On the Rademacher maximal function

Tools known as maximal functions are frequently used in harmonic analysis when studying local behaviour of functions. Typically they measure the suprema of local averages of non-negative functions. It is essential that the size (more precisely, the L^p-norm) of the maximal function is comparable to the size of the original function. When dealing with families of operators between Banach spaces we are often forced to replace the uniform bound with the larger R-bound. Hence such a replacement is also needed in the maximal function for functions taking values in spaces of operators. More specifically, the suprema of norms of local averages (i.e. their uniform bound in the operator norm) has to be replaced by their R-bound. This procedure gives us the Rademacher maximal function, which was introduced by Hytönen, McIntosh and Portal in order to prove a certain vector-valued Carleson's embedding theorem. They noticed that the sizes of an operator-valued function and its Rademacher maximal function are comparable for many common range spaces, but not for all. Certain requirements on the type and cotype of the spaces involved are necessary for this comparability, henceforth referred to as the “RMF-property”. It was shown, that other objects and parameters appearing in the definition, such as the domain of functions and the exponent p of the norm, make no difference to this. After a short introduction to randomized norms and geometry in Banach spaces we study the Rademacher maximal function on Euclidean spaces. The requirements on the type and cotype are considered, providing examples of spaces without RMF. L^p-spaces are shown to have RMF not only for p greater or equal to 2 (when it is trivial) but also for 1 < p < 2. A dyadic version of Carleson's embedding theorem is proven for scalar- and operator-valued functions. As the analysis with dyadic cubes can be generalized to filtrations on sigma-finite measure spaces, we consider the Rademacher maximal function in this case as well. It turns out that the RMF-property is independent of the filtration and the underlying measure space and that it is enough to consider very simple ones known as Haar filtrations. Scalar- and operator-valued analogues of Carleson's embedding theorem are also provided. With the RMF-property proven independent of the underlying measure space, we can use probabilistic notions and formulate it for martingales. Following a similar result for UMD-spaces, a weak type inequality is shown to be (necessary and) sufficient for the RMF-property. The RMF-property is also studied using concave functions giving yet another proof of its independence from various parameters.

Characterization of the molecular components and function of BARE-1, Hin-Mu and Mu transposition machineries

Transposable elements, transposons, are discrete DNA segments that are able to move or copy themselves from one locus to another within or between their host genome(s) without a requirement for DNA homology. They are abundant residents in virtually all the genomes studied, for instance, the genomic portion of TEs is approximately 3% in Saccharomyces cerevisiae, 45% in humans, and apparently more than 70% in some plant genomes such as maize and barley. Transposons plays essential role in genome evolution, in lateral transfer of antibiotic resistance genes among bacteria and in life cycle of certain viruses such as HIV-1 and bacteriophage Mu. Despite the diversity of transposable elements they all use a fundamentally similar mechanism called transpositional DNA recombination (transposition) for the movement within and between the genomes of their host organisms. The DNA breakage and joining reactions that underlie their transposition are chemically similar in virtually all known transposition systems. The similarity of the reactions is also reflected in the structure and function of the catalyzing enzymes, transposases and integrases. The transposition reactions take place within the context of a transposition machinery, which can be particularly complex, as in the case of the VLP (virus like particle) machinery of retroelements, which in vivo contains RNA or cDNA and a number of element encoded structural and catalytic proteins. Yet, the minimal core machinery required for transposition comprises a multimer of transposase or integrase proteins and their binding sites at the element DNA ends only. Although the chemistry of DNA transposition is fairly well characterized, the components and function of the transposition machinery have been investigated in detail for only a small group of elements. This work focuses on the identification, characterization, and functional studies of the molecular components of the transposition machineries of BARE-1, Hin-Mu and Mu. For BARE-1 and Hin-Mu transpositional activity has not been shown previously, whereas bacteriophage Mu is a general model of transposition. For BARE-1, which is a retroelement of barley (Hordeum vulgare), the protein and DNA components of the functional VLP machinery were identified from cell extracts. In the case of Hin-Mu, which is a Mu-like prophage in Haemophilus influenzae Rd genome, the components of the core machinery (transposase and its binding sites) were characterized and their functionality was studied by using an in vitro methodology developed for Mu. The function of Mu core machinery was studied for its ability to use various DNA substrates: Hin-Mu end specific DNA substrates and Mu end specific hairpin substrates. The hairpin processing reaction by MuA was characterized in detail. New information was gained of all three machineries. The components or their activity required for functional BARE-1 VLP machinery and retrotransposon life cycle were present in vivo and VLP-like structures could be detected. The Hin-Mu core machinery components were identified and shown to be functional. The components of the Mu and Hin-Mu core machineries were partially interchangeable, reflecting both evolutionary conservation and flexibility within the core machineries. The Mu core machinery displayed surprising flexibility in substrate usage, as it was able to utilize Hin-Mu end specific DNA substrates and to process Mu end DNA hairpin substrates. This flexibility may be evolutionarily and mechanistically important.

