Bio- ja ympäristötieteellinen tiedekunta

 

Recent Submissions

  • Fraixedas, Sara (Helsingin yliopisto, 2017)
    The combined effects of climate and land-use change constitute a major threat to global biodiversity. Accurate tools to track changes in biodiversity have been largely called upon in order to address global conservation targets. In response to this, a range of ecological indicators have been developed to measure the state of biodiversity in a changing world. Because of their sensitivity to environmental changes, birds are increasingly used in the construction of multi-species indicators, which represent a powerful tool for decision-makers to assess conservation effectiveness. This work aims to further our understanding of the general state of bird populations in Finland and the underlying ecological processes behind corresponding trends, covering different environments and with a special focus on some of the most threatened ecosystems of northern Europe. Using data on common bird species, the effects of climate change and anthropogenic habitat degradation on bird populations are quantified for different habitat types and seasons of the year. Habitat-specific indicators are also produced to deepen knowledge about large-scale impacts taking place in the environment while allowing an evaluation of the conservation status of bird populations, thus helping target the most critical conservation issues. Although the effects of climate and land-use change on bird populations vary significantly with the habitat type and the life-history traits of the species (e.g. migration strategy), the conservation status of nearly all studied communities is considerably deteriorating in both Finland and its neighbouring Northern European countries. Peatlands and forests are of particular concern, given that intensive management actions are severely impacting the inhabiting bird communities.
  • Kaartinen, Tanja (Helsingin yliopisto, 2017)
    T cells are a promising therapeutic target and remedy in modern medicine. Various ways of modifying T-cell response are under development with a view to treating cancer, autoimmune diseases, and transplantation-related complications. T-cell function can be steered by altering target recognition or cosignaling receptors as well as by inducing immunological memory or regulatory T cells (Tregs). Unwanted immune responses can be curtailed by administering Tregs and, perhaps, long-lasting immunological tolerance can be induced. Cytotoxic T cells can be directed against cancer cells. Considerable T-cell numbers are required for clinical efficacy. Therefore, in vitro cell expansion is often necessary and cultures are commonly supplemented with interleukin (IL)-2. As T-cell activation, proliferation, effector differentiation, and the development of memory are inherently coupled to each other, excessive stimulation during expansion may lead to exhaustion. Hence, cells with weaker therapeutic potency may be produced. In this thesis, various methods of T-cell activation and in vitro cell expansion were evaluated particularly in the context of personalized medicine and cell therapy. Good therapeutic response to T-cell therapy in cancer depends in part on the survival of T cells and T-cell memory. The present study demonstrated that the proportion of memory T cells could be increased by limiting the length of in vitro T-cell expansion and by reducing the amount of IL-2. This study further showed that as a result of in vitro expansion Tregs expressed higher levels of the Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) cosignaling receptor. CTLA4 is a central molecule for the Treg-mediated inhibition. The level of CTLA4 expression in Tregs correlated with higher inhibitory function of the cells. Apparently, high CTLA4 receptor expression after cell expansion was in part a result of changes in the alternative splicing of CTLA4 messenger RNA (mRNA). It was also found that the splicing preferences and the expression levels of CTLA4 mRNAs were associated with genetic variation in the T-cell cosignaling receptor gene region. This thesis provides new knowledge that can be applied in the evaluation of individual variation in T-cell immunity and the production of therapeutic T cells. The T-cell expansion method that was developed here is directly applicable in T-cell manufacturing, and the findings may have substantial clinical relevance.
  • Survila, Mantas (Helsingin yliopisto, 2017)
    To survive, plants must recognize the presence of danger and establish effective defenses against invading pathogens. Most plants are resistant to the majority of plant pathogens. This passive protection is provided primarily by the cell wall and waxy cuticular layer that limit the progress of most attackers. If these barriers are overcome, the second line of defense is triggered upon detection of pathogen-associated molecular patterns or damage-associated molecular patterns by pattern recognition receptors. The activation of PRRs induces multifaceted intracellular signaling pathways that ultimately initiate defense responses. Many molecular components by which plants perceive pathogens and the downstream signaling cascades have been characterized on a molecular level. However, the mechanisms by which plants protect themselves from phytopathogens (in particular necrotrophs) remain to be elucidated. Three aspects of plant immunity to phytopathogens are addressed in this thesis: (I) the role of glucosidase II β-subunit AtGCSIIβ in EFR receptor-mediated defense signaling, (II) the role of F-box protein AFB4 in plant innate immunity against necrotrophic pathogens, and (III) the role of class III peroxidases in cuticle formation that governs very strong and local resistance against necrotrophic bacterial and fungal pathogens. Plants exploit membrane-localized PRRs for specific and rapid detection of the potential pathogens. Many eukaryotic membrane-localized proteins undergo quality control during folding and maturation in the endoplasmic reticulum, a process termed endoplasmic reticulum quality control. The