Electrochemical formation of Cu/Ag surfaces and their applicability as heterogeneous catalysts

In this work the electrochemical formation of porous Cu/Ag materials is reported via the simple and quick method of hydrogen bubble templating. The bulk and surface composition ratio between Ag and Cu was varied in a systematic manner and was readily controlled by the concentration of precursor metal salts in the electrolyte. The incorporation of Ag within the Cu scaffold only affected the formation of well-defined pores at high Ag loading whereas the internal pore wall structure gradually transformed from dendritic to cube like and finally needle like structures, which was due to the concomitant formation of Cu2O within the structure. The materials were characterised by scanning electron microscopy (SEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). Their surface properties were further investigated by surface enhanced Raman spectroscopy (SERS) and electrochemically probed by recording the hydrogen evolution reaction (HER) which is highly sensitive to the nature of the surface. The effect of surface composition was then investigated for its influence on two catalytic reactions namely the reduction of ferricyanide ions with thiosulphate ions and the reduction of 4-nitrophenol with NaBH4 in aqueous solution where it was found that the presence of Ag had a beneficial effect in both cases but more so in the case of nitrophenol reduction. It is believed that this material may have many more potential applications in the area of catalysis, electrocatalysis and photocatalysis.

Molecular mechanisms of cisplatin resistance in lung cancer

Cisplatin is one of the most potent anticancer agents, displaying significant clinical activity against a variety of solid tumours. To date, cisplatin-based combination treatment remains the most effective systemic chemotherapy for non-small cell lung cancer (NSCLC) patients. Unfortunately, the outcome of cisplatin therapy in NSCLC has reached a plateau due to the development of both intrinsic and acquired resistance that have become a major obstacle in the use of cisplatin in the clinical setting. The molecular mechanisms that underlie chemoresistance are largely unknown. Mechanisms of acquired resistance to cisplatin include reduced intracellular accumulation of the drug, enhanced drug inactivation by metallothionine and glutathione, increased repair activity of DNA damage, and altered expression of oncogenes and regulatory proteins. Cisplatin-induced cytotoxicity is mediated through the induction of apoptosis and cell cycle arrest as a result of cisplatin-DNA adduct formation, which in turn, activates multiple signaling pathways and mediators. These include p53, Bcl-2 family, caspases, cyclins, CDKs, MAPK and PI3K/Akt. Increased expression of anti-apoptotic genes and mutations in the intrinsic apoptotic pathway may also contribute to the inability of cells to detect DNA damage or to induce apoptosis. This chapter will provide an insight into the mechanisms involved in cisplatin resistance and a better understanding of the molecular basis of the cellular response to cisplatin-based chemotherapy in lung cancer.

Distinguishing between mechanisms of cell aggregation using pair-correlation functions

Many cell types form clumps or aggregates when cultured in vitro through a variety of mechanisms including rapid cell proliferation, chemotaxis, or direct cell-to-cell contact. In this paper we develop an agent-based model to explore the formation of aggregates in cultures where cells are initially distributed uniformly, at random, on a two-dimensional substrate. Our model includes unbiased random cell motion, together with two mechanisms which can produce cell aggregates: (i) rapid cell proliferation, and (ii) a biased cell motility mechanism where cells can sense other cells within a finite range, and will tend to move towards areas with higher numbers of cells. We then introduce a pair-correlation function which allows us to quantify aspects of the spatial patterns produced by our agent-based model. In particular, these pair-correlation functions are able to detect differences between domains populated uniformly at random (i.e. at the exclusion complete spatial randomness (ECSR) state) and those where the proliferation and biased motion rules have been employed - even when such differences are not obvious to the naked eye. The pair-correlation function can also detect the emergence of a characteristic inter-aggregate distance which occurs when the biased motion mechanism is dominant, and is not observed when cell proliferation is the main mechanism of aggregate formation. This suggests that applying the pair-correlation function to experimental images of cell aggregates may provide information about the mechanism associated with observed aggregates. As a proof of concept, we perform such analysis for images of cancer cell aggregates, which are known to be associated with rapid proliferation. The results of our analysis are consistent with the predictions of the proliferation-based simulations, which supports the potential usefulness of pair correlation functions for providing insight into the mechanisms of aggregate formation.

