A central tenet of stem cell biology has been that proliferating tissues are maintained through a cellular hierarchy comprising of self-renewing stem cells at the apex, multiple lineage-restricted short-lived progenitor cells, and post-mitotic differentiated cells. The wide range of colony sizes in cultured human keratinocytes has been taken to support this hypothesis. Contrary to this model, researchers using genetic lineage tracing in mouse epidermis have inferred a single progenitor population for homeostasis, and a quiescent stem cell population activated upon wounding or genetic mutation. To study the proliferative behaviour of human keratinocytes, I used live imaging in vitro at single cell resolution. This shows two modes of proliferation: Type 1 cell division is stochastic with equal odds of generating dividing or non-dividing progeny, while Type 2 cell division predominantly produces two dividing daughters. These two modes are sufficient to explain the entire range of colony sizes seen after 7-12 days of culture and does not require a spectrum of proliferative ability. This insight provides a simple way to study the effects of external factors on cell fate. To exemplify this, I observed the effects of epidermal growth factor (EGF) and the Wnt agonist R-spondin on proliferation. Here I find proliferation in type 2 colonies changes by changing the proportion of cells dividing. This has implications for the limited success of EGF therapies in clinical trials following burns. To examine clonal contributions to wound repair, I used the mouse oesophageal epithelium which is exclusively composed of, and maintained by, a single progenitor population. I developed a micro-endoscopic wounding technique that produced localised superficial wounds. Here, I found that these wounds healed by uniform contribution from surrounding keratinocytes, demonstrating that reserve stem cells are not obligatory for wound repair. In summary, my work shows that human keratinocytes in vitro have two, and only two, modes of proliferation: a stochastic mode that is insensitive to external EGF signalling, and a EGF-sensitive exponential mode. Additionally, proliferation during wound repair can occur with stochastically dividing progenitors, and does not obligate stem cell recruitment in vivo.

<p>During osteoclast differentiation and bone resorption the redox status in the cell display a decrease in reduction and a shift to an oxidized state. Structure, metabolism and function are some of the extensive changes that cells undergo during differentiation which alters both the extra- and intracellular redox environment. Osteoclasts express enzymes such as TRAP and NADPH oxidase which generates reactive oxygen species (ROS). ROS are molecules formed by oxygen reduction which gives these radicals at least one unpaired electron and makes them very reactive and chemically unstable. These are factors which stimulates differentiation of osteoclasts and bone resorption. RAW 264.7 cells will differentiate to osteoclasts when stimulated with RANKL and to activated macrophages when stimulated with LPS.</p><p>The aim of this project was to analyze if the redox environment is affected during differentiation of RAW 264.7 cells to osteoclasts and macrophages. The reason for this was that we aimed to se if RAW 264.7 cells could be used as an in vitro system to study the effects of redox changes in osteoclasts and macrophages and their activation.</p><p>Results from Western blot showed that protein expression of the Cysteine/Glutamate transporter xCT was up regulated with LPS and downregulated with RANKL. Results from the GSH/Cys assay show that the treatments with redox modulators did not affect the levels of GSH and Cys to a measurable extent. However the levels increased for both intracellular and extracellular GSH and Cys forms at day 4 in the control and stimulated cells. Addition of the disulfide reductant DTT affected differentiation to osteoclasts, leading to smaller osteoclasts probably due to interference with fusion of mononuclear pre-osteoclasts. Thus, down regulation of the xCT transporter could be an important mechanism to maintain a low level of free thiols shown to interfere with the differentiation to osteoclasts.</p>

