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The vast majority of rivers in the developed world are affected by human alteration, which in turn negatively affects the species that rely on these highly diverse and species rich areas to survive. Homogenization of previously heterogenic areas due to a change in water discharge and the substrate availability in the rivers is often regarded as the main reason for the loss of species richness in rivers and the riparian zone. Because of this, there are a lot of restoration projects which main goal is to increase heterogeneity. The goal of this thesis is to compare hydrology and geomorphology variables between four types of reaches with high flow velocity (rapids in free-flowing reaches, rapids with regulated flow, impounded reaches and reaches in outlet channels below hydropower plants). In this thesis I wanted to find out how changes in geomorphology and hydrology affect fish species richness, riparian vegetation richness and aquatic vascular plants species richness. The result show that both fish species richness and riparian vegetation species richness are negatively affected by the changes in geomorphology and hydrology. There was no significant difference for aquatic vascular plants when comparing the regulation types. The most altered reach type, outlet channels, had significantly fewer fish species compared to the less altered reach type, impounded reaches. Outlet channels also had less riparian vegetation cover compared to all other regulation types, and fewer riparian vegetation species compared to both free-flowing reaches and impounded reaches. In conclusion, hydropower plants and timber floating have both negatively impacted the riverine ecosystem, and in turn caused a decrease in species richness for fish and riparian plants. The reaches studied in this thesis will continue to be altered and the species richness and species composition will change from an ecosystem that relied on the natural flow regime to an ecosystem more used to the flow regime created by the hydropower plants. 

South Africa has the largest occurrence of the human immune deficiency virus (HIV) in the world but has also implemented the largest antiretroviral (ARV) treatment programme. It was therefore of interest to determine the presence and concentrations of commonly used antiretroviral drugs (ARVDs) and, also, to determine the capabilities of wastewater treatment plants (WWTPs) for removing ARVDs. To this end, a surrogate standard based LC-MS/MS method was optimized and applied for the detection of thirteen ARVDs used in the treatment and management of HIV/acquired immune deficiency syndrome (HIV/AIDS) in two major and one modular WWTP in the eThekwini Municipality in KwaZulu-Natal, South Africa. The method was validated and the detection limits fell within the range of 2–20 ng L⁻¹. The analytical recoveries for the ARVDs were mainly greater than 50% with acceptable relative standard deviations. The concentration values ranged from <LOD – 53000 ng L⁻¹ (influent), <LOD – 34000 ng L⁻¹ (effluent) in a decentralized wastewater treatment facility (DEWATS); <LOD – 24000 ng L⁻¹ (influent), <LOD – 33000 ng L⁻¹ (effluent) in Northern WWTP and 61–34000 ng L⁻¹ (influent), <LOD – 20000 ng L⁻¹ (effluent) in Phoenix WWTP. Whilst abacavir, lamivudine and zidovudine were almost completely removed from the effluents, atazanavir, efavirenz, lopinavir and nevirapine persisted in the effluents from all three WWTPs. To estimate the ecotoxicological risks associated with the discharge of ARVDs, a countrywide survey focussing on the occurrence of ARVDs in WWTPs, surface and fresh water bodies, and aquatic organisms, is necessary.

L’Amarante des rochers Lagonosticta sanguinodorsalis dans les Monts Mandara, Nigeria du sud-est: une nouvelle sous-espèce ? Nous fournissons une description d’un mâle et d’une femelle, ainsi que desphotos d’un mâle, d’un amarante capturé dans les Monts Mandara, au nord-est du Nigeria. Le plumage et le cri de cet amarante sont identiques à ceux de l’Amarante des rochers Lagonosticta sanguinodorsalis, une espèce précédemment rapportée de la zone, excepté que le mâle n’a pas la couronne grise typique de l’espèce. Des travaux supplémentaires sur le terrain sont nécessaires pour déterminer s’il agit d’un individu aberrant ou d’une population de l’Amarante des rochers morphologiquement distincte.

Ethnopharmacological relevance: Present survey was conducted to explore ethnomedicinal uses and cultural importance of wild edible fruits species by the inhabitants of Lesser Himalayas-Pakistan. Materials and methods: Information was obtained through informed consent semi-structured interviews, questionnaires, market survey, focus group conversation, unceremonious dialogue and village walks with key informants. Cultural significance of each species was calculated based on use report by participants at each study site. Results: A total of 35 wild edible fruits belonging to 21 genera and 17 families were used for the treatment of various ailments and consumed. Rosaceae was found dominating family with (8 spp.), followed by Moraceae (6 spp.), Rhamnaceae (5 spp.), Palmae and Vitaceae (2 spp. each) and remaining families were represented by one species each. Fruits (48%) were found highly utilized plant parts, followed by leaves (34%), bark, flowers and seeds (4% each), branches, latex and roots (2% each). Water was used as a medium for preparation while milk, ghee, oil, egg and butter are used for application. Modes of preparation were fall into seven categories like fresh parts eaten raw (38%), powder (24%), decoction (20%), extract (12 %), paste (4%), juice and latex (2% each). Based on cultural important index (CI) Morus nigra was found most significant species within top ten fruit plants followed by Morus alba, Olea ferruginea, Berberis lycium, Pyrus pashia, Ficus carica, Ficus palmata, Ziziphus mauritiana, Diospyros lotus and Ziziphus nummularia. Conclusions: Traditional uses of wild edible plant depend mainly on socio-economic factors rather than climatic conditions or wealth of flora. Use reports and citation demonstrated that there is a common cultural heritage regarding the gathered food plants. Further investigation is required for Antioxidant study, essential and toxic components, pharmacological applications; dietary requirements and biotechnological techniques to improve yields.

(C) 2013 Elsevier Ireland Ltd. All rights reserved.

Mediator is a well-known transcriptional co-regulator and serves as an adaptor between gene-specific regulatory proteins and RNA polymerase II. Studies on the chromatin-bound form of Mediator revealed interactions with additional protein complexes involved in various transcription-related processes, such as the Lsm2–8 complex that is part of the spliceosomal U6 small nuclear ribonucleoprotein complex. Here, we employ Chromatin Immunoprecipitation sequencing (ChIP-seq) of chromatin associated with the Lsm3 protein and the Med1 or Med15 Mediator subunits. We identify 86 genes co-occupied by both Lsm3 and Mediator, of which 73 were intron-containing ribosomal protein genes. In logarithmically growing cells, Mediator primarily binds to their promoter regions but also shows a second, less pronounced occupancy at their 3́-exons. During the late exponential phase, we observe a near-complete transition of Mediator from these promoters to a position in their 3́-ends, overlapping the Lsm3 binding sites ∼250 bp downstream of their last intron–exon boundaries. Using an unbiased RNA sequencing approach, we show that transition of Mediator from promoters to the last exon of these genes correlates to reduction of both their messenger RNA levels and splicing ratios, indicating that the Mediator and Lsm complexes cooperate to control growth-regulated expression of intron-containing ribosomal protein genes at the levels of transcription and splicing.

Background: Natural colorants, including natural pigments, e.g., anthocyanins, carotenoids, and chlorophylls, in novel and attractive food matrixes have become a popular trend. They impart favorite colors to food products and provide significant therapeutic effects. This study is aimed at extracting and identifying some natural pigments from different plant sources and evaluating their ability as antibacterial, antioxidant, and anticancer activities.

Methods: The anthocyanin-rich extract (ARE) is derived from three natural plant sources: pomegranate peel (Punica granatum), chili pepper fruit (Capsicum annuum), and Bougainvillea flowers. Bougainvillea spectabilis are analyzed for biochemical composition, as well as antioxidant, antibacterial, and anticancer activity, HPLC, DPPH, FRAP, disc diffusion assay, MIC, MTT, VEGFR‐2, and caspase-9 assays.

