Characterization of the mitochondrial active-site serine protein LACTB: filaments in the mitochondrial intermembrane space

Mitochondria have evolved from endosymbiotic alpha-proteobacteria. During the endosymbiotic process early eukaryotes dumped the major component of the bacterial cell wall, the peptidoglycan layer. Peptidoglycan is synthesized and maintained by active-site serine enzymes belonging to the penicillin-binding protein and the β-lactamase superfamily. Mammals harbor a protein named LACTB that shares sequence similarity with bacterial penicillin-binding proteins and β-lactamases. Since eukaryotes lack the synthesis machinery for peptidoglycan, the physiological role of LACTB is intriguing. Recently, LACTB has been validated in vivo to be causative for obesity, suggesting that LACTB is implicated in metabolic processes. The aim of this study was to investigate the phylogeny, structure, biochemistry and cell biology of LACTB in order to elucidate its physiological function. Phylogenetic analysis revealed that LACTB has evolved from penicillin binding-proteins present in the bacterial periplasmic space. A structural model of LACTB indicates that LACTB shares characteristic features common to all penicillin-binding proteins and β-lactamases. Recombinat LACTB protein expressed in E. coli was recovered in significant quantities. Biochemical and cell biology studies showed that LACTB is a soluble protein localized in the mitochondrial intermembrane space. Further analysis showed that LACTB preprotein underwent proteolytic processing disclosing an N-terminal tetrapeptide motif also found in a set of cell death-inducing proteins. Electron microscopy structural studies revealed that LACTB can polymerize to form stable filaments with lengths ranging from twenty to several hundred nanometers. These data suggest that LACTB filaments define a distinct microdomain in the intermembrane space. A possible role of LACTB filaments is proposed in the intramitochondrial membrane organization and microcompartmentation. The implications of these findings offer novel insight into the evolution of mitochondria. Further studies of the LACTB function might provide a tool to treat mitochondria-related metabolic diseases.

Lätta stabila isotopfraktioner som verktyg i biologisk forskning

Det finns en mätbar entitet i all levande materia, nämligen förhållandet mellan ett atomslags lätta och tunga isotop. Denna entitet divideras med en definierad referens och ett värde erhålls som brukar anges i promille ( ). Värdet är ett mått på isotopfraktionering. Genom att studera ekologi, taxonomi, kretslopp, isotopeffekter, dokumenterad data och metabola effekter kan man skissera över vad isotopfraktionen betyder. Migrationsbeteende, trofinätverk, dieter, miljö- och klimatdata kan rekonstrueras i tid och rum. Vid rekonstruktioner av gången tid har de museala samlingarna en nyckelposition. I litteraturöversikten tas teori, praktik och forskning upp för att påvisa metodens faror och användningsområden.

From actin monomers to bundles: The roles of twinfilin and α-actinin in Drosophila melanogaster development