Structure-Function Studies of GDNF Family Ligand-RET signalling

Glial cell line-derived neurotrophic factor (GDNF) and its family members neurturin (NRTN), artemin (ARTN) and persephin (PSPN) are growth factors, which are involved in the development, differentiation and maintenance of many neuron types. In addition, they function outside of the nervous system, e.g. in the development of kidney, testis and liver. GDNF family ligand (GFL) signalling happens through a tetrameric receptor complex, which includes two glycosylphosphatidylinositol (GPI)-anchored GDNF family receptor (GFRα) molecules and two RET (rearranged during transfection) receptor tyrosine kinases. Each of the ligands binds preferentially one of the four GFRα receptors: GDNF binds to GFRα1, NRTN to GFRα2, ARTN to GFRα3 and PSPN to GFRα4. The signal is then delivered by RET, which cannot bind the GFLs on its own, but can bind the GFL-GFRα complex. Under normal cellular conditions, RET is only phosphorylated on the cell surface after ligand binding. At least the GDNF-GFRα1 complex is believed to recruit RET to lipid rafts, where downstream signalling occurs. In general, GFRαs consist of three cysteine-rich domains, but all GFRα4s except for chicken GFRα4 lack domain 1 (D1). We characterised the biochemical and cell biological properties of mouse PSPN receptor GFRα4 and showed that it has a significantly weaker capacity than GFRα1 to recruit RET to the lipid rafts. In spite of that, it can phosphorylate RET in the presence of PSPN and contribute to neuronal differentiation and survival. Therefore, the recruitment of RET to the lipid rafts does not seem to be crucial for the biological activity of all GFRα receptors. Secondly, we demonstrated that GFRα1 D1 stabilises the GDNF-GFRα1 complex and thus affects the phosphorylation of RET and contributes to the biological activity. This may be important in physiological conditions, where the concentration of the ligand or the soluble GFRα1 receptor is low. Our results also suggest a role for D1 in heparin binding and, consequently, in the biodistribution of released GFRα1 or in the formation of the GFL-GFRα-RET complex. We also presented the crystallographic structure of GDNF in the complex with GFRα1 domains 2 and 3. The structure differs from the previously published ARTN-GFRα3 structure in three significant ways. The biochemical data verify the structure and reveal residues participating in the interactions between GFRα1 and GDNF, and preliminarily also between GFRα1 and RET and heparin. Finally, we showed that, the precursor of the oncogenic MEN 2B (multiple endocrine neoplasia type 2) form of RET gets phosphorylated already during its synthesis in the endoplasmic reticulum (ER). We also demonstrated that it associates with Src homology 2 domain-containing protein (SHC) and growth factor receptor-bound protein (GRB2) in the ER, and has the capacity to activate several downstream signalling molecules.

