Comparative leaf anatomy and micromorphology of the Chilean Myrtaceae: Taxonomic and ecological implications

The family Myrtaceae in Chile comprises 26 species in 10 genera. The species occur in a diverse rangeof environments including humid temperate forests, swamps, riparian habitats and coastal xeromorphicshrublands. Most of these species are either endemic to Chile or endemic to the humid temperate forestsof Chile and Argentina. Although many taxa have very restricted distributions and are of conservationconcern, little is known about their biology and vegetative anatomy. In this investigation, we describe andcompare the leaf anatomy and micromorphology of all Chilean Myrtaceae using standard protocols forlight and scanning electron microscopy. Leaf characters described here are related to epidermis, cuticle,papillae, stomata, hairs, mesophyll, crystals, secretory cavities and vascular system. Nearly all the specieshave a typical mesophytic leaf anatomy, but some species possess xerophytic characters such as doubleepidermis, hypodermis, pubescent leaves, thick adaxial epidermis and straight epidermal anticlinal walls,which correlate with the ecological distribution of the species. This is the first report on leaf anatomyand micromorphology in most of these species. We identified several leaf characters with potential tax-onomic and ecological significance. Some combinations of leaf characters can reliably delimitate genera,while others are unique to some species. An identification key using micromorphological and anatomicalcharacters is provided to distinguish genera and species.

Estimation of gear tooth deflection by the finite element method

The finite element method (FEM) is used to determine for pitch-point, mid-point and tip loading, the deflection curve of a Image 1 diamentral pitch (DP) standard spur gear tooth corresponding to number of teeth of 14, 21, 26 and 34. In all these cases the deflection of the gear tooth at the point of loading obtained by FEM is in good agreement with the experimental value. The contraflexure in the deflection curve at the point of loading observed experimentally in the cases of pitch-point and mid-point loading, is predicted correctly by the FEM analysis.

Characterisation of the efficacy of endodontic medications using a three-dimensional fluorescent tooth model: An ex vivo study

The purpose of this study was to establish a three-dimensional fluorescent tooth model to investigate bacterial viability against intra-canal medicaments across the thickness and surface of root dentine. Dental microbial biofilms (Enterococcus faecalis and Streptococcus mutans) were established on the external root surface and bacterial kill was monitored over time against intra-canal medicament (Ca(OH)2 ) using fluorescent microscopy in conjunction with BacLight SYTO9 and propidium iodide stains. An Olympus digital camera fitted to SZX16 fluorescent microscope captured images of bacterial cells in biofilms on the external root surface. Viability of biofilm was measured by calculating the total pixel area of green (viable bacteria) and red (non-viable bacteria) for each image using ImageJ® software. All data generated were assessed for normality and then analysed using a Mann-Whitney t-test. The viability of S. mutans biofilm following Ca(OH)2 treatment showed a significant decline compared with the untreated group (P = 0.0418). No significant difference was seen for E. faecalis biofilm between the Ca(OH)2 and untreated groups indicating Ca(OH)2 medicament is ineffective against E. faecalis biofilm. This novel three-dimensional fluorescent biofilm model provides a new clinically relevant tool for testing of medicaments against dental biofilms.

Characterisation of wood properties and transverse anatomy for vacuum drying modelling of commercially important Australian hardwood species.

Characterisation and investigation of a number of key wood properties, critical for further modelling work, has been achieved. The key results were: • Morphological characterisation, in terms of fibre cell wall thickness and porosity, was completed. A clear difference in fibre porosity, size, wall thickness and orientation was evident between species. Results were consistent with published data for other species. • Viscoelastic properties of wood were shown to differ greatly between species and in the radial and tangential directions, largely due to anatomical and chemical variations. Consistent with published data, the radial direction shows higher stiffness, internal friction and glass transition temperature than the tangential directions. The loss of stiffness over the measured temperature range was greater in the tangential direction than the radial direction. Due to time dependant molecular relaxation, the storage modulus and glass transition temperature decreased with decreasing test frequency, approaching an asymptotic limit. Thus the viscoelastic properties measured at lower frequencies are more representative of static material. • Dynamic interactions between relative humidity, moisture content and shrinkage of four Australian hardwood timbers can be accurately monitored on micro-samples using a specialised experimental device developed by AgroParisTech – ENGREF. The device generated shrinkage data that varied between species but were consistent (repeatable) within a species. Collapse shrinkage was clearly evident with this method for Eucalyptus obliqua, but not with other species, consistent with industrial seasoning experience. To characterise the wood-water relations of this species, free of collapse, thinner sample sections (in the R-T plane) should be used.

