New [18F]Tracers for Alzheimer's Disease Imaging. Labeling Synthesis And Biological Testing

Alzheimer’s disease (AD) is the most common form of dementia. Characteristic changes in an AD brain are the formation of β-amyloid protein (Aβ) plaques and neurofibrillary tangles, though other alterations in the brain have also been connected to AD. No cure is available for AD and it is one of the leading causes of death among the elderly in developed countries. Liposomes are biocompatible and biodegradable spherical phospholipid bilayer vesicles that can enclose various compounds. Several functional groups can be attached on the surface of liposomes in order to achieve long-circulating target-specific liposomes. Liposomes can be utilized as drug carriers and vehicles for imaging agents. Positron emission tomography (PET) is a non-invasive imaging method to study biological processes in living organisms. In this study using nucleophilic 18F-labeling synthesis, various synthesis approaches and leaving groups for novel PET imaging tracers have been developed to target AD pathology in the brain. The tracers were the thioflavin derivative [18F]flutemetamol, curcumin derivative [18F]treg-curcumin, and functionalized [18F]nanoliposomes, which all target Aβ in the AD brain. These tracers were evaluated using transgenic AD mouse models. In addition, 18F-labeling synthesis was developed for a tracer targeting the S1P3 receptor. The chosen 18F-fluorination strategy had an effect on the radiochemical yield and specific activity of the tracers. [18F]Treg-curcumin and functionalized [18F]nanoliposomes had low uptake in AD mouse brain, whereas [18F]flutemetamol exhibited the appropriate properties for preclinical Aβ-imaging. All of these tracers can be utilized in studies of the pathology and treatment of AD and related diseases.

Photoelectric Measurements and Modeling of Bacteriorhodopsin

Recent technology has provided us with new information about the internal structures and properties of biomolecules. This has lead to the design of applications based on underlying biological processes. Applications proposed for biomolecules are, for example, the future computers and different types of sensors. One potential biomolecule to be incorporated in the applications is bacteriorhodopsin. Bacteriorhodopsin is a light-sensitive biomolecule, which works in a similar way as the light sensitive cells of the human eye. Bacteriorhodopsin reacts to light by undergoing a complicated series of chemical and thermal transitions. During these transitions, a proton translocation occurs inside the molecule. It is possible to measure the photovoltage caused by the proton translocations when a vast number of molecules is immobilized in a thin film. Also the changes in the light absorption of the film can be measured. This work aimed to develop the electronics needed for the voltage measurements of the bacteriorhodopsin-based optoelectronic sensors. The development of the electronics aimed to get more accurate information about the structure and functionality of these sensors. The sensors used in this work contain a thick film of bacteriorhodopsin immobilized in polyvinylalcohol. This film is placed between two transparent electrodes. The result of this work is an instrumentation amplifier which can be placed in a small space very close to the sensor. By using this amplifier, the original photovoltage can be measured in more detail. The response measured using this amplifier revealed two different components, which could not be distinguished earlier. Another result of this work is the model for the photoelectric response in dry polymer films.

Mechanistic population Models in Biology: Model Derivation and Application

In general, models of ecological systems can be broadly categorized as ’top-down’ or ’bottom-up’ models, based on the hierarchical level that the model processes are formulated on. The structure of a top-down, also known as phenomenological, population model can be interpreted in terms of population characteristics, but it typically lacks an interpretation on a more basic level. In contrast, bottom-up, also known as mechanistic, population models are derived from assumptions and processes on a more basic level, which allows interpretation of the model parameters in terms of individual behavior. Both approaches, phenomenological and mechanistic modelling, can have their advantages and disadvantages in different situations. However, mechanistically derived models might be better at capturing the properties of the system at hand, and thus give more accurate predictions. In particular, when models are used for evolutionary studies, mechanistic models are more appropriate, since natural selection takes place on the individual level, and in mechanistic models the direct connection between model parameters and individual properties has already been established. The purpose of this thesis is twofold. Firstly, a systematical way to derive mechanistic discrete-time population models is presented. The derivation is based on combining explicitly modelled, continuous processes on the individual level within a reproductive period with a discrete-time maturation process between reproductive periods. Secondly, as an example of how evolutionary studies can be carried out in mechanistic models, the evolution of the timing of reproduction is investigated. Thus, these two lines of research, derivation of mechanistic population models and evolutionary studies, are complementary to each other.