Causes and consequences of variation in nestling immune function

Defence against pathogens is a vital need of all living organisms that has led to the evolution of complex immune mechanisms. However, although immunocompetence the ability to resist pathogens and control infection has in recent decades become a focus for research in evolutionary ecology, the variation in immune function observed in natural populations is relatively little understood. This thesis examines sources of this variation (environmental, genetic and maternal effects) during the nestling stage and its fitness consequences in wild populations of passerines: the blue tit (Cyanistes caeruleus) and the collared flycatcher (Ficedula albicollis). A developing organism may face a dilemma as to whether to allocate limited resources to growth or to immune defences. The optimal level of investment in immunity is shaped inherently by specific requirements of the environment. If the probability of contracting infection is low, maintaining high growth rates even at the expense of immune function may be advantageous for nestlings, as body mass is usually a good predictor of post-fledging survival. In experiments with blue tits and haematophagous hen fleas (Ceratophyllus gallinae) using two methods, methionine supplementation (to manipulate nestlings resource allocation to cellular immune function) and food supplementation (to increase resource availability), I confirmed that there is a trade-off between growth and immunity and that the abundance of ectoparasites is an environmental factor affecting allocation of resources to immune function. A cross-fostering experiment also revealed that environmental heterogeneity in terms of abundance of ectoparasites may contribute to maintaining additive genetic variation in immunity and other traits. Animal model analysis of extensive data collected from the population of collared flycatchers on Gotland (Sweden) allowed examination of the narrow-sense heritability of PHA-response the most commonly used index of cellular immunocompetence in avian studies. PHA-response is not heritable in this population, but is subject to a non-heritable origin (presumably maternal) effect. However, experimental manipulation of yolk androgen levels indicates that the mechanism of the maternal effect in PHA-response is not in ovo deposition of androgens. The relationship between PHA-response and recruitment was studied for over 1300 collared flycatcher nestlings. Multivariate selection analysis shows that it is body mass, not PHA-response, that is under direct selection. PHA-response appears to be related to recruitment because of its positive relationship with body mass. These results imply that either PHA-response fails to capture the immune mechanisms that are relevant for defence against pathogens encountered by fledglings or that the selection pressure from parasites is not as strong as commonly assumed.

Microarrays in Lung Cancer Research: From Comparative Analyses to Verified Findings

Keuhkosyöpä on yleisimpiä syöpätauteja. Se jaetaan kahteen päätyyppiin: pienisoluiseen ja ei-pienisoluiseen keuhkosyöpään. Ei-pienisoluinen keuhkosyöpä jaetaan lisäksi alatyyppeihin, joista suurimmat ovat levyepiteeli-, adeno- ja suurisoluinen karsinooma. Keuhkosyövän tärkein riskitekijä on tupakointi, mutta muutkin työ- ja elinympäristön altisteet, kuten asbesti, voivat johtaa syöpään. Väitöstyössä tutkittiin kahdenlaisten keuhkosyöpäryhmien erityispiirteitä. Työssä kartoitettiin, onko löydettävissä muutoksia, jotka erottavat asbestikeuhkosyövät muista syövistä sekä luuytimeen varhaisessa vaiheessa leviävät keuhkosyövät leviämättömistä syövistä. Tutkimusten ensimmäisessä vaiheessa käytettiin mikrosirupohjaisia menetelmiä, jotka mahdollistavat jopa kaikkien geenien tarkastelun yhden kokeen avulla. Vertailevien mikrosirututkimusten avulla on mahdollista paikantaa geenejä tai kromosomialueita, joiden muutokset erottelevat ryhmät toisistaan. Asbestiin liittyvissä tutkimuksissa paikannettiin kuusi kromosomialuetta, joissa geenien kopiolukumäärän sekä ilmenemistason muutokset erottelivat potilaat altistushistorian mukaan. Riippumattomilla laboratoriomenetelmillä tehtyjen jatkoanalyysien avulla pystyttiin varmistamaan, että 19p-alueen häviämä oli yhteydessä asbestialtistukseen. Työssä osoitettiin myös, että 19p-alueen muutoksia voidaan indusoida altistamalla soluja asbestille in vitro. Tutkimuksessa saatiin lisäksi viitteitä asbestispesifisistä muutoksista signaalinvälitysreiteissä, sillä yhdessä toimivien geenien ilmentymisessä havaittiin eroja asbestille altistuneiden ja altistumattomien välillä. Vertailemalla luuytimeen syövän aikaisessa vaiheessa levinneiden ja leviämättömien keuhkoadenokarsinoomien muutosprofiileita toisiinsa, paikannettiin viisi aluetta, joilla geenien kopiolukumäärä- sekä ilmenemistason muutokset erottelivat ryhmät toisistaan. Jatkoanalyyseissä havaittiin, että 4q-alueen häviämää esiintyi adenokarsinoomien lisäksi levyepiteelikarsinoomiin, jotka olivat levinneet luuytimeen. Myös keuhkosyöpien aivometastaaseissa alue oli toistuvasti hävinnyt. Väitöstyön tutkimukset osoittavat, että vertailevien mikrosiruanalyysien avulla saadaan tietoa syöpäryhmien erityispiirteistä. Työssä saadut tulokset osoittavat, että 19p-alueen muutokset ovat tyypillisiä asbestikeuhkosyöville ja 4q-alueen muutokset luuytimeen aikaisessa vaiheessa leviäville keuhkosyöville.