Results: All three extracts had varying total phenolic contents, ranging from 14 to 466 mg GAE/g extract, where Punica granatum was the highest (466 mg GAE/g extract), followed by Bougainvillea spectabilis (180 mg GAE/g extract), and then Capsicum annuum (14 mg GAE/g extract). The antioxidant activity rose steadily with raising concentration. The ARE of pomegranate peels recorded highest value, followed by Bougainvillea flowers and chili pepper fruit. The MTT assay revealed an inhibitory action of the tested extracts on the proliferation of HCT-116, MCF-7, and HepG2 in a concentration-based manner. Gene expression of caspase-9 transcripts was considerably multiplied by the application of ARE of pomegranate peels. All the tested extracts inhibited VEGFR-2, and the inhibition (%) expanded gradually with increasing concentrations, achieving the highest value (80 %) at 10 μg/mL. The ARE of pomegranate peels scored highest antibacterial activity, followed by ARE of chili pepper fruit and Bougainvillea flowers. The inhibition zone diameter escalated gradually with rising concentrations of the tested samples.

Conclusion: The AREs of the three studied plant sources can be used as multifunctional products with antioxidant, anticancer, and antibacterial activities that are natural, safe, and cheap.

The main target of this work is to discover new protein fractions from natural resources with high antibacterial action. The 7S and 11S globulin fractions, as well as the basic subunit (BS), were isolated from lupine seeds (Lupinus termis), chemically characterized, and screened for antibacterial activity against seven pathogenic bacteria. SDS-PAGE revealed molecular weights ranging from 55 to 75 kDa for 7S globulin, 20–37 kD for 11S globulin, and 20 kD for the BS. 11S globulin and the BS migrated faster on Urea-PAGE toward the cathode compared to 7S globulin. FTIR and NMR showed different spectral patterns between the 7S and 11S globulins but similar ones between 11S globulin and the BS. The MICs of the BS were in the range of 0.05–2 μg/mL against Listeria monocytogenes, Klebsiella oxytoca, Proteus mirabilis, Staphylococcus aureus, Listeria ivanovii, Salmonella typhimurium, and Pseudomonas aeruginosa compared to higher values for 11S globulin. The BS surpassed 11S globulin in antibacterial action, while 7S globulin showed no effect. The MICs of 11S globulin and the BS represented only 5% and 2.5% of the specific antibiotic against L. monocytogenes, respectively. Scanning electron microscopy (SEM) demonstrated different signs of cellular deformation and decay in the protein-treated bacteria, probably due to interaction with the bacterial cell wall and membranes. 11S globulin and the BS can be nominated as effective food biopreservatives.

Reindeer are important drivers of ecosystem processes in arctic and subarctic ecosystems, changing nutrient conditions and influencing vegetation by grazing and trampling. Mountain birch forests are one of the ecosystems impacted by reindeer grazing, but the effect on the 3D structure of these forests is not well understood. Drones are revolutionising ecological studies, allowing to create high-resolution 3D point clouds at low costs. I investigated the effect of year-round and autumn reindeer grazing on mountain birch forest vegetation in historically separated grazing areas at the Finnish-Norwegian border, using a combination of field data and drone data. The two sampling techniques were further compared to evaluate the possibility to use photogrammetric point clouds to characterise mountain birch forests. I found lower productivity in the year-round grazing regime, coinciding with generally higher vegetation density. Vertically, higher densities were found above browsing height, while the understory showed lower densities compared to autumn grazed areas. These results suggest that mountain birches allocate more biomass to the canopy area, which can be direct or indirect grazing effects. Nevertheless, overall productivity is lowered by grazing indicating changes in vegetation biomass and composition. The point clouds generally matched field data, but the understory vegetation tended to be underrepresented, arising the question if found effects are ecological or technical. As this could not be disentangled, cautious interpretation of my results is required. I conclude that using photogrammetric point clouds is a promising technique for ecological studies, but needs further development to improve accuracy and reliability of results.

We hypothesized a geographical pattern of the plant performance (seedling development, biomass production, relative water content and chlorophyll content) as a result of response to the interaction between photoperiod and water availability in populations of the arctic-alpine Silene suecica from different latitudes, thus experiencing different photoperiods during the growing season. Particularly, we expected a lower drought sensitivity in northern compared to southern populations as a consequence of harsher conditions experienced by the northern populations in terms of water availability. The experiment was carried out under common garden conditions, manipulating the water availability (wet and dry) and the photoperiod (21 and 16 h). We found an interaction between photoperiod and water availability on plant height, leaves, growth, biomass and total chlorophyll. However, the photoperiod neither counteracted nor intensified the effect of drought. Plants exposed to drought compensated for decreasing water availability by reducing their shoot growth. Changes in the chlorophyll content and chlorophyll a/b ratio were observed. Northern populations showed a higher basal growth performance and a greater response to the changed water regime (from wet to dry) than the southern populations. Southern populations showed a reduced ability to respond to drought, but their low basal performance may be advantageous under low water availability, avoiding water loss. In contrast, northern populations showed a stronger plastic response that limited the negative effects of reduced water availability. This study highlights the possibility that the plant response to environmental constraints (specifically water availability) may follow a geographical pattern.

We investigated the microplastics (MP) abundance, characteristics and pollution severity in the six coral reef ecosystems along the southern and western coasts of Sri Lanka during the southwest monsoon. The small blue-coloured fibres dominated within coral reefs and reef environments. The average coral, surface water and surface sediment MP concentration were 806.64 ± 93.72 particles kg⁻¹ww, 23.42 ± 5.01 particles m⁻³, and 54.11 ± 8.04 particles kg⁻¹dw, respectively. Coral MP concentration was significantly correlated with surface water (r = 0.65) and surface sediments (r = 0.59) indicating that the reef environment was the source of coral MP enrichment. Coral MP concentration was spatially different (p < 0.05) which may likely be due to the changes in winds, waves, coral biology and plastic sources. MP Pollution Load Index indicated that low MP contamination hazard. Since the presence of MP often deteriorates coral's health, further investigations are necessary to understand the MP's effects on the health of coral reef ecosystems.

The diffusion of viruses at the cell membrane is essential to reach a suitable entry site and initiate subsequent internalization. Although many viruses take advantage of glycosaminoglycans (GAG) to bind to the cell surface, little is known about the dynamics of the virus–GAG interactions. Here, single-particle tracking of the initial interaction of individual herpes simplex virus 1 (HSV-1) virions reveals a heterogeneous diffusive behavior, regulated by cell-surface GAGs with two main diffusion types: confined and normal free. This study reports that different GAGs can have competing influences in mediating diffusion on the cells used here: chondroitin sulfate (CS) enhances free diffusion but hinders virus attachment to cell surfaces, while heparan sulfate (HS) promotes virus confinement and increases entry efficiency. In addition, the role that the viral mucin-like domains (MLD) of the HSV-1 glycoprotein C plays in facilitating the diffusion of the virus and accelerating virus penetration into cells is demonstrated. Together, our results shed new light on the mechanisms of GAG-regulated virus diffusion at the cell surface for optimal internalization. These findings may be extendable to other GAG-binding viruses.

Plant retinoblastoma-related (RBR) proteins are primarily considered as key regulators of G(1)/S phase transition, with functional roles in a variety of cellular events during plant growth and organ development. Polyclonal antibody against the C-terminal region of the Arabidopsis RBR1 protein also specifically recognizes the alfalfa 115 kDa MsRBR protein, as shown by the antigen competition assay. The MsRBR protein was detected in all cell cycle phases, with a moderate increase in samples representing G(2)/M cells. Antibody against the human phospho-pRb peptide (Ser807/811) cross-reacted with the same 115 kDa MsRBR protein and with the in vitro phosphorylated MsRBR protein C-terminal fragment. Phospho-MsRBR protein was low in G(1) cells. Its amount increased upon entry into the S phase and remained high during the G(2)/M phases. Roscovitine treatment abolished the activity of alfalfa MsCDKA1;1 and MsCDKB2;1, and the phospho-MsRBR protein level was significantly decreased in the treated cells. Colchicine block increased the detected levels of both forms of MsRBR protein. Reduced levels of the MsRBR protein in cells at stationary phase or grown in hormone-free medium can be a sign of the division-dependent presence of plant RBR proteins. Immunolocalization of the phospho-MsRBR protein indicated spots of variable number and size in the labelled interphase nuclei and high signal intensity of nuclear granules in prophase. Structures similar to phospho-MsRBR proteins cannot be recognized in later mitotic phases. Based on the presented western blot and immunolocalization data, the possible involvement of RBR proteins in G(2)/M phase regulation in plant cells is discussed.