Adventures in plant anatomy – not just a pigment of my imagination

An overview of teaching and research activities in the area of plant anatomy at QUT is provided. The current status of teaching of technical skills in plant anatomy is discussed briefly. Examples of applications of plant anatomy to a diverse range of fields are provided, including the crossover between art and science.

The function of Bmps and Runx2 in normal tooth development and in the pathogenesis of cleidocranial dysplasia

The development of many embryonic organs is regulated by reciprocal and sequential epithelial-mesenchymal interactions. These interactions are mediated by conserved signaling pathways that are reiteratively used. Cleidocranial dysplasia (CCD) is a congenital syndrome where both bone and tooth development is affected. The syndrome is characterized by short stature, abnormal clavicles, general bone dysplasia, and supernumerary teeth. CCD is caused by mutations in RUNX2, a transcription factor that is a key regulator of osteoblast differentiation and bone formation. The first aim of this study was to analyse the expression of a family of key signal molecules, Bone morphogenetic protein (Bmp) at different stages of tooth development. Bmps have a variety of functions and they were originally discovered as signals inducing ectopic bone formation. We performed a comparative in situ hybridisation analysis of the mRNA expression of Bmp2-7 from initiation of tooth development to differentiation of dental hard tissues. The expression patterns indicated that the Bmps signal between the epithelial and mesenchymal tissues during initiation and morphogenesis of tooth development, as well as during the differentiation of odontoblasts and ameloblasts. Furthermore, they are also part of the signalling networks whereby the enamel knot regulates the patterning of tooth cusps. The second aim was to study the role of Runx2 during tooth development and thereby to gain better understanding of the pathogenesis of the tooth phenotype in CCD. We analysed the tooth phenotype of Runx2 knockout mice and examined the patterns and regulation of Runx2 gene expression.. The teeth of wild-type and Runx2 mutant mice were compared by several methods including in situ hybridisation, tissue culture, bead implantation experiments, and epithelial-mesenchymal recombination studies. Phenotypic analysis of Runx2 -/- mutant tooth development showed that teeth failed to advance beyond the bud stage. Runx2 expression was restricted to dental mesenchyme between the bud and early bell stages of tooth development and it was regulated by epithelial signals, in particular Fgfs. We searched for downstream targets of Runx2 by comparative in situ hybridisation analysis. The expression of Fgf3 was downregulated in the mesenchyme of Runx2 -/- teeth. Shh expression was absent from the enamel knot in the lower molars of Runx2 -/- and reduced in the upper molars. In conclusion, these studies showed that Runx2 regulates key epithelial-mesenchymal interactions that control advancing tooth morphogenesis and histodifferentiation of the epithelial enamel organ. In addition, in the upper molars of Runx2 mutants extra buddings occured at the palatal side of the tooth bud. We suggest that Runx2 acts as an inhibitor of successional tooth formation by preventing advancing development of the buds. Accordingly, we propose that RUNX2 haploinsuffiency in humans causes incomplete inhibition of successional tooth formation and as a result supernumerary teeth.

Three-dimensional corner reflector of arbitrary apex angle

A method of analysing a 3-dimensional corner reflector antenna of arbitrary apex angle is given. Expressions have been obtained for the far field of the 3-dimensional corner reflector fed by a dipole. The radiation resistance and the directive gain of the antenna have been calculated. The method described is applicable even when the feed dipole is arbitrarily oriented. It is found that the radiation along a prescribed direction can be circularly polarised (right or left) by suitably orienting the feed dipole.