biogenesis of EFR, and to a lesser extent FLS2 receptors, is regulated by this mechanism. Study I demonstrated that the glucosidase II β-subunit AtGCSIIβ is pivotal for the function of the plant innate immunity receptor EFR. Loss-of-function in AtGCSIIβ results in elf18-insensitive phenotype, confirming the importance of AtGCSIIβ in biogenesis of the EFR receptor. F-box proteins are important components in plant hormone responses. They target regulatory proteins to the ubiquitin proteolytic machinery and mediate hormone signaling transduction. In study II, we demonstrated that auxin signaling F-box protein mutant plants are enhanced in their resistance to bacterial and fungal necrotrophic pathogens. This was accompanied with altered sensitivity to methyl jasmonate, indole-3-acetic acid, and abscisic acid phytohormones, thus providing evidence that ABF4-mediated signaling is involved in balancing growth and defense responses via coordination of hormone-mediated signaling pathways. The ability to maintain the barrier properties of the epidermis is largely due to the cell walls, which are covered with specialized lipids. This fine structure at the outermost region of the cell walls of epidermal cells is called the cuticle, which has been the subject of many studies. Plants perceive and ultimately activate defense mechanisms in response to cuticular and cell wall structural components e.g. oligogalacturonides released by the action of degradative enzymes secreted by pathogenic bacteria or fungi. Cuticle alterations induce a battery of reactions that often result in reactive oxygen species production and resistance to necrotrophic pathogens. However, the source of ROS generated upon altered cuticle status and the acute downstream defense signaling pathways involved in such defense remains elusive. Study III provides evidence that ROS produced by class III apoplastic peroxidases suppress the expression of cuticle-biosynthetic genes, and together with ABA, regulate the formation of the cuticle envelope. However, resistance to necrotrophic pathogens in cuticle-depleted plants is a result of activated OG signaling components and function independently of salicylic acid and jasmonic acid signaling pathways. This thesis demonstrates the use of Arabidopsis in studying the genetic basis of plant defense mechanisms. It provides novel insights on plant resistance to pathogens, and reveals how cuticular defects activate defense via OG signaling pathway.
  • Alanko, Teija (Helsingin yliopisto, 2017)
    Archaeobotany combines botany, archaeology and history, and studies useful plants and interactions between humans and plants in the past, including horticulture. Garden history has been studied in Finland mainly through historical sources, but not much with archaeological or archaeobotanical methods, although the importance of multidisciplinary work has been noted, since written sources available are often not sufficient. Archaeobotany in Finland has revealed garden plant remains, but garden soils have not been investigated much. Archaeobotanical material, obtained from soil samples, i.e. macrosubfossil plant remains, is interpreted in archaeological and historical contexts. Excavations are, however, often restricted for practical reasons, determining also sites for macrofossil analyses. An alternative sampling method may be one solution to carry out macrofossil studies in sites unlikely to be excavated, such as historical gardens. The aims of this study were to elucidate a part of Finnish and Swedish garden history by means of archaeobotany, and to test archaeobotanical sampling in gardens in the absence of excavations with a sampler and applying AMS-radiocarbon dating. The research comprises four case studies and a review from five sites; Naantali Cloister, Kumpula Manor, and academic gardens in Uppsala, Turku and Helsinki. The sites are partly linked historically to each other, and they reach from the 15th century to the 21st century. Soil samples were collected at four sites with a sampler from different levels from narrow pits, one by one in vertical series. At one site, samples came from excavations. The samples were floated and sieved in a laboratory, and macrofossil remains were identified and counted. Altogether 8,404 macrofossil plant remains belonging to 154 plant taxa were obtained. In total 30 AMS-radiocarbon dates were measured from seeds, charred grains, and pieces of charred wood. The oldest dated seeds and grains were medieval, the youngest were modern. Macrofossil plant remains included cereals, berries, ornamental, medicinal and garden plants, and cultural or garden weeds, indicating both consumption and garden cultivation at the sites. Other soil contents, such as fish scales and chips of wood and charcoal, referred to fertilization and thus also gardening. The sampling method worked reasonably well. Sampling was independent of excavations, and relatively quick. Still, the maximum size of a sample was limited, although larger samples could have yielded more macrofossils and species. Written sources were necessary for the background, but in the cases of historical gardens, the literature gave historical contexts well enough. Garden history can and should be studied with both written sources and archaeobotanical methods. Informative macrofossil sampling can be carried out both from excavations and straight from garden soil. Plant lists, when existing, give information of cultivated species, but not of plants consumed or having grown as garden weeds at the sites. Still, quite few species that were mentioned in the plant lists were obtained as macrofossils in this study, perhaps due to the relatively poor state of preservation of the seeds in garden soil, and the probable scarce accumulation of seeds of cultivated species into the garden soil. Nevertheless, in sites with no comprehensive plant lists, archaeobotany revealed valuable information of plants that could not be gained otherwise. The Naantali Cloister case showed the importance of searching remains of garden plants also from structures outside of gardens.