Factors Regulating Chondrogenic Differentiation

Chondrogenesis is a co-ordinated differentiation process in which mesenchymal cells condensate, differentiate into chondrocytes and begin to secrete molecules that form the extracellular matrix. It is regulated in a spatio-temporal manner by cellular interactions and growth and differentiation factors that modulate cellular signalling pathways and transcription of specific genes. Moreover, post-transcriptional regulation by microRNAs (miRNAs) has appeared to play a central role in diverse biological processes, but their role in skeletal development is not fully understood. Mesenchymal stromal cells (MSCs) are multipotent cells present in a variety of adult tissues, including bone marrow and adipose tissue. They can be isolated, expanded and, under defined conditions, induced to differentiate into multiple cell lineages including chondrocytes, osteoblasts and adipocytes in vitro and in vivo. Owing to their intrinsic capability to self-renew and differentiate into functional cell types, MSCs provide a promising source for cell-based therapeutic strategies for various degenerative diseases, such as osteoarthritis (OA). Due to the potential therapeutic applications, it is of importance to better understand the MSC biology and the regulatory mechanisms of their differentiation. In this study, an in vitro assay for chondrogenic differentiation of mouse MSCs (mMSCs) was developed for the screening of various factors for their chondrogenic potential. Conditions were optimized for pellet cultures by inducing mMSC with different bone morphogenetic proteins (BMPs) that were selected based on their known chondrogenic relevance. Characterization of the surface epitope profile, differentiation capacity and molecular signature of mMSCs illustrated the importance of cell population composition and the interaction between different populations in the cell fate determination and differentiation of MSCs. Regulation of Wnt signalling activity by Wnt antagonist sFRP-1 was elucidated as a potential modulator of lineage commitment. Delta-like 1 (dlk1), a factor regulating adipogenesis and osteogenesis, was shown to exhibit stage-specific expression during embryonic chondrogenesis and identified as a novel regulator of chondrogenesis, possibly through mediating the effect of TGF-beta1. Moreover, miRNA profiling demonstrated that MSCs differentiating into a certain lineage exhibit a specific miRNA expression profile. The complex regulatory network between miRNAs and transcription factors is suggested to play a crucial role in fine-tuning the differentiation of MSCs. These results demonstrate that commitment of mesenchymal stromal cells and further differentiation into specific lineages is regulated by interactions between MSCs, various growth and transcription factors, and miRNA-mediated translational repression of lineage-specific genes.

Studies on 68Ga-Based Agents for PET Imaging of Cancer and Inflammation

Studies on ⁶⁸Ga-Based Agents for PET Imaging of Cancer and Inflammation Positron emission tomography (PET) is based on the use of radiolabeled agents and facilitates in vivo imaging of biological processes, such as cancer. Because the detection of cancer is demanding and is often obscured by inflammation, there is a demand for better PET imaging agents. The aim was to preliminarily evaluate new PET agents for imaging cancer and inflammation using experimental models. ⁶⁸Ga-chloride and peptides, ⁶⁸Ga-labeled through 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), targeting matrix metalloproteinase-9 (MMP-9) were tested for tumor imaging. In addition, a ⁶⁸Ga-DOTA-conjugated peptide targeting vascular adhesion protein-1 (VAP-1), was tested for inflammation imaging. The ⁶⁸Ga-based imaging agents described here showed potential features by passing the essential in vitro tests, proceeding further to preclinical in vivo evaluation and being able to visualize the target. The target uptake and target-to-background ratios of ⁶⁸Ga-based agents were, however, not optimal. ⁶⁸Ga-chloride showed slow clearance caused by its binding to blood transferrin. In the case of ⁶⁸Ga-DOTA-peptides low in vivo stability and/or low lipophilicity led to too rapid blood clearance and urinary excretion. The properties of ⁶⁸Ga-labeled peptides are modifiable, as shown with matrix metalloproteinase-9 targeting ligands. In the conclusion of this PhD thesis, ⁶⁸Ga-based agents for PET imaging of cancer and inflammation could be applied in the development of drugs, earlier diagnostics and following-up of the efficacy of therapies.