In addition to malignant cells, solid tumors comprise supporting stromal tissue that consists of extra cellular matrix (ECM), connective tissue cells, inflammatory cells and blood vessels. The stromal compartment and the malignant cells together shape the tumor microenvironment that in turn determines tumor progression and efficacy of anti-tumor treatments. In this thesis, studies that investigate the roles of different kinds of interactions between tumor cells and stromal cells were undertaken. Further, growth factors that have important roles in interactions between tumor cells and stromal cells were investigated in a non-tumor environment. Tumor cells were found to modulate the response to the platelet derived growth factor  (PDGF) by microvascular pericytes, a cell type found in the vasculature of solid tumors. The importance of this growth factor in biology of tumors has earlier been shown, but here it was shown that PDGF also modulate the ECM phenotype of solid tumors. The ECM of tumors treated with an inhibitor of PDGF receptor (PDGFR) signaling induced a less fibrotic collagen scaffold, which could explain how PDGFR inhibition in earlier reports lowered tumor interstitial fluid pressure (IFP). Lowering the normally high IFP in tumors increases efficacy of chemotherapy. The integrin αVβ3 is activated downstream of PDGF-B in acute inflammations, and this integrin is important for raising IFP in loose connective tissue in such conditions. However, in tumors we found that lack of the β3 subunit lead to an increased IFP, which were attributed to a more fibrotic ECM phenotype. In addition to PDGF-B, transforming growth factor β (TGFβ) is an important growth factor in the biology of tumors. These two growth factors were separately overexpressed in mouse skin and they both induced an inflammatory response. Expressed in a tumor free context, they evoked a response that was in many ways reminiscent of what can be observed in the tumor microenvironment. This thesis contributes further understanding of how the complex tumor microenvironment affects the phenotype of solid tumors.

Tumor growth and progression not only depend on properties of the malignant cells but are strongly influenced by the tumor microenvironment. The tumor stroma consists of various cell types such as inflammatory cells, endothelial cells and fibroblasts, which can either inhibit or promote tumor growth. Consequently, therapeutic targeting of the tumor stroma is increasingly recognized as an important tool to fight cancer. Two particularly important processes that contribute to the pathology of most types of tumors are angiogenesis and inflammation. In order to target these processes specifically and efficiently, it is fundamental to identify and understand the factors and signaling pathways involved. This thesis initially describes the multiple functions of the small heat shock protein αB-crystallin in the tumor microenvironment. αB-crystallin was first identified in a screen of proteins specifically up-regulated in endothelial cells forming vessel-like structures. We found that αB-crystallin is expressed in a subset of tumor vessels and promotes angiogenesis by inhibiting endothelial apoptosis, suggesting that targeting of αB-crystallin might inhibit angiogenesis and thereby decrease tumor growth. However, we also discovered an important role of αB-crystallin in regulation of inflammatory processes. We show that αB-crystallin increases the surface levels of E-selectin, an important leukocyte-endothelial adhesion molecule. Thereby, αB-crystallin may alter leukocyte recruitment to inflamed tissues such as the tumor stroma. In addition, we found that αB-crystallin is expressed in immature myeloid cells that accumulate in the periphery and at the tumor site during tumor development. Importantly, lack of αB-crystallin resulted in increased accumulation of immature myeloid cells, which might increase tumor associated inflammation. Finally, through combining laser microdissection of vessels from human tissue and microarray analysis, we identified a gene expression signature specifically associated with vessels in high grade glioma. Blood vessels in malignant glioma are highly abnormal and contribute to the pathology of the disease. Thus, knowledge about the molecular set-up of these vessels might contribute to the development of future vascular normalizing treatments.