Earth’s life-sustaining oceans harbor diverse bacterial communities that display varying composition across time and space. While particular patterns of variation have been linked to a range of factors, unifying rules are lacking, preventing the prediction of future changes. Here, analyzing the distribution of fast- and slow-growing bacteria in ocean datasets spanning seasons, latitude, and depth, we show that higher seawater temperatures universally favor slower-growing taxa, in agreement with theoretical predictions of how temperature-dependent growth rates differentially modulate the impact of mortality on species abundances. Changes in bacterial community structure promoted by temperature are independent of variations in nutrients along spatial and temporal gradients. Our results help explain why slow growers dominate at the ocean surface, during summer, and near the tropics and provide a framework to understand how bacterial communities will change in a warmer world.

Photosynthetic organisms strictly depend on CO₂ availability and the CO₂:O₂ ratio, as both CO₂/O₂ compete for catalytic site of Rubisco. Green alga Chlamydomonas reinhardtii, can overcome CO₂ shortage by inducing CO₂-concentrating mechanism (CCM). Cells transferred to low-CO₂ are subjected to light-driven oxidative stress due to decrease in the electron sink. Response to environmental perturbations is mediated to some extent by changes in the lipid and carotenoid metabolism. We thus hypothesize that when cells are challenged with changes in CO₂ availability, changes in the lipidome and carotenoids profile occur. These changes expected to be transient, when CCM is activated, CO₂ limitation will be substantially ameliorated. In our experiments, cells were transferred from high (5%) to low (air equilibrium) CO₂. qPCR analysis of genes related to CCM and lipid metabolism was carried out. Lipidome was analyzed both in whole cells and in isolated lipid droplets. We characterized the changes in polar lipids, fatty acids and ketocarotenoids. In general, polar lipids significantly and transiently increased in lipid droplets during CCM. Similar pattern was observed for xanthophylls, ketocarotenoids and their esters. The data supports our hypothesis about the roles of lipids and carotenoids in tackling the oxidative stress associated with acclimation to sub-saturating CO₂.

Wood, or secondary xylem, is the product of xylogenesis, a developmental process that begins with the proliferation of cambial derivatives and ends with mature xylem fibers and vessels with lignified secondary cell walls. Fully mature xylem has undergone a series of cellular processes, including cell division, cell expansion, secondary wall formation, lignification and programmed cell death. A complex network of interactions between transcriptional regulators and signal transduction pathways controls wood formation. However, the role of metabolites during this developmental process has not been comprehensively characterized. To evaluate the role of metabolites during wood formation, we performed a high spatial resolution metabolomics study of the wood-forming zone of Populus tremula, including laser dissected aspen ray and fiber cells. We show that metabolites show specific patterns within the wood-forming zone, following the differentiation process from cell division to cell death. The data from profiled laser dissected aspen ray and fiber cells suggests that these two cell types host distinctly different metabolic processes. Furthermore, by integrating previously published transcriptomic and proteomic profiles generated from the same trees, we provide an integrative picture of molecular processes, for example, deamination of phenylalanine during lignification is of critical importance for nitrogen metabolism during wood formation.

Associations of salicylate-like phenolic glycosides (PGs) with biological activity have been reported in Salix and Populus trees, but only for a few compounds, and in relation to a limited number of herbivores. By considering the full diversity of PGs, we may improve our ability to recognize genotypes or chemotype groups and enhance our understanding of their ecological function. Here, we present a fast and efficient general method for salicylate determination in leaves of Eurasian aspen that uses ultra-high performance liquid chromatography-electrospray ionization/time-of-flight mass spectrometry (UHPLC-ESI/TOFMS). The time required for the liquid chromatography separations was 13.5 min per sample, compared to around 60 min per sample for most HPLC protocols. In leaf samples from identical P. tremula genotypes with diverse propagation and treatment histories, we identified nine PGs. We found the compound-specific mass chromatograms to be more informative than the UV-visible chromatograms for compound identification and when quantitating samples with large variability in PG content. Signature compounds previously reported for P. tremoloides (tremulacin, tremuloidin, salicin, and salicortin) always were present, and five PGs (2'-O-cinnamoyl-salicortin, 2'-O-acetyl-salicortin, 2'-O-acetyl-salicin, acetyl-tremulacin, and salicyloyl-salicin) were detected for the first time in P. tremula. By using information about the formic acid adduct that appeared for PGs in the LTQ-Orbitrap MS environment, novel compounds like acetyl-tremulacin could be tentatively identified without the use of standards. The novel PGs were consistently either present in genotypes regardless of propagation and damage treatment or were not detectable. In some genotypes, concentrations of 2'-O-acetyl-salicortin and 2'-O-cinnamoyl-salicortin were similar to levels of biologically active PGs in other Salicaceous trees. Our study suggests that we may expect a wide variation in PG content in aspen populations which is of interest both for studies of interactions with herbivores and for mapping population structure.

Klebsiella (K.) pneumoniae is a common cause of pneumonia-derived sepsis. Myeloid related protein 8 (MRP8, S100A8) and MRP14 (S100A9) are the most abundant cytoplasmic proteins in neutrophils. They can form MRP8/14 heterodimers that are released upon cell stress stimuli. MRP8/14 reportedly exerts antimicrobial activity, but in acute fulminant sepsis models MRP8/14 has been found to contribute to organ damage and death. We here determined the role of MRP8/14 in K. pneumoniae sepsis originating from the lungs, using an established model characterized by gradual growth of bacteria with subsequent dissemination. Infection resulted in gradually increasing MRP8/14 levels in lungs and plasma. Mrp14 deficient (mrp14(-/-)) mice, unable to form MRP8/14 heterodimers, showed enhanced bacterial dissemination accompanied by increased organ damage and a reduced survival. Mrp14(-/-) macrophages were reduced in their capacity to phagocytose Klebsiella. In addition, recombinant MRP8/14 heterodimers, but not MRP8 or MRP14 alone, prevented growth of Klebsiella in vitro through chelation of divalent cations. Neutrophil extracellular traps (NETs) prepared from wildtype but not from mrp14(-/-) neutrophils inhibited Klebsiella growth; in accordance, the capacity of human NETs to kill Klebsiella was strongly impaired by an anti-MRP14 antibody or the addition of zinc. These results identify MRP8/14 as key player in protective innate immunity during Klebsiella pneumonia.

Gene expression is spatially and temporally regulated at multiple levels. N⁶-methyladenosine (m⁶A) is the most prevalent internal modification in messenger RNA (mRNA) and long noncoding RNA (lncRNAs). m⁶A plays important roles in multiple cellular processes including stem cell pluripotency, adipogenesis, spermatogenesis, neurogenesis, circadian rhythm and development by modulating RNA splicing, export, stability, degradation and translation. Although aberrant m⁶A methylation has been reported in various types of cancer, the underlying molecular functions of METTL3, the solely catalytic subunit of the m⁶A-methylase complex, has yet to be defined.

m⁶A has been recently identified in nascent pre-mRNA, and more specifically intronic m⁶A has been linked to exon skipping events. The occurrence of impaired alternative splicing (AS) is frequently found during the development of cancer. We performed transcriptome wide analysis in breast cancer cell lines and explored AS events. Our results define an AS signature for breast tumorigenesis. We found that METTL3 modulates AS directly through m⁶A deposition at the intron-exon junctions or indirectly by the m⁶A deposition in transcripts encoding for splicing factors and transcription factors. In particular, we show that MYC mRNA harbours the m⁶A mark, suggesting that METTL3 regulates AS indirectly via the regulation of MYC expression. Indeed, the targets of MYC overlapped with METTL3-associated AS events. Importantly, five of the AS events identified and validated in vitro, are linked to a worse prognosis in breast cancer patients. Additionally, we show that METTL3 enhances the breast cancer phenotype through a dual mechanism depending on its sub-cellular localization. We find that the canonical nuclear function of METTL3 decorates transcripts that are involved in cell proliferation and migration. We observe that METTL3 is highly expressed in the cytoplasmic compartment of breast cancer cells from patients. Remarkably, we uncover that the cytoplasmic METTL3 interacts with subunits of the exocyst, whose subunit EXOC7 has been linked to cell adhesion, migration and invasion. Notably, we show that breast cancer cell lines depleted of METTL3 display less gelatinase activity and invadopodia formation, supporting the role of METTL3 in cell invasion via exocytosis.

m⁶A is a reversible modification, which can be demethylated by the erasers FTO and ALKBH5. Depletion of FTO has been shown to increase the level of m⁶A in mRNA, however recent studies have reported that FTO could demethylate N⁶,2´-O-dimethyladenosine (m⁶A_m), adjacent to the 7-methylguanosine cap on mRNA. In the cellular model of colorectal cancer CRC1, depletion of FTO leads to a cancer stem cell phenotype and confers chemotherapy resistance. By performing m⁶A-RNA immunoprecipitation followed by sequencing (MeRIP), we show that knockdown of FTO in CRC1 cells does not affect the global level of m⁶A in mRNA but of m⁶A_m level.

