Polypore assemblages in boreal old-growth forests, and associated Coleoptera

This thesis examines assemblages of wood-decaying fungi in Finnish old-growth forests, and patterns of species interactions between fruit bodies of wood-rotting Basidiomycetes and associated Coleoptera. The present work is a summary of four original publications and a manuscript, which are based on empirical observations and deal with the prevalence of polypores in old-growth forests, and fungicolous Coleoptera. The study area consists of eleven old-growth, mostly spruce- and pine-dominated, protected forests rich in dead wood in northern and southeastern Finland. Supplementary data on fungus beetle interactions were collected in southern Finland and the Åland Islands. 11251 observations of fruit bodies from 153 polypore species were made in 789 forest compartments. Almost a half of the polypore species demonstrated a distinct northern or southeastern trend of prevalence. Polypores with a northern prevalence profile were in extreme cases totally absent from the Southeast, although almost uniformly present in the North. These were Onnia leporina, Climacocystis borealis, Antrodiella pallasii, Skeletocutis chrysella, Oligoporus parvus, Skeletocutis lilacina, and Junghuhnia collabens. Species with higher prevalence in the southeastern sites were Bjerkandera adusta, Inonotus radiatus, Trichaptum pargamenum, Antrodia macra, and Phellinus punctatus. 198 (86%) species of Finnish polypores were examined for associated Coleoptera. Adult beetles were collected from polypore basidiocarps in the wild, while their larvae were reared to adulthood in the lab. Spatial and temporal parallels between the properties of polypore fruit body and the species composition of Coleoptera in fungus beetle interactions were discussed. New data on the biology of individual species of fungivorous Coleoptera were collected. 116 species (50% of Finnish polypore mycota) were found to host adults and/or larvae of 179 species from 20 Coleoptera families. Many new fungus beetle interactions were found among the 614 species pairs; these included 491 polypore fruit body adult Coleoptera species co-occurrences, and 122 fruit body larva interrelations. 82 (41%) polypore species were neither visited nor colonized by Coleoptera. The total number of polyporicolous beetles in Finland is expected to reach 300 species.

Maintaining plant species richness by cattle grazing - mesic semi-natural grasslands as focal habitats

Semi-natural grasslands are the most important agricultural areas for biodiversity. The present study investigates the effects of traditional livestock grazing and mowing on plant species richness, the main emphasis being on cattle grazing in mesic semi-natural grasslands. The two reviews provide a thorough assessment of the multifaceted impacts and importance of grazing and mowing management to plant species richness. It is emphasized that livestock grazing and mowing have partially compensated the suppression of major natural disturbances by humans and mitigated the negative effects of eutrophication. This hypothesis has important consequences for nature conservation: A large proportion of European species originally adapted to natural disturbances may be at present dependent on livestock grazing and / or mowing. Furthermore, grazing and mowing are key management methods to mitigate effects of nutrient-enrichment. The species composition and richness in old (continuously grazed), new (grazing restarting 3-8 years ago) and abandoned (over 10 years) pastures differed consistently across a range of spatial scales, and was intermediate in new pastures compared to old and abandoned pastures. In mesic grasslands most plant species were shown to benefit from cattle grazing. Indicator species of biologically valuable grasslands and rare species were more abundant in grazed than in abandoned grasslands. Steep S-SW-facing slopes are the most suitable sites for many grassland plants and should be prioritized in grassland restoration. The proportion of species trait groups benefiting from grazing was higher in mesic semi-natural grasslands than in dry and wet grasslands. Consequently, species trait responses to grazing and the effectiveness of the natural factors limiting plant growth may be intimately linked High plant species richness of traditionally mowed and grazed areas is explained by numerous factors which operate on different spatial scales. Particularly important for maintaining large scale plant species richness are evolutionary and mitigation factors. Grazing and mowing cause a shift towards the conditions that have occurred during the evolutionary history of European plant species by modifying key ecological factors (nutrients, pH and light). The results of this Dissertation suggest that restoration of semi-natural grasslands by private farmers is potentially a useful method to manage biodiversity in the agricultural landscape. However, the quality of management is commonly improper, particularly due to financial constraints. For enhanced success of restoration, management regulations in the agri-environment scheme need to be defined more explicitly and the scheme should be revised to encourage management of biodiversity.