  • Kärpänoja, Pauliina (Helsingin yliopisto, 2017)
    The increasing trend of antimicrobial resistance in bacteria is a global problem, although resistance varies between geographical regions significantly. Today, common bacterial pathogens can be resistant to all known antimicrobial agents. The growing resistance has been linked to increasing use of antimicrobials in humans, food industry ant veterinary medicine in several studies. The battle against antimicrobial resistance is highly dependent on the knowledge of resistance rates in different bacterial species and accurate methods to measure the resistance. Finnish laboratories provide resistance data annually for the national FINRES report. This data is forwarded to the European EARSNet database and from 2016 also to the Global Antimicrobial Resistance Surveillance System (GLASS, organised by WHO). The solidity and uniformity of this data depend on the primary results of laboratories. This thesis is composed of four studies, which cover methodolgical issues in the susceptibility testing of the main respiratory pathogens and the connection between antimicrobial use and resistance. Susceptibility testing methods and their quality were examined for Haemophilus influenzae and Streptococcus pneumoniae. The connection between sulfamethoxazole-trimethoprim use and resistance was investigated among H. influenzae, S. pneumoniae and Moraxella catarrhalis using the FINRES data and drug consumption data provided by Finnish Medical Agency. As a result of this study the susceptibility testing method for H. influenzae with good sensitivity and specificity was launched for the Finnish susceptibility testing guideline (FiRe standard), the focus was in identifying the difficult to detect non-β-lactamase mediated ampicillin resistance. In addition, evaluation of the quality of susceptibility testing in Finnish laboratories showed good reproducubility with two indicator organisms H. influenzae (ATCC49247) and S. pneumoniae (ATCC49619) when recommended guidelines were followed. The quality was assessed from internal quality control results of the laboratories. An automated method (Vitek2®, AST GP-74, bioMerieux) for susceptibility testing of S. pneumoniae provides highly comparable results with the reference broth dilution method. Time to results is considerabely shorter than with the traditional methods. Regional sulfamethoxazole-trimethoprim consumption was found to have a positive connection with resistance in S. pneumoniae but the change of resistance was not significant. The change in resistance over time in H. influenzae was border-line significant, but the drug use did not explain the change. Change in resistance among M. catarrhalis was not statistically significant and there was no significant connection between the drug consumption and resistance. Sulfa-trimethoprim consumption fell throughout the country during the investigation period. Conclusions: The accuracy of the susceptibility testing of bacteria requires evidence-based standardization and continuous quality controlling. Clinical laboratory automation can be implemented safely in pneumococcal susceptibility testing. The impact of sulfamethoxazole-trimethoprim consumption on resistance varies for different bacterial species. A reduction in its use in the long run has not led to a significant reduction in resistance.
  • Luhtanen, Anne-Mari (Helsingin yliopisto, 2017)
    Virus-host systems in sea ice Sea ice is one of the largest habitats on Earth. A specialized microbial community lives inside the narrow brine channels that are formed during freezing process, when salt and other components from sea water concentrate between ice crystals. These microbes have an active role in the biogeochemistry of the sea ice by primary production, degradation of material and excreation of compounds, which effect the gas exchange between the ocean and atmosphere and the nutrient status of the under ice sea. Sea ice microbial community consist of auto- and heterotrophic protists, prokaryotes and viruses. The main heterotrophs are the bacteria. Viruses are the most abundant lifeform on Earth. They are found everywhere where there is life and they infect all kinds of cells. Infections are crucial for viruses because they can reproduce only by using a host cell to produce new virus particles. Majority of the viruses infect the most numerous cells on Earth, the prokaryotes, i.e. bacteria and archaea. Viruses infecting bacteria (bacteriophages or phages) are a major factor in bacterial mortality. They can also control the community composition of bacteria because of the high specificity of the infection. Bacteria have different mechanisms to avoid phage infections and phages need to evolve to be able to reproduce. This arms race of phages and bacteria can lead to co-evolution. Although viruses are known to have significant effects on bacterial communities in various habitats, not much is known about the viruses in the sea ice. Before this project, only three isolates have been reported from the Arctic sea ice. The aim in this thesis was to get a better understanding of the phages and their role in sea ice. For that, isolation, cultivation and purification methods needed to be developed and optimized. Bacteria and phages were isolated from samples taken from Baltic and Antarctic sea ice. The phage particles were purified and characterized by their morphology, structural protein patterns and host range. The identities of the host bacteria were analyzed by their 16S rRNA gene sequence. Effect of temperature on the host bacterial growth and phage infections, and the adsorption and life cycle of the phages, was experimentally studied. The abundance of virus-like particles in Antarctic sea ice was analyzed using flow cytometry. The first phage-host systems were isolated from Baltic Sea ice and Antarctic sea ice. All of the phages infected bacterial strains belonging to genera that are typically abundant in sea ice i.e. Shewanella, Flavobacterium, Paraglaciecola and Octadecabacter. All the bacterial strains and phages were cold-active. The adsorption and life cycle of phages was suprisingly fast at tested 4 °C. The phage infections were specific to certain bacterial strains. A complex phage-host system network was seen among two of the phages and 15 closely related bacterial strains from Antarctica, which may be a result of co-evolution. The abundance of virus-like particles in melted Antarctic winter sea ice (105 106 particles ml-1 bulk ice) was high when considering that they are normally concentrated in the brine channels. The amount of virus-like particles in sea ice even during Antarctic winter, indicates that viruses are an active and important member of the sea ice microbial community. Adsorption and life cycle studies show that phage infections may be efficient in the closed and concentrated environment of sea ice brines. By the strain specificic infections the phages can control the bacterial community composition and this way effect the community functions. The co-evolution of phages and bacteria may be important factor in the bacterial evolution.
