Tumor heterogeneity favors tumor tissue to survive and resist drugs, leading to the failure of chemotherapeutic agents to induce a therapeutic response. In addition, the absorption mechanisms,... Show moreTumor heterogeneity favors tumor tissue to survive and resist drugs, leading to the failure of chemotherapeutic agents to induce a therapeutic response. In addition, the absorption mechanisms, metabolism and excretion of chemotherapeutic drugs, which are commonly used for cancer patients and the lack of specific targeting of these drugs can cause adverse effects on treated patients. Thus, the general objective of this thesis is to investigate the biological activity of targeted poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) as a drug delivery system (DDS) for carvedilol (CVDL) or oxaliplatin (OXA), in vitro and in vivo, to treat colorectal cancer (CRC). DDSs were formulated to achieve this goal. In chapters 2, 3 and 4, our studies were discussed in detail on the formulations and characterizations of NPs as DDSs with ideal characteristics to increase the therapeutic range of drugs at the tumor site. As well as the biological evaluation of these DDS when its anti-inflammatory activity (Chapter 2) and its antitumor activity in vitro (Chapters 2, 3 and 4) and in vivo (Chapters 3 and 4). Taken together, all the DDSs studied in this thesis were able to improve the chemotherapeutic efficiency of the drugs studied in Chapters 2, 3 and 4. Show less
Many of the recognition molecules and mechanisms involved in immune responses have no bias towards external stimuli, but also sense and respond to pathological and physiological changes of non... Show moreMany of the recognition molecules and mechanisms involved in immune responses have no bias towards external stimuli, but also sense and respond to pathological and physiological changes of non infectious origin taking place within the body. Aiming at defining potential immuno-therapeutic strategies to treat human atherosclerosis, the focus of this work was the modulation of immune processes determinant of atherosclerosis lesion progression or cessation in mice, such as hematopoiesis, diapedesis and intravasation, leukocyte differentiation, cholesterol uptake apoptosis and cell survival. Modulation of these processes, by using bone marrow transplantation of hematopoietic stem cells with genetic deficiencies or over-expressing human or mouse engineered genes, demonstrated to alter the fate of atherosclerotic lesions at the balance between macrophage accumulation and lesion vulnerability versus resolution of inflammation and wound healing. This thesis demonstrates that processes responsible for the development and progression of atherosclerosis are dynamic and can be modulated to induce lesion stabilization and disease resolution. These results are promising for the development of novel therapeutics and challenge the current notion that atherosclerosis has a predetermined fate towards lesion vulnerability to rupture, which in humans results in thrombosis and clinical manifestations such myocardial infarction or stroke and sudden death. Show less
The aim of the work included in this PhD thesis was to explore the diverse application possibility of using NIR fluorescent probes with specific properties to visualize and characterize cancer and... Show moreThe aim of the work included in this PhD thesis was to explore the diverse application possibility of using NIR fluorescent probes with specific properties to visualize and characterize cancer and cell death. In this thesis, we mainly focus on optical imaging and its application, both at microscopic and macroscopic level. Because we believe optical imaging in particular represents a technology that has unique potential to exploit further our knowledge in preclinical research. First, we imaged breast tumors and their metastases using combinations of four NIR fluorescent probes that possess different optical imaging properties. Then, we studied two different NIR fluorescent probes, PSVue and a heat shock protein-90 alkylator (NIR fluorescent conjugate of GSAO), which can be used to non-invasively imaging cell death with different optical modules in a mouse model of traumatic brain injury. Next, we employed the NIR fluorescently tagged GSAO as a biomarker for monitoring breast cancer cell death after chemotherapy. Moreover, we provides a general discussion about the advantages and the challenging that the state-of-art optical imaging is facing and shares some future prospective. This thesis ends with a summary that outlined the major findings of studies described in different chapters and explored the clinical implications. Show less
