Bio- ja ympäristötieteellinen tiedekunta

 

Recent Submissions

  • Araujo, César (Helsingin yliopisto, 2016)
    Prostatic acid phosphatase (PAP) was discovered during the mid-1930s, but the molecular mechanisms in which this protein is involved remain poorly understood. This enzyme was originally described as a highly-expressed protein in the human prostate that was secreted to the seminal fluid. It has always been associated to prostate cancer, since high levels of acid phosphatase activity were found in the sera of patients with metastatic disease. Therefore, for 40 years, research was focused on the improvement of biochemical assays in an effort to find specific substrates for clinical application. However, in the 1980s PSA (prostate specific antigen) superseded PAP as a biomarker for early detection of the disease and became the preferred marker for diagnosis. Later, in the mid-1990s with the advent of new molecular techniques such as cloning and high-scale protein purification it was possible to obtain high-quality crystals for 3D-structural determinations of PAP. In addition, new concepts emerged concerning the physiological role of the enzyme, with renewed speculation regarding the molecular mechanisms in which this protein could be involved. The fact that the serum levels of the enzyme are increased in prostate cancer patients with metastatic disease rendered this enzyme an attractive target for immunotherapies against advanced prostate cancer. However, this hypothesis has thus far neglected the existence of any potential isoforms that could be expressed in other organs and tissues. Therefore, as part of this thesis, the molecular mechanisms where PAP is involved will be investigated. For this purpose, two biological tools were employed: a PAP-knockout mouse model and stable virus-transfected LNCaP cell lines. A novel transmembrane type-I isoform of PAP (TMPAP) was first characterized as a product of alternative splicing of the same gene (ACPP) that encodes for the well-known secretory isoform (SPAP). TMPAP is distributed throughout mouse tissues, including prostate, lung, kidney, endometrium, salivary glands, and dorsal-root ganglia. The enzyme comprises an N-terminal domain containing the catalytic active site, a transmembrane helical domain, and a short C-terminal cytosolic domain that carries a tyrosine-based motif (Yxxφ) that targets the enzyme to the endosomal-lysosomal/exosomal pathway. This was confirmed by co-localization studies that revealed that PAP localizes to the plasma membrane as well as to the intracellular membranes of vesicles, lysosomes, and intraluminal vesicles of the multivesicular endosomes. Microarray experiments were performed on mouse tissues to study the differential gene expression profile between wild-type and PAP-knockout mice. The differential gene expression that was observed between the prostates of wild-type and PAP-knockout mice suggested that PAP is involved in secretory mechanisms, as many genes related to this process appeared dysregulated. Moreover, the results obtained from two-hybrid system experiments suggested that snapin (a SNARE-associated protein) was a likely candidate protein that could interact with TMPAP. This interaction was recently proved by co-localization and florescence resonance energy transfer (FRET) studies. The PAP-knockout mice developed prostate adenocarcinoma and showed dysregulation of genes related to vesicular traffic. Consequently, this investigation was focused on murine submandibulary glands (SMG) as a model of an exocrine organ. The expression of PAP in SMG was found to be even higher than in mouse prostate. In addition to microarrays and miRNA analyses, physiological and biochemical determinations help to demonstrate that there is an increased salivation volume in PAP-knockout mice upon stimulation with secretagogue drugs. This supports the hypothesis that PAP is involved in the regulation of secretory and exocytic processes. PAP was found to account for 50% of the total acid phosphatase activity in male mouse saliva and it is expressed by the granular convoluted tubular cells of the male SMG but not by the acinar cells. Unlike prostate gland, however, the mouse SMG does not develop signs of hyperplasia or adenocarcinoma in spite of an observed increased acinar cell proliferation. This discrepancy was explained by studying the degree of lymphocyte infiltration, the dysregulation of miRNAs, and the differentially expressed genes in microarray data. In SMGs of PAP-knockout mice, the innate immune system was shown to be responsive and able to remove proliferating acinar cells, which may explain the absence of adenocarcinoma. In addition, the upregulation of anti-inflammatory molecules may prevent the extension of tissue damage. Finally, we compared the effect of the overexpression of SPAP and TMPAP in LNCaP cells with empty-vector cells. As a result, the TMPAP-LNCaP cells exhibited slower growth than SPAP-LNCaP or empty-vector cells. Cells overexpressing either SPAP or TMPAP isoform showed increased 2D-projection area and increased HRP-uptake when compared with empty-vector cells. These two observations suggested an increased vesicular traffic in endo/exocytic pathways to maintain cell membrane homeostasis. Thus, vesicles loaded with TMPAP are most likely sorted to lysosomes by means of its Yxxφ motif. Consequently, there is an increased degradation of cargo molecules such as receptor tyrosine kinases expressed on the cell surface that could explain the observed slow growth of LNCaP cells that overexpress TMPAP. The molecular mechanisms identified in this study will definitely contribute to a better understanding of the physiological role of PAP in diseases and to a critical re-evaluation of existing immunotherapies. The knowledge of the molecular determinants responsible for the presence of TMPAP in the endo/exocytic pathway can also be exploited for the future development of radio-imaging and drug delivery protocols.