  • Lehti, Satu (Helsingin yliopisto, 2017)
    Aortic stenosis and athrosclerosis are slowly progressing diseases. Being clinically silent, as they start developing decades before they cause symptoms, cardiovascular diseases and atherosclerosis in particular, are the leading cause of morbitidy and mortality in the Western world. Lipid accumulation begins both in the artery walls and in the aortic valves before any clinical signs of atherosclerosis or aortic stenosis can be detected. While atherosclerotic lesions are characterized with cells filled with lipid droplets i.e. foam cells, and they are found to be calcified only in the late stages of atherosclerosis, the stenotic aortic valve leaflets contain both lipid droplets and calcified nodules already in early lesions. The proteoglycan matrix common to artery wall and aortic valve leaflets retains the entering lipoprotein particles that are then enzymatically and oxidatively modified. Such modifications have an ability to transform the non-inflammatory plasma lipoprotein particles into crystals and particles that can induce sterile inflammation in the various intimal and valvular cells. This thesis was set to study the distribution and characteristics of the extracellular lipid and to reveal the origin of the extracellular lipid particles. Prior to the analyses of the isolated extracellular lipid particles, the human carotid artery plaques were imaged with three-dimensional electron microscopy and sections of human coronary arteries were analyzed with imaging mass spectrometry to study the spatial distribution of lipids in different stages of atherosclerosis. In the human coronary arteries, the lipid domains found in advanced atherosclerotic lesions were different from the domains found in atheroma-stage artery sections, and even more different from healthy sections of coronary arteries. In the human carotid artery plaques, cholesterol crystals were found to be large sheets or needle-like structures, and they appeared to be growing out from large lipid particles in the intima. For the purpose of studying the chemical and physical characteristics of the extracellular lipid particles, the extracellular particles were isolated from aortic valve leaflets and coronary artery plaques. The lipid particles were examined with multiple tools to study their lipid composition, protein composition, protein structure, size, and density. The extracellular lipid particles, both in human aortic valve leaflets and human carotid arteries, were found to be derived from plasma lipoproteins, mainly from low density lipoprotein (LDL) or very low density lipoprotein (VLDL). Apart from multiple small exchangeable lipoproteins, like apolipoprotein (apo) E, apoA-family, and apoC-family, the particles contained mainly apolipoprotein B-100 (apoB-100), the integral protein of LDL and VLDL, and they contained features which suggest that they were multiply modified. Another goal was to find which modifications would change the intimal or valvular extracellular lipid particles to such fused and aggregated lipid particles that were found in the valves and intimal plaques, and which modifications would induce a sterile inflammatory response similar to the response the extracellular lipid particles induce. To study the possible culprit modifications, both the extracellular lipid particles, in vitro-generated cholesterol crystals, and LDL that was modified by lipolysis, proteolysis, and oxidation, were applied to human primary monocyte-derived macrophages. Both the isolated extracellular lipid particles and LDL modified with a combination of phospholipolysis by PLA2 and cholesterol esterase were found to be able to activate a multiprotein complex inflammasome in human primary monocyte-derived macrophages in vitro, and to induce the secretion of proinflammatory cytokines. According to the results of this thesis, the lipid particles in the arterial intima and in the aortic valve are active components of atherosclerosis and aortic stenosis. They can induce the cells in the intima and in the valve to produce inflammatory cytokines and thus can affect the progress of these diseases.
  • Etelälahti, Tiina (Helsingin yliopisto, 2017)
    Alcohol dependence and alcoholism are modulated by environmental factors and genetic predisposition. Rat models have been invaluable in the investigation of several aspects of alcoholism in humans. The rodents exhibit a wide range of genetically determined alcohol-drinking preferences. Selective rat breeding programs with different alcohol preference has produced stable lines of rats that reliably exhibit high and low voluntary alcohol consumption (termed here AA and ANA, respectively). Alcohol consumption is also strongly dependent on environmental conditions. Stressful events evoke an extensive multisystem and integrative physiological response, where a major component is the activation of the hypothalamic-pituitary-adrenal (HPA) axis. Testosterone has been implicated as mediator of the rewarding effect of alcohol, and the testosterone level is predictive of future alcohol consumption. The objective of the present thesis is to examine in greater detail the relations between alcohol, testosterone and neuroendocrine stress responses, and alcohol drinking. In the first study (I), the interrelations between endogenous and alcohol-mediated effects on testosterone and corticosterone levels and voluntary alcohol consumption were studied in a crossbred F2 generation of the original AA and ANA rat lines. The second study (II) was a reinvestigation of the effect of subchronic nandrolone decanoate treatment, which in an earlier study had been shown to increase alcohol consumption, and the relation of the effect on the HPA and hypothalamic-pituitary-gonadal (HPG) axes. The third study (III) examined the possible role of benzyl alcohol, present as a preservative in the nandrolone product used in the earlier study, as a confounding factor that could explain differences between the results of the earlier study and the present one. In the fourth study (IV) the interrelations between the effect of corticosterone on alcohol-mediated testosterone changes and alcohol consumption in AA, ANA, F2 and Wistar rats were examined. The results of Study I shows connections between testosterone elevation and increased alcohol drinking, as well as between testosterone reduction and decreased alcohol drinking, which is in line with the original data of the AA and ANA lines. Elevated endogenous testosterone levels and higher frequencies of alcohol-induced testosterone increases were found in high consumption groups compared to low consumption groups. In Study II, subchronic nandrolone administration led to a reduction in alcohol-mediated testosterone levels, correlating with reduced voluntary alcohol consumption in the alcohol-preferring AA rat line. On the other hand, Study III showed that benzyl alcohol increases voluntary alcohol intake at least in the low-consumption rats, which may explain earlier discrepancies among studies. The results of Study IV were consistent with the hypothesis that corticosterone is involved in the regulation of alcohol-mediated testosterone changes. In conclusion, our present results suggests that corticosterone is a balancer, which regulates both the alcohol-mediated testosterone increase followed by reinforcement and increased voluntary alcohol drinking in high-drinking rats, and the alcohol-mediated testosterone reduction followed by disinforcement and reduced alcohol intake in low-drinking rats.