Urban Growth Analysis Using Spatial and Temporal Data

Urban growth identification, quantification, knowledge of rate and the trends of growth would help in regional planning for better infrastructure provision in environmentally sound way. This requires analysis of spatial and temporal data, which help in quantifying the trends of growth on spatial scale. Emerging technologies such as Remote Sensing, Geographic Information System (GIS) along with Global Positioning System (GPS) help in this regard. Remote sensing aids in the collection of temporal data and GIS helps in spatial analysis. This paper focuses on the analysis of urban growth pattern in the form of either radial or linear sprawl along the Bangalore - Mysore highway. Various GIS base layers such as builtup areas along the highway, road network, village boundary etc. were generated using collateral data such as the Survey of India toposheet, etc. Further, this analysis was complemented with the computation of Shannon's entropy, which helped in identifying prevalent sprawl zone, rate of growth and in delineating potential sprawl locations. The computation Shannon's entropy helped in delineating regions with dispersed and compact growth. This study reveals that the Bangalore North and South taluks contributed mainly to the sprawl with 559% increase in built-up area over a period of 28 years and high degree of dispersion. The Mysore and Srirangapatna region showed 128% change in built-up area and a high potential for sprawl with slightly high dispersion. The degree of sprawl was found to be directly proportional to the distances from the cities.

Rice husk filtrate as a nutrient medium for the growth of Desulfotomaculum nigrificans: characterisation and sulfate reduction studies

The filtrate obtained by interacting a known amount of rice husk with deionised, Milli-Q water was assessed as a carbon source and nutrient medium for the growth of Desulfotomaculum nigrificans, a typical sulfate-reducing bacterium. The filtrate contained essential growth constituents such as magnesium, potassium, phosphorous apart from calcium, sodium, chloride and sulfate ions. Based on the 1H and 13C NMR characterization studies, the organic composition of the components dissolved from the rice husk, was found to be: (i) 66% lignocellulosic material, (ii) 24% xylose + arabinose and (iii) 10% galactose. The growth studies indicated a 15-fold increase in the bacterial cell number in about 20 days. Nearly 81% and 66% reduction in sulfate concentration could be achieved in about 28 days, from the solutions containing initial sulfate concentrations of 550 mg/l and 1200 mg/l respectively. In both the cases studied, the iron concentration could be reduced by over 85%.

Expression of Human Growth Hormone in Silkworm Larvae through RecombinantBombyx moriNuclear Polyhedrosis Virus

We have generated a recombinantBombyx morinuclear polyhedrosis virus, vBmhGH, harboring the full-length human growth hormone gene (2.4-kb genomic DNA, with four introns and the signal peptide sequences) under the control of the polyhedrin promoter. BmN cells in culture infected with the recombinant virus showed the presence of RNA corresponding to the authentic growth hormone mRNA as well as its incompletly processed precusor. Electrophoretic analysis and immunoprecipitation of proteins of recombinant virus-infected BmN cells revealed the presence of the growth hormone protein. Infection of silkworm larvae with vBmhGH led to the synthesis and efficient secretion of the protein into hemolymph. The recombinant human growth hormone was biologically active in a radioreceptor competition binding assay. The secreted protein was isolated and purified to homogeneity by a single step immunoaffinity chromatography, to a specific activity of 2.4 × 104U/mg. The recombinant hGH retained the immunological and biolological properties of the native peptide. We conclude that BmNPV vectors can be used successfully for expressing chromosomal genes harboring multiple introns.

