Faculty of Biological and Environmental Sciences


Recent Submissions

  • Kokki, Krista (Helsingin yliopisto, 2022)
    In order to survive, all organism require sugars, amino acids and lipids, which are used for energy and growth. The nutritional needs of an organism vary tremendously between species, depending on their environment and physiological period. Despite dietary differences, every organism must maintain their metabolic homeostasis, otherwise they will suffer harmful – and eventually deadly – complications. For example, excess sugar consumption has been linked to metabolic diseases such as metabolic syndrome and type II diabetes. The maintenance of metabolic homeostasis requires regulation and sensing of nutrient levels. Amino acids, lipids and sugars are sensed and monitored through distinct mechanisms that initiate transcriptional responses, which then activate corresponding downstream pathways. Intracellular sugars are sensed by the highly conserved transcription factor ChREBP and Mondo, which heterodimerize with Mlx. The complex then binds to the carbohydrate response elements of their target genes, regulating a variety of metabolic pathways, including glycolysis and lipogenesis. However, despite recent discoveries of new sugar sensors, our understanding of the ChREBP/Mondo-Mlx network remains incomplete. As metabolic regulation, including sugar sensing, is highly conserved between mammals and Drosophila, this organism has been widely used in in vivo studies. Drosophila provides an extensive genetic toolkit, lack of genetic redundancy and a fully sequenced, annotated genome. These features make Drosophila an optimal model for sugar sensing studies. Genome-wide sequencing allows researchers to study organisms’ entire genome at once. However, the organization and analysis of such a large amount of data requires computational methods. A variety of strategies, carried out by computational algorithms, have been developed to best analyze different types of biological data. The focus of this thesis is on usage of in silico methods to uncover novel genetic regulators in sugar sensing. By using in silico methods for both pre-existing and novel data we identified two new sugar responsive members of the Mondo-Mlx sugar sensing network in Drosophila: transcription factors Grain and Clockwork orange (Cwo). Further, we revealed that Grain converges on lipid metabolism with Sugarbabe, a previously known sugar responsive transcription factor. While binding to overlapping genomic sites, Grain contributes to lipogenic gene expression mainly on a low sugar diet, and Sugarbabe under high sugar conditions. We also uncovered tissue-specificity of the sugar-inducible expression of Grain, and established Cwo as a regulatory link between Mondo-Mlx and Myc, both in Drosophila and mouse hepatocytes. We also describe the connection between ChREBP, DEC1 and DEC2 (mammalian orthologs of Cwo) in the regulation of ribosome biogenesis. Lastly, we demonstrate reprogramming of carbohydrate pathways in a relatively short evolutionary timeframe within closely related species. Finally, we identified genes involved in genomic variation of sugar tolerance. All results were achieved through a close co-operation between computational and experimental research. In conclusion, the results of this thesis highlight the potential of computational analyses in driving the identification of new candidate regulators of sugar responsive gene regulation.
  • Cerejeira-Matos, Rita (Unigrafia, 2021)
    The survival of any organism depends on many factors, including their ability to adapt to novel environments. Nutrient accessibility in the environment is crucial for the development, growth, fertility, metabolism, and other biological functions of animals. Nutrients such as sugars, amino acids, and lipids are not only essential building blocks for the maintenance of a typical bioenergetic and cellular homeostasis of animals, but they also serve as signals to regulate cellular functions. The integration of such signals sent by the availability or scarcity of nutrients provided by the environment are sensed by nutrient sensors. Different nutrient sensors are activated or inactivated by specific nutrients, and ultimately mediate the physiological function of an animal through regulation of gene expression. The regulation of gene expression implicated in nutrient sensing can be studied using Drosophila melanogaster as a model organism. The fruit fly has several advantages for studying nutrient sensing and the impact of regulatory gene expression on the physiology of reproduction, growth, and metabolism. Examples of such advantages are reflected in the publicly available genetic toolkit, and the highly conserved molecular mechanisms to humans. This thesis used Drosophila melanogaster as a model to study novel regulatory mechanisms in gene expression. The first study demonstrated the physiological role of the heterodimeric basic-helix-loop-helix zipper (BHLH/Zip) transcription factor, Mondo-Mlx, in the adult reproductive system of fruit flies. Specifically, the impact of its loss of function in the fertility of female flies was shown for the first time. This result led to the discovery of several defects in the oogenesis progression. Finally, ribosome and oogenesis gene clusters were identified as target genes of Mlx. In the second study, evidence was shown for the role of the chromatin binding periodic tryptophan protein 1 homolog (PWP1) as a direct regulator of RNA Polymerase III (Pol III) target genes in a nutrient-dependent manner. Specifically, it was shown that PWP1 directly binds to the 5S rRNA gene, a well-known Pol III target, and induces its expression in a nutrient-dependent manner. By conducting this study, both in Drosophila melanogaster larvae and mammalian cells, it was shown that this biological role of PWP1 was conserved among organisms. The third study was the first to demonstrate the physiological role of actin in the reproductive system of adult flies and its requirement for Drosophila melanogaster oogenesis progression through direct regulation of RNA Polymerase II (Pol II) transcribed genes. In conclusion, using Drosophila melanogaster as a model, these three studies identified previously unknown mechanisms in gene expression that ultimately affect biological functions such as fertility and growth of the fruit fly.