Structural studies on enzymes of biotechnological and biomedical interest

Structural studies of proteins aim at elucidating the atomic details of molecular interactions in biological processes of living organisms. These studies are particularly important in understanding structure, function and evolution of proteins and in defining their roles in complex biological settings. Furthermore, structural studies can be used for the development of novel properties in biomolecules of environmental, industrial and medical importance. X-ray crystallography is an invaluable tool to obtain accurate and precise information about the structure of proteins at the atomic level. Glutathione transferases (GSTs) are amongst the most versatile enzymes in nature. They are able to catalyze a wide variety of conjugation reactions between glutathione (GSH) and non-polar components containing an electrophilic carbon, nitrogen or sulphur atom. Plant GSTs from the Tau class (a poorly characterized class) play an important role in the detoxification of xenobiotics and stress tolerance. Structural studies were performed on a Tau class fluorodifen-inducible glutathione transferase from Glycine max (GmGSTU4-4) complexed with GSH (2.7 Å) and a product analogue Nb-GSH (1.7 Å). The three-dimensional structure of the GmGSTU4-4-GSH complex revealed that GSH binds in different conformations in the two subunits of the dimer: in an ionized form in one subunit and a non-ionized form in the second subunit. Only the ionized form of the substrate may lead to the formation of a catalytically competent complex. Structural comparison between the GSH and Nb-GSH bound complexes revealed significant differences with respect to the hydrogen-bonding, electrostatic interaction pattern, the upper part of -helix H4 and the C-terminus of the enzyme. These differences indicate an intrasubunit modulation between the G-and Hsites suggesting an induced-fit mechanism of xenobiotic substrate binding. A novel binding site on the surface of the enzyme was also revealed. Bacterial type-II L-asparaginases are used in the treatment of haematopoietic diseases such as acute lymphoblastic leukaemia (ALL) and lymphomas due to their ability to catalyze the conversion of L-asparagine to L-aspartate and ammonia. Escherichia coli and Erwinia chrysanthemi asparaginases are employed for the treatment of ALL for over 30 years. However, serious side-effects affecting the liver and pancreas have been observed due to the intrinsic glutaminase activity of the administered enzymes. Structural studies on Helicobacter pylori L-asparaginase (HpA) were carried out in an effort to discover novel L-asparaginases with potential chemotherapeutic utility in ALL treatment. Detailed analysis of the active site geometry revealed structurally significant differences between HpA and other Lasparaginases that may be important for the biological activities of the enzyme and could be further exploited in protein engineering efforts.

Biogeochemistry in acid sulphate soil landscapes and small urban centres in Western Finland