  • Yan, Xu (Helsingin yliopisto, 2017)
    Progressive development of pathology in neuroanatomically connected brain regions is a common feature of many neurodegenerative diseases. The spread of disease pathology is suggested to be dependent on the transmissibility of disease-associated proteins, particularly soluble aggregates of misfolded proteins. Emerging evidence suggests that many disease-associated proteins such as α-synuclein (aSyn) and tau, in certain misfolded and aggregated states convert from physiologically normal proteins into forms that lead to progression of disease pathology in a template-dependent manner, which is also known as seeding . The propagation and the proteinopathy have been suggested to occur via cell-to-cell transmission. The exact mechanisms involved in the seeding and spreading process are incompletely understood. In this thesis work, three critical steps of the seeding pathway (a process involves multiple steps), the intracellular aggregation, cellular release and uptake of aSyn and tau, were carefully studied primarily via a newly developed platform based on protein-fragment complementation assay. The main findings of this thesis are: a)Prolyl oligopeptidase (PREP) is a serine peptidase that was previously known to accelerate the process of aSyn aggregation and suppress autophagy clearance in cells and transgenic aSyn mice. The results of this thesis show that PREP directly interacts with aSyn in neuro2A cells and cell-free environment, and enhances aSyn dimerization, which is an early event in aSyn aggregation pathway. In addition, the PREP-mediated aSyn dimerization can be antagonized by KYP-2047, a small-molecule PREP inhibitor. b) Late-onset Alzheimer s disease (LOAD) susceptibility genes affect the individual risk of developing Alzheimer s disease, which is one of the common tauopathies. In this work, the functional connection between selected LOAD susceptibility genes and cell-to-cell transmission of tau was studied in vitro. We observed that RNAi knockdown of CD2AP and FRMD4A reduced tau secretion, and knockdown of APOE reduced tau uptake in HEK293T cells. Further mechanistic studies revealed that FRMD4A modulates tau secretion via the FRMD4A-cytohesin-Arf6 signalling pathway and the Par6/aPKC polarity signalling complex. This data, for the first time, demonstrates a functional connection between LOAD risk genes and cell-to-cell propagation of tau. c) Following internalization, extracellular, hyperphosphorylated tau was found to be recruited to stress granules, transient non-membraneous cytosolic structures composed of RNA and self-aggregating RNA-binding proteins. Tau recruitment was dependent on TIA-1, an RNA-binding stress granule protein. Importantly, the stress granules induced by and containing internalized tau were resistant to normal clearance and associated with increased sensitivity of cells to other stresses. This data describe a previously unrecognized mechanism and pathological consequence of cell-to-cell propagation of tau-mediated by stress granules, which have previously been associated with the pathophysiology of various neurodegenerative diseases. Overall, the work described in this thesis provides several novel findings that improve our understanding of cellular mechanisms underlying the development and spreading of aSyn and tau-related neurodegenerative pathologies. These pieces of knowledge may be potential avenues towards the development of crucial therapeutics against aSyn and tau-related neurodegenerative diseases.