Effect of oxygen partial pressure on the growth of zinc micro and nanostructures

Zinc micro and nanostructures were synthesized in vacuum by condensing evaporated zinc on Si substrate at different gas pressures. The morphology of the grown Zn structures was found to be dependent on the oxygen partial pressure. Depending on oxygen partial pressure it varied from two-dimensional microdisks to one-dimensional nanowire. The morphology and structural properties of the grown micro and nanostructures were studied by scanning electron microscopy (SEM) and X-ray diffraction (XRD). Transmission electron microscopy (TEM) studies on the grown Zn nanowires have shown that they exhibit core/shell-like structures, where a thin ZnO layer forms the shell. A possible growth mechanism behind the formation of different micro and nanostructures has been proposed. In addition, we have synthesized ZnO nanocanal-like structures by annealing Zn nanowires in vacuum at 350 °C for 30 min.

Absence of parallelism between polyamine and nucleic acid contents during induced growth of cucumber cotyledons

The cytokinins (benzyladenine or benzyladenosine) decreased spermidine and spermine contents despite increasing putrescine content, when administered to isolated cotyledons of Cucumis sativus L. var. Guntur in organ culture. KCl decreased putrescine contents, although marginally increasing polyamine contents. The cytokinins and/or KCl augmented nucleic acid biosynthesis and accumulation, resulting in enhanced growth and differentiation of the isolated cotyledons. These observations show that polyamine accumulation and growth are not always coupled.

Mechanisms and molecular regulation of mammalian tooth replacement

In most non-mammalian vertebrates, such as fish and reptiles, teeth are replaced continuously. However, tooth replacement in most mammals, including human, takes place only once and further renewal is apparently inhibited. It is not known how tooth replacement is genetically regulated, and little is known on the physiological mechanism and evolutionary reduction of tooth replacement in mammals. In this study I have attempted to address these questions. In a rare human condition cleidocranial dysplasia, caused by a mutation in a Runt domain transcription factor Runx2, tooth replacement is continued. Runx2 mutant mice were used to investigate the molecular mechanisms of Runx2 function. Microarray analysis from dissected embryonic day 14 Runx2 mutant and wild type dental mesenchymes revealed many downstream targets of Runx2, which were validated using in situ hybridization and tissue culture methods. Wnt signaling inhibitor Dkk1 was identified as a candidate target, and in tissue culture conditions it was shown that Dkk1 is induced by FGF4 and this induction is Runx2 dependent. These experiments demonstrated a connection between Runx2, FGF and Wnt signaling in tooth development and possibly also in tooth replacement. The role of Wnt signaling in tooth replacement was further investigated by using a transgenic mouse model where Wnt signaling mediator β-catenin is continuously stabilized in dental epithelium. This stabilization led to activated Wnt signaling and to the formation of multiple enamel knots. In vitro and transplantation experiments were performed to examine the process of extra tooth formation. We showed that new teeth were continuously generated and that new teeth form from pre-existing teeth. A morphodynamic activator-inhibitor model was used to simulate enamel knot formation. By increasing the intrinsic production rate of the activator (β-catenin), the multiple enamel knot phenotype was reproduced by computer simulations. It was thus concluded that β-catenin acts as an upstream activator of enamel knots, closely linking Wnt signaling to the regulation of tooth renewal. As mice do not normally replace teeth, we used other model animals to investigate the physiological and genetic mechanisms of tooth replacement. Sorex araneus, the common shrew was earlier reported to have non-functional tooth replacement in all antemolar tooth positions. We showed by histological and gene expression studies that there is tooth replacement only in one position, the premolar 4 and that the deciduous tooth is diminished in size and disappears during embryogenesis without becoming functional. The growth rates of deciduous and permanent premolar 4 were measured and it was shown by competence inference that the early initiation of the replacement tooth in relation to the developmental stage of the deciduous tooth led to the inhibition of deciduous tooth morphogenesis. It was concluded that the evolutionary loss of deciduous teeth may involve the early activation of replacement teeth, which in turn suppress their predecessors. Mustela putorius furo, the ferret, has a dentition that resembles that of the human as ferrets have teeth that belong to all four tooth families, and all the antemolar teeth are replaced once. To investigate the replacement mechanism, histological serial sections from different embryonic stages were analyzed. It was noticed that tooth replacement is a process which involves the growth and detachment of the dental lamina from the lingual cervical loop of the deciduous tooth. Detachment of the deciduous tooth leads to a free successional dental lamina, which grows deeper into the mesenchyme, and later buds the replacement tooth. A careful 3D analysis of serial histological sections was performed and it was shown that replacement teeth are initiated from the successional dental lamina and not from the epithelium of the deciduous tooth. The molecular regulation of tooth replacement was studied and it was shown by examination of expression patterns of candidate regulatory genes that BMP/Wnt inhibitor Sostdc1 was strongly expressed in the buccal aspect of the dental lamina, and in the intersection between the detaching deciduous tooth and the successional dental lamina, suggesting a role for Sostdc1 in the process of detachment. Shh was expressed in the enamel knot and in the inner enamel epithelium in both generations of teeth supporting the view that the morphogenesis of both generations of teeth is regulated by similar mechanisms. In summary, histological and molecular studies on different model animals and transgenic mouse models were used to investigate tooth replacement. This thesis work has significantly contributed to the knowledge on the physiological mechanisms and molecular regulation of tooth replacement and its evolutionary suppression in mammals.