Alternative splicing (AS) enables differential inclusion of exons from a given transcript, thereby contributing to the transcriptome and proteome diversity. Aberrant AS patterns play major roles in the development of different pathologies, including breast cancer. N⁶-methyladenosine (m⁶A), the most abundant internal modification of eukaryotic mRNA, influences tumor progression and metastasis of breast cancer, and it has been recently linked to AS regulation. Here, we identify a specific AS signature associated with breast tumorigenesis in vitro. We characterize for the first time the role of METTL3 in modulating breast cancer-associated AS programs, expanding the role of the m⁶A-methyltransferase in tumorigenesis. Specifically, we find that both m⁶A deposition in splice site boundaries and in splicing and transcription factor transcripts, such as MYC, direct AS switches of specific breast cancer-associated transcripts. Finally, we show that five of the AS events validated in vitro are associated with a poor overall survival rate for patients with breast cancer, suggesting the use of these AS events as a novel potential prognostic biomarker.

Primordial follicles are the first small follicles to appear in the mammalian ovary. Women are born with a fixed number of primordial follicles in the ovaries. Once formed, the pool of primordial follicles serves as a source of developing follicles and oocytes. The first aim of this thesis was to investigate the functional role of the intra-oocyte signaling pathways, especially the phosphatidylinositol-3 kinase (PI3K) and mammalian target of rapamycin complex 1 (mTORC1) pathways in the regulation of primordial follicle activation and survival. We found that a primordial follicle remains dormant when the PI3K and mTORC1 signaling in its oocyte is activated to an appropriate level, which is just sufficient to maintain its survival, but not sufficient for its growth initiation. Hyperactivation of either of these signaling pathways causes global activation of the entire pool of primordial follicles leading to the exhaustion of all the follicles in young adulthood in mice. Mammalian oocytes, while growing within the follicles, remain arrested at prophase I of meiosis. Oocytes within the fully-grown antral follicles resume meiosis upon a preovulatory surge of leutinizing hormone (LH), which indicates that LH mediates the resumption of meiosis. The prophase I arrest in the follicle-enclosed oocyte is the result of low maturation promoting factor (MPF) activity, and resumption of meiosis upon the arrival of hormonal signals is mediated by activation of MPF. MPF is a complex of cyclin dependent kinase 1 (Cdk1) and cyclin B1, which is essential and sufficient for entry into mitosis. Although much of the mitotic cell cycle machinery is shared during meiosis, lack of Cdk2  in mice leads to a postnatal loss of all oocytes, indicating that Cdk2 is important for oocyte survival, and probably oocyte meiosis also. There have been conflicting results earlier about the role of Cdk2 in metaphase II arrest of Xenopus  oocytes. Thus the second aim of the thesis was to identify the specific Cdk that is essential for mouse oocyte meiotic maturation. We generated mouse models with oocytespecific deletion of Cdk1  or Cdk2  and studied the specific requirements of Cdk1 and Cdk2 during resumption of oocyte meiosis. We found that only Cdk1 is essential and sufficient for the oocyte meiotic maturation. Cdk1 does not only phosphorylate the meiotic phosphoproteins during meiosis resumption but also phosphorylates and suppresses the downstream protein phosphatase 1, which is essential for protecting the Cdk1 substrates from dephosphorylation.

To maintain the length of reproductive life in a woman, it is essential that most of her ovarian primordial follicles are maintained in a quiescent state to provide a continuous supply of oocytes. However, our understanding of the molecular mechanisms that control the quiescence and activation of primordial follicles is still in its infancy. In this study, we provide some genetic evidence to show that the tumor suppressor tuberous sclerosis complex 2 (Tsc2), which negatively regulates mammalian target of rapamycin complex 1 (mTORC1), functions in oocytes to maintain the dormancy of primordial follicles. In mutant mice lacking the Tsc2 gene in oocytes, the pool of primordial follicles is activated prematurely due to elevated mTORC1 activity in oocytes. This results in depletion of follicles in early adulthood, causing premature ovarian failure (POF). Our results suggest that the Tsc1-Tsc2 complex mediated suppression of mTORC1 activity is indispensable for maintenance of the dormancy of primordial follicles, thus preserving the follicular pool, and that mTORC1 activity in oocytes promotes follicular activation. Our results also indicate that deregulation of Tsc/mTOR signaling in oocytes may cause pathological conditions of the ovary such as infertility and POF.

Once formed, the pool of dormant primordial follicles serves as the source of developing follicles and fertilizable ova for the duration of a female’s reproductive life. Depending upon the species, primordial follicles can remain quiescent for months, years, or even decades, and the highly regulated process of primordial follicle activation ensures the availability of growing follicles throughout the reproductive period. We have recently begun to elucidate the molecular mechanisms underlying the maintenance of follicular quiescence and the activation of primordial follicles, mainly through the use of genetically modified mouse models. Both overactivation as well as the failure of activation of primordial follicles can lead to pathological conditions such as premature ovarian failure (POF) in the experimental models. A thorough understanding of the underlying mechanisms that regulate quiescence and activation of oocyte growth in primordial follicles will have important biological and clinical implications.

Viroids are non-coding RNA plant pathogens that are characterized by their possession of a high mutation level. Although the sequence heterogeneity in viroid infected plants is well understood, shifts in viroid population dynamics due to mutations over the course of infection remain poorly understood. In this study, the ten most abundant sequence variants of potato spindle tuber viroid RG1 (PSTVd) expressed at different time intervals in PSTVd infected tomato plants were identified by high-throughput sequencing. The sequence variants, forming a quasi-species, were subjected to both the identification of the regions favoring mutations and the effect of the mutations on viroid secondary structure and viroid derived small RNAs (vd-sRNA). At week 1 of PSTVd infection, 25% of the sequence variants were similar to the "master" sequence (i.e., the sequence used for inoculation). The frequency of the master sequence within the population increased to 70% at week 2 after PSTVd infection, and then stabilized for the rest of the disease cycle (i.e., weeks 3 and 4). While some sequence variants were abundant at week 1 after PSTVd infection, they tended to decrease in frequency over time. For example, the variants with insertions at positions 253 or 254, positions that could affect the Loop E as well as the metastable hairpin I structure that has been shown important during replication and viroid infectivity, resulted in decreased frequency. Data obtained byin silicoanalysis of the viroid derived small RNAs (vd-sRNA) was also analyzed. A few mutants had the potential of positively affecting the viroid's accumulation by inducing the RNA silencing of the host's defense related genes. Variants with mutations that could negatively affect viroid abundance were also identified because their derived vd-sRNA were no longer capable of targeting any host mRNA or of changing its target sequence from a host defense gene to some other non-important host gene. Together, these findings open avenues into understanding the biological role of sequence variants, this viroid's interaction with host components, stable and metastable structures generated by mutants during the course of infection, and the influence of sequence variants on stabilizing viroid population dynamics.