  • Guenther, Carla (Helsingin yliopisto, 2020)
    Our health is protected by the immune system, which maintains a carefully regulated balance between inflammation and immune suppression. To mediate homeostasis and inflammation, leukocytes continuously patrol the body and therefore operate in a dynamic mechanical landscape. Mechanical information is transduced into the cell via different proteins, including adhesion receptors called β2-integrins. These receptors mediate essential leukocyte processes, including phagocytosis, immune cell trafficking, adhesion under shear flow conditions of the blood stream and immunological synapse formation between antigen presenting cells and T-cells. However, β2-integrins can also restrict inflammatory processes, such as macrophage Toll-like receptor signalling and cytokine expression, as well as dendritic cell maturation and migration. The precise signalling pathways involved in β2-integrin-mediated leukocyte regulation are not yet fully unravelled. β2-integrin activity is regulated via conformational changes. β2-integrin conformational changes are mediated by interactions with cytoplasmic proteins, such as talin, filamin A and kindlin-3. Talin, filamin A and kindlin-3 are sensitive to mechanical forces and respond by changing their conformation. However, the precise roles of these two proteins in regulating β2-integrin mediated immune processes, especially in response to force, have not yet been fully investigated. The aim of this doctoral thesis was to study molecular mechanisms involved in β2-integrin-mediated leukocyte mechanotransduction and immune responses. We investigated the role of filamin A in β2-integrin-mediated primary neutrophil and T-cell adhesive functions using conditional filamin A knockout mice. In neutrophils, filamin A restricts β2-integrin mediated cell spreading and static adhesion. Furthermore, filamin A was found to inhibit neutrophil oxidative burst, but it was required for proper formation of neutrophil extracellular traps. In T-cells, filamin A restricts F-actin content in cells spreading on integrin ligands. Interestingly, filamin A plays a different role in T-cell adhesion than in neutrophils, as it is required for optimal adhesion under shear flow conditions, and for the generation of integrin-mediated traction forces. Furthermore, filamin A is necessary for T-cell homing in vivo and for T-cell trafficking to sites of inflammation. Together the studies therefore revealed a dual role for the mechanosensitive integrin-binding protein filamin A in regulating leukocyte adhesive processes. In dendritic cells, β2-integrins have previously been shown to regulate gene expression, and to restrict cell maturation, migration and dendritic cell-mediated T-cell activation. Here, we used a β2-integrin TTT/AAA knock-in (KI) mouse model, where the β2-integrins/kindlin-3 interaction has been disrupted, which leads to expressed but dysfunctional integrins. The aim of these studies was to identify integrin-mediated signalling pathways regulating the mature dendritic cell phenotype. In our studies, we found that the mechanoresponsive MRTF-A/SRF pathway is downstream of β2-integrins in dendritic cells. Furthermore, this pathway regulates cytoskeletal gene expression, which governs the ability of dendritic cells to adhere and to generate traction forces, but not dendritic cell 3D migration. We subsequently identified novel mechanotransductional mechanisms involved in integrin-mediated dendritic cell programming and function. We discovered a β2-integrin/actin/lamin link in dendritic cells that regulates global histone methylation in these cells, as well as chromatin accessibility and gene expression. Interestingly, targeting this mechanical link leads to a similar mature dendritic cell phenotype as that caused by abolishing β2-integrin function, and can be used to induce better dendritic cell-mediated tumour rejection in vivo. Taken together, these studies outline several β2-integrin mediated leukocyte mechanotransduction pathways, which we demonstrate are relevant for immune system function in vivo. The results highlight the importance of considering and studying how mechanical forces and information impact immune system function. This is especially important in diseases such as cancer and during aging where the mechanical properties of tissues change over time.
  • Heo, Jungok (Helsingin yliopisto, 2020)
    The Arabidopsis root meristem consists of concentrically arranged tissues that surround the central vasculature. While the outer cell layers, the epidermis and ground tissue (GT), contribute to the radial symmetry of the entire root, the internal vasculature displays bilateral symmetry. These spatial patterns are established by the activity of stem cells located at defined positions within the stem cell niche. Daughter cells undergo cell expansion and differentiation to form shootward files along the longitudinal axis of the root. In the first part of this thesis, I describe GT specification and maturation. GT comprises the endodermis and cortex, and their lineages bifurcate following the asymmetric cell division (ACD) of the cortex-endodermis initial. The ACD requires the function of SHORT-ROOT (SHR) and its downstream regulator SCARECROW (SCR), which both belong to the GRAS transcription factor (TF) family. GA in the endodermis modulates the timing of GT maturation by promoting a second periclinal cell division which gives rise to an additional cortical layer between the endodermis and cortex. This additional cortex layer is named the middle cortex, and this process also requires SHR/SCR activity. Here, we have genetically and molecularly demonstrated that another GRAS member, SCARECROW-LIKE 3 (SCL3), is a direct downstream target of SHR/SCR, and its expression is also under the direct control of REPRESSOR OF ga1-3 (RGA), one of the DELLA proteins that are Gibberellin (GA) signaling inhibitors. SCL3 is expressed exclusively in the endodermis, where it integrates the SHR/SCR-mediated developmental signal and the GA-mediated hormonal signal to fine-tune the timing of middle cortex formation. The second part of my thesis investigates phloem sieve element (SE) development. SEs form a conductive tissue in the phloem that mediates the long-distance transport of sucrose. In Arabidopsis roots, protophloem SEs (PSE) comprise 20-25 cells from the stem cell to the enucleating cell. This cell file can serve as a model system to study tissue morphogenesis at single-cell resolution. Using confocal time lapse imaging technology, we monitored single-cell behaviors along the PSE lineage and defined five discernible stages: quiescent (stem cell), active division (transit amplifying cells), transition, differentiation and nuclear degradation (enucleation). We then focused on PSE differentiation, which involves dynamic cellular rearrangement ending with enucleation. We discovered a regulatory cascade in which ALTERED PHLOEM DEVELOPMENT (APL) and two closely related NAC-domain containing TFs – NAC45 and NAC86 – play major roles. We also identified NAC45/86-DEPENDENT EXONUCLEASE-DOMAIN PROTEINs (NENs) that is involved in nuclear degradation downstream of the APL-NAC pathway. In order to comprehensively understand PSE development, we carried out phloem-specific transcriptome profiling by performing fluorescence activated cell sorting of various phloem reporter lines followed by RNA-seq. By analyzing the transcriptome data, we identified 925 phloem-abundant genes. We focused on a cluster of genes which initiate their expression early in the protophloem sieve element (PSE) lineage. Interestingly, this cluster includes six DOF transcription factor family members. Knocking out all six genes resulted in a very narrow vasculature and impaired phloem transport, indicating that these DOFs are required for phloem differentiation as well as specification. We also determined that these DOFs promote PSE differentiation by directly activating the APL pathway. As part of the root meristem, the PSE must differentiate in coordination with the surrounding tissues. In fact, defects in vascular tissue development often lead to systemic growth inhibition and even seedling lethality in extreme cases. In the final part of this thesis, I demonstrate that the global root meristem regulator PLETHORA (PLT) overrides DOF function in the early stages of phloem development by suppressing the expression of APL, thereby preventing premature PSE differentiation.
