Hematopoietic Stem Cell Development and Transcriptional Regulation

The continuous production of blood cells, a process termed hematopoiesis, is sustained throughout the lifetime of an individual by a relatively small population of cells known as hematopoietic stem cells (HSCs). HSCs are unique cells characterized by their ability to self-renew and give rise to all types of mature blood cells. Given their high proliferative potential, HSCs need to be tightly regulated on the cellular and molecular levels or could otherwise turn malignant. On the other hand, the tight regulatory control of HSC function also translates into difficulties in culturing and expanding HSCs in vitro. In fact, it is currently not possible to maintain or expand HSCs ex vivo without rapid loss of self-renewal. Increased knowledge of the unique features of important HSC niches and of key transcriptional regulatory programs that govern HSC behavior is thus needed. Additional insight in the mechanisms of stem cell formation could enable us to recapitulate the processes of HSC formation and self-renewal/expansion ex vivo with the ultimate goal of creating an unlimited supply of HSCs from e.g. human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPS) to be used in therapy. We thus asked: How are hematopoietic stem cells formed and in what cellular niches does this happen (Papers I, II)? What are the molecular mechanisms that govern hematopoietic stem cell development and differentiation (Papers III, IV)? Importantly, we could show that placenta is a major fetal hematopoietic niche that harbors a large number of HSCs during midgestation (Paper I)(Gekas et al., 2005). In order to address whether the HSCs found in placenta were formed there we utilized the Runx1-LacZ knock-in and Ncx1 knockout mouse models (Paper II). Importantly, we could show that HSCs emerge de novo in the placental vasculature in the absence of circulation (Rhodes et al., 2008). Furthermore, we could identify defined microenvironmental niches within the placenta with distinct roles in hematopoiesis: the large vessels of the chorioallantoic mesenchyme serve as sites of HSC generation whereas the placental labyrinth is a niche supporting HSC expansion (Rhodes et al., 2008). Overall, these studies illustrate the importance of distinct milieus in the emergence and subsequent maturation of HSCs. To ensure proper function of HSCs several regulatory mechanisms are in place. The microenvironment in which HSCs reside provides soluble factors and cell-cell interactions. In the cell-nucleus, these cell-extrinsic cues are interpreted in the context of cell-intrinsic developmental programs which are governed by transcription factors. An essential transcription factor for initiation of hematopoiesis is Scl/Tal1 (stem cell leukemia gene/T-cell acute leukemia gene 1). Loss of Scl results in early embryonic death and total lack of all blood cells, yet deactivation of Scl in the adult does not affect HSC function (Mikkola et al., 2003b. In order to define the temporal window of Scl requirement during fetal hematopoietic development, we deactivated Scl in all hematopoietic lineages shortly after hematopoietic specification in the embryo . Interestingly, maturation, expansion and function of fetal HSCs was unaffected, and, as in the adult, red blood cell and platelet differentiation was impaired (Paper III)(Schlaeger et al., 2005). These findings highlight that, once specified, the hematopoietic fate is stable even in the absence of Scl and is maintained through mechanisms that are distinct from those required for the initial fate choice. As the critical downstream targets of Scl remain unknown, we sought to identify and characterize target genes of Scl (Paper IV). We could identify transcription factor Mef2C (myocyte enhancer factor 2 C) as a novel direct target gene of Scl specifically in the megakaryocyte lineage which largely explains the megakaryocyte defect observed in Scl deficient mice. In addition, we observed an Scl-independent requirement of Mef2C in the B-cell compartment, as loss of Mef2C leads to accelerated B-cell aging (Gekas et al. Submitted). Taken together, these studies identify key extracellular microenvironments and intracellular transcriptional regulators that dictate different stages of HSC development, from emergence to lineage choice to aging.