Multiple sclerosis (MS) is a chronic inflammatory and neurodegenerative disease of the central nervous system, with a high rate of neurocognitive symptoms for which the molecular background is still uncertain. There is accumulating evidence for dysregulation of the kynurenine pathway (KP) in different psychiatric and neurodegenerative conditions. We here report the first comprehensive analysis of cerebrospinal fluid (CSF) kynurenine metabolites in MS patients of different disease stages and in relation to neurocognitive symptoms. Levels of tryptophan (TRP), kynurenine (KYN), kynurenic acid (KYNA) and quinolinic acid (QUIN) were determined with liquid chromatography mass spectrometry in cell-free CSF. At the group level MS patients (cohort 1; n = 71) did not differ in absolute levels of TRP, KYN, KYNA or QUIN as compared to non-inflammatory neurological disease controls (n = 20). Stratification of patients into different disease courses revealed that both absolute QUIN levels and the QUIN/KYN ratio were increased in relapsing-remitting MS (RRMS) patients in relapse. Interestingly, secondary progressive MS (SPMS) displayed a trend for lower TRP and KYNA, while primary progressive (PPMS) patients displayed increased levels of all metabolites, similar to a group of inflammatory neurological disease controls (n = 13). In the second cohort (n = 48), MS patients with active disease and short disease duration were prospectively evaluated for neuropsychiatric symptoms. In a supervised multivariate analysis using orthogonal projection to latent structures (OPLS-DA) depressed patients displayed higher KYNA/TRP and KYN/TRP ratios, mainly due to low TRP levels. Still, this model had low predictive value and could not completely separate the clinically depressed patients from the non-depressed MS patients. No correlation was evident for other neurocognitive measures. Taken together these results demonstrate that clinical disease activity and differences in disease courses are reflected by changes in KP metabolites. Increased QUIN levels of RRMS patients in relapse and generally decreased levels of TRP in SPMS may relate to neurotoxicity and failure of remyelination, respectively. In contrast, PPMS patients displayed a more divergent pattern more resembling inflammatory conditions such as systemic lupus erythematosus. The pattern of KP metabolites in RRMS patients could not predict neurocognitive symptoms.

Litter decomposition and nutrient mineralization in high-latitude peatlands are constrained by low temperatures. So far, little is known about the effects of seasonal components of climate change (higher spring and summer temperatures, more snow which leads to higher winter soil temperatures) on these processes. In a 4-year field experiment, we manipulated these seasonal components in a sub-arctic bog and studied the effects on the decomposition and N and P dynamics of leaf litter of Calamagrostis lapponica, Betula nana, and Rubus chamaemorus, incubated both in a common ambient environment and in the treatment plots. Mass loss in the controls increased in the order Calamagrostis < Betula < Rubus. After 4 years, overall mass loss in the climate-treatment plots was 10 % higher compared to the ambient incubation environment. Litter chemistry showed within each incubation environment only a few and species-specific responses. Compared to the interspecific differences, they resulted in only moderate climate treatment effects on mass loss and these differed among seasons and species. Neither N nor P mineralization in the litter were affected by the incubation environment. Remarkably, for all species, no net N mineralization had occurred in any of the treatments during 4 years. Species differed in P-release patterns, and summer warming strongly stimulated P release for all species. Thus, moderate changes in summer temperatures and/or winter snow addition have limited effects on litter decomposition rates and N dynamics, but summer warming does stimulate litter P release. As a result, N-limitation of plant growth in this sub-arctic bog may be sustained or even further promoted.

Membrane vesicles (MVs) contribute to various biological processes in bacteria, including virulence factor delivery, antimicrobial resistance, host immune evasion and cross-species communication. MVs are frequently released from the surface of both Gram-negative and Gram-positive bacteria during growth. In some Gram-positive bacteria, genes affecting MV biogenesis have been identified, but the mechanism of MV formation is unknown. In Enterococcus faecalis, a causative agent of life-threatening bacteraemia and endocarditis, neither mechanisms of MV formation nor their role in virulence has been examined. Since MVs of many bacterial species are implicated in host–pathogen interactions, biofilm formation, horizontal gene transfer, and virulence factor secretion in other species, we sought to identify, describe and functionally characterize MVs from E. faecalis. Here, we show that E. faecalis releases MVs that possess unique lipid and protein profiles, distinct from the intact cell membrane and are enriched in lipoproteins. MVs of E. faecalis are specifically enriched in unsaturated lipids that might provide membrane flexibility to enable MV formation, providing the first insights into the mechanism of MV formation in this Gram-positive organism.

The inhibitor of angiogenesis, thrombospondin 2 (TSP2), belongs to a group of matricellular proteins that are induced in response to injury and modulate the healing of dermal wounds. Thus, TSP-2-null mice display abnormal connective tissue architecture and increased angiogenesis in the dermis, and heal wounds at an accelerated rate. In this study, we report that the content of TSP2 is increased in the uninjured skin of aged mice. Furthermore, in primary dermal fibroblasts, TSP2 expression is increased both as a function of the age of the donor and days in culture. To determine the significance of the increased TSP2 in aged mice (two years or older), we performed full-thickness excisional wounds and compared their healing in aged and young (3–4 months) wild-type and TSP2-null mice. Gross morphological examination of wounds indicated that aged TSP2-null mice healed faster than their aged wild-type counterparts, but healing in aged mice was always sub-optimal in comparison to that in young animals. Surprisingly, despite the increase in TSP2, a potent inhibitor of angiogenesis, in wounds in aged mice, the vascular density of these wounds was not reduced in comparison to that in young animals. However, immunohistochemical analysis of healing wounds revealed a shift in the peak content of TSP2, from day 10 in young mice to day 14 or later in aged mice, and there was a corresponding delay in the expected increase in matrix metalloproteinase (MMP) 2 levels in aged TSP2-null mice. We suggest that the delay in expression of TSP2 and MMP2 in the wounds of aged mice could contribute to their impaired rate of wound healing.

Ecological significance of trees growing in urban and peri-urban settings is likely to increase in future land-use regimes, calling for better understanding of their role as potential reservoirs or stepping stones for associated biodiversity. We studied the diversity of fungal endophytes in woody tissues of asymptomatic even aged pedunculate oak trees, growing as amenity trees in a peri-urban setting. The trees were classified into three groups according to their phenotypic vitality (high, medium, and low). Endophytes were cultured on potato dextrose media from surface sterilized twigs and DNA sequencing was performed to reveal the taxonomic identity of the morphotypes. In xylem tissues, the frequency and diversity of endophytes was highest in oak trees showing reduced vitality. This difference was not found for bark samples, in which the endophyte infections were more frequent and communities more diverse than in xylem. In general, most taxa were shared across the samples with few morphotypes being recovered in unique samples. Leaf phenolic profiles were found to accurately classify the trees according to their phenotypic vitality. Our results confirm that xylem is more selective substrate for endophytes than bark and that endophyte assemblages in xylem are correlated to the degree of host vitality. Thus, high vitality of trees may be associated with reduced habitat quality to wood-associated endophytes.

1. Many riparian ecosystems in Mediterranean Europe are affected by land use and flow alteration by dams. We focused on understanding how these stressors and their components affect riparian forests in the region. We asked the following questions: (1) Are there well‐defined, responsive riparian guilds? (2) Do dam‐induced streamflows determine abundance and distribution of riparian guilds? (3) What are the main drivers governing composition and cover of riparian guilds in regulated rivers?

2. We inventoried the cover of riparian woody species in free‐flowing rivers and downstream of dams. We performed a cluster analysis and ordination to derive riparian guilds, using abundance data from 66 riparian woody species and 26 functional plant traits. We used a reduced set of principal components for the environment, land use and hydrology, and general linear modelling to explore the effect of these factors (separately and combined) on riparian guilds.

3. We found that: (1) four dominant guilds are responsive to disturbance in southwestern European streams, namely the obligate riparian, water‐stress tolerant, deciduous competitive and Mediterranean evergreen guilds; (2) a set of land use and hydrological variables differentially affect the diverse co‐occurring riparian guilds; (3) frequency and duration of high flow pulses and the low‐flow conditions were major drivers of change in landscapes dominated by intensive agriculture and forestry; (4) storage reservoirs reduced the cover of obligate riparian and Mediterranean evergreen guilds, and increased the abundance of water‐stress tolerant and deciduous competitive guilds, while run‐of‐river dams, having limited water storage, reduced both obligate and deciduous competitive guilds.