Co-Signaling by Neurotrophic Factors and the Extracellular Matrix for Axonal Growth and Neuronal Survival

Neurotrophic factors (NTFs) and the extracellular matrix (ECM) are important regulators of axonal growth and neuronal survival in mammalian nervous system. Understanding of the mechanisms of this regulation is crucial for the development of posttraumatic therapies and drug intervention in the injured nervous system. NTFs act as soluble, target-derived extracellular regulatory molecules for a wide range of physiological functions including axonal guidance and the regulation of programmed cell death in the nervous system. The ECM determines cell adhesion and regulates multiple physiological functions via short range cell-matrix interactions. The present work focuses on the mechanisms of the action of NTFs and the ECM on axonal growth and survival of cultured sensory neurons from dorsal root ganglia (DRG). We first examined signaling mechanisms of the action of the glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) on axonal growth. GDNF, neurturin (NRTN) and artemin (ART) but not persephin (PSPN) promoted axonal initiation in cultured DRG neurons from young adult mice. This effect required Src family kinase (SFK) activity. In neurons from GFRalpha2-deficient mice, NRTN did not significantly promote axonal initiation. GDNF and NRTN induced extensive lamellipodia formation on neuronal somata and growth cones. This study suggested that GDNF, NRTN and ARTN may serve as stimulators of nerve regeneration under posttraumatic conditions. Consequently we studied the convergence of signaling pathways induced by NTFs and the ECM molecule laminin in the intracellular signaling network that regulates axonal growth. We demonstrated that co-stimulation of DRG neurons with NTFs (GDNF, NRTN or nerve growth factor (NGF)) and laminin leads to axonal growth that requires activation of SFKs. A different, SFK-independent signaling pathway evoked axonal growth on laminin in the absence of the NTFs. In contrast, axonal branching was regulated by SFKs both in the presence and in the absence of NGF. We proposed and experimentally verified a Boolean model of the signaling network triggered by NTFs and laminin. Our results put forward an approach for predictable, Boolean logics-driven pharmacological manipulation of a complex signaling network. Finally we found that N-syndecan, the receptor for the ECM component HB-GAM was required for the survival of neonatal sensory neurons in vitro. We demonstrated massive cell death of cultured DRG neurons from mice deficient in the N-syndecan gene as compared to wild type controls. Importantly, this cell death could not be prevented by NGF the neurotrophin which activates multiple anti-apoptotic cascades in DRG neurons. The survival deficit was observed during first postnatal week. By contrast, DRG neurons from young adult N-syndecan knock-out mice exhibited normal survival. This study identifies a completely new syndecan-dependent type of signaling that regulates cell death in neurons.