Knowledge unlatched : A global library consortium model for funding Open Access scholarly books

Specialist scholarly books, including monographs, allow researchers to present their work, pose questions and to test and extend areas of theory through long-form writing. In spite of the fact that research communities all over the world value monographs and depend heavily on them as a requirement of tenure and promotion in many disciplines, sales of this kind of book are in free fall, with some estimates suggesting declines of as much as 90% over twenty years (Willinsky 2006). Cashstrapped monograph publishers have found themselves caught in a negative cycle of increasing prices and falling sales, with few resources left to support experimentation, business model innovation or engagement with digital technology and Open Access (OA). This chapter considers an important attempt to tackle failing markets for scholarly monographs, and to enable the wider adoption of OA licenses for book-length works: the 2012 – 2014 Knowledge Unlatched pilot. Knowledge Unlatched is a bold attempt to reconfigure the market for specialist scholarly books: moving it beyond the sale of ‘content’ towards a model that supports the services valued by scholarly and wider communities in the context of digital possibility. Its success has powerful implications for the way we understand copyright’s role in the creative industries, and the potential for established institutions and infrastructure to support the open and networked dynamics of a digital age.

Tumour necrosis factor receptor-associated periodic syndrome (TRAPS) : Identification of novel TNFRSF1A mutations and intracellular signalling defects

The systemic autoinflammatory disorders are a group of rare diseases characterized by periodically recurring episodes of acute inflammation and a rise in serum acute phase proteins, but with no signs of autoimmunity. At present eight hereditary syndromes are categorized as autoinflammatory, although the definition has also occasionally been extended to other inflammatory disorders, such as Crohn s disease. One of the autoinflammatory disorders is the autosomally dominantly inherited tumour necrosis factor receptor-associated periodic syndrome (TRAPS), which is caused by mutations in the gene encoding the tumour necrosis factor type 1 receptor (TNFRSF1A). In patients of Nordic descent, cases of TRAPS and of three other hereditary fevers, hyperimmunoglobulinemia D with periodic fever syndrome (HIDS), chronic infantile neurologic, cutaneous and articular syndrome (CINCA) and familial cold autoinflammatory syndrome (FCAS), have been reported, TRAPS being the most common of the four. Clinical characteristics of TRAPS are recurrent attacks of high spiking fever, associated with inflammation of serosal membranes and joints, myalgia, migratory rash and conjunctivitis or periorbital cellulitis. Systemic AA amyloidosis may occur as a sequel of the systemic inflammation. The aim of this study was to investigate the genetic background of hereditary periodically occurring fever syndromes in Finnish patients, to explore the reliability of determining serum concentrations of soluble TNFRSF1A and metalloproteinase-induced TNFRSF1A shedding as helpful tools in differential diagnostics, as well as to study intracellular NF-κB signalling in an attempt to widen the knowledge of the pathomechanisms underlying TRAPS. Genomic sequencing revealed two novel TNFRSF1A mutations, F112I and C73R, in two Finnish families. F112I was the first TNFRSF1A mutation to be reported in the third extracellular cysteine-rich domain of the gene and C73R was the third novel mutation to be reported in a Finnish family, with only one other TNFRSF1A mutation having been reported in the Nordic countries. We also presented a differential diagnostic problem in a TRAPS patient, emphasizing for the clinician the importance of differential diagnostic vigiliance in dealing with rare hereditary disorders. The underlying genetic disease of the patient both served as a misleading factor, which possibly postponed arrival at the correct diagnosis, but may also have predisposed to the pathologic condition, which led to a critical state of the patient. Using a method of flow cytometric analysis modified for the use on fresh whole blood, we studied intracellular signalling pathways in three Finnish TRAPS families with the F112I, C73R and the previously reported C88Y mutations. Evaluation of TNF-induced phosphorylation of NF-κB and p38, revealed low phosphorylation profiles in nine out of ten TRAPS patients in comparison to healthy control subjects. This study shows that TRAPS is a diagnostic possibility in patients of Nordic descent, with symptoms of periodically recurring fever and inflammation of the serosa and joints. In particular in the case of a family history of febrile episodes, the possibility of TRAPS should be considered, if an etiology of autoimmune or infectious nature is excluded. The discovery of three different mutations in a population as small as the Finnish, reinforces the notion that the extracellular domain of TNFRSF1A is prone to be mutated at the entire stretch of its cysteine-rich domains and not only at a limited number of sites, suggesting the absence of a founder effect in TRAPS. This study also demonstrates the challenges of clinical work in differentiating the symptoms of rare genetic disorders from those of other pathologic conditions and presents the possibility of an autoinflammatory disorder as being the underlying cause of severe clinical complications. Furthermore, functional studies of fresh blood leukocytes show that TRAPS is often associated with a low NF-κB and p38 phosphorylation profile, although low phosphorylation levels are not a requirement for the development of TRAPS. The aberrant signalling would suggest that the hyperinflammatory phenotype of TRAPS is the result of compensatory NF-κB-mediated regulatory mechanisms triggered by a deficiency of the innate immune response.

