+ regulatory T (Treg) cells and increased number of Foxp3- effector T (Teff) cells upon manipulating the ∆Np63-miR-181a-Sirt1 pathway. In conclusion, these findings provide novel mechanistic insights into how HCV uses cellular senescent pathways to regulate T cell functions, revealing new targets for rejuvenating impaired T cell responses during chronic viral infection.

T cells in chronic viral infections are featured by premature aging with accelerated telomere erosion, but the mechanisms underlying telomere attrition remain unclear. Here, we employed human CD4 T cells treated with KML001 (a telomere-targeting drug) as a model to investigate the role of telomere integrity in remodeling T cell senescence. We demonstrated that KML001 could inhibit cell proliferation, cytokine production, and promote apoptosis via disrupting telomere integrity and DNA repair machineries. Specifically, KML001-treated T cells increased dysfunctional telomere-induced foci (TIF), DNA damage marker γH2AX, and topoisomerase cleavage complex (TOPcc) accumulation, leading to telomere attrition. Mechanistically, KML001 compromised telomere integrity by inhibiting telomeric repeat binding factor 2 (TRF2), telomerase, topoisomerase I and II alpha (Top1/2a), and ataxia telangiectasia mutated (ATM) kinase activities. Importantly, these KML001-induced telomeric DNA damage and T cell senescent phenotype and machineries recapitulated our findings in patients with clinical HCV or HIV infection in that their T cells were also senescent with short telomeres and thus more vulnerable to KML001-induced apoptosis. These results shed new insights on the T cell aging network that is critical and essential in protecting chromosomal telomeres from unwanted DNA damage and securing T cell survival during cell crisis upon genomic insult.

Background: T cells play a key role in controlling viral infections; however, the underlying mechanisms regulating their functions during human viral infections remain incompletely understood. Here, we used CD4 T cells derived from individuals with chronic viral infections or healthy T cells treated with camptothecin (CPT) - a topoisomerase I (Top 1) inhibitor - as a model to investigate the role of DNA topology in reprogramming telomeric DNA damage responses (DDR) and remodeling T cell functions. Results: We demonstrated that Top 1 protein expression and enzyme activity were significantly inhibited, while the Top 1 cleavage complex (TOP1cc) was trapped in genomic DNA, in T cells derived from individuals with chronic viral (HCV, HBV, or HIV) infections. Top 1 inhibition by CPT treatment of healthy CD4 T cells caused topological DNA damage, telomere attrition, and T cell apoptosis or dysfunction via inducing Top1cc accumulation, PARP1 cleavage, and failure in DNA repair, thus recapitulating T cell dysregulation in the setting of chronic viral infections. Moreover, T cells from virally infected subjects with inhibited Top 1 activity were more vulnerable to CPT-induced topological DNA damage and cell apoptosis, indicating an important role for Top 1 in securing DNA integrity and cell survival. Conclusion: These findings provide novel insights into the molecular mechanisms for immunomodulation by chronic viral infections via disrupting DNA topology to induce telomeric DNA damage, T cell senescence, apoptosis and dysfunction. As such, restoring the impaired DNA topologic machinery may offer a new strategy for maintaining T cell function against human viral diseases.

The aim of this thesis was to investigate morphogenesis in the fruit fly Drosophila melanogaster using three in vitro tissue culture systems. Primary embryonic cultures derived from Drosophila melanogaster were used to study the effect of the moulting hormone ecdysone on cells in culture. The hypothesis was that the effect of ecdysone on these primary embryonic cells would parallel events which occur during metamorphosis in vivo and therefore the primary embryonic cultures could be used as an ‘in vitro’ model system. Transgenic fly lines expressing GFP were used to visualise and identify specific cell types and it was shown that cells in primary embryonic cultures respond to ecdysone morphologically. However due to the variability of cultures it was concluded that this culture system was not suitable for use as a model system. As defined cell types were observed the development of a protocol suitable for use with the primary embryonic culture system using dsRNA in order to demonstrate RNA interference was undertaken. Although this was unsuccessful, as cells in the primary embryonic cultures appeared to be resistant to dsRNA, some technical avenues remain to be explored. The Drosophila melanogaster cell line, Clone 8+, was used to investigate cell adhesion in tissue culture. Statistical analyses were carried out and it was established that derivatives of the parent cell line, Clone 8+, showed differential adhesion and proliferation characteristics. Analysis of microarray data was carried out in order to identify genes which may be responsible for the loss of cell adhesion in Clone 8+ cell lines and the potential roles of these genes in adhesion were discussed. A gene of interest, glutactin, was identified which may be responsible for loss of cell adhesion. Antibody staining was used to establish the expression of the protein glutactin in the Clone 8+ cell lines. The expression of glutactin suggested that the Clone 8+ cell line had maintained properties of the wing disc epithelial cell-type and disruption of cell polarity was considered as a possible mechanism. It was shown that f-actin colocalised with glutactin and the role of the cytoskeleton in glutactin secretion was discussed. It was concluded that glutactin was not responsible for loss of cell adhesion in the Clone 8+ cell lines. Further analysis of the microarray data revealed potential genes that could be responsible for the loss of cell polarity in the Clone 8+ cell lines and the possibility of cellular senescence was considered. It was hypothesised that the properties of adhesion and proliferation related to their ‘in vitro’ age. In the final investigation the movement of epithelial cells in Drosophila melanogaster third instar larval imaginal discs during morphogenesis was investigated. Firstly a lumen was identified in fixed imaginal disc tissue in association with cells expressing f-actin. This result was discussed in relation to the process of dorsal closure and wound healing. Further investigations involved live imaging of the dynamic process of evagination in the imaginal wing disc using transgenic flies expressing moesin-GFP. It was concluded that the lumen was not associated with the process of wound healing and it was concluded that the lumen appeared to be the mechanism directing peripodial epithelium contraction during morphogenesis of the imaginal wing disc. Dorsal closure and the process of invagination in relation to morphogenesis of the imaginal wing disc were discussed.