  • Yan, Yan (Hansaprint, 2018)
    The LKB1-AMPK signaling pathway is critical in cell growth control and energy metabolism homeostasis. As a energy sensor, AMPK regulates numerous downstream pathways to reprogram cellular energy metabolism, thereby restoring energy balance via direct phosphorylation of downstream substrates or indirect modulation of metabolic transcription programs. While the discoveries of AMPK-regulated pathways have been crucial in understanding how AMPK restores energy homeostasis, evidence of modulating AMPK activity in a substrate specific manner is lacking. The first study of this thesis provided the first evidence that AMPKα1, the catalytic subunit of the AMPK, is SUMOylated and that the activity of AMPKα1 towards mTORC1, a downstream pathway of AMPK and a central regulator of cell growth, is specifically regulated. SUMOylation of AMPKα1 is induced upon AMPK activation, and PIAS4 was identified as the E3 ligase catalyzing the AMPKα1 SUMOylation. Cells depleted of either the sole E2 enzyme Ubc9, or E3 ligase PIAS4, or that harbor only SUMOylation-defective AMPKα1 mutant, all exhibit significantly higher AMPKα1 activity specifically towards mTORC1. Taken together, these data suggest that SUMOylation of AMPKα1 by PIAS4 upon AMPK activation serves as a “braking system” to prevent over-repression of mTORC1. Importantly, depleting PIAS4 potentiates the ability of AMPK to suppress mTORC1 and cell proliferation of breast cancer cells, implying that inhibition of AMPK SUMOylation may provide a novel strategy to inhibit cancers with hyperactive mTORC1.  In contrast to well-established roles in maintaining cellular energy homeostasis, the functions of AMPK in non-metabolic cellular processes have been less investigated. The second study of this thesis examined the role of AMPK in cell adhesion and mechanotransduction, and identified AMPK as a negative regulator of integrin, a key mediator of cell-matrix interactions. AMPKα1 and AMPKα2 redundantly repress ß1-integrin activity, integrin-dependent fibrillar adhesion formation, cell spreading and traction stress. Mechanistically, AMPKα inhibits integrin activity by repressing the expression of integrin-binding proteins, Tensin1/3. The study demonstrated that repression of Tensin by the energy sensor AMPK is a novel mechanism that regulates the activity of integrin, an essential membrane receptor that mediates extracellular matrix (ECM)-cytoskeleton interactions.  While the primary function of Lkb1, the major upstream kinase of AMPK in response to energy stress, is to restrict cell division and thereby suppress neoplastic growth of many tissues, it has also been reported to play critical roles in tissue homeostasis, such as hematopoietic stem cells (HSCs) homeostasis where Lkb1 is essential for maintaining stem cell quiescence. The critical role of Lkb1 in HSCs raised an intriguing question: does Lkb1 play a similar role in all stem cell populations or is it restricted to stem cells that are predominantly quiescent? The third study of this thesis investigated the role of Lkb1 in actively cycling intestinal stem cells (ISCs) and identified Lkb1 as a critical factor for maintaining ISCs. Mechanistically, Lkb1 represses the secretory cell gatekeeper, Atoh1, to restrict ISCs and intestinal progenitors from differentiating into secretory cell lineages. Importantly, both the repression of Atoh1 and the maintenance of ISCs by Lkb1 are independent of the classic Dll-Notch mediated cell extrinsic mechanism, thus establishing Lkb1as a cell intrinsic regulator of Atoh1 and ISCs.
  • Jousimo, Jussi (2018)
    Environmental and ecological processes drive interactions between organisms in space and time. Inferences from observations, that are dependent and measure the processes of interest indirectly, require an appropriate statistical approach that accounts for the processes' autocorrelation and stochasticity. While acknowledged by ecologists, the presence of spatial dependencies is often ignored. We developed a spatio-temporally explicit statistical framework and considered three case studies in spatial population ecology. In the first study, we conducted a simulation study by mimicking a large-scale and long-term survey of a sparsely populated hypothetic species. The spatio-temporal approach provided density estimates which were found to be unbiased and more precise compared to the baseline models. Furthermore, the model was able to uncover missing observations if there were nearby observations. In addition, we developed a method to estimate population densities for habitat types that were not covered in the study by utilizing individual-level GPS movements data. During winter, the Eurasian red squirrel (Sciurus vulgaris) depends on cone seeds as its main source of nutrition and it is preyed on by several predators. In the second case study, we combined data from surveys of almost 30 years of multiple species in Finland and applied the spatio-temporal framework to find the effect of adjacent trophic interactions on the population dynamics of the red squirrel. The results showed that the population fluctuations of the red squirrel appears to be mostly driven by bottom-up effects such as the cone yield. Positive association with the predator abudance likely results from a shared preference for the habitat rather than predation, but local variation may exists. Powdery mildew (Podosphaera plantaginis) is a fungal disease that infects the plant Plantago lanceolata. In the Åland island of Finland, the plantago is found in a network of meadows and occupancy of the mildew was observed by a ten-year survey. The pathogen follows metapopulation dynamics with occasional colonizations and extinctions of local populations in the host network. Ecological theory predicts that the more isolated populations of a host plant are less susceptible to an infection. On the other hand, higher rates of gene flow in more connected areas facilitate the evolution of resistance. In the last study, by applying the spatio-temporal approach, we found the spatial connectivity to correlate negatively with the occupancies and colonizations and positively with the extinctions. A laboratory experiment confirmed that the plantago is more resistant in more dense sub-networks. We hypothesize this to be a result of a higher gene flow in more dense sub-networks and/or an ability to invest in resistance more in nutritionally rich areas.