Type III Secretion System of Phytopathogenic Bacterium Pseudomonas syringae:From Gene to Function

The type III secretion system (T3SS) is an essential requirement for the virulence of many Gram-negative bacteria which infect plants, animals and men. Pathogens use the T3SS to deliver effector proteins from the bacterial cytoplasm to the eukaryotic host cells, where the effectors subvert host defenses. The best candidates for directing effector protein traffic are the bacterial type III-associated appendages, called needles or pili. In plant pathogenic bacteria, the best characterized example of a T3SS-associated appendage is the HrpA pilus of the plant pathogen Pseudomonas syringae pv. tomato DC3000. The components of the T3SS in plant pathogens are encoded by a cluster of hrp (hypersensitive reaction and pathogenicity) genes. Two major classes of T3SS-secreted proteins are: harpin proteins such as HrpZ which are exported into extracellular space, and avirulence (Avr) proteins such as AvrPto which are translocated directly to the plant cytoplasm. This study deals with the structural and functional characterization of the T3SS-associated HrpA pilus and the T3SS-secreted harpins. By insertional mutagenesis analysis of HrpA, we located the optimal epitope insertion site in the amino-terminus of HrpA, and revealed the potential application of the HrpA pilus as a carrier of antigenic determinants for vaccination. By pulse-expression of proteins combined with immuno-electron microscopy, we discovered the Hrp pilus assembly strategy as addition of HrpA subunits to the distal end of the growing pilus, and we showed for the first time that secretion of HrpZ occurs at the tip of the pilus. The pilus thus functions as a conduit delivering proteins to the extracellular milieu. By using phage-display and scanning-insertion mutagenesis methods we identified a conserved HrpZ-binding peptide and localized the peptide-binding site to the central domain of HrpZ. We also found that the HrpZ specifically interacts with a host bean protein. Taken together, the current results provide deeper insight into the molecular mechanism of T3SS-associated pilus assembly and effector protein translocation, which will be helpful for further studies on the pathogenic mechanisms of Gram-negative bacteria and for developing new strategies to prevent bacterial infection.

Structure, function and intracellular dynamics of alphavirus replication complexes

Intracellular membrane alterations are hallmarks of positive-sense RNA (+RNA) virus replication. Strong evidence indicates that within these exotic compartments, viral replicase proteins engage in RNA genome replication and transcription. To date, fundamental questions such as the origin of altered membranes, mechanisms of membrane deformation and topological distribution and function of viral components, are still waiting for comprehensive answers. This study addressed some of the above mentioned questions for the membrane alterations induced during Semliki Forest virus (SFV) infection of mammalian cells. With the aid of electron and fluorescence microscopy coupled with radioactive labelling and immuno-cytochemistry techniques, our group and others showed that few hours after infection the four non structural proteins (nsP1-4) and newly synthesized RNAs of SFV colocalized in close proximity of small membrane invaginations, designated as spherules . These 50-70 nm structures were mainly detected in the perinuclear area, at the limiting membrane of modified endosomes and lysosomes, named CPV-I (cytopathic vacuoles type I). More rarely, spherules were also found at the plasma membrane (PM). In the first part of this study I present the first three-dimensional reconstruction of the CPV-I and the spherules, obtained by electron tomography after chemical or cryo-fixation. Different approaches for imaging these macromolecular assemblies to obtain better structure preservation and higher resolution are presented as unpublished data. This study provides insights into spherule organization and distribution of viral components. The results of this and other experiments presented in this thesis will challenge currently accepted models for virus replication complex formation and function. In a revisitation of our previous models, the second part of this work provides the first complete description of the biogenesis of the CPV-I. The results demonstrate that these virus-induced vacuoles, where hundreds of spherules accumulate at late stages during infection, represent the final phase of a journey initiated at the PM, which apparently serves as a platform for spherule formation. From the PM spherules were internalized by an endocytic event that required the activity of the class I PI3K, caveolin-1, cellular cholesterol and functional actin-myosin network. The resulting neutral endocytic carrier vesicle delivered the spherules to the membrane of pre-existing acidic endosomes via multiple fusion events. Microtubule based transport supported the vectorial transfer of these intermediates to the pericentriolar area where further fusions generated the CPV-I. A signal for spherule internalization was identified in one of the replicase proteins, nsP3. Infections of cells with viruses harbouring a deletion in a highly phosphorylated region of nsP3 did not result in the formation of CPV-Is. Instead, thousands of spherules remained at the PM throughout the infection cycle. Finally, the role of the replicase protein nsP2 during viral RNA replication and transcription was investigated. Three enzymatic activities, protease, NTPase and RNA-triphosphatase were studied with the aid of temperature sensitive mutants in vitro and, when possible, in vivo. The results highlighted the interplay of the different nsP2 functions during different steps of RNA replication and sub-genomic promoter regulation, and suggest that the protein could have different activities when participating in the replication complex or as a free enzyme.