  • Mattsson, Marja (Helsingin yliopisto, 2016)
    ABSTRACT The microbial community composition is among the most important factors to consider, when pesticide removal from the environment is planned by indigenous microbial consortia. The main interest of this study was to assess relationships between pesticide contaminations from the past, microbial community compositions, physico-chemical conditions, and pesticide dissipation. The microbial communities were studied by lipid biomarkers. Samples were collected from surface soils to deep drilling sediments and groundwater deposits, which had the long history of pesticides such as atrazine, 2,6-dichlorobenzamide, simazine, 1,1,1-trichloro-2,2-di(4-chlorophenyl)ethane (DDT), hexachlorobenzene (HCB), and pentachloroaniline (PeCA). The microbial lipid quantities and community compositions were generally dependent on organic matter (OM) content and soil type. Microbial biomass and cell numbers decreased with increasing depth and varied more on the top soil layers than in the deep sediments. Generally, the quantity of phospholipid fatty acids (PLFAs) was higher than that of glycolipid fatty acids (GLFAs) but lower than the quantity of neutral lipid fatty acids (NLFAs), when the amounts of main nutrients were balanced. However, in groundwater deposits and deep drilling sediments the phosphorous deficiency possibly caused higher GLFA quantities compared to PLFAs. The quantity of NLFAs seemed to increase with soil OM content and excess carbon. Microbes in all polluted samples seemed to be adapted to pesticides. Pesticide impacts on microbial community composition were few and no stress were found in the physiological indices of lipid profiles. The long-term contamination of atrazine in the groundwater, as well as DDT, HCB, and PeCA in surface soils seemed to mainly increase microbial activity compared to controls. The effects of DDT and its residues on microbial lipids were minor, though their persistence was high in soils with low OM content. HCB and PeCA were more persistent in soils with high OM content and their effects on microbial lipids were more numerous. The indigenous microbes of groundwater pipe A deposits were capable of atrazine biodegradation in aerobic conditions though nutrient levels were low, when C/N-ratio was near to optimal. Possibly Gram-positive bacteria and fungi were related to atrazine biodegradation.
  • Hällfors, Maria (Helsingin yliopisto, 2016)
    Global climate change will make the current habitat of many species unfavourable. It can also cause species suitable areas to shift or disappear. Under rapid climate change many species will not be able to adapt or disperse fast enough. Therefore, human-mediated dispersal of species has been proposed as a conservation method for mitigating the negative effects of climate change on biodiversity. In scientific discussions on the method, several different terms and definitions have been used. Such inconsistent use of terminology can lead to misunderstandings and to conflicting studies and evaluations of the method. In this thesis, assisted migration (AM) is suggested as the preferred term for the idea of translocating species threatened by climate change, and a concise definition that distinguishes it from other translocation practices is proposed. Should AM be accepted as a conservation tool, there is a demand for a readily applicable, rapid, and effective way of evaluating the species-specific benefit of AM. This thesis presents a method for estimating the need and potential of AM from predictions of changes in the range of species. The method is applied to several plant species and this thesis thus provides on of the first data-based estimations of the need to apply AM as a conservation strategy under different scenarios of climate change. The results indicate that the need and potential of AM for these species increases substantially with the strength of climate change and the temporal extent of climate change projections. Furthermore, this thesis suggests ways to deal with uncertainties in the process of obtaining range change predictions through species distribution models. Conventional assumptions concerning local adaptation within species, according to which species are treated as a single entity, may lead to erroneous predictions when applying species distribution models. Here, the magnitude of error in conservation guidance that can be introduced through opposing assumptions concerning local adaptation is explored. It is found that the assumption of local adaptation and, therefore, separate modelling of populations can provide different and more precautionary outcomes compared to the assumption of no local adaptation and, hence, modelling the species as a whole. To obtain insight into the presence of intraspecific local adaptation to climatic conditions, a translocation trial of two geographically separated populations of the same plant species was initiated. The preliminary results presented here indicate that one of the studied populations is less adapted to conditions in its home environment while the other population exhibits stronger local adaptation. Knowledge of this kind provided by experimental studies should be reflected in studies using species distribution modelling to reduce uncertainty in predictions and threat estimates based on the models. This thesis concludes that predictive tools such as species distribution models hold great potential in providing rough estimates of future trajectories for conservation of biodiversity and could provide a useful scientific basis for policy decisions. However, more in-depth knowledge, which can be gained through experimental approaches, is needed to detail how individual species and populations may respond to altered conditions in their environment and which conservation method is the most relevant.