4. Synthesis and applications. Future research in southwestern Europe should address the resilience of riparian guilds and the effects of interacting landscape factors and stressors on guild distribution. Streamflow regulations downstream of reservoirs should focus on specific flow components, namely the magnitude of flows, and frequency and duration of extreme flow events. For successful mitigation of the dam‐induced effects on riparian vegetation, river management plans must incorporate the environmental and land use site‐specific contexts.

Cryptochromes are widely dispersed flavoprotein photoreceptors that regulate numerous developmental responses to light in plants, as well as to stress and entrainment of the circadian clock in animals and humans. All cryptochromes are closely related to an ancient family of light-absorbing flavoenzymes known as photolyases, which use light as an energy source for DNA repair but themselves have no light sensing role. Here we review the means by which plant cryptochromes acquired a light sensing function. This transition involved subtle changes within the flavin binding pocket which gave rise to a visual photocycle consisting of light-inducible and dark-reversible flavin redox state transitions. In this photocycle, light first triggers flavin reduction from an initial dark-adapted resting state (FADox). The reduced state is the biologically active or ‘lit’ state, correlating with biological activity. Subsequently, the photoreduced flavin reoxidises back to the dark adapted or ‘resting’ state. Because the rate of reoxidation determines the lifetime of the signaling state, it significantly modulates biological activity. As a consequence of this redox photocycle Crys respond to both the wavelength and the intensity of light, but are in addition regulated by factors such as temperature, oxygen concentration, and cellular metabolites that alter rates of flavin reoxidation even independently of light. Mechanistically, flavin reduction is correlated with conformational change in the protein, which is thought to mediate biological activity through interaction with biological signaling partners. In addition, a second, entirely independent signaling mechanism arises from the cryptochrome photocycle in the form of reactive oxygen species (ROS). These are synthesized during flavin reoxidation, are known mediators of biotic and abiotic stress responses, and have been linked to Cry biological activity in plants and animals. Additional special properties arising from the cryptochrome photocycle include responsivity to electromagnetic fields and their applications in optogenetics. Finally, innovations in methodology such as the use of Nitrogen Vacancy (NV) diamond centers to follow cryptochrome magnetic field sensitivity in vivo are discussed, as well as the potential for a whole new technology of ‘magneto-genetics’ for future applications in synthetic biology and medicine.

Proteins function as worker molecules in the cell and their natural environment is crowded. How they fold in a cell-like environment and how they recognize their interacting partners in such conditions, are questions that underlie the work of this thesis.

Two distinct subjects were investigated using a combination of biochemical- and biophysical methods. First, the unfolding/dissociation of a heptameric protein (cpn10) in the presence of the crowding agent Ficoll 70. Ficoll 70 was used to mimic the crowded environment in the cell and it has been used previously to study macromolecular crowding effects, or excluded volume effects, in protein folding studies. Second, the conformational changes upon interaction between the Mediator subunit Med25 and the transcription factor Dreb2a from Arabidopsis thaliana. Mediator is a transcriptional co-regulator complex which is conserved from yeast to humans. The molecular mechanisms of its action are however not entirely understood. It has been proposed that the Mediator complex conveys regulatory signals from promoter-bound transcription factors (activators/repressors) to the RNA polymerase II machinery through conformational rearrangements.

The results from the folding study showed that cpn10 was stabilized in the presence of Ficoll 70 during thermal- and chemical induced unfolding (GuHCl). The thermal transition midpoint increased by 4°C, and the chemical midpoint by 0.5 M GuHCl as compared to buffer conditions. Also the heptamer-monomer dissociation was affected in the presence of Ficoll 70, the transition midpoint was lower in Ficoll 70 (3.1 μM) compared to in buffer (8.1 μM) thus indicating tighter binding in crowded conditions. The coupled unfolding/dissociation free energy for the heptamer increased by about 36 kJ/mol in Ficoll. Altogether, the results revealed that the stability effect on cpn10 due to macromolecular crowding was larger in the individual monomers (33%) than at the monomer-monomer interfaces (8%).

The results from the interaction study indicated conformational changes upon interaction between the A. thaliana Med25 ACtivator Interaction Domain (ACID) and Dreb2a. Structural changes were probed to originate from unstructured Dreb2a and not from the Med25-ACID. Human Med25-ACID was also found to interact with the plant-specific Dreb2a, even though the ACIDs from human and A. thaliana share low sequence homology. Moreover, the human Med25-interacting transcription factor VP16 was found to interact with A. thaliana Med25. Finally, NMR, ITC and pull-down experiments showed that the unrelated transcription factors Dreb2a and

VP16 interact with overlapping regions in the ACIDs of A. thaliana and human Med25.

The results presented in this thesis contribute to previous reports in two different aspects. Firstly, they lend support to the findings that the intracellular environment affects the biophysical properties of proteins. It will therefore be important to continue comparing results between in vitro and cell-like conditions to measure the magnitude of such effects and to improve the understanding of protein folding and thereby misfolding of proteins in cells. Better knowledge of protein misfolding mechanisms is critical since they are associated to several neurodegenerative diseases such as Alzheimer’s and Parkinson's. Secondly, our results substantiate the notion that transcription factors are able to bind multiple targets and that they gain structure upon binding. They also show that subunits of the conserved Mediator complex, despite low sequence homologies, retain a conserved structure and function when comparing evolutionary diverged species.

Mediator is an evolutionary conserved multi-protein complex present in all eukaryotes. It functions as a transcriptional coregulator by conveying signals from activators and repressors to the RNA polymerase II transcription machinery. The Arabidopsis thaliana Med25 (aMed25) ACtivation Interaction Domain (ACID) interacts with the Dreb2a activator which is involved in plant stress response pathways, while Human Med25-ACID (hMed25) interacts with the herpes simplex virus VP16 activator. Despite low sequence similarity, hMed25-ACID also interacts with the plant-specific Dreb2a transcriptional activator protein. We have used GST pull-down-, surface plasmon resonance-, isothermal titration calorimetry and NMR chemical shift experiments to characterize interactions between Dreb2a and VP16, with the hMed25 and aMed25-ACIDs. We found that VP16 interacts with aMed25-ACID with similar affinity as with hMed25-ACID and that the binding surface on aMed25-ACID overlaps with the binding site for Dreb2a. We also show that the Dreb2a interaction region in hMed25-ACID overlaps with the earlier reported VP16 binding site. In addition, we show that hMed25-ACID/Dreb2a and aMed25-ACID/Dreb2a display similar binding affinities but different binding energetics. Our results therefore indicate that interaction between transcriptional regulators and their target proteins in Mediator are less dependent on the primary sequences in the interaction domains but that these domains fold into similar structures upon interaction.

Cluster 5 picocyanobacteria significantly contribute to primary productivity in aquatic ecosystems. Estuarine populations are highly diverse and consist of many co-occurring strains, but their physiology remains largely understudied. In this study, we characterized 17 novel estuarine picocyanobacterial strains. Phylogenetic analysis of the 16S rRNA and pigment genes (cpcB and cpeBA) uncovered multiple estuarine and freshwater-related clusters and pigment types. Assays with five representative strains (three phycocyanin rich and two phycoerythrin rich) under temperature (10–30°C), light (10–190 μmol photons m−2 s−1), and salinity (2–14 PSU) gradients revealed distinct growth optima and tolerance, indicating that genetic variability was accompanied by physiological diversity. Adaptability to environmental conditions was associated with differential pigment content and photosynthetic performance. Amplicon sequence variants at a coastal and an offshore station linked population dynamics with phylogenetic clusters, supporting that strains isolated in this study represent key ecotypes within the Baltic Sea picocyanobacterial community. The functional diversity found within strains with the same pigment type suggests that understanding estuarine picocyanobacterial ecology requires analysis beyond the phycocyanin and phycoerythrin divide. This new knowledge of the environmental preferences in estuarine picocyanobacteria is important for understanding and evaluating productivity in current and future ecosystems.