<p>In this thesis, the shape of the distal humerus trochlea is analysed using landmark-based morphometrics and multivariate methods, with the aim of exploring locomotor evolution in carnivorans. Elbow joint morphology is used together with body size and craniodental morphology to characterize past and present carnivorans. Evolutionary implications are studied at the ordinal, familial, and species levels, testing specific hypotheses about scaling, morphological constraints, evolutionary trajectories, and potential for social pack-hunting behaviour. The circumference of the distal humerus trochlea is found to be highly correlated with body mass, and appears to scale similarly throughout the order Carnivora. A general predictive model for carnivoran bodymass is presented (a=0.601; b= 2.552; r2=0.952, SEE=0.136, p<0001, n=92), which removes the need for the investigator to actively choose between the diverging estimates that different predictors and their equations often produce. At the elbow joint, manual manipulation and locomotion appear to be conflicting functions, thus suggesting mutually exclusive lifestyles involving either forelimb grappling or pursuit. At large body sizes, carnivorans are distributed over a strongly dichotomised pattern (grappling or locomotion), a pattern coinciding with the postulated threshold in predator-prey size ratio at 21.5-25 kg. This pattern is compared to that of two carnivoran faunas from the Tertiary. In the Oligocene (33.7-23.8 Myr BP), the overall pattern is remarkably similar to that observed for extant Carnivora. In the Miocene (23.8-11.2 Myr BP) carnivores show a similarly dichotomised pattern as the Oligocene and Recent, although the whole pattern is shifted towards larger body sizes. This difference is suggested to be a reflection of the extraordinary species richness of browsing ungulates in the early Miocene of North America. Such an increase in prey spectrum would create a unique situation, in which large carnivores need not commit to a cursorial habitus in order to fill their nutritional requirements. Finally, the elbow joints and craniodental morphology (14 measurements) of fossil canids were examined with the aim of assessing the potential for pack-hunting in fossil canids. It is clear that small and large members of the Recent Caninae share similar craniodental morphologies. However, this pattern is not present in Borophaginae and Hesperocyoninae. In the latter, large representatives are characterized by being short-faced, with reduced anterior premolars and enlarged posterior premolars, thus approaching a “pantherine-like” craniodental configuration. These traits are interpreted as an adaptation for killing prey with canine bites. It is similarly determined that, unlike recent Caninae, all analyzed species of borophagines and hesperocyonines have retained the ability to supinate their forearms. It is therefore likely that manual manipulation was part of their hunting behaviour, thus removing an essential part of the argument for social pack-hunting in these forms, as the benefits of such a strategy become less obvious.</p>

<p>Ultrastructural studies of airway epithelium in airway disease are important for diagnosis and understanding the underlying pathology which helps clinicians to improve the patients' treatment.</p><p>Airway biopsies from a 5-month old boy with respiratory problems and gastro-oesophageal reflux were studied by transmission electron microscopy (TEM). The tracheal columnar cells showed accumulation of lamellar bodies, indicative of lysosomal storage disease. The patient was diagnosed with Gaucher disease type 2.</p><p>Shedding of airway epithelial cells is commonly found in asthma. The attachment of these cells to the basal lamina was investigated by TEM of biopsies from patients with asthma and healthy controls. The contact area between columnar cells and basal lamina in asthmatics was significantly less than in controls. Attachment of columnar cells to the basal lamina occurs mainly indirectly, via desmosomal attachment to basal cells. </p><p>Primary ciliary dyskinesia (PCD) is a congenital disease. It is important to differentiate PCD from acquired (secondary) ciliary dyskinesia (SCD). The number of dynein arms determined by TEM was 1.5 and 1.4 for outer and inner dynein arms, respectively in PCD, versus 7.9 and 5.2 for controls and 8.1 and 5.9 in SCD. Compared to PCD patients, SCD patients have more structurally abnormal cilia. A significant difference was found in orientation of the central microtubule pair between PCD and SCD, but also overlap. </p><p>Leukotriene receptor antagonists are a new treatment for asthma. Both corticosteroids and montelukast caused apoptosis and necrosis of airway epithelial cells, and reduced the expression of intercellular adhesion molecule-1. Treatment of cells with tumor necrosis factor-α or interferon-γ reduced the fraction of the lateral cell membrane occupied by desmosomes and this effect was counteracted by corticosteroids. </p>