Drought adaptation of stay-green sorghum is associated with canopy development, leaf anatomy, root growth, and water uptake

Stay-green sorghum plants exhibit greener leaves and stems during the grain-filling period under water-limited conditions compared with their senescent counterparts, resulting in increased grain yield, grain mass, and lodging resistance. Stay-green has been mapped to a number of key chromosomal regions, including Stg1, Stg2, Stg3, and Stg4, but the functions of these individual quantitative trait loci (QTLs) remain unclear. The objective of this study was to show how positive effects of Stg QTLs on grain yield under drought can be explained as emergent consequences of their effects on temporal and spatial water-use patterns that result from changes in leaf-area dynamics. A set of four Stg near-isogenic lines (NILs) and their recurrent parent were grown in a range of field and semicontrolled experiments in southeast Queensland, Australia. These studies showed that the four Stg QTLs regulate canopy size by: (1) reducing tillering via increased size of lower leaves, (2) constraining the size of the upper leaves; and (3) in some cases, decreasing the number of leaves per culm. In addition, they variously affect leaf anatomy and root growth. The multiple pathways by which Stg QTLs modulate canopy development can result in considerable developmental plasticity. The reduction in canopy size associated with Stg QTLs reduced pre-flowering water demand, thereby increasing water availability during grain filling and, ultimately, grain yield. The generic physiological mechanisms underlying the stay-green trait suggest that similar Stg QTLs could enhance post-anthesis drought adaptation in other major cereals such as maize, wheat, and rice.

Three-dimensional corner reflector of arbitrary apex angle

A method of analysing a 3-dimensional corner reflector antenna of arbitrary apex angle is given. Expressions have been obtained for the far field of the 3-dimensional corner reflector fed by a dipole. The radiation resistance and the directive gain of the antenna have been calculated. The method described is applicable even when the feed dipole is arbitrarily oriented. It is found that the radiation along a prescribed direction can be circularly polarised (right or left) by suitably orienting the feed dipole.

Characterisation of wood–water relationships and transverse anatomy and their relationship to drying degrade

Characterisation of a number of key wood properties utilising ‘state of the art’ tools was achieved for four commercial Australian hardwood species: Corymbia citriodora, Eucalyptus pilularis, Eucalyptus marginata and Eucalyptus obliqua. The wood properties were measured for input into microscopic (cellular level) and macroscopic (board level) vacuum drying models currently under development. Morphological characterisation was completed using a combination of ESEM, optical microscopy and a custom vector-based image analysis software. A clear difference in wood porosity, size, wall thickness and orientation was evident between species. Wood porosity was measured using a combination of fibre and vessel porosity. A highly sensitive microbalance and scanning laser micrometres were used to measure loss of moisture content in conjunction with directional shrinkage on micro-samples of E. obliqua to investigate the validity of measuring collapse-free shrinkage in very thin sections. Collapse-free shrinkage was characterised, and collapse propensity was verified when testing thicker samples. Desorption isotherms were calculated for each species using wood–water relations data generated from shrinkage experiments. Fibre geometry and wood shrinkage anisotropy were used to explain the observed difficulty in drying of the different species in terms of collapse and drying stress-related degrade.

Time to rest : Signals in shoot apex developmental transitions underlying dormancy