The overall purpose of this thesis was to increase the knowledge on the biogeochemistry of rural acid sulphate (AS) soil environments and urban forest ecosystems near small towns in Western Finland. In addition, the potential causal relationship between the distribution of AS soils and geographical occurence of multiple sclerosis (MS) disease was assessed based on a review of existing literature and data. Acid sulphate soils, which occupy an area of approximately 17–24 million hectare worldwide, are regarded as the nastiest soils in the world. Independent of the geographical locality of these soils, they pose a great threat to their surrounding environment if disturbed. The abundant metal-rich acid drainage from Finnish AS soils, which is a result of sulphide oxidation due to artificial farmland drainage, has significant but spatially and temporally variable ecotoxicological impacts on biodiversity and community structure of fish, benthic invertebrates and macrophytes. This has resulted in mass fish kills and even eradication of sensitive fish species in affected waters. Moreover, previous investigations demonstrated significantly enriched concentrations of Co, Ni, Mn and Al, metals which are abundantly mobilised in AS soils, in agricultural crops (timothy grass and oats) and approximately 50 times higher concentrations of Al in cow milk originating from AS soils in Western Finland. Nevertheless, the results presented here demonstrate, in general, relatively moderate metal concentrations in oats and cabbage grown on AS soils in Western Finland, although some of the studied fields showed anomalous values of metals (e.g. Co and Ni) in both the soil and target plants (especially oats), similar to that of the previous investigations. The results indicated that the concentrations of Co, Ni, Mn and Zn in oats and Co and Zn in cabbage were governed by soil geochemistry as these metals were correlated with corresponding concentrations extracted from the soil by NH4Ac-EDTA and NH4Ac, respectively. The concentrations of Cu and Fe in oats and cabbage were uncorrelated to that of the easily soluble concentrations in the soils, suggesting that biological processes (e.g. plant-root processes) overshadow geochemical variation. The concentrations of K and Mg in cabbage, which showed a low spread and were strongly correlated to the NH4Ac extractable contents in the soil, were governed by both the bioavailable fractions in the topsoil and plant-uptake mechanisms. The plant´s ability to regulate its uptake of Ca and P (e.g. through root exudates) seemed to be more important than the influence of soil geochemistry. The distribution of P, K, Ca, Mg, Mn and S within humus, moss and needles in and around small towns was to a high degree controlled by biological cycling, which was indicated by the low correlation coefficients for P, K, Ca, Mg and S between humus and moss, and the low spread of these nutrients in moss and needles. The concentration variations of elements in till are mainly due to natural processes (e.g. intrusions, weathering, mineralogical variations in the bedrock). There was a strong spatial pattern for B in humus, moss and needles, which was suggested to be associated with anthropogenic emissions from nearby town centres. Geogenic dust affected the spatial distribution of Fe and Cr in moss, while natural processes governed the Fe anomaly found in the needles. The spatial accumulation patterns of Zn, Cd, Cu, Ni and Pb in humus and moss were strong and diverse, and related to current industry, the former steel industry, coal combustion, and natural geochemical processes. An intriguing Cu anomaly was found in moss. Since it was located close to a main railway line and because the railway line´s electric cables are made of Cu, it was suggested that the reason for the Cu anomaly is corrosion of these cables. In Western Finland, where AS soils are particularly abundant and enrich the metal concentrations of stream waters, cow milk and to some extent crops, an environmental risk assessment would be motivated to elucidate if the metal dispersion affect human health. Within this context, a topic of concern is the distribution of multiple sclerosis as high MS prevalence rates are found in the main area of AS soils. Regionally, the AS soil type in the Seinäjoki area has been demonstrated to be very severe in terms of metal leaching, this area also shows one of the highest MS rates reported worldwide. On a local scale, these severe AS soil types coincide well with the corresponding MS clustering along the Kyrönjoki River in Seinäjoki. There are reasons to suspect that these spatial correlations are causal, as multiple sclerosis has been suggested to result from a combination of genetic and environmental factors.