  • Hasygar, Kiran (Painosalama Oy, 2017)
    The ability to dynamically adapt to fluctuating nutrient availability determines the survival of living organisms. While the capacity to grow rapidly and build energy stores on a nutritious diet can provide a selective advantage, being able to endure nutrient scarcity is also key to survival. Insulin/Insulin-like Signaling (IIS) is a conserved anabolic pathway which drives growth and energy storage during nutrient abundance. However, the IIS needs to be suppressed during starvation to readjust growth and energy homeostasis. The molecular factors which regulate the IIS to determine the optimal balance between anabolism and catabolism, aiding the survival of animals in a wide range of nutrient conditions remain poorly characterized. Using Drosophila melanogaster as a model organism, we aimed to identify novel nutrient responsive signaling pathways that coordinate animal growth and metabolic homeostasis. Conservation of the IIS pathway, availability of extensive genetic toolkit, and little genetic redundancy make Drosophila an attractive model to study growth and energy metabolism. Insulin secretion is dynamically regulated by nutrition in flies, and multiple nutrient sensing pathways converge on the Insulin Producing Cells (IPCs) to modulate Insulin secretion according to the nutrient status. Hence, we aimed to perform a tissue specific RNAi screen in the IPCs to identify novel regulators of the IIS in vivo. In this thesis, we discover a novel role for a ribosome surveillance pathway in starvation response. We show that suppression of ribosome biogenesis in the IPCs leads to activation of a ribosome surveillance response, resulting in inhibition of Insulin secretion and animal growth. Also, we identify tumor suppressor p53 and an atypical MAP kinase ERK7 as essential components of this pathway. We demonstrate that ERK7 is expressed in the IPCs upon starvation, and its expression is sufficient and essential to inhibit Insulin secretion and growth. Moreover, inhibition of p53 or ERK7 in the IPCs results in a blunted starvation response, highlighting the importance of this cell intrinsic pathway. Further, we establish that another component of the secretory pathway, the Exocyst complex is essential for Insulin secretion in flies. In addition to its function in the IPCs, we show that ERK7 expression is elevated upon starvation in fat body, the fly counterpart of liver and adipose tissue, to regulate growth and metabolism. While ERK7 in the fat body limits animal growth through inhibiting the expression of an insulinotropic peptide, Stunted, it inhibits de novo lipogenesis and triacylglycerol (TAG) storage by suppressing the expression of a transcription factor, Sugarbabe. Also, we have generated ERK7 mutant flies, which display accelerated growth rate and store more TAG on a nutrient rich diet, consistent with ERK7 being an inhibitor of growth and lipid synthesis. However, ERK7 mutants fail to limit their growth, and sufficiently mobilize lipid stores during nutrient scarcity, and consequently survive poorly upon starvation. Further, we show that ERK7 regulates a majority of the starvation-induced gene expression on a genome wide scale, thus establishing ERK7 as a major determinant of systemic starvation response. Thus, we demonstrate that ERK7 is a starvation induced molecular factor regulating growth and metabolic homeostasis. Through inhibiting anabolic processes such as the Insulin signaling and lipid synthesis, and activating catabolic processes such as lipid breakdown, ERK7 facilitates survival of animals upon starvation.
  • Koskela, Katja (Juvenes Print, 2017)
    A biological threat is an epidemic or its threat caused by a microbe or biological material of a magnitude that would overwhelm healthcare services due to the contagiousness or wide distribution of infections. A biological threat can be naturally occurring, such as the West African Ebola epidemic of 2014-2016, or the consequence of an intentional release of a microbe or toxin. The aim of this thesis was to develop and use molecular methods in order to reliably and rapidly identify potential biological threat agents. The focus was on the detection and typing of biological threat agents, whether they are naturally occurring or intentionally released. Different molecular methods were used: polymerase chain reaction (PCR) to detect and differentiate pathogenic from non-pathogenic bacterial strains, 16S ribosomal RNA (rRNA) gene sequencing to investigate polymicrobial samples, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) spacer comparison for bacterial strain typing. Cholera, a disease caused by Vibrio cholerae bacterium, is a major public health problem worldwide and a potential bioterrorism agent, according to the Centers for Disease Control and Prevention (CDC). In this thesis, an accurate PCR-based method was developed to detect V. cholerae strains: one assay for pathogenic strains and another for all V. cholerae strains. In addition, three different PCR platforms were compared. The PCR assays proved to be suitable for the reliable identification and differentiation of V. cholerae strains. The PCR platforms gave identical results, which indicate that the assays can be transferred between the platforms while maintaining sufficient sensitivity and specificity. Two 16S rRNA gene-based detection methods, using Sanger sequencing or pyrosequencing, were employed to study the presence of bacterial residues in carotid artery tissue samples and in livers of splenomegalic voles. The objectives were to observe the utility of the two methods and compare their performance. Both methods were found to be convenient approaches to detect and identify bacterial species present in different matrices and thus could be employed when investigating polymicrobial samples. In addition, the two methods gave similar results which emphasises the reliability of the methods and their results. The Yersinia genus includes three human pathogens; Y. pestis, the causal agent of plague and a potential biothreat agent, as well as Y. enterocolitica and Y. pseudotuberculosis, which commonly cause self-limiting enteritis. Due to the high level of DNA similarity between Y. pestis and Y. pseudotuberculosis, typing of Yersinia species and distinguishing them from each other has been challenging. Here, CRISPR spacers were used for typing Yersinia pseudotuberculosis complex strains. This method proved to be a promising tool, although the large diversity of different spacer sequences hindered the clustering of different strains. In addition, CRISPR data of Y. pseudotuberculosis and Y. pestis were compared to examine phylogenetic relationships, but surprising lack of shared spacers limited any further resolution from being made. In this thesis, molecular methods were developed and used to detect, identify, and type potential biological threat agents. PCR assays developed can be transferred to a field-deployable instrument and thus employed close to the patient, for example, during epidemics. PCR results were ready within a few hours, enabling a rapid and appropriate medical response. The 16S rRNA gene-based methods can be utilized in detection of biological agents, which are challenging or laborious to identify using traditional methods. The CRISPR-based sequencing method can be used for typing different strains of Y. pseudotuberculosis, if a comprehensive reference database is made available. DNA sequencing and recently next-generation sequencing have become powerful tools to identify and type biological agents. Sequencing methods can also be utilized in epidemiological investigations and source tracking. Different molecular methods have evolved recently and detection has become fast and more reliable. Rapid detection of microbes enables swift medical countermeasures, and accurate identification and typing methods facilitate the ability to distinguish a natural outbreak from an intentional release.