The actin cytoskeleton is essential for a large variety of cell biological processes. Actin exists in either a monomeric or a filamentous form, and it is very important for many cellular functions that the local balance between these two actin populations is properly regulated. A large number of proteins participate in the regulation of actin dynamics in the cell, and twinfilin, one of the proteins examined in this thesis, belongs to this category. The second level of regulation involves proteins that crosslink or bundle actin filaments, thereby providing the cell with a certain shape. α-Actinin, the second protein studied, mainly acts as an actin crosslinking protein. Both proteins are conserved in organisms ranging from yeast to mammals. In this thesis, the roles of twinfilin and α-actinin in development were examined using Drosophila melanogaster as a model organism. Twinfilin is an actin monomer binding protein that is structurally related to cofilin. In vitro, twinfilin reduces actin polymerisation by sequestering actin monomers. The Drosophila twinfilin (twf) gene was identified and found to encode a protein functionally similar to yeast and mammalian twinfilins. A strong hypomorphic twf mutation was identified, and flies homozygous for this allele were viable and fertile. The adult twf mutant flies displayed reduced viability, a rough eye phenotype and severely malformed bristles. The shape of the adult bristle is determined by the actin bundles that are regularly spaced around the perimeter of the developing pupal bristles. Examination of the twf pupal bristles revealed an increased level of filamentous actin, which in turn resulted in splitting and displacement of the actin bundles. The bristle defect was rescued by twf overexpression in developing bristles. The Twinfilin protein was localised at sites of actin filament assembly, where it was required to limit actin polymerisation. A genetic interaction between twinfilin and twinstar (the gene encoding Cofilin) was detected, consistent with the model predicting that both proteins act to limit the amount of filamentous actin. α-Actinin has been implicated in several diverse cell biological processes. In Drosophila, the only function for α-actinin yet known is in the organisation of the muscle sarcomere. Muscle and non-muscle cells utilise different α-actinin isoforms, which in Drosophila are produced by alternative splicing of a single gene. In this work, novel α-actinin deletion alleles, including ActnΔ233, were generated, which specifically disrupted the transcript encoding the non-muscle α-actinin isoform. Nevertheless, ActnΔ233 homozygous mutant flies were viable and fertile with no obvious defects. By comparing α-actinin protein distribution in wild type and ActnΔ233 mutant animals, it could be concluded that non-muscle α-actinin is the only isoform expressed in young embryos, in the embryonic central nervous system and in various actin-rich structures of the ovarian germline cells. In the ActnΔ233 mutant, α-actinin was detected not only in muscle tissue, but also in embryonic epidermal cells and in certain follicle cell populations in the ovaries. The population of α-actinin protein present in non-muscle cells of the ActnΔ233 mutant is referred to as FC-α-actinin (Follicle Cell). The follicular epithelium in the Drosophila ovary is a well characterised model system for studies on patterning and morphogenesis. Therefore, α-actinin expression, regulation and function in this tissue were further analysed. Examination of the α-actinin localisation pattern revealed that the basal actin fibres of the main body follicle cells underwent an organised remodelling during the final stages of oogenesis. This involved the assembly of a transient adhesion site in the posterior of the cell, in which α-actinin and Enabled (Ena) accumulated. Follicle cells genetically manipulated to lack all α-actinin isoforms failed to remodel their cytoskeleton and translocate Ena to the posterior of the cell, while the actin fibres as such were not affected. Neither was epithelial morphogenesis disrupted. The reorganisation of the basal actin cytoskeleton was also disturbed following ectopic expression of Decapentaplegic (Dpp) or as a result of a heat shock. At late oogenesis, the main body follicle cells express both non-muscle α-actinin and FC-α-actinin, while the dorsal anterior follicle cells express only non-muscle α-actinin. The dorsal anterior cells are patterned by the Dpp and Epidermal growth factor receptor (EGFR) signalling pathways, and they will ultimately secrete the dorsal appendages of the egg. Experiments involving ectopic activation of EGFR and Dpp signalling showed that FC-α-actinin is negatively regulated by combined EGFR and Dpp signalling. Ubiquitous overexpression of the adult muscle-specific α-actinin isoform induced the formation of aberrant actin bundles in migrating follicle cells that did not normally express FC-α-actinin, provided that the EGFR signalling pathway was activated in the cells. Taken together, this work contributes new data to our knowledge of α-actinin function and regulation in Drosophila. The cytoskeletal remodelling shown to depend on α-actinin function provides the first evidence that α-actinin has a role in the organisation of the cytoskeleton in a non-muscle tissue. Furthermore, the cytoskeletal remodelling constitutes a previously undescribed morphogenetic event, which may provide us with a model system for in vivo studies on adhesion dynamics in Drosophila.

Cooperation and conflict in conspecific brood parasitism : an alternative reproductive tactic