Inhibition of homogenous formation of calcium carbonate by poly (acrylic acid). The effect of molar mass and end-group functionality

The ability of poly(acrylic acid) (PAA) with different end groups and molar masses prepared by Atom Transfer Radical Polymerization (ATRP) to inhibit the formation of calcium carbonate scale at low and elevated temperatures was investigated. Inhibition of CaCO3 deposition was affected by the hydrophobicity of the end groups of PAA, with the greatest inhibition seen for PAA with hydrophobic end groups of moderate size (6–10 carbons). The morphologies of CaCO3 crystals were significantly distorted in the presence of these PAAs. The smallest morphological change was in the presence of PAA with long hydrophobic end groups (16 carbons) and the relative inhibition observed for all species were in the same order at 30 °C and 100 °C. As well as distorting morphologies, the scale inhibitors appeared to stabilize the less thermodynamically favorable polymorph, vaterite, to a degree proportional to their ability to inhibit precipitation.

Effect of culture conditions and calcium phosphate coating on ectopic bone formation

This study investigated the effect of a calcium phosphate (CaP) coating onto a polycaprolactone melt electrospun scaffold and in vitro culture conditions on ectopic bone formation in a subcutaneous rat model. The CaP coating resulted in an increased alkaline phosphatase activity (ALP) in ovine osteoblasts regardless of the culture conditions and this was also translated into higher levels of mineralisation. A subcutaneous implantation was performed and increasing ectopic bone formation was observed over time for the CaPcoated samples previously cultured in osteogenic media whereas the corresponding non-coated samples displayed a lag phase before bone formation occurred from 4 to 8 weeks post-implantation. Histology and immunohistochemistry revealed bone fill through the scaffolds 8 weeks post-implantation for coated and non-coated specimens and that ALP, osteocalcin and collagen 1 were present at the ossification front and in the bone tissues. Vascularisation in the vicinity of the bone tissues was also observed indicating that the newly formed bone was not deprived of oxygen and nutrients.We found that in vitro osteogenic induction was essential for achieving bone formation and CaP coating accelerated the osteogenic process. We conclude that high cell density and preservation of the collagenous and mineralised extracellular matrix secreted in vitro are factors of importance for ectopic bone formation.

The effect of electrode material on the electrochemical formation of porous copper surfaces using hydrogen bubble templating

The electrodeposition of copper onto copper, gold, palladium and glassy carbon (GC) electrodes via a hydrogen bubble templating method is reported. It is found that the composition of the underlying electrode material significantly influences the morphology of the copper electrodeposit. Highly ordered porous structures are achieved with Cu and Au electrodes, however on Pd this order is disrupted and a rough randomly oriented surface is formed whereas on GC a bubble templating effect is not observed. Chronopotentiograms recorded during the electrodeposition process allows bubble formation and detachment from the surface to be monitored where distinctly different potential versus time profiles are observed at the different electrodes. The porous Cu surfaces are characterised with scanning electron microscopy, X-ray diffraction and cyclic voltammetric measurements recorded under alkaline conditions. The latter demonstrates that there are active sites present on electrodeposited copper whose coverage and reactivity depend on the underlying electrode material. The most active Cu surface is achieved at a Pd substrate for both the hydrogen evolution reaction and the catalytic reduction of ferricyanide ions with thiosulphate ions. This demonstrates that the highly ordered porous structure on the micron scale which typifies the morphology that can be achieved with the hydrogen bubbling template method is not required in producing the most effective material.