Molecular Genetics of Age-related Macular Degeneration

Age-related macular degeneration (AMD; OMIM # 603075) is an eye disease of the elderly, signs of which appear after the age of 50. In the Western world it is a leading cause of permanent visual loss with a prevalence of 8.5% in persons under 54 years of age and of 37% in persons over 75 years of age. Early forms of AMD may be asymptomatic, but in the late forms usually a central scotoma in the visual field follows severely complicating daily tasks. Smoking, age, and genetic predisposition are known risk factors for AMD. Until recently no true susceptibility genes had been identified though the composition of drusen deposits, the hallmarks of AMD, has suggested that the complement system might play a role in the pathogenesis of AMD. When four groups reported in March 2005, that, on chromosome 1q32, a Y402H variant in the complement factor H (CFH) gene confers risk for AMD in independent Caucasian samples, a new period in the field of genetic research of AMD started. CFH is a key regulator of the complement system. Thus, it is logical to speculate, that it plays a role in the pathogenesis of AMD. We performed a case-control association study to analyse whether the CFH Y402H variant contain a risk for AMD in the Finnish population. Although the population of Finland represents a genetic isolate, the CFH Y402H polymorphism was associated with AMD also in our patient sample with similar risk allele frequencies as in the other Caucasian populations. We further evaluated the effects of this variant, but no association between lesion subtype (predominantly classic, minimally classic or occult lesion) or lesion size of neovascular AMD and the CFH Y402H variant was detected. Neither did the variant have an effect on the photodynamic therapy (PDT) outcome. The patients that respond to PDT carried the risk genotype as frequently as those who did not respond, and no difference was found in the number of PDT sessions needed in patients with or without the risk genotypes of CFH Y402H. Functional analyses, however, showed that the binding of C-reactive protein (CRP) to CFH was significantly reduced in patients with the risk genotype of Y402H. In the past two years, the LOC387715/ high-temperature requirement factor A1 (HTRA1) locus on 10q26 has also been repeatedly associated with AMD in several populations. The recent discovery of the LOC387715 protein on the mitochondrial outer membrane suggests that the LOC387715 gene, not HTRA1, is the true predisposing gene in this region, although its biological function is still unknown. In our Finnish patient material, patients with AMD carried the A69S risk genotype of LOC387715 more frequently than the controls. Also, for the first time, an interaction between the CFH Y402H and the LOC387715 A69S variants was found. The most recently detected susceptibilty gene of AMD, the complement component 3 (C3) gene, encodes the central component of the complement system, C3. In our Finnish sample, an additive gene effect for the C3 locus was detected, though weaker than the effects for the two main loci, CFH and LOC387715. Instead, the hemicentin-1 or the elongation of very long chain fatty acids-like 4 genes that have also been suggested as candidate genes for AMD did not carry a risk for AMD in the Finnish population. This was the first series of molecular genetic study of AMD in Finland. We showed that two common risk variants, CFH Y402H and LOC387715 A69S, represent a high risk of AMD also in the isolated Finnish population, and furthermore, that they had a statistical interaction. It was demonstrated that the CFH Y402H risk genotype affects the binding of CFH to CRP thus suggesting that complement indeed plays an important role in the pathogenesis of AMD.

Structural analysis of the roles of influenza A virus membrane-associated proteins in assembly and morphology