Chemical modifications of RNA provide a direct and rapid way to manipulate the existing transcriptome, allowing rapid responses to the changing environment further enriching the regulatory capacity of RNA. N-6-Methyladenosine(m(6)A) has been identified as the most abundant internal modification of messenger RNA in eukaryotes, linking external stimuli to an intricate network of transcriptional, post-transcriptional and translational processes. M(6)A modification affects a broad spectrum of cellular functions, including maintenance of the pluripotency of embryonic stem cells (ESCs) and the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). In this review, we summarize the most recent findings on m(6)A modification with special focus on the different studies describing how m(6)A is implicated in ESC self-renewal, cell fate specification and iPSC generation.

THAP1 (THAP [Thanatos-associated protein] domain-containing, apoptosis-associated protein 1) is a ubiquitously expressed member of a family of transcription factors with highly conserved DNA-binding and protein-interacting regions. Mutations in THAP1 cause dystonia, DYT6, a neurologic movement disorder. THAP1 downstream targets and the mechanism via which it causes dystonia are largely unknown. Here, we show that wild-type THAP1 regulates embryonic stem cell (ESC) potential, survival, and proliferation. Our findings identify THAP1 as an essential factor underlying mouse ESC survival and to some extent, differentiation, particularly neuroectodermal. Loss of THAP1 or replacement with a disease-causing mutation results in an enhanced rate of cell death, prolongs Nanog, Prdm14, and/or Rex1 expression upon differentiation, and results in failure to upregulate ectodermal genes. ChIP-Seq reveals that these activities are likely due in part to indirect regulation of gene expression.

Two groups of polyunsaturated fatty acids (PUFA), termed omega-3 and omega-6 in food (or here as n-3 and n-6 PUFA, respectively), are essential for all vertebrates and probably also for nearly all invertebrates. The absolute concentrations of the different PUFA are important, as is an appropriate balance between the two. The optimal ratio of n-3/n-6 is not known for most organisms but is anticipated to be more or less species-specific (Sargent et al. 1995). The three most important PUFA in vertebrates are eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (ARA, 20:4n-6). Both EPA and ARA are precursors for biologically active eicosanoids that are vital components of cell membranes and play many dynamic roles in mediating and controlling a wide array of cellular activities (Crawford et al. 1989; Harrison 1990; Henderson et al. 1996; see Chap. 9). Since n-3 and n-6 PUFA cannot be synthesized de novo by most metazoans, they must be included in the diet, either as EPA, DHA and ARA, or as their precursors, such as α-linolenic acid (ALA, 18:3n-3, precursor of EPA and DHA) and linoleic acid (LIN, 18:2n-6, precursor of ARA) (Bell et al. 1986; Sargent et al. 1995). Both ALA and LIN are produced in the thylacoid membranes of algae and plants with chlorophyll (Sargent at al. 1987).

Intensified land use can disturb water quality, potentially increasing the abundance of bacterial pathogens, threatening public access to clean water. This threat involves both direct contamination of faecal bacteria as well as indirect factors, such as disturbed water chemistry and microbiota, which can lead to contamination. While direct contamination has been well described, the impact of indirect factors is less explored, despite the potential of severe downstream consequences on water supply. To assess direct and indirect downstream effects of buildings, farms, pastures and fields on potential water sources, we studied five Swedish lakes and their inflows. We analysed a total of 160 samples in a gradient of anthropogenic activity spanning four time points, including faecal and water-quality indicators. Through species distribution modelling, Random Forest and network analysis using 16S rRNA amplicon sequencing data, our findings highlight that land use indirectly impacts lakes via inflows. Land use impacted approximately one third of inflow microbiota taxa, in turn impacting ∌20–50 % of lake taxa. Indirect effects via inflows were also suggested by causal links between e.g. water colour and lake bacterial taxa, where this influenced the abundance of several freshwater bacteria, such as Polynucleobacter and Limnohabitans. However, it was not possible to identify direct effects on the lakes based on analysis of physiochemical- or microbial parameters. To avoid potential downstream consequences on water supply, it is thus important to consider possible indirect effects from upstream land use and inflows, even when no direct effects can be observed on lakes. Legionella (a genus containing bacterial pathogens) illustrated potential consequences, since the genus was particularly abundant in inflows and was shown to increase by the presence of pastures, fields, and farms. The approach presented here could be used to assess the suitability of lakes as alternative raw water sources or help to mitigate contaminations in important water catchments. Continued broad investigations of stressors on the microbial network can identify indirect effects, avoid enrichment of pathogens, and help secure water accessibility.

Inbreeding depression (ID) is a fundamental selective pressure that shapes mating systems and population genetic structures in plants. Although it has been shown that ID varies over the life stages of shorter-lived plants, less is known about how the fitness effects of inbreeding vary across life stages in long-lived species. We conducted a literature survey in the Pinaceae, a tree family known to harbour some of the highest mutational loads ever reported. Using a meta-regression model, we investigated distributions of inbreeding depression over life stages, adjusting for effects of inbreeding levels and the genetic differentiation of populations within species. The final dataset contained 147 estimates of ID across life stages from 41 studies. 44 Fst estimates were collected from 40 peer-reviewed studies for the 18 species to aid genetic differentiation modelling. Partitioning species into fragmented and well-connected groups using Fst resulted in the best way (i.e. trade-off between high goodness-of-fit of the model to the data and reduced model complexity) to incorporate genetic connectivity in the meta-regression analysis. Inclusion of a life stage term and its interaction with the inbreeding coefficient (F) dramatically increased model precision. We observed that the correlation between ID and F was significant at the earliest life stage. Although partitioning of species populations into fragmented and well-connected groups explained little of the between-study heterogeneity, the inclusion of an interaction between life stage and population differentiation revealed that populations with fragmented distributions suffered lower inbreeding depression at early embryonic stages than species with well-connected populations. There was no evidence for increased ID in late life stages in well-connected populations, although ID tended to increase across life stages in the fragmented group. These findings suggest that life stage data should be included in inbreeding depression studies and that inbreeding needs to be managed over life stages in commercial populations of long-lived plants.

Predation-resistance has been suggested to be a key for persistence of pathogenic bacteria in aquatic environments. Little is known about driving factors for different types of protozoa resistant bacteria (PRB). We studied if presence of PRB is linked to specific plankton taxa, the aquatic nutrient state, or predation pressure on bacteria. Nineteen freshwater systems were sampled and analyzed for PRB, plankton composition and physicochemical variables. Three PRB genera were identified; Pseudomonas, Mycobacterium and Rickettsia. Use of minimum entropy decomposition algorithm and phylogenetic analysis showed that different nodes (representing OTUs of high taxonomic resolution) matched to environmental isolates of the three genera. Links between the PRB genera and specific plankton taxa were found, but showed different relationships depending on if 18S rRNA OTU or microscopy data were used in the analysis. Mycobacterium spp. was negatively correlated to aquatic nutrient state, while Pseudomonas showed the opposite pattern. Rickettsia spp. was positively related to predation pressure on bacteria. Both Mycobacterium and Rickettsia were more abundant in systems with high eukaryotic diversity, while Pseudomonas occurred abundantly in waters with low prokaryotic diversity. The different drivers may be explained by varying ecological strategies, where Mycobacterium and Rickettsia are slow growing and have an intracellular life style, while Pseudomonas is fast growing and opportunistic. Here we give an insight to the possibilities of newly advanced methods such as sequencing and oligotyping to link potential pathogens with biomarkers. This as a tool to assist predictions of the occurrence and persistence of environmental pathogens.

Earlier studies of the diet of frogs indicate that most adult frogs are mainly insectivorous. Overall, frogs are viewed more as generalists than specialists in terms of their diet. However, despite earlier studies, there are still gaps in our knowledge regarding what frogs tend to eat and the degree of specialization. The aim of this study was to investigate the diet choice of frogs in a tropical ecosystem. The present study was conducted in a well-known hotspot for frogs with 66 of the 156 known frog species in Borneo found in a protected area comprising of primary rainforest.