In this thesis, the shape of the distal humerus trochlea is analysed using landmark-based morphometrics and multivariate methods, with the aim of exploring locomotor evolution in carnivorans. Elbow joint morphology is used together with body size and craniodental morphology to characterize past and present carnivorans. Evolutionary implications are studied at the ordinal, familial, and species levels, testing specific hypotheses about scaling, morphological constraints, evolutionary trajectories, and potential for social pack-hunting behaviour. The circumference of the distal humerus trochlea is found to be highly correlated with body mass, and appears to scale similarly throughout the order Carnivora. A general predictive model for carnivoran bodymass is presented (a=0.601; b= 2.552; r2=0.952, SEE=0.136, p&lt;0001, n=92), which removes the need for the investigator to actively choose between the diverging estimates that different predictors and their equations often produce. At the elbow joint, manual manipulation and locomotion appear to be conflicting functions, thus suggesting mutually exclusive lifestyles involving either forelimb grappling or pursuit. At large body sizes, carnivorans are distributed over a strongly dichotomised pattern (grappling or locomotion), a pattern coinciding with the postulated threshold in predator-prey size ratio at 21.5-25 kg. This pattern is compared to that of two carnivoran faunas from the Tertiary. In the Oligocene (33.7-23.8 Myr BP), the overall pattern is remarkably similar to that observed for extant Carnivora. In the Miocene (23.8-11.2 Myr BP) carnivores show a similarly dichotomised pattern as the Oligocene and Recent, although the whole pattern is shifted towards larger body sizes. This difference is suggested to be a reflection of the extraordinary species richness of browsing ungulates in the early Miocene of North America. Such an increase in prey spectrum would create a unique situation, in which large carnivores need not commit to a cursorial habitus in order to fill their nutritional requirements. Finally, the elbow joints and craniodental morphology (14 measurements) of fossil canids were examined with the aim of assessing the potential for pack-hunting in fossil canids. It is clear that small and large members of the Recent Caninae share similar craniodental morphologies. However, this pattern is not present in Borophaginae and Hesperocyoninae. In the latter, large representatives are characterized by being short-faced, with reduced anterior premolars and enlarged posterior premolars, thus approaching a “pantherine-like” craniodental configuration. These traits are interpreted as an adaptation for killing prey with canine bites. It is similarly determined that, unlike recent Caninae, all analyzed species of borophagines and hesperocyonines have retained the ability to supinate their forearms. It is therefore likely that manual manipulation was part of their hunting behaviour, thus removing an essential part of the argument for social pack-hunting in these forms, as the benefits of such a strategy become less obvious.

Ultrastructural studies of airway epithelium in airway disease are important for diagnosis and understanding the underlying pathology which helps clinicians to improve the patients' treatment. Airway biopsies from a 5-month old boy with respiratory problems and gastro-oesophageal reflux were studied by transmission electron microscopy (TEM). The tracheal columnar cells showed accumulation of lamellar bodies, indicative of lysosomal storage disease. The patient was diagnosed with Gaucher disease type 2. Shedding of airway epithelial cells is commonly found in asthma. The attachment of these cells to the basal lamina was investigated by TEM of biopsies from patients with asthma and healthy controls. The contact area between columnar cells and basal lamina in asthmatics was significantly less than in controls. Attachment of columnar cells to the basal lamina occurs mainly indirectly, via desmosomal attachment to basal cells. Primary ciliary dyskinesia (PCD) is a congenital disease. It is important to differentiate PCD from acquired (secondary) ciliary dyskinesia (SCD). The number of dynein arms determined by TEM was 1.5 and 1.4 for outer and inner dynein arms, respectively in PCD, versus 7.9 and 5.2 for controls and 8.1 and 5.9 in SCD. Compared to PCD patients, SCD patients have more structurally abnormal cilia. A significant difference was found in orientation of the central microtubule pair between PCD and SCD, but also overlap. Leukotriene receptor antagonists are a new treatment for asthma. Both corticosteroids and montelukast caused apoptosis and necrosis of airway epithelial cells, and reduced the expression of intercellular adhesion molecule-1. Treatment of cells with tumor necrosis factor-α or interferon-γ reduced the fraction of the lateral cell membrane occupied by desmosomes and this effect was counteracted by corticosteroids.