The assembly of influenza A virus at the plasma membrane of infected cells leads to release of enveloped virions that are typically round in tissue culture-adapted strains but filamentous in strains isolated from patients. The viral proteins hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1), and M2 ion channel all contribute to virus assembly. When expressed individually or in combination in cells, they can all, under certain conditions, mediate release of membrane-enveloped particles, but their relative roles in virus assembly, release, and morphology remain unclear. To investigate these roles, we produced membrane-enveloped particles by plasmid-derived expression of combinations of HA, NA, and M proteins (M1 and M2) or by infection with influenza A virus. We monitored particle release, particle morphology, and plasma membrane morphology by using biochemical methods, electron microscopy, electron tomography, and cryo-electron tomography. Our data suggest that HA, NA, or HANA (HA plus NA) expression leads to particle release through nonspecific induction of membrane curvature. In contrast, coexpression with the M proteins clusters the glycoproteins into filamentous membrane protrusions, which can be released as particles by formation of a constricted neck at the base. HA and NA are preferentially distributed to differently curved membranes within these particles. Both the budding intermediates and the released particles are morphologically similar to those produced during infection with influenza A virus. Together, our data provide new insights into influenza virus assembly and show that the M segment together with either of the glycoproteins is the minimal requirement to assemble and release membrane-enveloped particles that are truly virus-like.

Energy efficient area monitoring using information coverage in wireless sensor networks

In this paper, we are concerned with energy efficient area monitoring using information coverage in wireless sensor networks, where collaboration among multiple sensors can enable accurate sensing of a point in a given area-to-monitor even if that point falls outside the physical coverage of all the sensors. We refer to any set of sensors that can collectively sense all points in the entire area-to-monitor as a full area information cover. We first propose a low-complexity heuristic algorithm to obtain full area information covers. Using these covers, we then obtain the optimum schedule for activating the sensing activity of various sensors that maximizes the sensing lifetime. The scheduling of sensor activity using the optimum schedules obtained using the proposed algorithm is shown to achieve significantly longer sensing lifetimes compared to those achieved using physical coverage. Relaxing the full area coverage requirement to a partial area coverage (e.g., 95% of area coverage as adequate instead of 100% area coverage) further enhances the lifetime.

Novel fluorescence detection technique for non-contact temperature sensing in microchip PCR

DNA amplification using Polymerase Chain Reaction (PCR) in a small volume is used in Lab-on-a-chip systems involving DNA manipulation. For few microliters of volume of liquid, it becomes difficult to measure and monitor the thermal profile accurately and reproducibly, which is an essential requirement for successful amplification. Conventional temperature sensors are either not biocompatible or too large and hence positioned away from the liquid leading to calibration errors. In this work we present a fluorescence based detection technique that is completely biocompatible and measures directly the liquid temperature. PCR is demonstrated in a 3 ILL silicon-glass microfabricated device using non-contact induction heating whose temperature is controlled using fluorescence feedback from SYBR green I dye molecules intercalated within sensor DNA. The performance is compared with temperature feedback using a thermocouple sensor. Melting curve followed by gel electrophoresis is used to confirm product specificity after the PCR cycles. (c) 2007 Elsevier B.V. All rights reserved.

Scale-up criteria of square tank surface aerator

Oxygen transfer rate and the corresponding power requirement to operate the rotor are vital for design and scale-up of surface aerators. Present study develops simulation or scale-up criterion correlating the oxygen transsimulation fer coefficient and power number along with a parameter governing theoretical power per unit volume (X, which is defined as equal to (FR1/3)-R-4/3, where F and R are impellers' Fronde and Reynolds number, respectively). Based on such scale-up criteria, design considerations are developed to save energy requirements while designing square tank surface aerators. It has been demonstrated that energy can be saved substantially if the aeration tanks are run at relatively higher input powers. It is also demonstrated that smaller sized tanks are more energy conservative and economical when compared to big sized tanks, while aerating the same volume of water, and at the same time by maintaining a constant input power in all the tanks irrespective of their size. An example illustrating how energy can be reduced while designing different sized aerators is given. The results presented have a wide application in biotechnology and bioengineering areas with a particular emphasis on the design of appropriate surface aeration systems.

Scale-up criteria of square tank surface aerator

Oxygen transfer rate and the corresponding power requirement to operate the rotor are vital for design and scale-up of surface aerators. Present study develops simulation or scale-up criterion correlating the oxygen transsimulation fer coefficient and power number along with a parameter governing theoretical power per unit volume (X, which is defined as equal to (FR1/3)-R-4/3, where F and R are impellers' Fronde and Reynolds number, respectively). Based on such scale-up criteria, design considerations are developed to save energy requirements while designing square tank surface aerators. It has been demonstrated that energy can be saved substantially if the aeration tanks are run at relatively higher input powers. It is also demonstrated that smaller sized tanks are more energy conservative and economical when compared to big sized tanks, while aerating the same volume of water, and at the same time by maintaining a constant input power in all the tanks irrespective of their size. An example illustrating how energy can be reduced while designing different sized aerators is given. The results presented have a wide application in biotechnology and bioengineering areas with a particular emphasis on the design of appropriate surface aeration systems.