Frogs were caught in the field and their stomachs were flushed. The stomach content was retrieved, sorted to prey categories, and the diet analysed. In addition, the frogs were identified to species level. The frogs belonged to five families: Bufonidae, Dicroglossidae, Megophryidae, Microhylidae and Ranidae. My results show that the most common food source was ants, which constituted 63.7 % of the total food for all studied frog families. Termites, beetles and spiders made up 11.7 %, 4.2 % and 2.8 % of the total prey, respectively. The results from the analysis of Shannon’s diversity index supported two diet specialist families, the Bufonidae and Megophridae, which had a significantly lower mean diversity index compared to the generalist Dicroglossidae. To better reveal differences in frog’s diet in this ecosystem, further studies using larger sample size are needed.

Intrinsically disordered proteins (IDPs) do not have a well-defined and stable 3-dimensional fold. Some IDPs can function as either transient or permanent binders of other proteins and may interact with an array of ligands by adopting different conformations. A novel outer membrane lipoprotein, bacterial interleukin receptor I (BilRI) of the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans binds a key gatekeeper proinflammatory cytokine interleukin (IL)-1β. Because the amino acid sequence of the novel lipoprotein resembles that of fibrinogen binder A of Haemophilus ducreyi, BilRI could have the potential to bind other proteins, such as host matrix proteins. However, from the tested host matrix proteins, BilRI interacted with neither collagen nor fibrinogen. Instead, the recombinant non-lipidated BilRI, which was intrinsically disordered, bound various pro/anti-inflammatory cytokines, such as IL-8, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-10. Moreover, BilRI played a role in the in vitro sensing of IL-1β and IL-8 because low concentrations of cytokines did not decrease the amount of extracellular DNA in the matrix of bilRI− mutant biofilm as they did in the matrix of wild-type biofilm when the biofilms were exposed to recombinant cytokines for 22 hours. BilRI played a role in the internalization of IL-1β in the gingival model system but did not affect either IL-8 or IL-6 uptake. However, bilRI deletion did not entirely prevent IL-1β internalization, and the binding of cytokines to BilRI was relatively weak. Thus, BilRI might sequester cytokines on the surface of A. actinomycetemcomitans to facilitate the internalization process in low local cytokine concentrations.

Urinary bladder cancer (UBC) is one of the most frequent cancer diseases with 380 000 new cases diagnosed worldwide and about 150 000 deaths yearly. To dissect the role of T helper (Th) cell responses in UBC we investigate the T helper cell subpopulations; Th1, Th2, Th17 and T regulatory cells (Tregs) and their lineage commitment in draining (sentinel) and non-draining lymph nodes and blood from patients subjected to transurethral resection of the bladder (TUR-B) and/or Cystectomy. By analyzing methylation in signature genes IFNG, IL13, IL17a and FOXP3 we measure the epigenetic stability of these T helper cells.

In most patients IFNG is more demethylated in sentinel nodes compared to non-sentinel nodes and blood, suggesting a Th1 activation in nodes in contact with the tumor. Aside from that, the distribution of subpopulations in all tissues investigated is highly variable in between patients. All subsets are represented, although there seem to be no or little Th17 cells in nodes. After neoadjuvant treatment (given in between the TUR-B and cystectomy) a temporary increase in methylation of IFNG locus is seen in blood, which could suggest a translocation of activated Th cells from the blood to the tumor area, but also de novo synthesis of Th cells.

By analyzing the intra-patient variations in distribution and relative amount of Th cell subpopulations in blood and sentinel nodes we hope to draw conclusions on differences in outcome. The long-term goal is to be able to identify which patients could respond well to immune modulatory treatments.

Background: Cellulose, a 1,4 beta-glucan polysaccharide, is produced by a variety of organisms including bacteria. Although the production of cellulose has a high biological, ecological and economical impact, regulatory mechanisms of cellulose biosynthesis are mostly unknown. Family eight cellulases are regularly associated with cellulose biosynthesis operons in bacteria; however, their function is poorly characterized. In this study, we analysed the role of the cellulase BcsZ encoded by the bcsABZC cellulose biosynthesis operon of Salmonella enterica serovar Typhimurium (S. Typhimurium) in biofilm related behavior. We also investigated the involvement of BcsZ in pathogenesis of S. Typhimurium including a murine typhoid fever infection model. Result: In S. Typhimurium, cellulase BcsZ with a putative periplasmic location negatively regulates cellulose biosynthesis. Moreover, as assessed with a non-polar mutant, BcsZ affects cellulose-associated phenotypes such as the rdar biofilm morphotype, cell clumping, biofilm formation, pellicle formation and flagella-dependent motility. Strikingly, although upregulation of cellulose biosynthesis was not observed on agar plate medium at 37 degrees C, BcsZ is required for efficient pathogen-host interaction. Key virulence phenotypes of S. Typhimurium such as invasion of epithelial cells and proliferation in macrophages were positively regulated by BcsZ. Further on, a bcsZ mutant was outcompeted by the wild type in organ colonization in the murine typhoid fever infection model. Selected phenotypes were relieved upon deletion of the cellulose synthase BcsA and/or the central biofilm activator CsgD. Conclusion: Although the protein scaffold has an additional physiological role, our findings indicate that the catalytic activity of BcsZ effectively downregulates CsgD activated cellulose biosynthesis. Repression of cellulose production by BcsZ subsequently enables Salmonella to efficiently colonize the host.

BACKGROUND: Obesity is a complex disease caused by the interplay of genetic and lifestyle factors, but identification of gene-lifestyle interactions in obesity has remained challenging. Few large-scale studies have reported use of genome-wide approaches to investigate gene-lifestyle interactions in obesity. METHODS: In the Pakistan Risk of Myocardial Infraction Study, a cross-sectional study based in Pakistan, we calculated body mass index (BMI) variance estimates (square of the residual of inverse-normal transformed BMI z-score) in 14 131 participants and conducted genome-wide heterogeneity of variance analyses (GWHVA) for this outcome. All analyses were adjusted for age, age(2), sex and genetic ancestry. RESULTS: The GWHVA analyses identified an intronic variant, rs140133294, in the FLJ33544 gene in association with BMI variance (P-value = 3.1 x 10(-8)). In explicit tests of gene x lifestyle interaction, smoking was found to significantly modify the effect of rs140133294 on BMI (Pinteraction = 0.0005), whereby the minor allele (T) was associated with lower BMI in current smokers, while positively associated with BMI in never smokers. Analyses of ENCODE data at the FLJ33534 locus revealed features indicative of open chromatin and high confidence DNA-binding motifs for several transcription factors, providing suggestive biological support for a mechanism of interaction. CONCLUSIONS: In summary, we have identified a novel interaction between smoking and variation at the FLJ33534 locus in relation to BMI in people from Pakistan.

Many organisms studied by evolutionary biologists have different sexes, and the evolution of separate sexes and sexual dimorphisms in morphology and behaviour are central questions in evolutionary biology. Considering scientists to be embedded in a social and cultural context, we are also subjected to the risk of gender-biased assumptions and stereotypical thinking to appear when working on topics related to sexual reproduction and sexual dimorphism. Here we present, for continued discussion, a set of good-practice guidelines aimed at (1) helping to improve researchers’ awareness of gender-biased assumptions underlying language use, generalizations, and interpretation of observations; and (2) providing recommendations to increase transparency, avoid problematic terminology, and improve study designs.

Background FtsH is an ATP-dependent membrane-bound metalloprotease. A. thaliana contains 12 FtsH proteases localized in membranes of chloroplasts and mitochondria where they form homo- or hetero-hexameric complexes. FtsH11 – the main subject of this thesis – is located in the chloroplast envelope.

Methods

• Field studies with A. thaliana to determine Darwinian fitness. A growth under outdoor conditions often allows discovering of phenotypes that are unascertainable in the controlled environment of growth chambers.
• Proteomic methods to discover fragments of substrate proteins (limited proteolysis) and changes in the proteome of FtsH protease deficient mutants.

Results ftsh11 has increased amount of: RuBisCO activase, several Calvin cycle enzymes, two enzymes involved in starch synthesis and some chaperons. Some of those enzymes have been identified as possible substrates of FtsH11. Under long photoperiods ftsh11 develops a chlorotic phenotype accompanied by decreasing NADP⁺/NADPH ratio and increase of ROS damaged proteins.