With the ever-increasing life expectancies of people, the prevalence of aging-related diseases is also increasing. We studied a unique cohort of people; offspring of nonagenarian siblings with the... Show moreWith the ever-increasing life expectancies of people, the prevalence of aging-related diseases is also increasing. We studied a unique cohort of people; offspring of nonagenarian siblings with the propensity for longevity were also recruited and compared to their partners, representing the general population. The offspring (~60 year old) already show lower prevalence of cardiovascular disease and diabetes. In the thesis __Cellular stress in vitro and longevity on vivo__ it is reported how cells (dermal fibroblasts) derived from these offspring were compared with cells from their partners. It was shown that they react differenly in vitro under both non stressed and under conditions of oxidative stress. Future research is warranted to further elucidate the genes involved in these differences. Show less
Estrogen is known to play an important role in longitudinal bone growth and growth plate maturation, but the mechanism by which estrogens exert their effect is not fully understood. In this thesis... Show moreEstrogen is known to play an important role in longitudinal bone growth and growth plate maturation, but the mechanism by which estrogens exert their effect is not fully understood. In this thesis this role is further explored. Chapter 1 contains a general introduction to longitudinal bone growth and the regulation of the growth plate in respect to relevant topics further studied in this thesis. Estrogen can act through a genomic or a nongenomic pathway. Both pathways are explored in rats at the onset of maturation in chapter 2. Estrogen stimulates VEGF expression in uterus and bone, which is an important growth factor for chondrocyte differentiation and chondrocytes survival in the growth plate. In chapter 3 the effect of estrogen on VEGF expression in the growth plate was studied in the rat and human growth plate. Another effect of estrogen is that it accelerates growth plate senescence. Senescence is one of the postulated intrinsic mechanisms by which the growth plate matures and finally fuses. In chapter 4 we investigated senescence in relation to proliferation, by investigating a cell cycle inhibitor p27Kip1. In animal models, catch-up growth is suggested to be caused by delayed growth plate senescence. In chapter 5 this hypothesis was further tested in humans. With puberty estrogen levels increase, the growth plate matures and at the end growth ceases with epiphyseal fusion through mechanisms not yet completely understood. In order to further explore growth plate maturation we subjected two growth plate tissues of the same patient, but with one year and one pubertal Tanner stage in between, to microarray analyses. Gene expression patterns and transcription factor binding sides in relation to pubertal maturation were studied in a longitudinal study within this single patient in chapter 6. In addition, we collected extra prepubertal and pubertal growth plate tissues and studied these samples with microarray techniques as well in chapter 7. In chapter 8 the process of epiphyseal fusion and apoptosis was studied in human growth plates. Animal models are frequently used but not fully representative for the human growth plate. Therefore we investigated a promising human in vitro model with multipotent mesenchymal stem cells (MSCs) that can differentiate into chondrocytes. MSCs can be isolated from various tissues. In chapter 9 we investigated the chondrogenic potential of MSCs from different origins and in chapter 10 we compared this model with the epiphyseal growth plate by analyzing gene expression patterns and pathways with micro-array analyses. Chapter 11 contains general conclusions and a discussion regarding the results. Show less
In this thesis the role of several apoptosis regulating proteins in the development of atherosclerosis and atherosclerotic plaque stability is investigated. Apoptosis of different cell types in... Show moreIn this thesis the role of several apoptosis regulating proteins in the development of atherosclerosis and atherosclerotic plaque stability is investigated. Apoptosis of different cell types in atherosclerotic plaques, such as macrophages and smooth muscle cells may inhibit or promote plaque development or stability depending on the stage of atherosclerosis. As many of these apoptosis regulating proteins also display immune-modulating features, we have particularly investigated effects of modulation of apoptosis regulating proteins on plaque and systemic inflammation. We performed a number of studies in mouse models of atherosclerosis. First gene expression profiles of stable and unstable atherosclerotic plaques were compared in order to identify genes or pathways that are associated with plaque vulnerability. We further developed transgenic mice partially or wholly lacking genes involved in apoptosis and/or inflammation such as Bcl-2 family members and focal adhesion kinase, both systemically or in the leukocyte subset. The studies described in this thesis show amongst other things that Bim and Mcl-1, both members of the Bcl-2 family of apoptosis regulators, regulate specific cell death and inflammatory processes relevant to atherosclerosis. Show less