Le cancer de la prostate est la forme de cancer la plus fréquente et la troisième cause de décès par cancer chez l’homme dans les sociétés occidentales. Alors que la plupart des cancers de la prostate localisés sont éradiqués chirurgicalement, la plupart des tumeurs métastatiques répondant initialement aux thérapies par privation d’androgènes deviennent résistantes au traitement, causant généralement le décès du patient. Les gènes suppresseurs de tumeur PTEN et p53 étant fréquemment mutés dans les cancers de la prostate métastatiques et résistants à la castration, le laboratoire d’accueil a généré des modèles murins dans lesquels Pten et/ou Trp53 sont sélectivement invalidés à l’âge adulte dans les cellules épithéliales prostatiques dans le but de déterminer les évènements clés conduisant à la progression du cancer de la prostate. Notre étude révèle que l’invalidation de PTEN stimule la prolifération des cellules épithéliales prostatiques et conduit à des néoplasmes prostatiques intraépithéliaux en quelques mois. Cette hyper-prolifération induit un stress réplicatif et une réponse aux dommages de l’ADN qui va conduire à un arrêt progressif de la croissance cellulaire et une entrée en sénescence. Les cellules sénescentes sécrètent de nombreuses cytokines et de chimiokines, et peuvent accumuler des mutations contribuant ainsi à la progression de la tumeur. Il est notable qu’en l’absence de Trp53, les épithéliums prostatiques dépourvus de Pten développent des néoplasmes prostatiques intraépithéliaux entrant en sénescence. Cependant, la formation d’adénocarcinomes est accélérée et des tumeurs sarcomatoïdes pouvant générer à long terme des métastases apparaissent. En l’absence de Pten, certaines cellules épithéliales prostatiques perdent leur identité moléculaire en exprimant des marqueurs caractéristiques de cellules souches et différenciation neuroendocrinienne. Nous avons également mis en évidence des cellules épithéliales prostatiques déficientes en PTEN et p53 résistantes à la castration exprimant à la fois des marqueurs de cellules basales et luminales. En conclusion, nos travaux ont permis une avancée dans la compréhension des mécanismes conduisant à des formes incurables de cancer de la prostate. Les traitements actuels ayant des effets secondaires importants et pouvant générer des résistances, le développement de nouvelles stratégies thérapeutiques permettant l’élimination des cellules sénescentes mais aussi des cellules épithéliales prostatiques exprimant des marqueurs de cellules basales et luminales dans les lésions précancéreuses représente des perspectives intéressantes pour traiter le cancer de la prostate. / Prostate cancer (PCa) is a leading cause of male cancer death worldwide. While most locally PCa are curable, metastatic tumors initially respond to androgen deprivation therapy but ultimately relapse to castration-resistant prostate cancer (CRPC), which is a lethal disease. Since the tumor suppressor genes PTEN and p53 are frequently mutated in metastatic and CRPC, the host laboratory generated mouse models in which Pten and/or Trp53 are selectively ablated in adult prostatic epithelial cells (PECs) in order to unravel the key events leading to prostate cancer progression. Our study reveals that Pten ablation stimulates PECs proliferation forming prostatic intraepithelial neoplasia (PIN) within a few months. This hyper-proliferation induces replicative stress and a DNA damage response (DDR), which in turn leads to a progressive growth arrest with characteristics of cell senescence. As senescent cells secrete a large number of cytokines and chemokines, and can accumulate other mutations, they might contribute to tumor progression. Importantly, in the absence of Trp53, most Pten-null PECs develop PINs that enter senescence. However partial loss of PECs identity is detected as we show enhanced stemness and focal neuroendocrine differentiation of luminal Pten-null PECs. In some cases, adenocarcinoma and sarcomatoid tumors are formed, and more than one-third of the latter develop metastases. Strikingly, we also show formation of a castrate-resistant cell entity of both Pten and Pten/Trp53-null PECs sharing luminal and basal markers. Taken together, as current treaments lead to side effects and resistance, the development of therapeutic strategies to eliminate senescent cells/and or PECs expressing luminal and basal/stem progenitor in pre- cancerous lesions represents promising option for prostate cancer treatment.