  • Verbeeren, Jens (Helsingin yliopisto, 2016)
    The protein coding information in our genome is located on genes which are very often interrupted by non-coding regions called introns. For proper gene expression, introns must be removed accurately and the remaining protein coding parts, the exons, must be rejoined. This reaction, termed splicing, is carried out by an enormous macromolecular machine called the spliceosome, and is one of the most crucial steps in gene expression. Two different intron types have been identified in eukaryotes, each removed by their own dedicated spliceosome; the U2-type (or major) introns, which constitute the majority of introns, and the U12-type (or minor) introns, of which ca. 700-800 have been identified in the human genome. The presence of a second type of intron and spliceosome has always been enigmatic. However, studies investigating U12-type intron removal have provided us with an important clue; it appears that U12-type introns are spliced less efficiently than U2-type introns. This suggests that their removal could be rate-limiting for the expression of the genes that harbor these introns, and it also offers the intriguing possibility that the activity of the minor spliceosome could be altered in response to changing cellular conditions. These implications could offer a valuable explanation for the extraordinary conservation of the U12-type introns and the components that catalyze their excision. There is currently not much known about the regulation of the minor spliceosome and this study aimed to address this issue. I have investigated the characteristics of a negative feedback loop that regulates the expression level of two essential and unique protein components of the minor spliceosome, the U11-48K and the U11/U12-65K proteins. In the genes that encode these proteins, an ultraconserved sequence element can be found which consists of a tandem repeat of U12-type 5ʹ splice sites. We uncovered that binding of U11/U12 di-snRNPs on these elements leads to alternative splicing where an mRNA isoform is produced that is targeted for degradation or nuclear retention. The presence of such enhancer elements is conserved from plants to animals, highlighting an extreme selection pressure for this regulatory mechanism. I further investigated the role of the U11-35K protein, another protein uniquely associated with the minor spliceosome, in alternative splicing, and the functional requirements for enhancer binding. Furthermore, I uncovered the molecular mechanism by which the level of translational-competent U11/U12-65K mRNA is downregulated through U11/U12 di-snRNP enhancer binding.
  • Murtomäki, Aino (Helsingin yliopisto, 2016)
    Mammals have two parallel vascular systems, the blood and the lymphatic vascular system. The blood vascular system transports oxygen and nutrients to tissues and metabolic wastes from the tissues to excretory organs. Lymphatic vessels collect excess fluid from the interstitial space and return it back into the bloodstream, thus maintaining fluid homeostasis. Like veins, lymphatic collecting ducts contain intraluminal valves that ensure unidirectional flow. Notch signaling is an evolutionary conserved signaling pathway that mediates cell fate decisions and regulates cellular functions through its modulation of downstream targets. Using transgenic mouse models, we studied the role of Notch in embryonic lymphatic development and postnatal blood vascular development. Better understanding of these basic mechanisms is crucial for developing new treatments for diseases associated with abnormal blood or lymphatic vessels function. Embryonically, the lymphatic system develops as a subset of endothelial cells in the cardinal veins start expressing Prox1 and other lymphatic markers and become committed to the lymphatic lineage. We show that loss of Notch during the initiation phase leads to an increase in lymphatic progenitor cells emerging from the cardinal vein and lymphatic overgrowth. Thus, Notch is required in the cardinal vein to limit the number of endothelial cells adopting the lymphatic endothelial fate. Lymphatic valve development occurs during collecting duct maturation as subsets of lymphatic endothelial cells in the lymphatic duct walls adopt a lymphatic valve fate. We show that loss of Notch signaling in lymphatic endothelial cells at the time of valve initiation results in a decrease in the number of valves, abnormal valve morphology and reduced expression of valve-specific markers in valve-forming lymphatic endothelial cells. Thus, Notch signaling is required for proper lymphatic valve development. Blood endothelial cells interact with contractile smooth muscle cells and non-contractile pericytes, which are collectively called mural cells. Endothelial cell-mural cell interactions provide mechanical support to vessels as well as regulate many vessel functions that are crucial for vascular integrity such as permeability, sprouting and quiescence. Notch1 is expressed in both endothelial cells and mural cells while Notch3 is restricted to vascular mural cells. We show that global Notch1 heterozygocity combined with global Notch3 deficiency results in impaired vascular smooth muscle cell recruitment in the mouse retina leading to abnormal vascular development. We also demonstrate that biological inhibition of Notch signaling using soluble Notch1 decoys results in defective vascular smooth muscle coverage in the mouse retina. Our data show that both Notch1 and Notch3 are required for proper vascular smooth muscle cell function during vascular development and thus report a novel role for Notch1 in mural cells.