  • Kulashekhar, Shrikanth (2017)
    Working memory is used to maintain information for cognitive operations, and its deficits are associated with several neuropsychological disorders. Human functional magnetic resonance imaging (fMRI) f isolated key brain areas associated with the maintenance of sensory and duration information. However, the systems-level mechanisms coordinating the collective neuronal activity in these brain areas have remained elusive. It has been suggested that synchronized oscillations could regulate communication in neuronal networks and could hence serve such coordination, but their role in the maintenance of sensory and duration information has remained largely unknown. In this thesis, combined magnetoencephalography (MEG) and electroencephalography (EEG) together with minimum norm estimate (MNE) based source modelling was used to study the oscillatory dynamics underlying visual and temporal working memory. In Publication I, we developed a neuro-informatics approach to understand the anatomical and dynamic structures of network synchrony supporting visual working memory (VWM). VWM was associated with a sustained and stable inter-areal phase synchrony among frontoparietal and visual areas in alpha- (10 13 Hz), beta- (18 24 Hz), and gamma- (30 40 Hz) frequency bands. In this study, the subjects' individual behavioural VWM capacity was predicted by synchrony in a network in which the intraparietal sulcus was the most central hub. In Publication II, we characterised the oscillatory amplitude dynamics associated with the VWM maintenance. Increasing VWM load was associated with strengthened oscillation amplitudes in the occipital and occipitotemporal cortical areas, in the alpha (8 14 Hz) beta- (15 30 Hz), gamma- (30-50 Hz), and high-gamma- (50 150 Hz) frequency bands. In Publication III, we addressed the functional significance of local neuronal synchronization, as indexed by the amplitudes of cortical oscillations, in the estimation and maintenance of duration information. The estimation of durations in the seconds range was associated with stronger beta-band (14 30 Hz) oscillations in cortical regions that have earlier been associated with temporal processing. The encoding of duration information was associated with strengthened gamma- (30 120 Hz), and the retrieval and comparison with alpha-band (8 14 Hz) oscillations. Further, the maintenance of stimulus duration was associated with stronger theta- and alpha-band (5 14Hz) frequencies. These data suggested that both local and large-scale phase synchrony in the alpha-, beta-, and gamma-frequency bands in the frontoparietal and visual regions could be a systems level mechanism for coordinating and regulating the maintenance of visual information in VWM. In addition, it suggested that beta-band oscillations may provide a mechanism for estimating short temporal durations, while gamma, alpha and theta-alpha oscillations support their encoding, retrieval, and maintenance in working memory, respectively.
  • von Ossowski, Lotta (Helsingin yliopisto, 2017)
    Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors are glutamate-gated cation channels and mediators of fast excitatory neurotransmission in the mammalian central nervous system. Trafficking and functional regulation of AMPA receptors GluA1-4 is carried out through numerous intracellular protein interactions and post-translational modifications. The aim of this thesis work was to study the selective interaction between AMPA receptor subunit GluA1 and synapse-associated protein 97 (SAP97), a protein scaffold belonging to the protein family of membrane associated guanylate kinase homologs. The interaction between SAP97 and GluA1 has been implicated in AMPA receptor trafficking, neuronal development and synaptic plasticity, while disturbances in normal levels of both GluA1 and SAP97 have been linked to neuropathologies such as Alzheimer s disease and schizophrenia. In the present study, a combination of biochemical and structural work was employed to gain detailed information on the selective interaction of GluA1 with SAP97 identifying molecular determinants involved in and regulating the interaction. X-ray crystallization screens of the second PDZ domain of SAP97 (SAP97PDZ2) yielded well-diffracting crystals both for the apo and ligand bound form. The solved crystal structure of the SAP97PDZ2-GluA1 peptide complex conformed to a conventional class I PDZ interaction with hydrogen bonds forming between the carboxylate group of the ultimate C-terminal residue of the peptide and main chain nitrogens in the carboxylate binding loop of the PDZ domain, and a hydrogen bond between the antepenultimate residue of the peptide and a conserved histidine in the αB helix lining the peptide binding groove. Beside these typical PDZ interactions, as a novel finding we observed contacts within the PDZ domain reorganizing upon peptide binding leading to a slight opening of the peptide binding groove facilitating better accommodation of the ligand. In vitro binding analysis of isolated PDZ domains and short GluA1 peptides showed that, in addition to the prototypic PDZ interaction, a C-terminal cysteine, C893 located upstream from the short PDZ binding motif on GluA1 participated in the interaction through a disulfide bond formed with cysteine C378 of SAP97 under in vitro conditions. Streptavidin pull-down experiments with full-length molecules expressed in cultured cells showed that the C893S mutation leads to a substantial reduction in binding of GluA1 to SAP97, confirming the involvement of C893 in the regulation of the interaction in live cells. Reactive cysteines, like C893, can in addition to disulfide bonds participate in other thiol modifications. In our work we constructed several deletion and cysteine replacement mutants of GluA1 and tested their sensitivity to S-nitrosylating agents nitrosoglutathione and nitrosocysteine. Out of the three C-terminal cysteine residues in GluA1, we identified C893 as the sole cysteine residue sensitive to a post-translational modification by nitric oxide (NO). Furthermore, we found evidence of a physical link between GluA1 and the NO generating neuronal enzyme nitric oxide synthase nNOS via SAP97. The results of the present study provide, for the first time, detailed structural information on the interaction between GluA1 AMPA receptor and SAP97. In addition to a canonical PDZ interaction, the association with SAP97 involves a reactive cysteine residue, C893, in GluA1 C-terminal tail, a potential regulatory target for nitric oxide and oxidative conditions.