Suppression of cluster ions during particle formation events in the atmosphere

Cluster ions and charged and neutral nanoparticle concentrations were monitored using a neutral cluster and air ion spectrometer (NAIS) over a period of one year in Brisbane, Australia. The study yielded 242 complete days of usable data, of which particle formation events were observed on 101 days. Small, intermediate and large ion concentrations were evaluated in real time. In the diurnal cycle, small ion concentration was highest during the second half of the night while large ion concentrations were a maximum during the day. The small ion concentration showed a decrease when the large ion concentration increased. Particle formation was generally followed by a peak in the intermediate ion concentration. The rate of increase of intermediate ions was used as the criteria for identifying particle formation events. Such events were followed by a period of growth to larger sizes and usually occurred between 8 am and 2 pm. Particle formation events were found to be related to the wind direction. The gaseous precursors for the production of secondary particles in the urban environment of Brisbane have been shown to be ammonia and sulfuric acid. During these events, the nanoparticle number concentrations in the size range 1.6 to 42 nm, which were normally lower than 1x104 cm-3, often exceeded 5x104 cm-3 with occasional values over 1x105 cm-3. Cluster ions generally occurred in number concentrations between 300 and 600 cm-3 but decreased significantly to about 200 cm-3 during particle formation events. This was accompanied by an increase in the large ion concentration. We calculated the fraction of nanoparticles that were charged and investigated the occurrence of possible overcharging during particle formation events. Overcharging is defined as the condition where the charged fraction of particles is higher than in charge equilibrium. This can occur when cluster ions attach to neutral particles in the atmosphere, giving rise to larger concentrations of charged particles in the short term. Ion-induced nucleation is one of the mechanisms of particle formation in the atmosphere, and overcharging has previously been considered as an indicator of this process. The possible role of ions in particle formation was investigated.

Vibration based baseline updating method to localize crack formation and propagation in reinforced concrete members

Structural Health Monitoring (SHM) schemes are useful for proper management of the performance of structures and for preventing their catastrophic failures. Vibration based SHM schemes has gained popularity during the past two decades resulting in significant research. It is hence evitable that future SHM schemes will include robust and automated vibration based damage assessment techniques (VBDAT) to detect, localize and quantify damage. In this context, the Damage Index (DI) method which is classified as non-model or output based VBDAT, has the ability to automate the damage assessment process without using a computer or numerical model along with actual measurements. Although damage assessment using DI methods have been able to achieve reasonable success for structures made of homogeneous materials such as steel, the same success level has not been reported with respect to Reinforced Concrete (RC) structures. The complexity of flexural cracks is claimed to be the main reason to hinder the applicability of existing DI methods in RC structures. Past research also indicates that use of a constant baseline throughout the damage assessment process undermines the potential of the Modal Strain Energy based Damage Index (MSEDI). To address this situation, this paper presents a novel method that has been developed as part of a comprehensive research project carried out at Queensland University of Technology, Brisbane, Australia. This novel process, referred to as the baseline updating method, continuously updates the baseline and systematically tracks both crack formation and propagation with the ability to automate the damage assessment process using output only data. The proposed method is illustrated through examples and the results demonstrate the capability of the method to achieve the desired outcomes.

Top-down influences on lexical selection during spoken word production: A 4T fMRI investigation of refractory effects in picture naming

Spoken word production is assumed to involve stages of processing in which activation spreads through layers of units comprising lexical-conceptual knowledge and their corresponding phonological word forms. Using high-field (4T) functional magnetic resonance imagine (fMRI), we assessed whether the relationship between these stages is strictly serial or involves cascaded-interactive processing, and whether central (decision/control) processing mechanisms are involved in lexical selection. Participants performed the competitor priming paradigm in which distractor words, named from a definition and semantically related to a subsequently presented target picture, slow picture-naming latency compared to that with unrelated words. The paradigm intersperses two trials between the definition and the picture to be named, temporally separating activation in the word perception and production networks. Priming semantic competitors of target picture names significantly increased activation in the left posterior temporal cortex, and to a lesser extent the left middle temporal cortex, consistent with the predictions of cascaded-interactive models of lexical access. In addition, extensive activation was detected in the anterior cingulate and pars orbitalis of the inferior frontal gyrus. The findings indicate that lexical selection during competitor priming is biased by top-down mechanisms to reverse associations between primed distractor words and target pictures to select words that meet the current goal of speech.