  • Mätlik, Kert (Helsingin Yliopisto, 2017)
    Acute ischemic stroke is among the leading causes of long-term disability in developed countries. Among the contributing factors are our inability to limit brain damage in the acute-phase of stroke and the infrequency of complete spontaneous functional recovery. Effective pharmacological therapies that would protect neurons from ischemia-induced death are not available. Moreover, as of now, there are no proven pharmacological treatments to enhance the limited neurological recovery process in stroke patients. Neurotrophic factors MANF (mesencephalic astrocyte-derived neurotrophic factor) and CDNF (cerebral dopamine neurotrophic factor) are currently among the most promising molecules for the treatment for Parkinson s disease (PD). Namely, both have shown great therapeutic potential in rodent models that mimic the most conspicuous and debilitating symptoms of PD, caused by the progressive degeneration of dopaminergic (DA) neurons in the substantia nigra (SN). However, in these preclinical studies the fate of CDNF and MANF after their therapeutic intracerebral administration has been poorly characterized. While MANF has been shown to protect cortical neurons from death in a rat model of ischemic brain injury, it is unknown whether CDNF has a similar effect. Moreover, the neurorestorative effects CDNF and MANF have been shown to have in animal models of PD raise the question of whether these factors could act similarly after cerebral ischemic damage and enhance functional recovery. In order to have a better understanding of what happens to recombinant human CDNF (rhCDNF) in brain tissue, we studied its distribution, intracellular localization and clearance after infusion into rat brain. We present the pharmacokinetic properties of striatally infused rhCDNF and describe the main intracellular localization patterns after its neuronal uptake. We also present data that after intrastriatal infusion, the rhCDNF found in the SN is almost exclusively localized to the DA neurons, thus showing that it is retrogradely transported. To shed light on the possible neuroprotective potential of CDNF for ischemic stroke, we studied the effect of CDNF in the rat model of ischemic brain injury achieved by temporary unilateral occlusion of the middle cerebral artery. We show that CDNF is protective against cortical ischemic injury when administered as a recombinant protein, but unlike MANF, not when delivered via a CDNF-expressing adeno-associated virus vector (AAV-CDNF). Our findings thus unmask an important difference in MANF and CDNF s capacity for therapeutic action. Using the same rat model of ischemic stroke, we studied the effect of delayed AAV-MANF administration on post-stroke behavioural recovery, taking advantage of the delivery method we have developed in order to target the expression of AAV vector-delivered genes to the peri-infarct area. We report that AAV-MANF, delivered to the peri-infarct area two days after transient ischemia, accelerates the reversal of ischemia-induced behavioral deficits without affecting lesion size. While histological analyses of brain tissue could not point out which cellular process mediates MANF s effect, an unbiased transcriptomics approach implies modulation of the activity of innate immune cells as a possible mechanism. In summary, MANF should be considered as a possible therapeutic agent or a drug target for promoting functional recovery after stroke. In addition, we show that in cultured peripheral neurons MANF needs to be in the endoplasmic reticulum in order to exert its intracellular survival-promoting activity. We also present data on the importance of two sequence motifs of MANF for its intracellular survival-promoting activity and the neuroprotective efficacy that extracellularly applied recombinant human MANF has in the rat model of cortical ischemic injury. In summary, this work has extended the knowledge on MANF and CDNF s capacity for therapeutic action, and the pharmacokinetic and structural properties important for their therapeutic use in further studies.
  • Bulanova, Daria (Helsingin yliopisto, 2016)
    Breast cancer is the most common malignancy in women. Familial history and inherited genetic mutations are the most important risk factors for the development of this type of neoplasia. However, according to current estimations, known highly penetrant germline mutations (e.g. in BRCA1 and BRCA2 genes) explain the origin of less than 30% of familial breast cancer cases 2 5. It urges for identification of the novel genetic determinants of breast carcinogenesis. In the presented work, we performed a whole exome sequencing-based search for new breast cancer-predisposing genetic mutations. We identified a germline inactivating mutation c.183delG [p. Arg61fs] in an orphan G protein-coupled receptor-encoding gene GPRC5A as a novel genetic determinant of breast cancer, which is highly prevalent in BRCA1-associated familial tumors. Following this finding, we revealed that GPRC5A modulates BRCA1 expression and function in homologous recombination-mediated DNA repair, suggesting that GPRC5A may act as a genetic modifier of BRCA1-mediated breast cancer progression. GPRC5A has been implicated in the epithelial carcinogenesis, but so far its function remains controversial. Exploring the effect of GPRC5A deficiency or excessive expression on different aspects of cancer progression, we discovered that GPRC5A plays a role in cancer cell-matrix interactions, and found that it functions as a positive modulator of integrin β1-mediated adhesion. The data suggest that GPRC5A may play a role in progression of particular types of epithelial tumors. We found that GPRC5A associated with two more matrix interaction regulators, Ephrin receptor A2 (EphA2) and hyaluronic acid receptor RHAMM; the effect of GPRC5A on RHAMM expression was BRCA1-dependent. Finally, we revealed that GPRC5A expression in mammary glands is estrogen-dependent and correlates with proliferative capacity of estrogen receptor-positive (ER+) mammary tumors, proposing GPRC5A as a biomarker of ER+ tumors progression. We propose that GPRC5A is a determinant of breast carcinogenesis, whose function depends on the context of the genetic background (presence of BRCA1 mutation) and the hormonal status of the tumor.
  • Siligato, Riccardo (2016)
    Plants possess the rare capability to shape the own architecture according to biotic and abiotic stimuli received from the environment. Spatially defined groups of cells, called meristems, contribute to the division and differentiation processes continuously occurring inside the organism. Meristems can be classified as primary meristems, if they are specified during embryogenesis, or secondary meristems, if they form from undifferentiated, quiescent cells outside the primary meristems. Primary meristems, like the Root Apical Meristem (RAM) and the Shoot Apical Meristem (SAM), coordinate the apical growth of the plant in opposite directions, while secondary meristems shape the radial architecture, regulating the thickness and branching of the primary root and shoot. Cambium is a secondary meristem which produces the vascular tissues xylem and phloem. Xylem transports water and minerals from the root to the photosynthetic tissues; it comprises lignified dead conducting cells called tracheary elements, living parenchyma cells, and lignified dead cells, called fibres, which confer mechanical support and strength. Phloem distributes glucose, RNA, viruses, and proteins from the photosynthetic sources to the sink cells; it consists of empty living sieve elements, supporting companion cells, and parenchyma cells. In order to investigate the regulation of primary and secondary growth, we developed a new chemically inducible system to control the timing and location of the induction of an effector or gene of interest. This enables us to avoid deleterious effects such as seed lethality or sterility when studying the role of a gene in a particular cell type. For example, the meristem cambium is difficult to access through normal techniques, since mutations affecting cambial cell divisions often inhibit the primary growth, too. We developed the inducible system by combining the Multi-Site Gateway cloning technology with the already extant XVE inducible system. This system was used to perform part of the research presented in the thesis. Phytohormones are involved in virtually every aspect of plant life, from development to stress response. They are small molecules which act cellautonomously or non-cell-autonomously to mediate the majority of developmental and environmental responses and, consequently, the activity of the meristems throughout the plant life cycle. Auxin and cytokinins, which were among the first phytohormones discovered, regulate almost every aspect of plant life, such as the division and differentiation processes occurring continuously in the RAM and SAM. The two phytohormones have long been known to interact, and recent studies have uncovered significant crosstalk on the level of biosynthesis, transport, signalling and degradation. We investigated the dynamic role of auxin in maintaining the balance between division, elongation, differentiation in the RAM of the model organism Arabidopsis thaliana. Our results confirm that an optimal level of auxin response is required for division and elongation, while differentiation mechanisms require just a minimal concentration of auxin to proceed normally. We discovered that auxin and cytokinin responses interact synergistically to specify the stem cells and to regulate the timing of divisions in the cambium of Arabidopsis thaliana. The auxin and cytokinin signalling pathways both have a positive role in triggering secondary growth, but the hierarchy of the crosstalk between them is still unclear. Finally, auxin transported via the AUX1/LAX auxin influx carriers regulates the differentiation of vessel elements in the later stages of root cambium development. In summary, we confirm that auxin and cytokinins behave as master regulators of meristematic activities throughout the root, as the signalling pathways associated with both phytohormones heavily influence primary and secondary growth.