Colorectal cancer (CRC) is a complicated disease in which both genetic pre-desposition and environmental factors are important. Patients with inflammatory bowel disease (IBD) have an increased risk... Show moreColorectal cancer (CRC) is a complicated disease in which both genetic pre-desposition and environmental factors are important. Patients with inflammatory bowel disease (IBD) have an increased risk of developing CRC, and it is believed that treatment of IBD patients with 5-Aminosalicylic acid (5-ASA) reduces the CRC risk. The general purpose of the studies described in this thesis was to evaluate the effect of 5-ASA on the development of CRC, as well as determining the feasibility of introducing 5-ASA as an adjuvant therapy for CRC patients. Animal research showed that chronic 5-ASA medication has the ability to prevent colitis-associated CRC, confirming results from 5-ASA medication in IBD patients. Although 5-ASA was not able to prevent the development of sporadic CRC, 5-ASA treatment was found to hold a great promise for the treatment of CRC, by exerting CRC growth inhibiting, anti-progression and cell death inducing effects, and should be considered to be implemented in a future treatment strategy to CRC. Besides this, the determination of cell death products in the circulation of CRC patients and within the tumour, holds a great promise for selection of CRC patient treatment and patient folluw-up. Show less
The work presented in this thesis has focused on the role of Mitogen Activated Protein Kinases (MAPKs) and their major downstream targets, the AP-1 transcription factors, in particular the AP-1... Show moreThe work presented in this thesis has focused on the role of Mitogen Activated Protein Kinases (MAPKs) and their major downstream targets, the AP-1 transcription factors, in particular the AP-1 components ATF3, Fra1, c-Jun, ATF-2 and c-Fos. Chapter II provides information on the signaling pathways involved in the activation of ATF-2 and ATF3 in the response of primary human fibroblasts to ionizing radiation. In chapter III c-Jun and ATF3, the MAPK JNK and the MAPK-phosphatase MKP-1 are identified as important sensors of UV-induced-DNA damage in transcribed genes. Chapter IV shows that ATF3 acts as an antiapoptotic JNK target in T98G glioblastoma cells, whereas Fra1 seems to act as a proapoptotic effector of both JNK and ERK. In addition, it is shown that ATF3 and Fra1 have opposite effects on cisplatin-induced S phase arrest. Chapter V shows that Fra1 also can exhibit a pro-apoptotic function in UV-irradiated fibroblasts. Furthermore, this chapter reports an as yet unknown function of JNK: repression of the transactivating activity of c-Jun/Fos(- like) dimers, mediated via hyper-phosphorylation of the c-Jun transactivation domain. The data further emphasizes that c-Jun/Fos(-like) and c-Jun/ATF dimers and their respective target genes can exhibit opposite functions in DNA damage responses. Show less
One of the major problems in the treatment of patients with acute leukemia is failure of therapy due to acquired resistance, which may partially be caused by defects in the apoptotic machinery of... Show moreOne of the major problems in the treatment of patients with acute leukemia is failure of therapy due to acquired resistance, which may partially be caused by defects in the apoptotic machinery of these leukemic cells. Apoptosis is induced and regulated by a complex network of proteins connected via various signal transduction cascades, eventually leading to death of the target cell. The results described in this thesis demonstrate that apoptosis induction in leukemic cells after treatment with chemotherapy or (cellular) immunotherapy is very complex and frequently dependent on the target cell studied, and on the interaction between target and effector cell. G0 cells derived from patients with B-CLL compared to G0 cells from patients with acute leukemia responded differently to Ara-C treatment, which may be partially explained by the different mechanisms of action exerted by Ara-C. The role of the death receptor pathway in both chemotherapy-induced and CTL-induced apoptosis of leukemic cells was also unraveled in more detail. It will be worthwhile to focus future strategies in the treatment of leukemia on modulation or activation of proteins that are common in multiple apoptotic pathways, like caspase-8, since this approach will sensitize the leukemic cells simultaneously to chemotherapy and cellular immunotherapy. Show less