Perfusion fMRI evidence for priming of shared feature-to-lexical connections during cumulative semantic interference in spoken word production

The speed at which target pictures are named increases monotonically as a function of prior retrieval of other exemplars of the same semantic category and is unaffected by the number of intervening items. This cumulative semantic interference effect is generally attributed to three mechanisms: shared feature activation, priming and lexical-level selection. However, at least two additional mechanisms have been proposed: (1) a 'booster' to amplify lexical-level activation and (2) retrieval-induced forgetting (RIF). In a perfusion functional Magnetic Resonance Imaging (fMRI) experiment, we tested hypotheses concerning the involvement of all five mechanisms. Our results demonstrate that the cumulative interference effect is associated with perfusion signal changes in the left perirhinal and middle temporal cortices that increase monotonically according to the ordinal position of exemplars being named. The left inferior frontal gyrus (LIFG) also showed significant perfusion signal changes across ordinal presentations; however, these responses did not conform to a monotonically increasing function. None of the cerebral regions linked with RIF in prior neuroimaging and modelling studies showed significant effects. This might be due to methodological differences between the RIF paradigm and continuous naming as the latter does not involve practicing particular information. We interpret the results as indicating priming of shared features and lexical-level selection mechanisms contribute to the cumulative interference effect, while adding noise to a booster mechanism could account for the pattern of responses observed in the LIFG.

Excessive bone formation in a mouse model of ankylosing spondylitis is associated with decreases in Wnt pathway inhibitors

Introduction: Ankylosing spondylitis (AS) is unique in its pathology where inflammation commences at the entheses before progressing to an osteoproliferative phenotype generating excessive bone formation that can result in joint fusion. The underlying mechanisms of this progression are poorly understood. Recent work has suggested that changes in Wnt signalling, a key bone regulatory pathway, may contribute to joint ankylosis in AS. Using the proteoglycan-induced spondylitis (PGISp) mouse model which displays spondylitis and eventual joint fusion following an initial inflammatory stimulus, we have characterised the structural and molecular changes that underlie disease progression. Methods: PGISp mice were characterised 12 weeks after initiation of inflammation using histology, immunohistochemistry (IHC) and expression profiling. Results: Inflammation initiated at the periphery of the intervertebral discs progressing to disc destruction followed by massively excessive cartilage and bone matrix formation, as demonstrated by toluidine blue staining and IHC for collagen type I and osteocalcin, leading to syndesmophyte formation. Expression levels of DKK1 and SOST, Wnt signalling inhibitors highly expressed in joints, were reduced by 49% and 63% respectively in the spine PGISp compared with control mice (P < 0.05) with SOST inhibition confirmed by IHC. Microarray profiling showed genes involved in inflammation and immune-regulation were altered. Further, a number of genes specifically involved in bone regulation including other members of the Wnt pathway were also dysregulated. Conclusions: This study implicates the Wnt pathway as a likely mediator of the mechanism by which inflammation induces bony ankylosis in spondyloarthritis, raising the potential that therapies targeting this pathway may be effective in preventing this process.

Fracture healing via periosteal callus formation requires macrophages for both initiation and progression of early endochondral ossification

The distribution, phenotype, and requirement of macrophages for fracture-associated inflammation and/or early anabolic progression during endochondral callus formation were investigated. A murine femoral fracture model [internally fixed using a flexible plate (MouseFix)] was used to facilitate reproducible fracture reduction. IHC demonstrated that inflammatory macrophages (F4/80+Mac-2+) were localized with initiating chondrification centers and persisted within granulation tissue at the expanding soft callus front. They were also associated with key events during soft-to-hard callus transition. Resident macrophages (F4/80+Mac-2neg), including osteal macrophages, predominated in the maturing hard callus. Macrophage Fas-induced apoptosis transgenic mice were used to induce macrophage depletion in vivo in the femoral fracture model. Callus formation was completely abolished when macrophage depletion was initiated at the time of surgery and was significantly reduced when depletion was delayed to coincide with initiation of early anabolic phase. Treatment initiating 5 days after fracture with the pro-macrophage cytokine colony stimulating factor-1 significantly enhanced soft callus formation. The data support that inflammatory macrophages were required for initiation of fracture repair, whereas both inflammatory and resident macrophages promoted anabolic mechanisms during endochondral callus formation. Overall, macrophages make substantive and prolonged contributions to fracture healing and can be targeted as a therapeutic approach for enhancing repair mechanisms. Thus, macrophages represent a viable target for the development of pro-anabolic fracture treatments with a potentially broad therapeutic window...