  • Mönttinen, Heli (2015)
    The right-hand-shaped polymerases comprising the DNA/RNA polymerase superfamily represent at least six different protein families containing replicases, transcriptases and repair proteins from all three domains of life as well as from their viruses. All of these polymerases have at least three subdomains: fingers, palm and thumb, which form together a structure resembling a right-hand. The catalytic site is located in the palm subdomain, in which polymerization process is catalysed by two Mg2+ ions. There can also be additional ions such as the non-catalytic ion in the RNA-dependent RNA polymerase of phage φ6, which is located approximately 6 Ångströms from the catalytic site. The phylogeny and common regions of the right-hand-shaped polymerases have been previously studied mainly using amino acid sequence alignments. However, the sequence similarity between polymerases belonging into different protein families is low and therefore, the structure alignment provides a potential alternative because protein structures can retain similarity longer in evolution than amino acid sequences. In this thesis, the common structural features between and within the families of the right-hand-shaped polymerases are described, and based on the structural cores the phylogenetic trees are deduced. In addition, the phylogenetic relationships between the right-hand-shaped polymerases and other structurally related proteins are described. As results, it is shown that a phylogenetic tree following the established boundaries of protein families is possible to construct based on structural core sharing no sequence identity. This tree, illustrating long distance phylogenetic relationships suggests that the known right-hand-shaped polymerase families are not the closest relatives to each other. The phylogenies within polymerase families suggest that the relationships among the polymerases do not always follow the evolution of the corresponding organism, which implies horizontal gene transfer between cells, and cells and viruses. The phylogeny of RNA virus RNA polymerases seem to be dependent on the priming mechanism and it does not follow the virion architecture or the Baltimore classification. In addition, the viral RNA-dependent RNA polymerases seem to share a third ion binding site in the proximity of catalytic site.
  • Jaakkola, Salla (Helsingin yliopisto, 2014)
    Extremely saline environments include salt lakes, evaporation ponds, and terrestrial environments, such as salt deserts and underground halite deposits. They are inhabited by halophilic microbes that require salt for living. Cell densities in hypersaline waters can be as high as 107-108 cfu/ml, and most of the cells are archaeal. The number of viruses can be ten times higher than that of the cells. In buried halite, the cell counts are generally low, but viable bacteria and archaea have been isolated from samples up to Permian in age (250-280 million years). Icosahedral tailless virus types seem to be common in hypersaline waters, based on microscopic studies. However, only few such viruses have been isolated and studied. In this thesis Haloarcula hispanica icosahedral virus 2 (HHIV-2) was studied using virological, biochemical, sequencing, lipidomic, and cryo-electron microscopy methods. HHIV-2 infects a halophilic euryarchaeal host and is virulent. It is icosahedral, tailless, and contains an inner membrane. The properties of HHIV-2 were compared to two structurally related viruses: haloarchaeal virus SH1 and thermophilic phage P23-77. The comparison revealed the evolutionary stability of the virion capsid structure, in contrast to the host-interacting structures of viruses. It was also established that different virus capsid assembly pathways can lead to identical capsid architecture. Drill core samples from deeply buried halite deposits were used for isolating halophilic microbes. Nine novel unique archaeal strains belonging to Halobacterium and Halolamina were obtained. No bacteria or viruses could be isolated. Three archaeal isolates from 40 million years old halite were found to be polyploid. Polyploidy is connected to higher mutation resistance, which might positively affect the survival of cells inside halite deposits. One unique isolate was obtained from 123 million years old halite. The complete genomic sequence of this isolate was resolved. Based on sequence data and DNA-DNA hybridization, the isolate represented a novel species, and was named Halobacterium hubeiense. The isolate was found to be closely related to halophilic archaea residing in surface habitats.
  • Dopie, Joseph (2014)
    Actin controls numerous nuclear events including transcription factor activity, chromatin remodeling and RNA polymerase activity. As a component of the cytoskeleton in the cytoplasm, actin traditionally influences cell motility, cell division, cell shape and intracellular transport. In the cytoplasm, actin-binding proteins (ABPs) regulate the dynamic interplay between actin polymerization into filaments and depolymerization into monomers, a process that is central to the cytoplasmic functions of actin. Details of the nuclear functions of actin are unclear and it remains unknown whether actin polymerization and depolymerization in the nucleus is directly linked to the nuclear functions of actin. Many cytoplasmic ABPs have also been identified in the nucleus and shown to influence gene expression, yet their nuclear function in relation to actin is not clear. Actin utilizes an active mechanism to exit the nucleus, however, the nuclear import mechanism for actin has not been characterized. This work provides evidence to support an active nuclear shuttling mechanism for actin and identify novel regulators of nuclear actin. Our live cell imaging data show that actin actively and constantly shuttles between the nucleus and the cytoplasm. Using RNA interference (RNAi) mediated loss-of-function analysis, we show that unphosphorylated cofilin, an ABP, and importin 9, a member of the karyopherin β family of transport receptors, are required for nuclear localization of actin. Protein interaction experiments show that importin 9, cofilin and actin form an import complex that mediates nuclear localization of actin to promote efficient transcriptional activity. Our genome-wide RNAi screens have identified novel and conserved regulators of nucleocytoplasmic transport of actin. Notably, we identified cell division cycle (CDC)73, also known as parafibromin, a component of the RNA polymerase II associated factor homolog (PAF)1 complex and cyclin-dependent kinase 13 (CDK13), a protein that controls cell fate, as regulators of nuclear export of actin. On the other hand our data implicate protein kinase activated gamma subunit 1 (PRKAG1), a regulatory subunit of the AMP-activated protein kinase (AMPK) and RAB18, a member of the ras-related protein family, as factors that promote nuclear import of actin. Also, we identify novel regulators of cofilin phosphorylation that influence nuclear localization of actin. These include; Capping protein B (CPB), an actin filament barbed end capping protein; shibire (SHI)/dynamin, involved in endocytosis; BTB and CNC homology 2 (BACH2), a Pox virus and Zinc finger domain-containing transcriptional regulator; receptor for protein kinase C 1 (RACK1) and structure-specific recognition protein (SSRP), a member of the facilitates chromatin transcription (FACT) complex. BACH2 promotes cofilin dephosphorylation via repression of Lim kinase (LIMK) and testis-specific kinase (TESK) expression, while CPB promotes cofilin dephosphorylation via activation of the expression of slingshot. This study has thus provided essential insights into the regulatory mechanism of nuclear actin and identified several routes through which nuclear actin levels could be regulated.