Several associations between 14-3-3 proteins and members of the p53 family have been revealed. However, numerous questions regarding 14-3-3 proteins, p53 family members and the relationships... Show moreSeveral associations between 14-3-3 proteins and members of the p53 family have been revealed. However, numerous questions regarding 14-3-3 proteins, p53 family members and the relationships between thetwo families remain. This thesis contributes to answer these questions. Downregulation of 14-3-3_ increased the propensity for DNA-damage induced apoptosis and increased cell-cell contacts, two characteristics that are often lost in cancer cells. Differences between 14-3-3_ and 14-3-3_ in nucleocytoplasmic shuttling have been described. The difference innucleocytoplasmic shuttling likely forms a basis for specialized biological functions of the 14-3-3 isoforms. Keratinocyte specific p53 regulated G2cell-cycle arrest and the lack of a contribution for 14-3-3_ in this cell-type has been observed, whereas 14-3-3_ induction clearly plays a distinct role in G2 arrest in other cell-types. An improved method for detecting p53 induced apoptosis by flow cytometry is portrayed by expressing a duplicated GFP protein. This double GFP protein can be used for future research in the fields of 14-3-3 and p53. A widely used method to detect and quantify p73 splice-variant expression is critically assessed. It turns out that, using this method it is not possible to accurately quantify p73splice-variants. Show less
For a tumor cell to propagate, it must survive extremely stressful conditions that would normally trigger the cell to die. Cancer cells however survive, probably due to evasion of the apoptotic... Show moreFor a tumor cell to propagate, it must survive extremely stressful conditions that would normally trigger the cell to die. Cancer cells however survive, probably due to evasion of the apoptotic cell death pathway. It follows that a detailed understanding of the regulation of the apoptotic pathways in cancer cells can improve the anti-cancer treatments. Part 1 of this thesis describes our in vitro studies regarding the regulation of apoptosis in melanoma cells, since melanoma is a form of cancer that is highly resistant to anti-cancer therapies. c-Myc enhances the apoptosis sensitivity of the cells. The protein Apaf-1 is not involved in this sensitivity. A yet unidentified serine protease plays an important role in the initiation of apoptosis upon DNA damage. Part 2 of this thesis describes our studies regarding both the regulation of apoptosis in rectal carcinoma and its prognostic value for rectal cancer patients. To evaluate the impact of (radiation-induced) tumor cell apoptosis on clinical outcome of cancer patients, the level of apoptosis have been determined in non-irradiated and irradiated rectal carcinoma samples. The level of tumor cell apoptosis is scored by immunohistochemical stainings of the carcinoma samples, and by measuring caspase-3 activity. Both studies show that high levels of apoptosis is associated with a low local recurrence risk. A genetic approach is used to identify factors that play a role in the regulation of apoptosis in rectal carcinoma in vivo. After evaluation two microarray procedures, the most convenient procedure is used to compare the gene expression profiles of tumors with high levels of apoptosis with low-apoptotic tumors. The difference in expression of several of the identified genes are confirmed on protein expression level by immunohistochemistry, and show two subsets of high-apoptotic tumors. These data suggest two different regulations of apoptosis in vivo. The prognostic value of one of the identified proteins, HLA-DR, has been studied in more detail and epithelial HLA-DR expression is significantly associated with lower recurrences and better survival for rectal cancer patients. Show less
The major targets in Systemic lupus erythematosus (SLE, a systemic autoimmune disease) are nuclear components (DNA, histones, ribonucleoproteins), which are mainly derived from dying cells ... Show moreThe major targets in Systemic lupus erythematosus (SLE, a systemic autoimmune disease) are nuclear components (DNA, histones, ribonucleoproteins), which are mainly derived from dying cells (apoptotic and necrotic cells). Defective clearance of dying cells by phagocytes may lead to the breakdown of peripheral tolerance and initiation of autoimmune SLE. I have investigated the role of the innate immune system in the processing of dying cells and its immunological consequences. I found that a subset of macrophages driven by M-CSF have intrinsic anti-inflammatory properties and are potent phagocytes that have the unique capacity to preferentially bind and ingest early apoptotic cells in a silent manner. I also identified that human peritoneal macrophages freshly isolated from patients on peritoneal dialysis resemble functionally the in vitro-generated M-CSF-driven macrophages. I further showed that the anti-inflammatory and pro-inflammatory macrophages co-exist but can re-differentiate towards opposing phenotype depending on the local cytokine environment. Next to the phagocyte system, I investigated the role of components of the innate immune system in the processing of dying cells. I found that one of the complement regulators called properdin, binds predominantly to late apoptotic and necrotic cells independently of C3b, resulting alternative pathway complement activation. Show less