Studies of solar and stellar flares using multi-instrument data

Solar flares were first observed by plain eye in white light by William Carrington in England in 1859. Since then these eruptions in the solar corona have intrigued scientists. It is known that flares influence the space weather experienced by the planets in a multitude of ways, for example by causing aurora borealis. Understanding flares is at the epicentre of human survival in space, as astronauts cannot survive the highly energetic particles associated with large flares in high doses without contracting serious radiation disease symptoms, unless they shield themselves effectively during space missions. Flares may be at the epicentre of man s survival in the past as well: it has been suggested that giant flares might have played a role in exterminating many of the large species on Earth, including dinosaurs. Having said that prebiotic synthesis studies have shown lightning to be a decisive requirement for amino acid synthesis on the primordial Earth. Increased lightning activity could be attributed to space weather, and flares. This thesis studies flares in two ways: in the spectral and the spatial domain. We have extracted solar spectra using three different instruments, namely GOES (Geostationary Operational Environmental Satellite), RHESSI (Reuven Ramaty High Energy Solar Spectroscopic Imager) and XSM (X-ray Solar Monitor) for the same flares. The GOES spectra are low resolution obtained with a gas proportional counter, the RHESSI spectra are higher resolution obtained with Germanium detectors and the XSM spectra are very high resolution observed with a silicon detector. It turns out that the detector technology and response influence the spectra we see substantially, and are important to understanding what conclusions to draw from the data. With imaging data, there was not such a luxury of choice available. We used RHESSI imaging data to observe the spatial size of solar flares. In the present work the focus was primarily on current solar flares. However, we did make use of our improved understanding of solar flares to observe young suns in NGC 2547. The same techniques used with solar monitors were applied with XMM-Newton, a stellar X-ray monitor, and coupled with ground based Halpha observations these techniques yielded estimates for flare parameters in young suns. The material in this thesis is therefore structured from technology to application, covering the full processing path from raw data and detector responses to concrete physical parameter results, such as the first measurement of the length of plasma flare loops in young suns.

High-Throughput Flexible Constraint Length Viterbi Decoders on De Bruijn, Shuffle-Exchange and Butterfly Connected Architectures

Flexible constraint length channel decoders are required for software defined radios. This paper presents a novel scalable scheme for realizing flexible constraint length Viterbi decoders on a de Bruijn interconnection network. Architectures for flexible decoders using the flattened butterfly and shuffle-exchange networks are also described. It is shown that these networks provide favourable substrates for realizing flexible convolutional decoders. Synthesis results for the three networks are provided and a comparison is performed. An architecture based on a 2D-mesh, which is a topology having a nominally lesser silicon area requirement, is also considered as a fourth point for comparison. It is found that of all the networks considered, the de Bruijn network offers the best tradeoff in terms of area versus throughput.

Intent Based Clustering of Search Engine Query Log

The keyword based search technique suffers from the problem of synonymic and polysemic queries. Current approaches address only theproblem of synonymic queries in which different queries might have the same information requirement. But the problem of polysemic queries,i.e., same query having different intentions, still remains unaddressed. In this paper, we propose the notion of intent clusters, the members of which will have the same intention. We develop a clustering algorithm that uses the user session information in query logs in addition to query URL entries to identify cluster of queries having the same intention. The proposed approach has been studied through case examples from the actual log data from AOL, and the clustering algorithm is shown to be successful in discerning the user intentions.