  • Ojala, Teija (Helsingin yliopisto, 2016)
    Modern DNA sequencing technologies have opened up new possibilities to study bacteria. These methods have not only enabled the characterization of the genetic capacities of bacteria at previously unseen scale but have also provided a wealth of information about bacterial transcriptomes. In this thesis, sequencing and subsequent analysis approaches were applied to study Lactobacillus crispatus and Propionibacterium freudenreichii. Specifically, the aim was to uncover how these two Gram-positive species of human relevance can live in and interact with their environments. L. crispatus is a prominent member of the human vaginal flora and important for urogenital health. In this thesis, an annotated genome sequence was produced for L. crispatus ST1 and analyzed in conjunction with publicly available genome sequences of nine vaginal L. crispatus isolates. The common ortholog groups of the ten isolates captured approximately 57% of the ortholog groups of each isolate and provided a good estimation of the final set of core features of this central urogenital species. Notably, several of the detected L. crispatus core features were of putative relevance to vaginal health. Among these features was a previously characterized adhesin, which was in this thesis identified as a likely antagonist to the harmful vaginal bacterium Gardnerella vaginalis. Altogether, the study revealed a notable functional similarity between the L. crispatus strains and established the role of L. crispatus core proteins in maintaining vaginal health. P. freudenreichii, in turn, is an industrially important dairy culture. In this thesis, the cheese starter P. freudenreichii ssp. shermanii JS was subjected to transcriptome and genome sequencing to gain a deeper understanding of the role of this bacterium in industrial cheese ripening. The genome of strain JS encoded several enzymes and metabolic pathways involved in the formation of flavor compounds and was highly similar to those of the other P. freudenreichii strains. Transcriptome analysis of industrial cheese samples revealed nearly 15% of the 2,377 protein-coding genes of strain JS to be significantly differentially expressed between the warm and cold room ripening of cheese. Several of the flavor-associated genes exhibited higher expression levels in the warm compared to the cold room samples, corroborating the hypothesis that P. freudenreichii contributes more to the cheese flavor development during warm than cold room ripening. Automated function prediction of bacterial protein sequences greatly facilitated the genomics investigations of L. crispatus and P. freudenreichii in this thesis, providing functional descriptions for a majority of the predicted coding sequences of strains ST1 and JS. Moreover, re-annotation of the coding sequences of the nine publicly available vaginal L. crispatus isolates significantly increased the portion of the L. crispatus coding sequences with functional descriptions in the comparative genomics study of L. crispatus. The different methods varied in their prediction capabilities and were often complementary, supporting the use of more than one function prediction method in a bacterial genome project. Moreover, extremely strict thresholds in the homology searches were noted to unnecessarily restrict the pool of hits available for annotation transfer, hampering both the annotation quality and the fraction of coding sequences with a functional classification. Taken together, the utilized sequencing approaches coupled with suitable downstream analyses proved effective in deciphering the physiology of lactobacilli and propionibacteria and offered novel insights into the health-promoting properties of L. crispatus and flavor-forming capabilities of P. freudenreichii.
  • Batchu, Krishna chaithanya (Helsingin yliopisto, 2016)
    The aim of this thesis was to study the substrate specificity of A-type phospholipases (PLAs) that belong to different sub-families in order to understand the key factors that regulate their activity. The experimental part of this thesis consists of three studies each one focusing on PLAs that belong to a specific subgroup. In the first study, we developed a mass-spectrometric (MS) assay and implemented it to study in detail the effect of acyl chain length and unsaturation of glycerophospholipids (GPLs) on their hydrolysis by three different secretory PLA2s (sPLA2s) from various sources. The key finding of this study was that efflux of the GPL substrate from the bilayer largely determines the rate of hydrolysis by these PLAs. In micelles, accommodation of the GPL acyl chains in the catalytic active site seems to be more important for substrate specificity. In the next study we used the MS assay developed in study I, to investigate whether substrate efflux propensity regulates the activity of the Ca2+ -independent PLA-Beta (iPLAβ). Our results strongly suggest that the activity of iPLAβ is also determined by the efflux of the GPL substrate from the membrane bilayer. Our last study was on the cytoplasmic PLA-alpha (cPLA2α) that has been implicated in the initiation of the inflammatory lipid-mediator cascade generating eicosanoids and platelet-activating factor. The study was carried out to understand to what extent accommodation in the catalytic site determines specificity for arachidonic acid (AA) and if efflux propensity plays a role in the substrate specificity of cPLA2α. Our results indicate that while accommodation of the substrate in the active site greatly contributes to the preferential hydrolysis of AA-containing GPLs by cPLA2α, efflux of the substrate from a membrane bilayer also plays a significant role. In summary, these studies not only provide information on the factors regulating the substrate specificity of various PLAs but also indicate that lateral arrangement of GPLs could be a key regulator of homeostatic PLAs like iPLAβ.
  • Peltola, Marjaana (Helsingin yliopisto, 2016)
    Schizophrenia is a devastating psychiatric illness afflicting approximately 1% of the world s population. Currently, the disease mechanism is poorly understood and the pharmacological interventions relieve only some of the symptoms. Schizophrenia is highly heritable and genetic factors contribute to about 65-80% of the liability to the illness. However, the genetic etiology is complex and remains largely unknown. Potassium channels are key determinants of neuronal excitability. Kv2.1 is a widely-expressed voltage-gated potassium channel α-subunit. Kv2.1 channels constitute an essential component of the somatodendritic delayed rectifier current (IK) in several neuronal types and regulate excitability, especially during periods of high-frequency firing. This study outlines the identification and characterization of a novel neuronal transmembrane protein AMIGO, which contains extracellular immunoglobulin (Ig) and leucine-rich repeat (LRR) domains. AMIGO was shown to be widely expressed in cerebral neurons and localized to distinctive clusters in the neuronal plasma membrane, restricted to the cell soma and proximal part of neurites. AMIGO was further identified as an auxiliary subunit of the Kv2.1 potassium channel. AMIGO and Kv2.1 were shown to display extensive spatial and temporal colocalization and association in brain. AMIGO was also shown to modify the voltage-dependent activation of Kv2.1 and neuronal delayed rectifier current (IK). To further understand the physiological role of AMIGO in brain, a mouse line lacking the Amigo gene was created and characterized as part of this study. Absence of AMIGO clearly reduced the amount of the Kv2.1 channel protein in mouse brain and altered the voltage-dependent activation of neuronal IK. These changes were accompanied by behavioral and pharmacological abnormalities reminiscent of those identified in schizophrenia. Concomitantly, the rare KV2.1 variant was found to be associated with human schizophrenia. These findings demonstrate the involvement of the AMIGO-Kv2.1 channel complex in schizophrenia-related behavioral domains in mice and establish KV2.1 as a susceptibility gene for schizophrenia spectrum disorders in humans. In the current study, AMIGO was identified as an integral component of the Kv2.1 channel complex in brain. The convergent findings in humans and mice suggest a role for the AMIGO-Kv2.1 potassium channel complex in the pathophysiology of schizophrenia. Furthermore, these findings suggest AMIGO and Kv2.1 may represent potential new targets for schizophrenia treatment development.