A cell spot microarray method for high-throughput biology

High-throughput screening of cellular effects of RNA interference (RNAi) libraries is now being increasingly applied to explore the role of genes in specific cell biological processes and disease states. However, the technology is still limited to specialty laboratories, due to the requirements for robotic infrastructure, access to expensive reagent libraries, expertise in high-throughput screening assay development, standardization, data analysis and applications. In the future, alternative screening platforms will be required to expand functional large-scale experiments to include more RNAi constructs, allow combinatorial loss-of-function analyses (e.g. genegene or gene-drug interaction), gain-of-function screens, multi-parametric phenotypic readouts or comparative analysis of many different cell types. Such comprehensive perturbation of gene networks in cells will require a major increase in the flexibility of the screening platforms, throughput and reduction of costs. As an alternative for the conventional multi-well based high-throughput screening -platforms, here the development of a novel cell spot microarray method for production of high density siRNA reverse transfection arrays is described. The cell spot microarray platform is distinguished from the majority of other transfection cell microarray techniques by the spatially confined array layout that allow highly parallel screening of large-scale RNAi reagent libraries with assays otherwise difficult or not applicable to high-throughput screening. This study depicts the development of the cell spot microarray method along with biological application examples of high-content immunofluorescence and phenotype based cancer cell biological analyses focusing on the regulation of prostate cancer cell growth, maintenance of genomic integrity in breast cancer cells, and functional analysis of integrin protein-protein interactions in situ.

Functional Study of Oncogenic Transcription Factor ERG and its Signaling in Prostate Cancer

TMPRSS2–ERG is the most frequent type of genomic rearrangement present in prostate tumors, in which the 5- prime region of the TMPRSS2 gene is fused to the ERG oncogene. TMPRSS2, containing androgen response elements (AREs), is regulated by androgens in the prostate. The truncated TMPRSS2-ERG fusion transcript is overexpressed in half of the prostate cancer patients. The formation of TMPRSS2-ERG transcript is an early event in prostate carcinogenesis and previous in vivo and in vitro studies have shown ectopic ERG expression to be associated with increased cell invasion. However, the molecular function of ERG and its role in cell signaling is poorly understood. In this study, genomic rearrangement of ERG with TMPRSS2 was studied by using comparative genomic hybridization (CGH) in prostate cancer samples. The biological processes associated with the ERG oncogene expression in prostate epithelial cells were studied, and the results were compared with findings observed in clinical prostate tumor samples. The gene expression data indicated that increased WNT signaling and loss of cell adhesion were a characteristic of TMPRSS2- ERG fusion positive prostate tumor samples. Up- regulation of WNT pathway genes were present in ERG positive prostate tumors, with frizzled receptor 4 (FZD4) presenting with the highest association with ERG overexpression, as verified by quantitative reverse transcription-PCR, immunostaining, and immunoblotting in TMPRSS2-ERG positive VCaP prostate cancer cells. Furthermore, ERG and FZD4 silencing increased cell adhesion by inducing active β1-integrin and E-cadherin expression in VCaP cells. Furthermore, we found a novel inhibitor, 4-(chloromethyl) benzoyl chloride which inhibited the WNT signaling and induced similar phenotypic effects as observed after ERG or FZD4 down regulation in VCaP cells. In conclusion, this work deepens our understanding on the complex oncogenic mechanisms of ERG in prostate cancer that may help in developing drugs against TMPRSS2-ERG positive tumors.

Methods for construction and analysis of computational models in systems biology : applications to the modelling of the heat shock response and the self-assembly of intermediate filaments

Systems biology is a new, emerging and rapidly developing, multidisciplinary research field that aims to study biochemical and biological systems from a holistic perspective, with the goal of providing a comprehensive, system- level understanding of cellular behaviour. In this way, it addresses one of the greatest challenges faced by contemporary biology, which is to compre- hend the function of complex biological systems. Systems biology combines various methods that originate from scientific disciplines such as molecu- lar biology, chemistry, engineering sciences, mathematics, computer science and systems theory. Systems biology, unlike “traditional” biology, focuses on high-level concepts such as: network, component, robustness, efficiency, control, regulation, hierarchical design, synchronization, concurrency, and many others. The very terminology of systems biology is “foreign” to “tra- ditional” biology, marks its drastic shift in the research paradigm and it indicates close linkage of systems biology to computer science. One of the basic tools utilized in systems biology is the mathematical modelling of life processes tightly linked to experimental practice. The stud- ies contained in this thesis revolve around a number of challenges commonly encountered in the computational modelling in systems biology. The re- search comprises of the development and application of a broad range of methods originating in the fields of computer science and mathematics for construction and analysis of computational models in systems biology. In particular, the performed research is setup in the context of two biolog- ical phenomena chosen as modelling case studies: 1) the eukaryotic heat shock response and 2) the in vitro self-assembly of intermediate filaments, one of the main constituents of the cytoskeleton. The range of presented approaches spans from heuristic, through numerical and statistical to ana- lytical methods applied in the effort to formally describe and analyse the two biological processes. We notice however, that although applied to cer- tain case studies, the presented methods are not limited to them and can be utilized in the analysis of other biological mechanisms as well as com- plex systems in general. The full range of developed and applied modelling techniques as well as model analysis methodologies constitutes a rich mod- elling framework. Moreover, the presentation of the developed methods, their application to the two case studies and the discussions concerning their potentials and limitations point to the difficulties and challenges one encounters in computational modelling of biological systems. The problems of model identifiability, model comparison, model refinement, model inte- gration and extension, choice of the proper modelling framework and level of abstraction, or the choice of the proper scope of the model run through this thesis.