The current thesis discusses the use of molecular and biological tumor markers to predict clinical outcome. By studying several key processes in the develepment of cancer as regulation of cell... Show moreThe current thesis discusses the use of molecular and biological tumor markers to predict clinical outcome. By studying several key processes in the develepment of cancer as regulation of cell motility (non-receptor protein tyrosin adesion kinases, FAK, Src and paxillin, Apoptosis (caspase-3 activity and M30 expression) and regulation of cell growth (COX-2 expression). In addition the use of selective COX-2 inhibitors for the treatment of colorectal cancer liver metastases is investigated and discussed. The main outcomes of the thesis are that combined FAK/Src immunohistochemical expression is predictive of tumor recurrence in colorectal cancer, but is not overexpressed in liver metastases. Increased tumor cell apoptosis can have a positive or a negative impact on survival and local recurrence, depending on the location of the tumor in the large bowel . In rectal cancer caspase-3 activity can be used to preoperatively select patients who will not benefit from radiation therapy. COX-2 expression in rectal cancer is only of prognostic significance in irradiated rectal cancer patients, not in non-irradiated. Liver metastases in an animal model show deminished growth in the abcence of COX-2 expression and prostaglandin production. Show less
The development of multicellular organisms involves an important balance between cell growth, cell division and cell death. In animals, programmed cell death (PCD) plays a key role by forming and... Show moreThe development of multicellular organisms involves an important balance between cell growth, cell division and cell death. In animals, programmed cell death (PCD) plays a key role by forming and deleting structures, controlling cell numbers and eliminating abnormal damaged cells. Caspases were found to be the key executioner of the cell suicide pathway. In plants, PCD plays an important role in development and in the responses to pathogens and abiotic stresses. Some common features of PCD were found to be conserved in both plants and animals (cytoplasm shrinkage, cytochrome c leakage out of mitochondria, chromatin condensation, altered nuclear morphology, DNA fragmentation in large fragments and DNA laddering). This indicates that a similar apoptotic machinery may be present in plants. After the elucidation of the complete sequences of Arabidopsis and rice, it has become clear that no genes for the caspases are present in plants. In this thesis, three model systems are described to determine the existence of caspase-like activities in plants during PCD. The first model used is a T-DNA integration arabidopsis mutant that shows necrotic spots on its leaves. The second model used is barley, during the transition from multicellular structures to globular embryos during androgenesis. The third model used was an artificial model of rice suspension cells with heat-shock as a PCD inducer. In these models, PCD was described and caspase-like activity was measured. In addition, the development and implementation of the protocol to purify plant caspase-3 like activity is described. Show less
Head and Neck Paragangliomas (HNP) are hypervascular tumours characterised by a slow growth pattern and a strong hereditary context that originate from the neural crest derived paraganglia, which... Show moreHead and Neck Paragangliomas (HNP) are hypervascular tumours characterised by a slow growth pattern and a strong hereditary context that originate from the neural crest derived paraganglia, which are associated with the autonomous nervous system and are situated at several locations in the head and neck region. Inactivating mutations in subunits of complex II (SDH) of the mitochondrial respiratory chain are responsible for hereditary tumours and have lead to a novel concept of mitochondrial tumoursupressor-genes and further insight in the intricate association of cellular oxygen sensing mechanisms and (pseudo)-hypoxia as environmental risk factors. However, further characterisation of the tumour biology is warranted for better understanding of the natural behaviour of HNP and possible identification clinicopathological parameters that could aid the clinician in his treatment decisions. In this thesis several studies on the molcular pathology of HNP are discussed including genotype-fenotype relations, the role of bFGF in tumourgenisis, the interplay between proliferation, cell cycle activity and apoptosis, and the nature of sustentacular cells in these apparent biphasic tumours. Additionally, in a clincal study the prevalence of synchronic or metachronic pheochromocytomas in patients with SDHD-linked HNP was determined. Show less