  • Andreevskaya, Margarita (Helsingin yliopisto, 2017)
    Lactic acid bacteria (LAB) play a dual role in food manufacturing. While being indispensable for food fermentations and preservation, they are also involved in spoilage of foods and beverages, and some food-borne LAB are pathogens. Particularly, they became the main spoilage organisms in the cold-stored modified atmosphere packaged (MAP) foods. LAB species composition and their relative abundances depend on the nature of food products and preservation technology. However, two LAB species, Leuconostoc gelidum and Lactococcus piscium, have frequently been predominating at the end of shelf life in a variety of packaged and refrigerated foods of animal and plant origin. Besides the predominant species, spoilage LAB communities contain less abundant and slower growing species, such as Lactobacillus oligofermentans, the role of which in food spoilage is unclear. Taking into account the increased popularity of MAP technology combined with cold storage for preservation of minimally processed fresh foods, the need to obtain more information on the metabolism, genomics, ecology and interactions of psychrotrophic food-spoilage-associated LAB is clear. In this thesis a genomic approach was used to study these LAB. In order to characterize spoilage community members, we sequenced and annotated genomes of Lc. piscium MKFS47 and Lb. oligofermentans LMG 22743T, both isolated from broiler meat, and seven strains of Le. gelidum, isolated from vegetable-based foods. The analysis of their gene contents and their comparison with gene repertoire of other close related species allowed us to predict putative factors that might facilitate their survival in their habitats and increase competitiveness in the spoilage microbial communities. No major differences in the gene contents of the vegetable and meat Le. gelidum strains were observed that would suggest niche-specificity, therefore, indicating that the absence of strain dissemination between vegetable- and meat-processing chains is a more likely factor responsible for the reported strain segregation between vegetable and meat-based products. Lc. piscium MKFS47 was identified as an efficient producer of buttery off-odors compounds from glucose under aerobic conditions, which is in agreement with the previous inoculation studies. Time course glucose catabolism-based transcriptome profiles revealed the presence of classical carbon catabolite repression mechanism for the regulation of carbohydrate catabolism, which was relieved along with decreasing concentration of glucose. During the same time, the shift from homolactic to heterolactic fermentation mode was observed. For Lb. oligofermentans, a pentose-preferring obligate heterofermentative LAB, the induction of efficient utilization of hexoses was confirmed indicating that it has flexible carbohydrate catabolism, which can be adjusted depending on the carbohydrate sources available in the environment. Unexpectedly, transcriptome responses of Lb. oligofermentans during growth on glucose and xylose were more alike than during fermentation of ribose in the early exponential growth phase. In addition, cross induction of glucose and xylose catabolic genes by either glucose or xylose was observed. These phenomena could be governed by the CcpA transcriptional regulator, the regulation mechanism of which remains to be determined. Transcriptome-based study of interspecies interactions between three above mentioned LAB species revealed their different survival strategies to cope with competition for the common resources. Le. gelidum was shown to enhance its nutrient- (mainly carbohydrates) scavenging and growth capabilities under glucose limitation conditions when competing with the other LAB species, while the opposite was observed for Lc. piscium and Lb. oligofermentans. Such behavior might explain the competitive success and, hence, the predominance of Le. gelidum in spoilage microbial communities. Peculiarly, interspecies interactions activated expression of prophages and restriction modification systems in Lc. piscium and Lb. oligofermentans, but not in Le. gelidum. The downregulation of stress protection-related genes in all the LAB at the early growth stage was unexpected, and it requires further studies. Finally, overexpression of the numerous putative adhesins in Lb. oligofermentans during growth with other LAB could be one of the factors explaining its survival in actively growing communities in meat.
  • Rosti, Katja Marjukka (Helsingin yliopisto, 2016)
    This thesis work comprises the characterization of proteins from two different neuronal membrane receptor protein families: the growth factor receptor -type (GFRa) of protein, growth arrest specific-1 (GAS1) and the synaptic leucine rich repeat adhesion proteins, leucine-rich-repeat transmembrane-2 (LRRTM2), and synaptic adhesion-like molecules 1 and 5 (SALM1 and SALM5). The GAS1 project has focused on the structural characterization of the recombinant human GAS1 protein, and on the possible interaction and effect of GAS1 on the tyrosine kinase receptor protein, re-arranged during transfection (RET), signaling. GAS1 has two different types of interactions, GAS1-RET signaling participates in neuronal survival and maintenance and GAS1-Patched1-Sonic hedgehog (SHH) signaling is needed both for cell survival and in the development of the enteric nervous system during early development. As a conclusion GAS1 has very different structure from other members of the protein family and can interact directly with RET unlike the other GFRa co-receptors. Indicating a different biological role. The study of leucine-rich-repeat transmembrane proteins (LRRTMs), and the synaptic adhesion-like molecules (SALMs) concentrated characterization of an engineered variant of LRRTM2, SALM1 and SALM5. These proteins are involved in neurite outgrowth, branching and synapse formation. They are mainly expressed in brain, and their malfunction is connected to familial schizophrenia, bipolar disorder and autism. We developed a new strategy for protein structure solution and utilized a statistical sequence-based protein engineering method for generation of a stabilized protein for structural studies. We believe this method will have wider use in structural studies of difficult proteins. The goals of this thesis work were to produce these proteins, solve their structures, test their interactions with ligands and do functional characterization studies using a variety of methods. The results will contribute to a better understanding of the molecular structure and the roles these proteins in neuronal tissue, and possibly generate new research into the cellular phenomena, including diseases, linked to these proteins, and aid in future drug development.