  • Nykänen, Niko-Petteri (Helsingin yliopisto, 2016)
    Abnormal regulation of various posttranslational modifications (PTMs) of microtubule- associated protein tau induce its self-aggregation, which is a hallmark pathophysiological process of neurodegenerative diseases (NDDs) collectively called as tauopathies including Alzheimer s disease (AD) and frontotemporal dementia. Increased tau phosphorylation is a key PTM in conversion of tau into more toxic species in cells, which is regulated by interactions of various protein kinases and phosphatases. However, the exact mechanism(s) of how various combinatory PTMs affect aggregation and cell-to-cell propagation of tau are poorly understood. We developed a novel live cell reporter system based on protein-fragment complementation assay (PCA) and studied dynamic protein- protein interactions of tau in native cellular environment. The PCA was further validated on investigating cellular secretion and uptake of tau in live cells. A proof-of-concept screen was performed using PCA platform revealed several GABAA receptor activators that altered the interaction of tau-Pin1. Pin1 act as a critical facilitator of tau dephosphorylation by catalyzing the isomerization of cis/trans peptidyl-prolyl bond at phosphorylated Thr231-Pro motif of tau. Additionally, we showed that screen-identified GABAA receptor modulators increased tau phosphorylation at the AT8 phosphoepitope in cultures of mature primary cortical neurons and remained at elevated level 24 h after washout of the drugs. Mechanistic studies suggested that enhanced GABAA receptor- induced tau phosphorylation was associated with decreased interaction of tau and protein phosphatase 2A (PP2A) without any reduction in enzymatic activity of PP2A and involved CDK5 kinase. Furthermore, the assessment of expression and splicing status of late-onset AD (LOAD) susceptibility genes in our neuropathologically validated AD cohort of post mortem brain samples revealed increased expression of MS4A6A and decreased expression of FRMD4A in regards to increased AD-related neurofibrillary pathology according to Braak staging. Moreover, the expression level of FRMD4A was functionally associated with amyloidogenic APP processing and increased tau phosphorylation in vitro. FRMD4A expression levels also correlated with cellular tau secretion assessed by PCA-based assay platform using siRNA-mediated gene silencing. Subsequent mechanistic studies on secretion showed a more general involvement of cell polarity complex signaling including Par3/Par6/aPKCζ complex-induced activation of Arf6 via cytohesins. These novel connections of altered FRMD4A expression level in AD brain and its impact on cellular tau secretion further corroborate the suggested role of FRMD4A in LOAD pathogenesis and pathophysiology. Here, for the first time, we assessed a functional association between LOAD-related susceptibility gene and cell-to- cell propagation of tau, and also showed the decreased expression of FRMD4A related to increasing disease severity according to Braak staging.
  • Idänheimo, Niina (Helsingin yliopisto, 2015)
    Responses to environmental changes are mediated via complex signaling networks in plants. Overlapping signaling pathways guarantee information flow from many simultaneous stress factors leading to both synergistic and antagonistic responses in order to maintain the most optimal conditions for growth under non- or suboptimal conditions. Adaptation to stressful environmental conditions is based on flexible interactions between hormone and reactive oxygen species (ROS) signaling. During the recent years researchers have started to understand the complexity of the crosstalk needed for stress tolerance. However, there are still many fundamental questions unanswered. For example, how are the intertwined signal transduction networks regulated, and how are ROS sensed and signaling specificity achieved? Receptor-like protein kinases (RLKs) are plasma membrane proteins which have a role in signal sensing. RLKs have been linked to many different physiological processes, such as plant development, pathogen defense and abiotic stress response. RLKs are involved in ROS signaling and it has been suggested that members of the cysteine-rich protein kinase (CRK) subfamily could be involved in direct ROS sensing due to the redox regulation possibilities in their extracellular protein domain. The large number of CRKs and their protein similarity suggests partly overlapping functions and possibilities for fine-tuning the stress responses. In this study, Arabidopsis CRKs, especially CRK6 and CRK7, have been characterized and their involvement in ROS signaling studied. Based on the presence of conserved kinase subdomains, this study suggests that Arabidopsis CRKs are active kinases and verifies kinase activity for CRK6 and CRK7 in vitro. This study shows that in addition to stress responses, as previously suggested, CRKs are involved also in many important developmental processes, such as germination and senescence. This novel finding broadens our understanding of the role of CRKs in plants. Despite the observed redundancy in crk phenotypes due to sequence similarity, some crk mutants, such as crk2 and crk5, showed clear individual phenotypes suggesting specific functions for these CRKs. crk6 and crk7 phenotypes were partly disguised by redundancy effect. Based on the obtained results and proposed redox regulation possibilities of CRK ectodomain, it can be proposed that CRKs are essential regulatory elements of cellular redox circuits that relay environmental information to the cell. Therefore the role of CRKs in cellular crosstalk is essential for maintaining the delicate balance between growth and defense. The loss of CRK function disrupts the information flow and leads to impaired stress tolerance. Thus, the obtained results suggest protective roles for CRKs. Furthermore, the large number of CRKs and their specific yet partly overlapping functions could bring operational reliability to the signal transduction and suggests specificity and fine tuning opportunities for signal transduction.