Conformal Field Theory Methods for Variants of Schramm-Loewner Evolutions

This thesis consists of an introduction, four research articles and an appendix. The thesis studies relations between two different approaches to continuum limit of models of two dimensional statistical mechanics at criticality. The approach of conformal field theory (CFT) could be thought of as the algebraic classification of some basic objects in these models. It has been succesfully used by physicists since 1980's. The other approach, Schramm-Loewner evolutions (SLEs), is a recently introduced set of mathematical methods to study random curves or interfaces occurring in the continuum limit of the models. The first and second included articles argue on basis of statistical mechanics what would be a plausible relation between SLEs and conformal field theory. The first article studies multiple SLEs, several random curves simultaneously in a domain. The proposed definition is compatible with a natural commutation requirement suggested by Dubédat. The curves of multiple SLE may form different topological configurations, ``pure geometries''. We conjecture a relation between the topological configurations and CFT concepts of conformal blocks and operator product expansions. Example applications of multiple SLEs include crossing probabilities for percolation and Ising model. The second article studies SLE variants that represent models with boundary conditions implemented by primary fields. The most well known of these, SLE(kappa, rho), is shown to be simple in terms of the Coulomb gas formalism of CFT. In the third article the space of local martingales for variants of SLE is shown to carry a representation of Virasoro algebra. Finding this structure is guided by the relation of SLEs and CFTs in general, but the result is established in a straightforward fashion. This article, too, emphasizes multiple SLEs and proposes a possible way of treating pure geometries in terms of Coulomb gas. The fourth article states results of applications of the Virasoro structure to the open questions of SLE reversibility and duality. Proofs of the stated results are provided in the appendix. The objective is an indirect computation of certain polynomial expected values. Provided that these expected values exist, in generic cases they are shown to possess the desired properties, thus giving support for both reversibility and duality.

Sulkeuman ja neuvottelun ehdoilla : asiantuntija-aseman rakentuminen työelämässä

Closure and negotiation constructing professional position in working life The aim of the thesis is to analyse how professional positions are constructed in working life. A professional position refers to a formal professional membership, but also to a position at a work site. Formal jurisdiction provides resources for supporting a position, but the relations, practices and processes at the work site strongly shape it as well. Professional membership includes two gates: obtaining a professional diploma and access to a professional post. The concept of a professional position is based on two sub-concepts: legitimation and authority. Legitimation is society-level jurisdiction over professioning. Legitimation can be claimed in legislation, in the public space and the media, and at the work site. Authority requires constructing professional work territories and practicing authority in work-related decision making processes. The thesis is based on five articles which deal with the following topics: gendered professional careers; organising professional work; the impact of the social and cultural backgrounds when striving for professional positions; and models of research work. The articles represent two types of sociological research: the structural approach with quantitative methodology and the approach of micro-social analysis with qualitative methodology. The first approach was suitable for analyzing professional career formation and its social and ethnic conditions. The second approach has been applied in the articles dealing with the organization of professional work and models of research work. I have combined and analysed the results of these studies under the theoretical frame of the professional position in working life. Legislation is the most powerful form of legitimation. Professional membership is strongly regulated in disciplines where a degree requirement is defined by law. In addition, closures related to social conditions still affect professional positions, but their character is loose and changing. The closures related to social conditions are based on many mutually overlapping principles: social, cultural and ethnic backgrounds and gender. Despite the closures, professional experts have to negotiate their positions, particularly when the situation in the work sites and society changes. Professional authority is reinforced at the organizational level by legislation; when the institutional status of a public sector professional organization is defined by law, it reinforces the professional position of the employees. In the business line of new media, the employees need to negotiate with the management, other professional groups and clients when striving for reinforce their professional position.

Molten globule-like state of peanut lectin monomer retains its carbohydrate specificity - Implications in protein folding and legume lectin oligomerization

A central question in biological chemistry is the minimal structural requirement of a protein that would determine its specificity and activity, the underlying basis being the importance of the entire structural element of a protein with regards to its activity vis a vis the overall integrity and stability of the protein. Although there are many reports on the characterization of protein folding/ unfolding intermediates, with considerable secondary structural elements but substantial loss of tertiary structure, none of them have been reported to show any activity toward their respective ligands. This may be a result of the conditions under which such intermediates have been isolated or due to the importance of specific structural elements for the activity. In this paper we report such an intermediate in the unfolding of peanut agglutinin that seems to retain, to a considerable degree, its carbohydrate binding specificity and activity. This result has significant implications on the molten globule state during the folding pathway(s) of proteins in general and the quaternary association in legume lectins in particular, where precise subunit topology is required for their biologic activities.