Interactions among individuals give rise to both cooperation and conflict. Individuals will behave selfishly or altruistically depending on which gives the higher payoff. The reproductive strategies of many animals are flexible and several alternative tactics may be present from which the most suitable one is applied. Generally, alternative reproductive tactics may be defined as a response to competition from individuals of the same sex. These alternative reproductive tactics are means by which individuals may fine-tune their fitness to the reigning circumstances and which are shaped by the environment individuals are occupying as well as by the behaviour of other individuals sharing the environment. By employing such alternative ways of achieving reproductive output, individuals may alleviate competition from others. Conspecific brood parasitism (CBP) is an alternative reproductive strategy found in several egg laying animal groups, and it is especially common among waterfowl. Within this alternative reproductive strategy, four reproductive options can be identified. These four options represent a continuum from low reproductive effort coupled with low fitness returns, to high reproductive effort and consequently high benefits. It may not be evident how individuals should allocate reproductive effort between eggs laid in their own nest vs. in nests of others, however. Limited fecundity will constrain the number of eggs donated by a parasite, but also the tendency for hosts to accept parasitic eggs may affect the allocation decision. Furthermore, kinship, individual quality and the costs of breeding may play a role in complicating the allocation decision. In this thesis, I view the seemingly paradoxical effects of kinship on conflict resolution in the context of alternative reproductive tactics, examining the resulting features of cooperation and conflict. Conspecific brood parasitism sets the stage for investigating these questions. By using both empirical and theoretical approaches, I examine the nature of CBP in a brood parasitic duck, the Barrow's goldeneye (Bucephala islandica). The theoretical chapter of this thesis gives rise to four main conclusions. Firstly, variation in individual quality plays a central role in shaping breeding strategies. Secondly, kinship plays a central role in the evolution of CBP. Thirdly, egg recognition ability may affect the prevalence of parasitism. If egg recognition is perfect, higher relatedness between host and parasite facilitates CBP. Finally, I show that the relative costs of egg laying and post-laying care play a so far underestimated role in determining the prevalence of parasitism. The costs of breeding may outweigh possible inclusive fitness benefits accrued from receiving eggs from relatives. Several of the patterns brought out by the theoretical work are then confirmed empirically in the following chapters. Findings include confirmation of the central role of relatedness in determining the extent of parasitism as well as inducing a counterintuitive host clutch reduction. Furthermore, I demonstrate a cost of CBP inflicted on hosts, as well as results suggesting that host age reflects individual quality, affecting the ability to overcome costs inflicted by CBP. In summary, I demonstrate both theoretically and empirically the presence of cooperation and conflict in the interactions between conspecific parasites and their hosts. The field of CBP research has traditionally been divided, but the first steps have now been taken toward the acceptance of the opposite side of the divide. Especially the theoretical findings of chapter 1 offer the possibility to view seemingly contrasting results of various studies within the same framework, and may direct future research toward more general features underlying differences in the patterns of CBP between populations or species.

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.

Äldres upplevelser av att flytta till ett servicehus

Mänga äldre är med om en förändring i deras hemmiljö. Ofta sker denna förändring dä den äldre inte klarar av att bo hemma längre. Jag är intresserad av hur äldre upplever denna förändring och om flytten päverkar känslan av att anpassa sig till ett nytt hem. Jag är ocksä intresserad av att undersöka vad den omgivande miljön betyder för den äldre och om hurudana känslor ett miljöombyte väcker hos den äldre. Jag har undersökt detta genom att halla en diskussionsgrupp för äldre som bor pä det finskspräkiga, kommunala servicehuset i Östra centrum. Diskussionsgruppen träffades en gang i veckan under en tidsperiod pä tvä mänader. Fyra äldre deltog aktivt i diskussionsgruppen. Som forskningsmetod har jag använt mig av narrativa livsberättelser. Jag bad de äldre i diskussionsgruppen om skrivna livsberättelser. Som forskningsmaterial användes dessutom de äldres skrivna dikter och tankar, bade nya men också gamla texter. Jag använde mig av narrativ analys enligt det paradigmatiska tänkandet. Jag farm sex olika huvudteman i mitt material: hemmets betydelse, miljöns betydelse, upplevelsen av att flytta till ett servicehus, sociala samverkan, värden och upplevelsen av älderdom. De äldre i min undersökning uppgav att miljöns betydelse hade ökat för dem efter att de flyttade tili servicehuset. De äldre uppskattade särskilt känslan av lugn och stillhet dä de vistades ute i naturen. Hemmets betydelse hade däremot sjunkit totalt. Ingen av de äldre uppgav lägenheten pä servicehuset vara ett hem för dem, utan främst använde de ordet rum. Upplevelsen av att flytta tili servicehuset hade för dem flesta i diskussionsgruppen värit positiv. Det som de äldre uppgav att de längtade mest efter pä servicehuset handlade förutom smä konkreta saker främst om dagliga hemsysslor. De äldre kände sig ocksä ensamma och en orsak tili detta var att den sociala samverkan uppgavs vara mycket fattig pä servicehuset. De viktigaste kalloina som jag har använt i denna undersökning är doktorsavhandlingarna av Anni Vilkko, Omaelämänkerta kohtaamispaikkana. Naisen elämän kerronta ja luenta (1997) och Annukka Klemola, Omasta kodista hoitokotiin. Etnografia keskipohjalaisten vanhusten siirtymävaiheesta (2006). Jag har ocksä utgått från Sinikka Hakonens Kirjoittaminen merkityksellistää elämää (2003).