  • Zhao, Xiang (Helsingin yliopisto, 2016)
    Vertebrate brain is one of the most complex and mysterious objects for biological research. Embryonic brain development involves stereotypic brain structure formation, and a vast number of precise intercellular connections are established for the generation of the highly complex circuitry of the brain. This work aims at explaining HMGB1 and AMIGO1 function in modulating vertebrate brain development. Hmgb1 knockdown zebrafish morphants produced by injection of morpholino oligonucleotides display severe defects in the forebrain and gross deteriorated catecholaminergic system. The morphant is also deficient in survival and proliferation of neural progenitors. Similar central nervous system (CNS) developmental defects have been observed in HMGB1 knockout mouse embryo. The HMGB1 null mouse embryonic brain cells showed much lower proliferating and differentiating activities compared to wild type animals. HMGB1 knockdown and knockout model respectively from zebrafish and mouse have confirmed that AMIGO1 expression is directly regulated by HMGB1. AMIGO1 regulates expression of Kv2.1 potassium channel during development, but the colocalization of AMIGO1 and Kv2.1 has only been observed in mouse and zebrafish adult brain. Furthermore, knockdown of amigo1 expression using morpholino oligonucleotides impairs the formation of fasciculated tracts in early fiber scaffolds of brain. The same defect can be also induced by mRNA-mediated expression of the Amigo1 ectodomain that inhibits adhesion mediated by the full-length protein. The impaired formation of neural circuitry is reflected in enhanced locomotor activity and attenuated escape responses. Our data demonstrate that HMGB1 is a critical factor for embryonic CNS development involved in many important developmental events. HMGB1 is essential for the neurogenesis and differentiation occurring at the developmental stage when forebrain structures are forming. Amigo1 is required for the development of neural circuits under the regulation of HMGB1. The mechanism involves homophilic interactions within the developing fiber tracts and regulation of the Kv2.1 potassium channel to form functional neural circuitry that controls locomotion. HMGB1 and AMIGO1 are both crucial for embryonic brain development and neural circuit formation.
  • Helenius, Laura (Helsingin yliopisto, 2015)
    Zooplankton are considered an important link between the zooplanktivores that consume them and the lower trophic levels, yet their specific ecological role in littoral brackish ecosystems is still relatively unstudied. In general, ecological interest in zooplankton derives from their roles as grazers or as a food source. As grazers, their role is coupled with predicting future densities and composition of the algal community, while alternatively as prey they provide information on fish stocks in terms of the zooplankton production available to fish. This thesis aimed to unravel aspects related to these roles in the littoral zones of the Baltic Sea, by shedding light on the themes of zooplankton composition and diversity, the interactions between zooplankton and their predators, and the general feeding ecology of components of zooplankton. The first objective of the thesis was to gain baseline information on zooplankton community composition and diversity in the littoral Baltic Sea via field sampling on a salinity gradient. The thesis also aimed to investigate how salinity and other abiotic factors, such as turbidity, temperature and wave exposure, affect zooplankton communities and the predation that structures them. Predation via two types of feeding by zooplanktivorous fish was studied experimentally as a regulator of zooplankton communities. Finally, the thesis investigated the role of copepod nauplii as grazers in laboratory conditions. Salinity was found to be the most significant abiotic driver of spatial patterns of composition and diversity of zooplankton. Turbidity/chl a also influenced community structure to a lesser extent. The spatial patterns of species heterogeneity remained relatively constant regardless of temporal turnover, and there was an abrupt change in species composition at an intermediate salinity of 4 (on the Practical Salinity Scale) on the salinity gradient. Functional diversity of zooplankton was found to be related to salinity, but also to factors related to productivity after a certain threshold. Zooplankton diversity was also affected by predation, but this effect was regulated by the initial composition of the zooplankton community, which was in turn directly related to seasonality. Predation itself was found to structure the community through direct removal of crustacean zooplankton, as well as cascading effects on microzooplankton. These effects, as well as the extent of the disturbance generated by turbidity on zooplanktivorous feeding, were all closely related to predator type. In the final section of the thesis, which concentrated on zooplankton as consumers, the functional responses of stage NII nauplii of the calanoid copepod Paracartia grani to various microalgae were found to be either Holling type II or III responses. Highest maximum clearance rates were found on a diatom and a dinoflagellate of a size of ~12 µm, indicating an optimal prey:predator size ratio of 0.08. In plurialgal mixtures, feeding patterns were largely dependent on prey type. Zooplankton are irrevocably linked to phytoplankton and fish through food web interactions. Changes in the abiotic environment inevitably lead to a response in the biotic environment as well, and a bottom-up resource level response reflects on top predators, in this case the littoral fish. Therefore, understanding the abiotic and biotic factors determining zooplankton diversity and density is a precondition to understanding the links between phytoplankton, zooplankton, and coastal zooplanktivores.