Vuodenajat rytmittävät monivuotisten kasvien elämää pohjoisella pallonpuoliskolla, jolla varmin merkki lähestyvästä talvikaudesta on asteittain lyhenevä päivänpituus. Kun päivänpituus on lyhentynyt tiettyyn raja-arvoon saakka, kasvu hiipuu ja kasvin kehityksessä tapahtuu suuria muutoksia. Väitöskirjatyössäni tutkittiin mekanismeja, jotka liittyvät pituuskasvun päättymiseen, silmujen lepotilan kehittymiseen ja kärkisilmun muodostumiseen hybridihaavan ja koivuntaimilla lyhyen päivänpituuden seurauksena kasvihuoneolosuhteissa. Vain lepotilaiset silmut selviytyvät luonnossa ankaran talvikauden yli, joten etenkin lepotilan kehittymisen tutkiminen on keskeistä pyrittäessä selvittämään monivuotisille kasveille tyypillisen kasvutavan mekanismeja. Jo pitkään on tiedetty, että täysikasvuiset lehdet vastaanottavat tiedon päivänpituudesta ja lähettävät signaaleja varren johtojänteissä ylöspäin kohti kasvin kärkiosaa. Sen sijaan varren kärjen ja kärkikasvupisteen roolia lepotilan kehittymisessä on selvitetty vain vähän. Kuitenkin juuri kärkikasvupisteen selviytyminen vuodesta toiseen on tärkeää, koska sen jakautumiskykyiset solukot tuottavat kasvin maanpäälliset osat. Tässä työssä tehdyissä varttamiskokeissa osoitettiin, että varren kärki ei ainoastaan vastaanota signaaleja lehdistä ja ajoita toimintaansa niiden mukaan, vaan myös kärjellä itsellään on aktiivinen rooli lepotilan kehittymisessä. Erityisesti kiinnitettiin huomiota kärkikasvupisteen eri alueiden, ns. apikaalimeristeemin ja rib-meristeemin erilaisiin tehtäviin ja pääteltiin, että molemmat vaikuttavat lepotilan kehittymiseen. Kokeissa käytettiin normaalien hybridihaapojen lisäksi siirtogeenisiä hybridihaapoja, jotka eivät lopeta kasvuaan lyhyt päivä –olosuhteissa. Siirtogeeniset hybridihaavat ilmensivät voimakkaasti fytokromi A -nimistä valon vastaanottajamolekyyliä rib-meristeemin alueella, mikä saattoi osaltaan vaikuttaa poikkeavaan pituuskasvukäyttäytymiseen. Myös useiden lepotilan kehittymiseen liittyvien geenien ilmenemisessä havaittiin poikkeavuuksia verrattuna ei-siirtogeenisiin kontrolleihin, joiden silmuissa kehittyi lepotila lyhyt päivä –altistuksen seurauksena. Väitöskirjatyössäni havaittiin, että myös kaasumainen kasvihormoni etyleeni toimii viestinvälittäjänä silmujen lepotilan kehittymisessä ja vaikuttaa etenkin lepotilan oikeaan ajoittumiseen. Etyleenillä huomattiin olevan määräävä rooli päätesilmun muodostumisessa: siirtogeeniset koivut, jotka eivät aisti etyleeniä, eivät muodostaneet päätesilmua. Silti siirtogeeniset koivut vaipuivat lepotilaan, joskin myöhemmin kuin ei-siirtogeeniset kontrollit. Tämän perusteella todettiin, että lepotilan ja päätesilmun kehittyminen ovat erillisiä kehitystapahtumia, vaikka ne saattavatkin ajoittua osaksi päällekkäin.

Molecular genetics of tooth agenesis

Congenital missing of teeth, tooth agenesis or hypodontia, is one of the most common developmental anomalies in man. The common forms in which one or a few teeth are absent, may cause occlusal or cosmetic harm, while severe forms which are relatively rare always require clinical attention to support and maintain the dental function. Observation of tooth agenesis is also important for diagnosis of malformation syndromes. Some external factors may cause developmental defects and agenesis in dentition. However, the role of inheritance in the etiology of tooth agenesis is well established by twin and family studies. Studies on familial tooth agenesis as well as mouse null mutants have also identified several genetic factors. However, these explain syndromic or rare dominant forms of tooth agenesis, whereas the genes and defects responsible for the majority of cases of tooth agenesis, especially the common and less severe forms, are largely unknown. In this study it was shown, that a dominant nonsense mutation in PAX9 was responsible for severe tooth agenesis (oligodontia) in a Finnish family. In a study of tooth agenesis associated with Wolf-Hirschhorn syndrome, it was shown that severe tooth agenesis was present if the causative deletion in 4p spanned the MSX1 locus. It was concluded that severe tooth agenesis was caused by haploinsufficiency of these transcription factors. A summary of the phenotypes associated with known defects in MSX1 and PAX9 showed that, despite similarities, they were significantly different, suggesting that the genes, in addition to known interactions, also have independent roles during the development of human dentition. The original aim of this work was to identify gene defects that underlie the common incisor and premolar hypodontia. After excluding several candidate genes, a genome-wide search was conducted in seven Finnish families in which this phenotype was inherited in an autosomal dominant manner. A promising locus for second premolar agenesis was identified in chromosome 18 in one family and this finding was supported by results from other families. The results also implied the existence of other loci both for second premolar agenesis and for incisor agenesis. On the other hand the results did not lend support for comprehensive involvement of the most obvious candidate genes in the etiology of incisor and premolar hypodontia. Rather, they suggest remarkable genetic heterogeneity of tooth agenesis. The available evidence suggests that quantitative defects during tooth development predispose to a failure to overcome a developmental threshold and to agenesis. The results of the study increase the understanding of the etiology and heredity of tooth agenesis. Further studies may lead to identification of novel genes that affect the development of teeth.

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.