Äldres upplevelser av att flytta till ett servicehus

Många äldre är med om en förändring i deras hemmiljö. Ofta sker denna förändring då den äldre inte klarar av att bo hemma längre. Jag är intresserad av hur äldre upplever denna förändring och om flytten påverkar känslan av att anpassa sig till ett nytt hem. Jag är också intresserad av att undersöka vad den omgivande miljön betyder för den äldre och om hurudana känslor ett miljöombyte väcker hos den äldre. Jag har undersökt detta genom att hålla en diskussionsgrupp för äldre som bor på det finskspråkiga, kommunala servicehuset i Östra centrum. Diskussionsgruppen träffades en gång i veckan under en tidsperiod på två månader. Fyra äldre deltog aktivt i diskussionsgruppen. Som forskningsmetod har jag använt mig av narrativa livsberättelser. Jag bad de äldre i diskussionsgruppen om skrivna livsberättelser. Som forskningsmaterial användes dessutom de äldres skrivna dikter och tankar, både nya men också gamla texter. Jag använde mig av narrativ analys enligt det paradigmatiska tänkandet. Jag fann sex olika huvudteman i mitt material: hemmets betydelse, miljöns betydelse, upplevelsen av att flytta till ett servicehus, sociala samverkan, vården och upplevelsen av ålderdom. De äldre i min undersökning uppgav att miljöns betydelse hade ökat för dem efter att de flyttade till servicehuset. De äldre uppskattade särskilt känslan av lugn och stillhet då de vistades ute i naturen. Hemmets betydelse hade däremot sjunkit totalt. Ingen av de äldre uppgav lägenheten på servicehuset vara ett hem för dem, utan främst använde de ordet rum. Upplevelsen av att flytta till servicehuset hade för dem flesta i diskussionsgruppen varit positiv. Det som de äldre uppgav att de längtade mest efter på servicehuset handlade förutom små konkreta saker främst om dagliga hemsysslor. De äldre kände sig också ensamma och en orsak till detta var att den sociala samverkan uppgavs vara mycket fattig på servicehuset. De viktigaste källorna som jag har använt i denna undersökning är doktorsavhandlingarna av Anni Vilkko, Omaelämänkerta kohtaamispaikkana. Naisen elämän kerronta ja luenta (1997) och Annukka Klemola, Omasta kodista hoitokotiin. Etnografia keskipohjalaisten vanhusten siirtymävaiheesta (2006). Jag har också utgått från Sinikka Hakonens Kirjoittaminen merkityksellistää elämää (2003).

Gauge Field Theories, Quantum Space-Time and Some Applications

In this thesis, the possibility of extending the Quantization Condition of Dirac for Magnetic Monopoles to noncommutative space-time is investigated. The three publications that this thesis is based on are all in direct link to this investigation. Noncommutative solitons have been found within certain noncommutative field theories, but it is not known whether they possesses only topological charge or also magnetic charge. This is a consequence of that the noncommutative topological charge need not coincide with the noncommutative magnetic charge, although they are equivalent in the commutative context. The aim of this work is to begin to fill this gap of knowledge. The method of investigation is perturbative and leaves open the question of whether a nonperturbative source for the magnetic monopole can be constructed, although some aspects of such a generalization are indicated. The main result is that while the noncommutative Aharonov-Bohm effect can be formulated in a gauge invariant way, the quantization condition of Dirac is not satisfied in the case of a perturbative source for the point-like magnetic monopole.