  • Nurmi, Katariina (Helsingin yliopisto, 2015)
    The innate immune system responds to infection or injury by initiating nonspecific inflammation, which functions to limit the spread of harmful microbes or the damage caused by tissue injury. The cells of the innate immune system are the first to encounter danger signals, and they mediate the rapid local immune response. Inflammatory reactions are normally beneficial for the host, and inflammation is usually resolved when the threat has been removed. However, in chronic inflammatory diseases, the danger signals either are not cleared or continue to be formed. Pathogen-derived molecules and danger signals induce the activation of pattern recognition receptors (PRRs) in the innate immune cells. Several families of PRRs exist, and their interplay is needed for the induction of efficient immune defense reactions. Nucleotide-binding domain and leucine-rich repeat-containing receptors (NLRs) are intracellular receptors that respond to a plethora of danger- and pathogen-associated molecular patterns. Their activation induces the assembly and activation of cytosolic multiprotein complexes called inflammasomes. Inflammasomes act as a primary checkpoint for the activation and secretion of the strong proinflammatory cytokines interleukin (IL)-1β and IL-18. Similar to other cellular functions, innate immune responses are operated via a complicated interplay between signalosomes. To become activated inflammasomes require the coordinated activation of Toll-like receptors (TLRs) and NLRs, which induce the activation and assembly of inflammasome complexes. The consequent secretion of inflammasome-derived cytokines is, in turn, modulated by autophagy. Inflammasome activation and autophagy also interact with cellular death pathways. Cellular death acts to limit the spread of intracellular pathogens by denying a protective niche to these pathogens, thereby inhibiting their replication and predisposing them for detection by the immune system. The aim of this study was to investigate the roles of ethanol and hemin in the modulation of innate immune cell functions, as well as the mechanisms underlying the reported protective effects of ethanol and hemin against chronic inflammatory diseases. Alcohol is the most commonly and widely used drug in the world. The consequences of alcohol consumption depend on both the pattern of consumption and the amounts consumed. Alcohol abuse predisposes to more frequent and severe infections, whereas the light to moderate consumption of alcoholic beverages has been associated with a reduced incidence of chronic inflammatory conditions, such as cardiovascular diseases and rheumatoid arthritis. These seemingly different responses may both derive from attenuated reactions of innate immunity. In the present thesis study, ethanol was shown to reduce the viability and proliferation of mast cells. This reduced viability resulted from the immunologically silent apoptotic death of mast cells. In macrophages, ethanol reduced the pyroptotic cell death induced by inflammasome activation and instead directed cell death toward apoptosis. Excessive inflammasome activation is a prominent feature of several chronic inflammatory diseases. The mechanisms that restrain inflammasome activation were studied in greater detail in cultured macrophages. Ethanol dose-dependently inhibited inflammasome activation and the secretion of IL-1β in human macrophages. It was further shown that the inhibitory effect of ethanol was mediated by a reduction in lysosomal disruption and the release of active cathepsin B, which thus contributed to diminished inflammasome assembly. The majority of mammalian cells are constantly renewed. Enormous numbers of senescent red blood cells are phagocytosed daily by macrophages. In certain pathologies, such as malarial infection, massive hemolysis occurs that exceeds the capacity of the scavenging and degradation systems of hemoglobin. As a consequence, free heme and hemin are released into the circulation. Free heme and hemin are cytotoxic and proinflammatory compounds. However, heme and hemin are also potent inducers of the heme oxygenase-1 (HO-1) enzyme, which possesses anti-inflammatory and cytoprotective effects. In the present thesis study, hemin and its synthetic derivative cobalt protoporphyrin (CoPP) blocked inflammasome activation and assembly. Decreased secretion of IL-1β was also observed in vivo in a nucleotide-binding domain and leucine-rich repeat-containing family, pyrin domain-containing 3 (NLRP3)-dependent peritonitis model in mice. The inhibitory effects of hemin and CoPP were partially dependent on the induction of HO-1 transcription by NF-E2-related factor-2 (Nrf2) and the enzymatic activity of HO-1. The inhibitory effects of hemin and CoPP were mediated by increased degradation of inflammasome components, which was due to elevated autophagy. Overall, the results of this study demonstrate that ethanol and hemin markedly prevent inflammatory cellular responses in macrophages and mast cells. This inhibition may contribute to the cardioprotective effect of ethanol and the anti-inflammatory effects of hemin. An enhanced molecular understanding of the mechanisms by which ethanol and hemin inhibit inflammation may help reveal new therapeutic options in the treatment of chronic inflammatory diseases.