MicroRNAs as novel regulators of skeletal homeostasis

The human skeleton is composed of bone and cartilage. The differentiation of bone and cartilage cells from their bone marrow progenitors is regulated by an intrinsic network of intracellular and extracellular signaling molecules. In addition, cells coordinate their differentiation and function through reciprocal cell‐to‐cell interactions. MicroRNAs (miRNAs) are small, single‐stranded RNA molecules that inhibit protein translation by binding to messenger RNAs (mRNAs). Recent evidence demonstrates the involvement of miRNAs in multiple biological processes. However, their role in skeletal development and bone remodeling is still poorly understood. The aim of this thesis was to elucidate miRNA‐mediated gene regulation in bone and cartilage cells, namely in osteoblasts, osteoclasts, chondrocytes and bone marrow adipocytes. Comparison of miRNA expression during osteogenic and chondrogenic differentiation of bone marrow‐derived mesenchymal stem cells (MSCs) revealed several miRNAs with substantial difference between bone and cartilage cells. These miRNAs were predicted to target genes essentially involved in MSC differentiation. Three miRNAs, miR‐96, miR‐124 and miR‐199a, showed marked upregulation upon osteogenic, chondrogenic or adipogenic differentiation. Based on functional studies, these miRNAs regulate gene expression in MSCs and may thereby play a role in the commitment and/or differentiation of MSCs. Characterization of miRNA expression during osteoclastogenesis of mouse bone marrow cells revealed a unique expression pattern for several miRNAs. Potential targets of the differentially expressed miRNAs included many molecules essentially involved in osteoclast differentiation. These results provide novel insights into the expression and function of miRNAs during the differentiation of bone and cartilage cells. This information may be useful for the development of novel stem cell‐based treatments for skeletal defects and diseases.

Adaptive Markov chain Monte Carlo and Bayesian filtering for state space models

This thesis is concerned with the state and parameter estimation in state space models. The estimation of states and parameters is an important task when mathematical modeling is applied to many different application areas such as the global positioning systems, target tracking, navigation, brain imaging, spread of infectious diseases, biological processes, telecommunications, audio signal processing, stochastic optimal control, machine learning, and physical systems. In Bayesian settings, the estimation of states or parameters amounts to computation of the posterior probability density function. Except for a very restricted number of models, it is impossible to compute this density function in a closed form. Hence, we need approximation methods. A state estimation problem involves estimating the states (latent variables) that are not directly observed in the output of the system. In this thesis, we use the Kalman filter, extended Kalman filter, Gauss–Hermite filters, and particle filters to estimate the states based on available measurements. Among these filters, particle filters are numerical methods for approximating the filtering distributions of non-linear non-Gaussian state space models via Monte Carlo. The performance of a particle filter heavily depends on the chosen importance distribution. For instance, inappropriate choice of the importance distribution can lead to the failure of convergence of the particle filter algorithm. In this thesis, we analyze the theoretical Lᵖ particle filter convergence with general importance distributions, where p ≥2 is an integer. A parameter estimation problem is considered with inferring the model parameters from measurements. For high-dimensional complex models, estimation of parameters can be done by Markov chain Monte Carlo (MCMC) methods. In its operation, the MCMC method requires the unnormalized posterior distribution of the parameters and a proposal distribution. In this thesis, we show how the posterior density function of the parameters of a state space model can be computed by filtering based methods, where the states are integrated out. This type of computation is then applied to estimate parameters of stochastic differential equations. Furthermore, we compute the partial derivatives of the log-posterior density function and use the hybrid Monte Carlo and scaled conjugate gradient methods to infer the parameters of stochastic differential equations. The computational efficiency of MCMC methods is highly depend on the chosen proposal distribution. A commonly used proposal distribution is Gaussian. In this kind of proposal, the covariance matrix must be well tuned. To tune it, adaptive MCMC methods can be used. In this thesis, we propose a new way of updating the covariance matrix using the variational Bayesian adaptive Kalman filter algorithm.