Molecular Alterations in Asbestos-Related Lung Cancer

Background: Asbestos is a well known cancer-causing mineral fibre, which has a synergistic effect on lung cancer risk in combination with tobacco smoking. Several in vitro and in vivo experiments have demonstrated that asbestos can evoke chromosomal damage and cause alterations as well as gene expression changes. Lung tumours, in general, have very complex karyotypes with several recurrently gained and lost chromosomal regions and this has made it difficult to identify specific molecular changes related primarily to asbestos exposure. The main aim of these studies has been to characterize asbestos-related lung cancer at a molecular level. Methods: Samples from asbestos-exposed and non-exposed lung cancer patients were studied using array comparative genomic hybridization (aCGH) and fluorescent in situ hybridization (FISH) to detect copy number alterations (CNA) as well as microsatellite analysis to detect allelic imbalance (AI). In addition, asbestos-exposed cell lines were studied using gene expression microarrays. Results: Eighteen chromosomal regions showing differential copy number in the lung tumours of asbestos-exposed patients compared to those of non-exposed patients were identified. The most significant differences were detected at 2p21-p16.3, 5q35.3, 9q33.3-q34.11, 9q34.13-q34.3, 11p15.5, 14q11.2 and 19p13.1-p13.3 (p<0.005). The alterations at 2p and 9q were validated and characterized in detail using AI and FISH analysis in a larger study population. Furthermore, in vitro studies were performed to examine the early gene expression changes induced by asbestos in three different lung cell lines. The results revealed specific asbestos-associated gene expression profiles and biological processes as well as chromosomal regions enriched with genes believed to contribute to the common asbestos-related responses in the cell lines. Interestingly, the most significant region enriched with asbestos-response genes was identified at 2p22, close to the previously identified region showing asbestos-related CNA in lung tumours. Additionally, in this thesis, the dysregulated biological processes (Gene Ontology terms) detected in the cell line experiment were compared to dysregulated processes identified in patient samples in a later study (Ruosaari et al., 2008a). Commonly affected processes such as those related to protein ubiquitination, ion transport and surprisingly sensory perception of smell were identified. Conclusions: The identification of specific CNA and dysregulated biological processes shed some light on the underlying genes acting as mediators in asbestos-related lung carcinogenesis. It is postulated that the combination of several asbestos-specific molecular alterations could be used to develop a diagnostic method for the identification of asbestos-related lung cancer.

Exposure-related human cancer: Molecular changes in sinonasal cancer and lung cancer, with focus on TP53 mutations