Mitochondrial Malfunction in Tumor Formation and Neuromuscular Diseases : Consequences of defects in fumarate hydratase and in the respiratory chain

The mitochondrion is an organelle of outmost importance, and the mitochondrial network performs an array of functions that go well beyond ATP synthesis. Defects in mitochondrial performance lead to diseases, often affecting nervous system and muscle. Although many of these mitochondrial diseases have been linked to defects in specific genes, the molecular mechanisms underlying the pathologies remain unclear. The work in this thesis aims to determine how defects in mitochondria are communicated within - and interpreted by - the cells, and how this contributes to disease phenotypes. Fumarate hydratase (FH) is an enzyme of the citrate cycle. Recessive defects in FH lead to infantile mitochondrial encephalopathies, while dominant mutations predispose to tumor formation. Defects in succinate dehydrogenase (SDH), the enzyme that precedes FH in the citrate cycle, have also been described. Mutations in SDH subunits SDHB, SDHC and SDHD are associated with tumor predisposition, while mutations in SDHA lead to a characteristic mitochondrial encephalopathy of childhood. Thus, the citrate cycle, via FH and SDH, seems to have essential roles in mitochondrial function, as well as in the regulation of processes such as cell proliferation, differentiation or death. Tumor predisposition is not a typical feature of mitochondrial energy deficiency diseases. However, defects in citrate cycle enzymes also affect mitochondrial energy metabolism. It is therefore necessary to distinguish what is specific for defects in citrate cycle, and thus possibly associated with the tumor phenotype, from the generic consequences of defects in mitochondrial aerobic metabolism. We used primary fibroblasts from patients with recessive FH defects to study the cellular consequences of FH-deficiency (FH-). Similarly to the tumors observed in FH- patients, these fibroblasts have very low FH activity. The use of primary cells has the advantage that they are diploid, in contrast with the aneuploid tumor cells, thereby enabling the study of the early consequences of FH- in diploid background, before tumorigenesis and aneuploidy. To distinguish the specific consequences of FH- from typical consequences of defects in mitochondrial aerobic metabolism, we used primary fibroblasts from patients with MELAS (mitochondrial encephalopathy with lactic acidosis and stroke-like episodes) and from patients with NARP (neuropathy, ataxia and retinitis pigmentosa). These diseases also affect mitochondrial aerobic metabolism but are not known to predispose to tumor formation. To study in vivo the systemic consequences of defects in mitochondrial aerobic metabolism, we used a transgenic mouse model of late-onset mitochondrial myopathy. The mouse contains a transgene with an in-frame duplication of a segment of Twinkle, the mitochondrial replicative helicase, whose defects underlie the human disease progressive external ophthalmoplegia. This mouse model replicates the phenotype in the patients, particularly neuronal degeneration, mitochondrial myopathy, and subtle decrease of respiratory chain activity associated with mtDNA deletions. Due to the accumulation of mtDNA deletions, the mouse was named deletor. We first studied the consequences of FH- and of respiratory chain defects for energy metabolism in primary fibroblasts. To further characterize the effects of FH- and respiratory chain malfunction in primary fibroblasts at transcriptional level, we used expression microarrays. In order to understand the in vivo consequences of respiratory chain defects in vivo, we also studied the transcriptional consequences of Twinkle defects in deletor mice skeletal muscle, cerebellum and hippocampus. Fumarate accumulated in the FH- homozygous cells, but not in the compound heterozygous lines. However, virtually all FH- lines lacked cytoplasmic FH. Induction of glycolysis was common to FH-, MELAS and NARP fibroblasts. In deletor muscle glycolysis seemed to be upregulated. This was in contrast with deletor cerebellum and hippocampus, where mitochondrial biogenesis was in progress. Despite sharing a glycolytic pattern in energy metabolism, FH- and respiratory chain defects led to opposite consequences in redox environment. FH- was associated with reduced redox environment, while MELAS and NARP displayed evidences of oxidative stress. The deletor cerebellum had transcriptional induction of antioxidant defenses, suggesting increased production of reactive oxygen species. Since the fibroblasts do not represent the tissues where the tumors appear in FH- patients, we compared the fibroblast array data with the data from FH- leiomyomas and normal myometrium. This allowed the determination of the pathways and networks affected by FH-deficiency in primary cells that are also relevant for myoma formation. A key pathway regulating smooth muscle differentiation, SRF (serum response factor)-FOS-JUNB, was found to be downregulated in FH- cells and in myomas. While in the deletor mouse many pathways were affected in a tissue-specific basis, like FGF21 induction in the deletor muscle, others were systemic, such as the downregulation of ALAS2-linked heme synthesis in all deletor tissues analyzed. However, interestingly, even a tissue-specific response of FGF21 excretion could elicit a global starvation response. The work presented in this thesis has contributed to a better understanding of mitochondrial stress signalling and of pathways interpreting and transducing it to human pathology.