Sphingosine-1-phosphate-evoked invasion of follicular thyroid cancer cells : evidence for the involvement of HIF-Iα, MMP2 and MMP9

Sphingolipids are widely expressed molecules, which traditionally were considered to have majorly structural properties. Nowadays, however, they are implicated in a wide range of different biological processes. The bioactive lipid sphingosine 1-phosphate (S1P) has emerged during the past decade as one of the most studied molecules due to its proliferative and pro-migratory abilities both during normal physiology and in the pathology of a subset of different diseases. Migration and invasion of cancer cells require changes in cell behavior and modulation of the tissue microenvironment. Tumor aggressiveness is markedly enhanced by hypoxia, in which hypoxia inducible transcription factors 1-2α (HIF-1-2α) are activated to promote metabolism, proliferation and migration. Invasion requires degradation of the extracellular matrix (ECM) achieved by several degrading and remodeling enzymes. Matrix metalloproteinases (MMPs) are broadly expressed and well accepted as proteolytic enzymes with essential roles both in normal physiology and in pathology. Previously, S1P was shown to strongly evoke migration of follicular ML-1 thyroid cancer cells. The objective of this study was to further investigate and understand the mechanisms behind this regulation. In the first project it was demonstrated that S1P enhances the expression and activity of HIF-1α. S1P enhanced the expression of HIF-1α by increasing its synthesis and stability. The S1P-increased HIF-1α was mediated via S1P3, Gi/0, PI3K, PKCβI, ERK1/2, mTOR and translation factors p70S6K and eIF4E. Finally, it was shown that HIF-1α mediated S1P-induced migration. The ECM is constituted of a complex and coordinated assembly of many types of proteins. In order to be able to invade, cells need to break down the ECM, therefore several key players in this event were investigated in the second project. S1P increased the secretion and activity of MMP2 and MMP9 via S1P-receptor 1 and 3 and that these MMPs participated in the S1P-facilitated invasion of ML-1 cells. In this interplay, calpains and Rac1 were involved, both of which are crucial players in migration and invasion. The prognosis for some types of thyroid cancer is relatively good. However, there are forms of thyroid cancers, for which there are no treatments or the current available treatments are inefficient. Thus, new medical interventions are urgently needed. In the third project the significance of the S1P-receptor modulating drug FTY720, which is currently used for the treatment of multiple sclerosis (MS), was studied. The effect of FTY720 was tested on several thyroid cancer cell lines, and it inhibited the proliferation and invasion of all cancer cell lines tested. In ML-1 cells, FTY720 attenuated invasion by blocking signaling intermediates important for migration and invasion of the cells. Moreover, FTY720 inhibited the proliferation of ML-1 cells by increasing the expression of p21 and p27, hence, inducing cell arrest in G1 phase of the cell cycle. Thus, it can be suggested that FTY720 could be used in the treatment of thyroid cancer.