Cancer is a leading cause of death worldwide and the total number of cancer cases continues to increase. Many cancers, for example sinonasal cancer and lung cancer, have clear external risk factors and so are potentially preventable. The occurrence of sinonasal cancer is strongly associated with wood dust exposure and the main risk factor for lung cancer is tobacco smoking. Although the molecular mechanisms involved in lung carcinogenesis have been widely studied, very little is known about the molecular changes leading to sinonasal cancer. In this work, mutations in the tumour suppressor TP53 gene in cases of sinonasal cancer and lung cancer and the associations of these mutations with exposure factors were studied. In addition, another important mechanism in many cancers, inflammation, was explored by analyzing the expression of the inflammation related enzyme, COX-2, in sinonasal cancer. The results demonstrate that TP53 mutations are frequent in sinonasal cancer and lung cancer and in both cancers they are associated with exposure. In sinonasal cancer, the occurrence of TP53 mutation significantly increased in relation to long duration and high level of exposure to wood dust. Smoking was not associated with the overall occurrence of the TP53 mutation in sinonasal cancer, but was associated with multiple TP53 mutations. Furthermore, inflammation appears to play a part in sinonasal carcinogenesis as indicated by our results showing that the expression of COX-2 was associated with adenocarcinoma type of tumours, wood dust exposure and non-smoking. In lung cancer, we detected statistically significant associations between TP53 mutations and duration of smoking, gender and histology. We also found that patients with a tumour carrying a G to T transversion, a mutation commonly found in association with tobacco smoking, had a high level of smoking-related bulky DNA adducts in their non-tumorous lung tissue. Altogether, the information on molecular changes in exposure induced cancers adds to the observations from epidemiological studies and helps to understand the role and impact of different etiological factors, which in turn can be beneficial for risk assessment and prevention.

Structure and function of GDNF receptor alpha splice variants

The growth factors of the glial cell line-derived neurotrophic factor (GDNF) family consisting of GDNF, neurturin (NRTN), artemin (ARTN) and persephin (PSPN), are involved in the development, differentiation and maintenance of many types of neurons. They also have important functions outside the nervous system in the development of kidney, testis and thyroid gland. Each of these GFLs preferentially binds to one of the glycosylphosphatidylinositol (GPI)-anchored GDNF family receptors α (GFRα). GDNF binds to GFRα1, NRTN to GFRα2, ARTN to GFRα3 and PSPN to GFRα4. The GFLs in the complex with their cognate GFRα receptors all bind to and signal through the receptor tyrosine kinase RET. Alternative splicing of the mouse GFRα4 gene yields three splice isoforms. These had been described as putative GPI-anchored, transmembrane and soluble forms. My goal was to characterise the function of the different forms of mouse GFRα4. I firstly found that the putative GPI-anchored GFRα4 (GFRα4-GPI) is glycosylated, membrane-bound, GPI-anchored and interacts with PSPN and RET. We also showed that mouse GFRα4-GPI mediates PSPN-induced phosphorylation of RET, promotes PSPN-dependent neuronal differentiation of the rat pheochromocytoma cell line PC6-3 and PSPN-dependent survival of cerebellar granule neurons (CGN). However, although this receptor can mediate PSPN-signalling and activate RET, GFRα4-GPI does not recruit RET into lipid rafts. The recruitment of RET into lipid rafts has previously been thought to be a crucial event for GDNF- and GFL-mediated signalling via RET. I secondly demonstrated that the putative transmembrane GFRα4 (GFRα4-TM) is indeed a real transmembrane GFRα4 protein. Although it has a weak binding capacity for PSPN, it can not mediate PSPN-dependent phosphorylation of RET, neuronal differentiation or survival. These data show that GFRα4-TM is inactive as a receptor for PSPN. Surprisingly, GFRα4-TM can negatively regulate PSPN-mediated signalling via GFRα4-GPI. GFRα4-TM interacts with GFRα4-GPI and blocks PSPN-induced phosphorylation of RET, neuronal differentiation as well as survival. Taken together, our data show that GFRα4-TM may act as a dominant negative inhibitor of PSPN-mediated signaling. The most exciting part of my work was the finding that the putative soluble GFRα4 (GFRα4-sol) can form homodimers and function as an agonist of the RET receptor. In the absence of PSPN, GFRα4-sol can promote the phosphorylation of RET, trigger the activation of the PI-3K/AKT pathway, induce neuronal differentiation and support the survival of CGN. Our findings are in line with a recent publication showing the GFRα4-sol might contribute to the inherited cancer syndrome multiple endocrine neoplasia type 2. Our data provide an explanation to how GFRα4-sol may cause or modify the disease. Mammalian GFRα4 receptors all lack the first Cys-rich domain which is present in other GFRα receptors. In the final part of my work I have studied the function of this particular domain. I created a truncated GFRα1 construct lacking the first Cys-rich domain. Using binding assays in both cellular and cell-free systems, phosphorylation assays with RET, as well as neurite outgrowth assays, we found that the first Cys-rich domain contributes to an optimal function of GFRα1, by stabilizing the interaction between GDNF and GFRα1.