Endoplasmic reticulum stress and cell death mechanisms in the cell model of Huntington’s disease

This thesis clarifies important molecular pathways that are activated during the cell death observed in Huntington’s disease. Huntington’s disease is one of the most common inherited neurodegenerative diseases, which is primarily inherited in an autosomal dominant manner. HD is caused by an expansion of CAG repeats in the first exon of the IT15 gene. IT15 encodes the production of a Huntington’s disease protein huntingtin. Mutation of the IT15 gene results in a long stretch of polyQ residues close to the amino-terminal region of huntingtin. Huntington’s disease is a fatal autosomal neurodegenerative disorder. Despite the current knowledge of HD, the precise mechanism behind the selective neuronal death, and how the disease propagates, still remains an enigma. The studies mainly focused on the control of endoplasmic reticulum (ER) stress triggered by the mutant huntingtin proteins. The ER is a delicate organelle having essential roles in protein folding and calcium regulation. Even the slightest perturbations on ER homeostasis are effective enough to trigger ER stress and its adaptation pathways, called unfolded protein response (UPR). UPR is essential for cellular homeostasis and it adapts ER to the changing environment and decreases ER stress. If adaptation processes fail and stress is excessive and prolonged; irreversible cell death pathways are engaged. The results showed that inhibition of ER stress with chemical agents are able to decrease cell death and formation of toxic cell aggregates caused by mutant huntingtin proteins. The study concentrated also to the NF-κB (nuclear factor-kappaB) pathway, which is activated during ER stress. NF-κB pathway is capable to regulate the levels of important cellular antioxidants. Cellular antioxidants provide a first line of defence against excess reactive oxygen species. Excess accumulation of reactive oxygen species and subsequent activation of oxidative stress damages motley of vital cellular processes and induce cell degeneration. Data showed that mutant huntingtin proteins downregulate the expression levels of NF-κB and vital antioxidants, which was followed by increased oxidative stress and cell death. Treatment with antioxidants and inhibition of oxidative stress were able to counteract these adverse effects. In addition, thesis connects ER stress caused by mutant huntingtin to the cytoprotective autophagy. Autophagy sustains cellular balance by degrading potentially toxic cell proteins and components observed in Huntington’s disease. The results revealed that cytoprotective autophagy is active at the early points (24h) of ER stress after expression of mutant huntingtin proteins. GADD34 (growth arrest and DNA damage-inducible gene 34), which is previously connected to the regulation of translation during cell stress, was shown to control the stimulation of autophagy. However, GADD34 and autophagy were downregulated at later time points (48h) during mutant huntingtin proteins induced ER stress, and subsequently cell survival decreased. Overexpression GADD34 enhanced autophagy and decreased cell death, indicating that GADD34 plays a critical role in cell protection. The thesis reveales new interesting data about the neuronal cell death pathways seen in Huntington’s disease, and how cell degeneration is partly counteracted by various therapeutic agents. Expression of mutant huntingtin proteins is shown to alter signaling events that control ER stress, oxidative stress and autophagy. Despite that Huntington’s disease is mainly an untreatable disorder; these findings offer potential targets and neuroprotective strategies in designing novel therapies for Huntington’s disease.