Skeletal muscle satellite cells located between the plasma membrane and the basal lamina of muscle fibres, could for many years, only be studied in situ by electron microscopy. The introduction of immunohistochemistry and the discovery of molecular markers of satellite cells then made them accessible for light microscopic studies and a wealth of information is today available. Satellite cells are myogenic stem cells that can be activated from a quiescent state to proliferate for self-renewal or differentiate into myogenic cells. The satellite cells are involved in muscle growth during fetal and postnatal development and play a key role in repair and regeneration of damaged muscle fibres. The satellite cells are also essential for muscle fibre hypertrophy and maintenance of muscle mass in the adult. When the present thesis was initiated, studies on satellite cells in human skeletal muscle relied on the neuronal cell adhesion molecule (NCAM) as a marker for satellite cell identification. The results from different studies varied markedly. Therefore the aims of the present thesis were i) to develop a highly reliable method using light microscopy for satellite cell identification and quantification in biopsies of human skeletal muscle in normal and pathological conditions. A molecular marker for the myofibre basal lamina or plasma membrane to enhance the reliability of myonuclei and satellite cell identification were to be included. Furthermore unbiased morphometric methods should be used in the quantification process. ii) to evaluate which molecular markers which had been described for satellite cell and stem cell identification in different cell states (quiescence, activated or differentiated) are the most useful for studies on human skeletal muscle. iii) to further explore the function and heterogeneity of satellite cells with respect to different markers in human skeletal muscle by studying the effects of strength-training, intake of anabolic substances and pathological conditions. A new immunofluorescence method was developed where in the same tissue section, two satellite cell markers, the basal lamina and nuclei were monitored. From the evaluation of different markers it was found that both NCAM and Pax7 identified the majority of satellite cells but that both markers were needed for reliable identification. The members of the myogenic regulatory family were evaluated and by using the new method MyoD and myogenin were found to be useful markers to identify activated and differentiated satellite cells. Upon re-examination of biopsies from power-lifters, power-lifters using anabolic substances and untrained subjects it was observed that the new results on satellite cell frequency were significantly different from those obtained when using staining for NCAM and nuclei alone. In addition three subtypes of satellite cells (94.4% NCAM+/Pax7+, 4.2% NCAM+/Pax7– and 1.4% NCAM–/Pax7+) were observed. Thus the multiple marker method gave more information about satellite cells heterogeneity in human muscle and we propose that this is more reliable than previous methods. Low numbers of MyoD or myogenin stained satellite cells were observed in both untrained and strength trained subjects. Other markers such as DLK1/FA1, a member of the EGF-like family and c-Met, the receptor for hepatocyte growth factor showed that satellite cell heterogeneity in human muscle is far greater than previously shown. Furthermore, new evidence is presented for so called fibre splitting observed in hypertrophic muscle fibres to be due to defect regeneration of partially damaged fibres.

Gliomas are the most common primary tumors of the central nervous system believed to arise from glial cells. Invasive growth and inherent propensity for malignant progression make gliomas incurable despite extensive treatment. I have developed a life-like orthotopic glioma model and used this and other in vivo models to study basic mechanisms of glioma development and treatment. Previous studies had indicated that experimental gliomas could arise from glial stem cells and astrocytes. The present thesis describes the making and characterization of a novel mouse model, Ctv-a, where gliomas are induced from oligodendrocyte progenitor cells (OPCs). Our study shows that OPCs have the capacity to give rise to gliomas and suggests in light of previous data that the differentiation state of the cell of origin affects tumor malignancy. CDKN2A encodes p16INK4a and p14ARF (p19Arf in mouse) commonly inactivated in malignant glioma. Their roles in experimental glioma have been extensively studied and both proteins have tumor suppressor functions in glial stem cells and astrocytes. Here, we demonstrate that p19Arf only could suppress gliomagenesis in OPCs while p16Ink4a had no tumor suppressive effect. Functional DNA repair is pivotal for maintaining genome integrity, eliminating unsalvageable cells and inhibiting tumorigenesis. We have studied how RAD51, a central protein of homology-directed repair, affected experimental glioma development and have found that expression of RAD51 may protect against genomic instability and tumor development. Angiogenesis, the formation of new blood vessels from pre-existing ones, is a central feature of malignant progression in glioma. Antiangiogenic treatment by inhibition of vascular endothelial growth factor receptor signaling is used in the clinic for treatment of some cancers. We have investigated the effect of an alternative antiangiogenic protein, histidine-rich glycoprotein (HRG), on glioma development and found that HRG could inhibit the formation of malignant gliomas and completely prevent the formation of glioblastoma.