Äldres upplevelser av att flytta till ett servicehus

Många äldre är med om en förändring i deras hemmiljö. Ofta sker denna förändring då den äldre inte klarar av att bo hemma längre. Jag är intresserad av hur äldre upplever denna förändring och om flytten påverkar känslan av att anpassa sig till ett nytt hem. Jag är också intresserad av att undersöka vad den omgivande miljön betyder för den äldre och om hurudana känslor ett miljöombyte väcker hos den äldre. Jag har undersökt detta genom att hålla en diskussionsgrupp för äldre som bor på det finskspråkiga, kommunala servicehuset i Östra centrum. Diskussionsgruppen träffades en gång i veckan under en tidsperiod på två månader. Fyra äldre deltog aktivt i diskussionsgruppen. Som forskningsmetod har jag använt mig av narrativa livsberättelser. Jag bad de äldre i diskussionsgruppen om skrivna livsberättelser. Som forskningsmaterial användes dessutom de äldres skrivna dikter och tankar, både nya men också gamla texter. Jag använde mig av narrativ analys enligt det paradigmatiska tänkandet. Jag fann sex olika huvudteman i mitt material: hemmets betydelse, miljöns betydelse, upplevelsen av att flytta till ett servicehus, sociala samverkan, vården och upplevelsen av ålderdom. De äldre i min undersökning uppgav att miljöns betydelse hade ökat för dem efter att de flyttade till servicehuset. De äldre uppskattade särskilt känslan av lugn och stillhet då de vistades ute i naturen. Hemmets betydelse hade däremot sjunkit totalt. Ingen av de äldre uppgav lägenheten på servicehuset vara ett hem för dem, utan främst använde de ordet rum. Upplevelsen av att flytta till servicehuset hade för dem flesta i diskussionsgruppen varit positiv. Det som de äldre uppgav att de längtade mest efter på servicehuset handlade förutom små konkreta saker främst om dagliga hemsysslor. De äldre kände sig också ensamma och en orsak till detta var att den sociala samverkan uppgavs vara mycket fattig på servicehuset. De viktigaste källorna som jag har använt i denna undersökning är doktorsavhandlingarna av Anni Vilkko, Omaelämänkerta kohtaamispaikkana. Naisen elämän kerronta ja luenta (1997) och Annukka Klemola, Omasta kodista hoitokotiin. Etnografia keskipohjalaisten vanhusten siirtymävaiheesta (2006). Jag har också utgått från Sinikka Hakonens Kirjoittaminen merkityksellistää elämää (2003).

Les fleuves dans l'œuvre romanesque de Jean-Marie Gustave Le Clézio

Relying on Merleau-Ponty's phenomenology of perception and on Mircea Eliade's works on the Sacred and the Profane, this study explores the river as a perceptual space and as the sacred Center in a cosmic vision of the world in twelve of Jean-Marie Gustave Le Clézio's fictional works, from The Interrogation (1963) to Revolutions (2003). In the first chapter, after introducing the field of study, I discuss the relation between the radical subjectivity and the evasiveness of perceiving subjects in Le Clézio's fiction. Next are some thoughts on the relation between Merleau-Ponty's and Le Clézio's ideas. The second chapter studies the river as an experience in the text, first as a topographical space, then as a sound world. The investigations move on to its water as a visual and a tactile phenomenon. Then follows the human use of the river, the (absence of) baths, and the river as a traveling space. The chapter closes with the study of the metaphorical use of the word, occurring mainly in urban space and for phenomena in the sky. The third chapter is organized around the river as the Center of the world in a religious cosmogony, where the river represents the origin of the world and of the human race. The core analysis shows how the middle of the river is a symbolic space of a new beginning. As a sacred space, the river abolishes time as the object of contemplation and as relative immobility from the point of view of a person drifting downstream. The functions of a new beginning and of abolition of time are combined in the symbolic immersions in the water. Finally, the dissertation explores other symbolical spaces, such as the unknown destination of the drift, and the river as the Center of a utopia. The chapter closes with the existential agony as a result of the elimination of the Center in the urban environment. In the final chapter, the river is compared to other watercourses : the creek, the brook and the rapids. The river is more of a spatial entity, whereas the actual water is more important in the smaller watercourses. The river is more common than the other watercourses as a topographical element in the landscape, whereas the minor watercourses invite the characters to a closer contact with their element, in immersions and in drinking their water. Finally, the work situates the rivers in a broader context of different fictional spaces in Le Clézio's text.