The effects of mutated cationic amino acid transporter y+LAT1 at the cellular and systemic level

y+LAT1 is a transmembrane protein that, together with the 4F2hc cell surface antigen, forms a transporter for cationic amino acids in the basolateral plasma membrane of epithelial cells. It is mainly expressed in the kidney and small intestine, and to a lesser extent in other tissues, such as the placenta and immunoactive cells. Mutations in y+LAT1 lead to a defect of the y+LAT1/4F2hc transporter, which impairs intestinal absorbance and renal reabsorbance of lysine, arginine and ornithine, causing lysinuric protein intolerance (LPI), a rare, recessively inherited aminoaciduria with severe multi-organ complications. This thesis examines the consequences of the LPI-causing mutations on two levels, the transporter structure and the Finnish patients’ gene expression profiles. Using fluorescence resonance energy transfer (FRET) confocal microscopy, optimised for this work, the subunit dimerisation was discovered to be a primary phenomenon occurring regardless of mutations in y+LAT1. In flow cytometric and confocal microscopic FRET analyses, the y+LAT1 molecules exhibit a strong tendency for homodimerisation both in the presence and absence of 4F2hc, suggesting a heterotetramer for the transporter’s functional form. Gene expression analysis of the Finnish patients, clinically variable but homogenic for the LPI-causing mutation in SLC7A7, revealed 926 differentially-expressed genes and a disturbance of the amino acid homeostasis affecting several transporters. However, despite the expression changes in individual patients, no overall compensatory effect of y+LAT2, the sister y+L transporter, was detected. The functional annotations of the altered genes included biological processes such as inflammatory response, immune system processes and apoptosis, indicating a strong immunological involvement for LPI.

Unconventional integrin-ligand interactions

Integrins are cell surface adhesion and signaling receptors. Cells use integrins to attach to the extracellular matrix and to other cells, as well as for sensing their environment. In addition to adhesion and migration, integrins have been shown to be important for many biological processes including apoptosis, cell proliferation, and differentiation into specific tissues. Many important next generation biological drugs inhibit integrin functions. Thus, research into interactions between integrins and their ligands under different physiological and pathological conditions is not only of academic interest, but is also important for the field of drug discovery. In this Ph.D. project, the functions of integrin-ligand interactions were studied under different physiologically interesting conditions including 1) human echovirus 1 binding to integrin α2β1, 2) integrin α2β1 binding to collagen under flow conditions, 3) integrin α2β1 binding to a ligand in the presence of the angiogenesis inhibitor histidine rich glycoprotein (HRG) and 4) integrin binding to posttranslationally citrullinated ligands. As a result of the project, we could show that for each condition the integrin-ligand interaction is somewhat unconventional. 1) Echovirus 1 binds only to non-activated conformations of integrin α2β1. 2) Surprisingly, the non-activated conformation is also the primary conformation of integrin α2β1 when it binds to collagen under flow conditions, like when platelets adhere to subendothelial collagen in vascular injuries. In addition, the pre-activation of integrin α2β1 does not increase adhesion under flow. 3) HRG binds to integrin α2β1 through a low-affinity interaction that inhibits integrin binding to collagen. This shows that low affinity interactions could be biologically relevant and possibly regulate angiogenesis. 4) The citrullination of collagen, a posttranslational modification reported to occur in rheumatoid arthritis, specifically inhibits the binding of integrin α10β1 and α11β1, but does not affect the binding of α1β1 ja α2β1. On the other hand, the citrullination of isoDGR in fibronectin and RGD in pro-TGF- β:n inhibit integrin binding completely. Citrullination seems to be an inflammation related process and integrin ligands become citrullinated frequently in vivo. This Ph.D. thesis suggests that unconventional interaction mechanisms between integrins and their ligands, such as posttranslational modifications, low affinity interactions, and non-activated integrin conformations, can have an important role in pathological processes. The study of these kinds of integrin-ligand interactions is important for understanding biological phenomena more deeply. The research might also be beneficial for the development of integrin based therapies for treating diseases.