Growth factor-loaded microparticles for tissue engineering: The discrepancies of in vitro characterization assays

Efficient and effective growth factor (GF) delivery is an ongoing challenge for tissue regeneration therapies. The accurate quantification of complex molecules such as GFs, encapsulated in polymeric delivery devices, is equally critical and just as complex as achieving efficient delivery of active GFs. In this study, GFs relevant to bone tissue formation, vascular endothelial growth factor (VEGF) and bone morphogenetic protein 7 (BMP-7), were encapsulated, using the technique of electrospraying, into poly(lactic-co-glycolic acid) microparticles that contained poly(ethylene glycol) and trehalose to assist GF bioactivity. Typical quantification procedures, such as extraction and release assays using saline buffer, generated a significant degree of GF interactions, which impaired accurate assessment by enzyme-linked immunosorbent assay (ELISA). When both dry BMP-7 and VEGF were processed with chloroform, as is the case during the electrospraying process, reduced concentrations of the GFs were detected by ELISA; however, the biological effect on myoblast cells (C2C12) or endothelial cells (HUVECs) was unaffected. When electrosprayed particles containing BMP-7 were cultured with preosteoblasts (MC3T3-E1), significant cell differentiation into osteoblasts was observed up to 3 weeks in culture, as assessed by measuring alkaline phosphatase. In conclusion, this study showed how electrosprayed microparticles ensured efficient delivery of fully active GFs relevant to bone tissue engineering. Critically, it also highlights major discrepancies in quantifying GFs in polymeric microparticle systems when comparing ELISA with cell-based assays.

Sol-Gel Template Synthesis, Structural Characterization and Growth Mechanism of Barium Zirconate Nanotubes

In this paper, we report the synthesis of barium zirconate, BaZrO3, (BZ) nanotubes fabricated by the modified sol-gel method within the nanochannels of anodic aluminum oxide (AAO) templates. The morphology, structure, and composition of as prepared nanotubes were characterized by means of X-ray diffraction (XRD), field emission scanning electron microscope (FE-SEM), transmission electron microscope (TEM), selected-area electron diffraction ( SAED), high resolution TEM (HRTEM) and energy-dispersive X-ray spectroscopy (EDX). The results of XRD and SAED indicated that postannealed (at 650 degrees C for 1 h) BZ nanotubes (BZNTs) exhibited a polycrystalline cubic perovskite crystal structure. SEM and TEM analysis revealed that BZNTs possessed a uniform length and diameter (similar to 200 nm) and the thickness of the wall of the BZNTs was about 20 nm. Y-junctions, multiple branching and typical T-junctions were also observed in some BZNTs. EDX analysis demonstrated that stoichiometric BaZrO3 was formed. HRTEM image confirmed that the obtained BZNTs were composed of nanoparticles in the range of 5-10 nm. The possible formation mechanism of BZNTs was discussed.

Selective growth of ZnO nanoneedles by thermal oxidation of Zn microstructures

Zinc film containing hexagonal plate stack and tower-like micro structures were grown on Si substrates at high temperature by thermal evaporation. Thermal oxidation studies on these micro structures have shown that ZnO nanoneedles selectively grow from the facets of the zinc microstructure at temperature above 300 degrees C in atmosphere TEM analysis showed that single crystalline and bicrystalline nanoneedles were formed in this oxidation process and the growth direction of these nanoneedles was identified along the [1 1 (2) overbar 0]. Based on the structural studies and morphological observation, we have proposed a possible mechanism for the selective growth of ZnO nanoneedles during thermal oxidation.

Pituitary gigantism treated successfully with the growth hormone receptor antagonist, pegvisomant

A Caucasian male aged 15 years presented with 2 years accelerated linear growth. He was 202 cm tall at presentation, with calculated mid-parental height of 173 cm. There were no features of hypopituitarism or acral growth. His visual fields and optic discs were normal...