  • Havula, Essi (Helsingin yliopisto, 2017)
    SUMMARY Sugars, amino acids and lipids provide the energy and building blocks for growth and maintenance in all animals. However, animal species display great variation in their dietary preferences. The optimal diet of even closely related species can vary tremendously. Also human populations and individuals vary in their dietary behavior and physiological responses to dietary interventions. Moreover, the high amounts of refined sugars in the modern human diet are suggested to contribute to the development of metabolic diseases such as metabolic syndrome and type 2 diabetes. Multicellular animals sense and control their energy homeostasis continuously by integrating nutritional, hormonal and neuronal inputs from their internal and external environment. For example, the counteracting hormones Insulin and Glucagon maintain the levels of circulating glucose constant during fluctuating nutritional conditions. At the cellular level, macronutrients are sensed by distinct mechanisms. The nutrient sensors and their downstream signaling pathways are activated in response to specific nutrients, and they ensure the metabolic homeostasis. Sugars are sensed by highly conserved transcription factor paralogs ChREBP (Carbohydrate-responsive element-binding protein) and MondoA, which share the same heterodimerization partner Mlx. ChREBP/MondoA-Mlx heterodimers regulate the majority of sugar-induced transcription in mammals, including genes of the glycolytic and lipogenic pathways. Dysregulation of ChREBP has been associated with the development of type 2 diabetes, and polymorphisms of ChREBP with circulating triglyceride levels and increased risk of coronary artery disease. The Drosophila genome encodes single orthologues for ChREBP/MondoA and Mlx called Mondo and Mlx, respectively. The function of the mammalian ChREBP/MondoA-Mlx has been largely studied in vitro. The aim of this thesis was to characterize the in vivo function of Drosophila Mondo-Mlx and to identify its target genes and their roles in regulating sugar metabolism. Due to the lack of genetic redundancy and with the extensive toolkit available, the Drosophila provides an optimal in vivo model for studying the role of Mondo-Mlx in nutrient sensing and metabolism. In this thesis, I demonstrate the physiological importance of Mondo-Mlx for organismal sugar tolerance. The mlx null mutant animals display severe sugar intolerance and a gene expression profile that confirms the role of Mondo-Mlx as a key regulator of glycolytic and lipogenic genes also in Drosophila. Furthermore, we expand the role of Mondo-Mlx as a metabolic regulator by showing that it directly controls the expression of several key enzymes of lipid storage, pentose phosphate pathway and amino acid metabolism in response sugars. We also show that Mondo-Mlx is a master regulator of a gene regulatory network composed of a secondary tier of transcriptional effectors including GLI similar transcription factor Sugarbabe and Krüppel-like factor Cabut. The metabolic profiling of the mlx null mutant animals revealed that in addition to being hyperglycaemic, the mutants show signs of amino acid catabolism and elevated ceramide levels that indicate lipotoxicity. This thesis demonstrates the use of Drosophila in studying the genetic basis of dietary sugar tolerance and metabolism. It reveals a number of new metabolic pathways and downstream effectors regulated by Mondo-Mlx, broadening its role as a master regulator of sugar-induced transcription.
  • Shirokova, Vera (Helsingin yliopisto, 2016)
    Development of the organ and its regeneration in adulthood are highly related events and are often guided by similar molecular cues. The hair follicle undergoes repetitive cycles of growth and represents an ideal model for studying both development and regeneration. Hair follicle is an ectodermally-derived organ formed trough interaction of embryonic epithelium and mesenchyme. Once morphogenesis is accomplished, hair follicle undergoes subsequent cycles of growth, regression and rest. Hair regeneration is ensured by the population of hair follicle stem cells which are set aside early during embryonic development. The most quiescent stem cells reside in the bulge, while the stem cells fated for the new growth are in the hair germ. Transcription factors control gene expression and regulate the variety of cell fate choices. Transcription factor Foxi3 is a causative gene for hairless phenotype in several dog breeds, but little else is known about its function in the hair follicle. In this thesis work, I have studied the role of Foxi3 in hair follicle development and postnatal regeneration. To address this question, several Foxi3-deficient mouse lines were used, as well as gene expression analysis, tissue culture techniques and molecular biology methods. Foxi3 is expressed in the developing hair follicle and in the hair germ, exclusively in the activated stem cells. Foxi3 is essential for specification of the hair follicle stem cells and for their activation at the beginning of the hair growth. Foxi3-deficient mice have delayed hair follicle downgrowth during embryogenesis. In the absence of Foxi3, postnatal natural hair regeneration and hair regrowth after depilation are both impaired due to poor activation of stem cells and decrease in their number. Genome-wide profiling, quantitative PCR and immunostaining showed downregulation of several stem cell associated genes upon Foxi3 loss. Thus, Foxi3 is a novel regulator of hair follicle stem cell specification, maintenance and activation.