  • Ylä-Anttila, Päivi (Helsingin yliopisto, 2015)
    Eukaryotic cells contain membrane-bound organelles to carry out specialized cellular functions. These organelles are inherited in cell division as templates and are augmented by proliferation through production of protein and lipid components by the cell, and the trafficking of these components within the cell. Autophagy is an evolutionarily conserved degradation pathway for cells to maintain homeostasis, produce nutrients for energy production, degrade misfolded proteins or damaged whole organelles, and fight against intruding pathogens. The process of autophagy entails the isolation of cargo by a specialized organelle, called the phagophore, which closes to form a sealed double membrane bound autophagosome. This organelle then undergoes maturation by fusion with endosomes and lysosomes to obtain its degradation capacity. Hence, there are many dynamic membrane modifications that need to take place during the autophagic process. The origin of the autophagic limiting membrane, as well as the clearance of the degradative structures, are yet to be defined. This study utilized high resolution electron microscopic methods and three dimensional modeling to reveal nanometer scale interactions of phagophores and autophagosomes with other organelles. Immunolabeling techniques at both light and electron microscopy level were utilized to determine which organelles should be sampled at an ultrastructural level. Direct membrane communication was detected between the phagophore and endoplasmic reticulum (ER), (putative) ER exit sites, mitochondria, the Golgi complex, as well as late endosomes or lysosomes. ER was the most frequent proximal organelle to phagophores and autophagosomes and this suggests an involvement of ER in the nucleation process of phagophores. This study also reveales a role of the small GTP-binding protein RAB24 in the clearance of autophagic structures in cells. Biochemical and microscopic methods in combination showed that RAB24 is needed in the clearance of autophagic structures in nutrient rich conditions i.e. during basal autophagy. RAB24 was confirmed to localize in both of the autophagosome limiting membranes. GTP binding and prenylation of RAB24 were found to be necessary for the targeting of the protein to LC3 positive autophagic structures, whereas tyrosine phosphorylation was less important for this targeting. Electron microscopy revealed that autolysosome-like structures accumulate in cells when RAB24 is silenced, suggesting that it has a role in the clearance of autolysosomes.
  • Milardi, Marco (Helsingin yliopisto, 2015)
    Fish introductions, unlike many other clades, are often carried out purposefully. This is a worldwide practice, which has been ongoing since ancient times and has found new powerful ways to increase in magnitude and scope through the development of more effective transport and with increasing population wealth. Even though the literature is building up pace fast with this relatively recent phenomenon, information is still lacking on the mechanisms and results of the impact that introduced fish have on freshwater ecosystems. Remote small lakes at high-latitudes can be used to evaluate some of these impacts, as they are of limited size and often host relatively simple food webs with a single species of introduced fish, which makes them ideal model systems. Many areas are not as easy to investigate, as they present much more complex ecosystems where complex species interactions take place. For example, artificial lotic systems with a high number of species interactions can be particularly challenging to tackle as other anthropogenic/environmental stressors might be challenging to disentangle. This thesis focused on three main themes which addressed the reliance of introduced fish on terrestrial energy, their cascading effects on the food web and their interaction with native species. The first two themes used brown trout introduced in remote small lake ecosystems at high latitudes as a model, whereas the last theme used a complex lotic system at low latitudes where multiple species were introduced at different times. The chapters of this thesis used neo- and paleolimnological techniques to investigate the impacts of introduced fish, sometimes in combination. In particular, the first theme was tackled through the use of stomach content, fatty acids and stable isotopes to unravel the feeding ecology of introduced brown trout. The second theme was instead addressed through multiple paleo-proxies in combination with neolimnological analyses and models. The last theme was investigated through the analysis of long-term environmental and fish-assemblage data. Our findings suggest that terrestrial sources could be highly important in supporting introduced brown trout populations in small lakes at high latitudes. Despite challenges inherent to the turnover rates of fish liver and muscle tissues, which were longer than previously thought, this support did not vary across different years or fish densities, but was affected by seasonal factors over the course of the open-water season. Our results also suggest that introduced brown trout affect the food web of host lakes through trophic cascades, altering the abundance of pelagic and benthic micro- and macroinvertebrates, probably through a modification of the distribution of macro-invertebrate communities. According to a bioenergetic and a mass balance model, introduced brown trout nutrient regeneration should have increased lake productivity; however paleo-proxies indicated no such change but rather a shift from pelagic to benthic productivity. Finally, our results suggest that, in artificial lotic systems at low latitudes, environmental chemistry did not play a role in the decline of the native fish species communities. Rather, this was a result of the interaction between some of the introduced fish species and the native ones, enhanced by the habitat simplification and the peculiar water fluctuation regime.