  • Yuan, Qiang (Qiang Yuan, 2012)
    SNARE Complex Regulation in Membrane Fusion SNAREs are membrane associated proteins essential for intracellular protein trafficking and membrane fusion. There are at least 24 SNAREs in the yeast Saccharomyces cerevisiae and more than 35 SNAREs in mammalian cells. Syntaxin1, SNAP25 and VAMP in mammalian cells can form a SNARE complex and function for the synaptic vesicle docking and fusion at neuronal synapses. The yeast cells contain two syntaxin homologous SNAREs, Sso1p and Sso2p that together with Sec9p and Snc1/2pmediate membrane fusion during exocytosis at the plasma membrane. Based on new phosphorylation mass spectrometry data, the contribution of phosphorylation on Sso protein in vivo function was assessed. Basal or overexpression of phosphomimicking or putative non-phosphorylated Sso1p or Sso2p mutants resulted in no obvious growth phenotype. Sso1p is specifically required for de novo formation of the prospore membrane during meiosis in sporulating cells. Cells expressing only mutant versions of Sso1p, however, did not display any detectable sporulation defects. In addition to sporulation, pseudohyphal and invasive growth modes are regulated by the availability of nutrients. Deletion of SSO1 or SSO2, or expression of the phospho-mutant versions of SSO1 or SSO2 as the sole copies of SSO genes caused no defects in haploid or diploid pseudohyphal and invasive growth. The results indicate that the tested phosphorylation sites in Sso1p or Sso2p are not functional in vivo. In addition to the SNAREs, the Sec1/Munc18 (SM) protein family and other proteins also function in the membrane fusion by facilitating the SNARE complex assembly. However, the molecular interactions for these proteins are not well understood. This study shows that Mso1p, a yeast Sec1p binding protein, interacts with cellular membranes through two biochemically distinct sites. The N-terminal region can insert into the lipid bilayer whereas the C-terminal region of the protein binds phospholipids mainly through electrostatic interactions and may associate with secretory vesicles. Mso1p lipid binding is essential for the plasma membrane localization of the Mso1p-Sec1p complex and for the function of Mso1p in membrane fusion in vivo. The results suggest the existence of a conserved mode of molecular interactions between SM protein binding proteins, Rab GTPases and lipid binding in SNARE mediated membrane fusion. We used a novel Sec1p mutant to study how Sec1p interacts with other proteins involved in SNARE assembly. The results indicate that Sec9p displays a novel type of interaction mode with Sec1p. In addition, the results suggest that Sec1p is required for the plasma membrane interaction of Sec9p-Sec4p-Sro7p complexes. Collectively, the results in the thesis suggest novel interaction modes and previously unknown regulation for the SNARE complex formation for exocytosis in yeast.
  • Sarin, Peter (2010)
    Double-stranded RNA (dsRNA) viruses encode only a single protein species that contains RNA-dependent RNA polymerase (RdRP) motifs. This protein is a central component in the life cycle of a dsRNA virus, carrying out both RNA transcription and replication. The architecture of viral RdRPs resembles that of a 'cupped right hand' with fingers, palm and thumb domains. Those applying de novo initiation have additional structural features, including a flexible C-terminal domain that constitutes the priming platform. Moreover, viral RdRPs must be able to interact with the incoming 3'-terminus of the template and position it so that a productive binary complex is formed. Bacteriophage phi6 of the Cystoviridae family is to date one of the best studied dsRNA viruses. The purified recombinant phi6 RdRP is highly active in vitro and possesses both RNA replication and transcription activities. The extensive biochemical observations and the atomic level crystal structure of the phi6 RdRP provides an excellent platform for in-depth studies of RNA replication in vitro. In this thesis, targeted structure-based mutagenesis, enzymatic assays and molecular mapping of phi6 RdRP and its RNA were used to elucidate the formation of productive RNA-polymerase binary complexes. The positively charged rim of the template tunnel was shown to have a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. This work demonstrated that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the phi6 RdRP can be greatly enhanced. Furthermore, proteolyzed phi6 RdRPs that possess a nick in the polypeptide chain at the hinge region, which is part of the extended loop, were better suited for catalysis at higher temperatures whilst favouring back-primed initiation. The clipped C-terminus remains associated with the main body of the polymerase and the hinge region, although structurally disordered, is involved in the control of C-terminal domain displacement. The accumulated knowhow on bacteriophage phi6 was utilized in the development of two technologies for the production of dsRNA: (i) an in vitro system that combines the T7 RNA polymerase and the phi6 RdRP to generate dsRNA molecules of practically unlimited length, and (ii) an in vivo RNA replication system based on restricted infection with phi6 polymerase complexes in bacterial cells to produce virtually unlimited amounts of dsRNA. The pools of small interfering RNAs derived from dsRNA produced by these systems were validated and shown to efficiently decrease the expression of both exogenous and endogenous targets.