Mechanisms and molecular regulation of mammalian tooth replacement

In most non-mammalian vertebrates, such as fish and reptiles, teeth are replaced continuously. However, tooth replacement in most mammals, including human, takes place only once and further renewal is apparently inhibited. It is not known how tooth replacement is genetically regulated, and little is known on the physiological mechanism and evolutionary reduction of tooth replacement in mammals. In this study I have attempted to address these questions. In a rare human condition cleidocranial dysplasia, caused by a mutation in a Runt domain transcription factor Runx2, tooth replacement is continued. Runx2 mutant mice were used to investigate the molecular mechanisms of Runx2 function. Microarray analysis from dissected embryonic day 14 Runx2 mutant and wild type dental mesenchymes revealed many downstream targets of Runx2, which were validated using in situ hybridization and tissue culture methods. Wnt signaling inhibitor Dkk1 was identified as a candidate target, and in tissue culture conditions it was shown that Dkk1 is induced by FGF4 and this induction is Runx2 dependent. These experiments demonstrated a connection between Runx2, FGF and Wnt signaling in tooth development and possibly also in tooth replacement. The role of Wnt signaling in tooth replacement was further investigated by using a transgenic mouse model where Wnt signaling mediator β-catenin is continuously stabilized in dental epithelium. This stabilization led to activated Wnt signaling and to the formation of multiple enamel knots. In vitro and transplantation experiments were performed to examine the process of extra tooth formation. We showed that new teeth were continuously generated and that new teeth form from pre-existing teeth. A morphodynamic activator-inhibitor model was used to simulate enamel knot formation. By increasing the intrinsic production rate of the activator (β-catenin), the multiple enamel knot phenotype was reproduced by computer simulations. It was thus concluded that β-catenin acts as an upstream activator of enamel knots, closely linking Wnt signaling to the regulation of tooth renewal. As mice do not normally replace teeth, we used other model animals to investigate the physiological and genetic mechanisms of tooth replacement. Sorex araneus, the common shrew was earlier reported to have non-functional tooth replacement in all antemolar tooth positions. We showed by histological and gene expression studies that there is tooth replacement only in one position, the premolar 4 and that the deciduous tooth is diminished in size and disappears during embryogenesis without becoming functional. The growth rates of deciduous and permanent premolar 4 were measured and it was shown by competence inference that the early initiation of the replacement tooth in relation to the developmental stage of the deciduous tooth led to the inhibition of deciduous tooth morphogenesis. It was concluded that the evolutionary loss of deciduous teeth may involve the early activation of replacement teeth, which in turn suppress their predecessors. Mustela putorius furo, the ferret, has a dentition that resembles that of the human as ferrets have teeth that belong to all four tooth families, and all the antemolar teeth are replaced once. To investigate the replacement mechanism, histological serial sections from different embryonic stages were analyzed. It was noticed that tooth replacement is a process which involves the growth and detachment of the dental lamina from the lingual cervical loop of the deciduous tooth. Detachment of the deciduous tooth leads to a free successional dental lamina, which grows deeper into the mesenchyme, and later buds the replacement tooth. A careful 3D analysis of serial histological sections was performed and it was shown that replacement teeth are initiated from the successional dental lamina and not from the epithelium of the deciduous tooth. The molecular regulation of tooth replacement was studied and it was shown by examination of expression patterns of candidate regulatory genes that BMP/Wnt inhibitor Sostdc1 was strongly expressed in the buccal aspect of the dental lamina, and in the intersection between the detaching deciduous tooth and the successional dental lamina, suggesting a role for Sostdc1 in the process of detachment. Shh was expressed in the enamel knot and in the inner enamel epithelium in both generations of teeth supporting the view that the morphogenesis of both generations of teeth is regulated by similar mechanisms. In summary, histological and molecular studies on different model animals and transgenic mouse models were used to investigate tooth replacement. This thesis work has significantly contributed to the knowledge on the physiological mechanisms and molecular regulation of tooth replacement and its evolutionary suppression in mammals.