Cholesteatoma is a benign, gradually expanding destructive epithelial lesion of the temporal bone, often accompanied with inflammation. The complications can be severe, e.g., destruction of the... Show moreCholesteatoma is a benign, gradually expanding destructive epithelial lesion of the temporal bone, often accompanied with inflammation. The complications can be severe, e.g., destruction of the ossicular chain and otic capsule with consecutive hearing loss. Several hypotheses for the pathogenesis of human cholesteatoma have been proposed but are still controversial. In this thesis, protein signaling pathways were investigated which are involved in different aspects of cholesteatoma pathogenesis, such as hyperproliferation, aberrant differentiation, and extra-cellular matrix deposition. In cholesteatoma epithelium increased expressions of those proteins were found which are involved in proliferation (Ki-67), cell cycle arrest (p53, p21), early terminal differentiation (involucrin) and survival (pAKT). The proteins of which the expressions were decreased were those concerning late terminal differentiation (filaggrin) and apoptosis (active caspase 3). Moreover, increased activation of MAPK- and its association with TGF_ cellular signaling cascades were demonstrated. In cholesteatoma stroma, increased extracellular matrix deposition, visualized by accumulation of EDA-Fibronectin, was present. These results were placed in the context of the interconnected signaling processes of wound healing. Concluding: cholesteatoma behaves as a chronic wound in which a persistent inflammation appears to be a contributing factor to its chronicity. Research into pharmacological interventions aimed at control of inflammation is recommended. Show less
The work described in this thesis was aimed at identifying the role of cell cycle and apoptosis genes in atherosclerosis. Atherosclerosis is the primary cause of cardiovascular disease, a disorder... Show moreThe work described in this thesis was aimed at identifying the role of cell cycle and apoptosis genes in atherosclerosis. Atherosclerosis is the primary cause of cardiovascular disease, a disorder occurring in the large and medium-sized arteries of the body. Although in the beginning 90s promising lipid lowering therapies predicted a strong reduction in cardiovascular deaths, in westernized societies it is still the underlying cause of about 40% of all deaths, indicating that treatment of atherosclerosis goes beyond lipid lowering solely. In addition to lipids, continuous cell growth (cell cycle) and cell death (i.e. apoptosis and necrosis) processes play a central role in the development of atherosclerosis. To investigate the role of several cell cycle and apoptosis genes (i.e. p53, Rb and Mdm2) in atherosclerosis we generated and characterized several mouse models based on site-specific recombinase (SSR) technology. The studies described in this thesis show that next to therapies aiming at lifestyle interventions, lipid therapies and regulation of inflammation, targeting cell cycle and apoptosis genes on lesional or cellular level might prove the most effective way to reduce the burden of atherosclerosis. Show less
The heart is built for life-long uninterrupted function, supporting blood flow through the organism. During life, the organism as well as its organs will become challenged by exogenous and... Show moreThe heart is built for life-long uninterrupted function, supporting blood flow through the organism. During life, the organism as well as its organs will become challenged by exogenous and endogenous stresses that should be coped with. To be able to adapt to stresses such as altered loading conditions, changes in myocardial perfusion and exposure to toxic agents, the heart possesses ample capabilities. This thesis deals with the question how cardiac cells react to different stresses in an attempt to adapt to the new circumstances. The effect of mechanical stress as well as integrin stimulation on cardiac cells was examined and described. Besides we investigated the reaction of cardiac cells to DNA damage caused by either ionizing radiation or UV-irradiation. The studies reported in this thesis confirm that cardiac cells, both myocytes and fibroblasts have the capacity to respond adequately to exogenous stresses. The responses that have been studied in detail demonstrate that these cells possess systems that sense the stress, transmit its message to the cell interior, and alter gene expression, leading to appropriate reactions. Show less