  • Kolari, Pasi (2010)
    The forest vegetation takes up atmospheric carbon dioxide (CO2) in photosynthesis. Part of the fixed carbon is released back into the atmosphere during plant respiration but a substantial part is stored as plant biomass, especially in the stems of trees. Carbon also accumulates in the soil as litter and via the roots. CO2 is released into the atmosphere from these carbon stocks in the decomposition of dead biomass. Carbon balance of a forest stand is the difference between the CO2 uptake and CO2 efflux. This study quantifies and analyses the dynamics of carbon balance and component CO2 fluxes in four Southern Finnish Scots pine stands that covered the typical economic rotation time of 80 years. The study was based on direct flux measurements with chambers and eddy covariance (EC), and modelling of component CO2 fluxes. The net CO2 exchange of the stand was partitioned into component fluxes: photosynthesis of trees and ground vegetation, respiration of tree foliage and stems, and CO2 efflux from the soil. The relationships between the component fluxes and the environmental factors (light, temperature, atmospheric CO2, air humidity and soil moisture) were studied with mathematical modelling. The annual CO2 balance varied from a source of about 400 g C/m2 at a recently clearcut site to net CO2 uptake of 200 300 g C/m2 in a middle-aged (40-year-old) and a mature (75-year-old) stand. A 12-year-old sapling site was at the turning point from source to a sink of CO2. In the middle-aged stand, photosynthetic production was dominated by trees. Under closed pine canopies, ground vegetation accounted for 10 20% of stand photosynthesis whereas at the open sites the proportion and also the absolute photosynthesis of ground vegetation was much higher. The aboveground respiration was dominated by tree foliage which accounted for one third of the ecosystem respiration. Rate of wood respiration was in the order of 10% of total ecosystem respiration. CO2 efflux from the soil dominated the ecosystem respiratory fluxes in all phases of stand development. Instantaneous and delayed responses to the environmental driving factors could predict well within-year variability in photosynthetic production: In the short term and during the growing season photosynthesis follows primarily light while the seasonal variation is more strongly connected to temperature. The temperature relationship of the annual cycle of photosynthesis was found to be almost equal in the southern boreal zone and at the timberline in the northern boreal zone. The respiratory fluxes showed instantaneous and seasonal temperature relationships but they could also be connected to photosynthesis at an annual timescale.
  • Mattsson, Tuija (2010)
    The terrestrial export of dissolved organic matter (DOM) is associated with climate, vegetation and land use, and thus is under the influence of climatic variability and human interference with terrestrial ecosystems, their soils and hydrological cycles. The present study provides an assessment of spatial variation of DOM concentrations and export, and interactions between DOM, catchment characteristics, land use and climatic factors in boreal catchments. The influence of catchment characteristics, land use and climatic drivers on the concentrations and export of total organic carbon (TOC), total organic nitrogen (TON) and dissolved organic phosphorus (DOP) was estimated using stream water quality, forest inventory and climatic data from 42 Finnish pristine forested headwater catchments, and water quality monitoring, GIS land use, forest inventory and climatic data from the 36 main Finnish rivers (and their sub-catchments) flowing to the Baltic Sea. Moreover, the export of DOM in relation to land use along a European climatic gradient was studied using river water quality and land use data from four European areas. Additionally, the role of organic and minerogenic acidity in controlling pH levels in Finnish rivers and pristine streams was studied by measuring organic anion, sulphate (SO4) and base cation (Ca, Mg, K and Na) concentrations. In all study catchments, TOC was a major fraction of DOM, with much lower proportions of TON and DOP. Moreover, most of TOC and TON was in a dissolved form. The correlation between TOC and TON concentrations was strong and TOC concentrations explained 78% of the variation in TON concentrations in pristine headwater streams. In a subgroup of 20 headwater catchments with similar climatic conditions and low N deposition in eastern Finland, the proportion of peatlands in the catchment and the proportion of Norway spruce (Picea abies Karsten) of the tree stand had the strongest correlation with the TOC and TON concentrations and export. In Finnish river basins, TOC export increased with the increasing proportion of peatland in the catchment, whereas TON export increased with increasing extent of agricultural land. The highest DOP concentrations and export were recorded in river basins with a high extent of agricultural land and urban areas, reflecting the influence of human impact on DOP loads. However, the most important predictor for TOC, TON and DOP export in Finnish rivers was the proportion of upstream lakes in the catchment. The higher the upstream lake percentage, the lower the export indicating organic matter retention in lakes. Molar TOC:TON ratio decreased from headwater catchments covered by forests and peatlands to the large river basins with mixed land use, emphasising the effect of the land use gradient on the stoichiometry of rivers. This study also demonstrated that the land use of the catchments is related to both organic and minerogenic acidity in rivers and pristine headwater streams. Organic anion dominated in rivers and streams situated in northern Finland, reflecting the higher extent of peatlands in these areas, whereas SO4 dominated in southern Finland and on western coastal areas, where the extent of fertile areas, agricultural land, urban areas, acid sulphate soils, and sulphate deposition is highest. High TOC concentrations decreased pH values in the stream and river water, whereas no correlation between SO4 concentrations and pH was observed. This underlines the importance of organic acids in controlling pH levels in Finnish pristine headwater streams and main rivers. High SO4 concentrations were associated with high base cation concentrations and fertile areas, which buffered the effects of SO4 on pH.
  • Jaatinen, Silja (Silja Jaatinen, 2009)
    In this thesis three icosahedral lipid-containing double-stranded (ds) deoxyribonucleic acid (DNA) bacteriophages have been studied: PRD1, Bam35 and P23-77. The work focuses on the entry, exit and structure of the viruses. PRD1 is the type member of the Tectiviridae family, infecting a variety of Gram-negative bacteria. The PRD1 receptor binding complex, consisting of the penton protein P31, the spike protein P5 and the receptor binding protein P2 recognizes a specific receptor on the host surface. In this study we found that the transmembrane protein P16 has an important stabilization function as the fourth member of the receptor binding complex and protein P16 may have a role in the formation of a tubular membrane structure, which is needed in the ejection of the genome into the cell. Phage Bam35 (Tectiviridae), which infects Gram-positive hosts, has been earlier found to resemble PRD1 in morphology and genome organization The uncharacterized early and late events in the Bam35 life cycle were studied by electrochemical methods. Physiological changes in the beginning of the infection were found to be similar in both lysogenic and nonlysogenic cell lines, Bam35 inducing a temporal decrease of membrane voltage and K+ efflux. At the end of the infection cycle physiological changes were observed only in the nonlysogenic cell line. The strong K+ efflux 40 min after infection and the induced premature cell lysis propose that Bam35 has a similar holin-endolysin lysis system to that of PRD1. Thermophilic icosahedral dsDNA Thermus phages P23-65H, P23-72 and P23-77 have been proposed to belong to the Tectiviridae family. In this study these phages were compared to each other. Analysis of structural protein patterns and stability revealed these phages to be very similar but not identical. The most stable of the studied viruses, P23-77, was further analyzed in more detail. Cryo-electron microscopy and three-dimensional image reconstruction was used to determine the structure of virus to 14 Å resolution. Results of thin layer chromatography for neutral lipids together with analysis of the three dimensional reconstruction of P23-77 virus particle revealed the presence of an internal lipid membrane. The overall capsid architecture of P23-77 is similar to PRD1 and Bam35, but most closely it resembles the structure of the capsid of archaeal virus SH1. This complicates the classification of dsDNA, internal lipid-containing icosahedral viruses.

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