Dissertationen zum Thema „Biology“

Life-like behaviors such as fission, fusion and movement can be artificially re-created exploiting highly simplified protocell systems. This thesis is mainly focused on chemotaxis protocell systems and their integration with biological systems in order to show potential future applications. 1-Decanol droplets, formed in an aqueous medium containing decanoate at high pH, become chemotactic when a chemical gradient is placed in the external aqueous environment. We investigated the behavior of these droplets, their ability to transport and deposit living and non-living objects and to interface them with biofilms. To make the artificial system compatible with natural living systems we developed a partially hydrophobic alginate capsule as a protective unit that can be precisely embedded in a droplet, transported along chemical gradients and deposited. We developed a system that was able to transport: Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae. Both bacteria survived the transport. However, yeast survived but not in a consistent and repeatable way. Next, we evolved the system to transport human cell lines. We found that A549 cells survive encapsulation but not the transport. A549 cells are in fact very sensitive to toxic 1-decanol. We however found out that this cell line secretes compounds able to decrease the surface tension and to increase the capsule-droplet affinity. Finally we discuss future solutions for the effective transport of human cells.

Life-like behaviors such as fission, fusion and movement can be artificially re-created exploiting highly simplified protocell systems. This thesis is mainly focused on chemotaxis protocell systems and their integration with biological systems in order to show potential future applications. 1-Decanol droplets, formed in an aqueous medium containing decanoate at high pH, become chemotactic when a chemical gradient is placed in the external aqueous environment. We investigated the behavior of these droplets, their ability to transport and deposit living and non-living objects and to interface them with biofilms. To make the artificial system compatible with natural living systems we developed a partially hydrophobic alginate capsule as a protective unit that can be precisely embedded in a droplet, transported along chemical gradients and deposited. We developed a system that was able to transport: Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae. Both bacteria survived the transport. However, yeast survived but not in a consistent and repeatable way. Next, we evolved the system to transport human cell lines. We found that A549 cells survive encapsulation but not the transport. A549 cells are in fact very sensitive to toxic 1-decanol. We however found out that this cell line secretes compounds able to decrease the surface tension and to increase the capsule-droplet affinity. Finally we discuss future solutions for the effective transport of human cells.

Essential oils (EO) are plant secondary metabolites that are known for their fragrance and food flavour properties. They consist of a complex mixture of mono- and sesquiterpenes, phenyl propanoids and oxygenated compounds. EOs can be present in different plant organs and materials, and their storage is related to specialised secretory structures. The yield of EOs from plant raw materials by distillation or pressing may on average vary from 0.1 – 1%, thus restricting the major EO production to the plant group of aromatic plants. Due to their function as signalling compounds between different types of organisms and diverse biological systems, their general antimicrobial and antioxidative effects and medicinal activity, EOs offer a promising potential for future applications within the fields of agriculture, medicine, pharmaceutical industry and biotechnology.

Changed consumer demands and raised interest in natural product compounds, especially essential oils, have formed the basis for initiating the research project “Norwegian Herb Production (Norsk Urteproduksjon NUP)” to encourage the cultivation, processing, marketing and distribution of aromatic and medicinal plants. The production, composition and quality characteristics of EOs (yield and terpene composition) from chamomile, lemon balm, oregano, peppermint, sachalinmint, thyme and yarrow have been investigated in the project period between 1994-1998.

Much focus has been put on the application of solid-phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC-MS) for the analysis of EO volatiles from various aromatic and medicinal plants. SPME is a fast, solvent-free and non- destructive sample preparation technique where the analytes are extracted from fluid or solid matrices by headspace (HS) or direct immersion sampling (DI). Apart from EO isolation by common distillation, the applicability and sensitivity of the SPME fibre has made it feasible to carry out qualitative and semi-quantitative HS analyses of aromatic plants with regard to changes of EO metabolism during ontogenesis and plant development.

Based on NUP-results from field trials in the period between 1995-1996, the mint species peppermint (Mentha × piperita L.) and sachalinmint (Mentha sachalinensis (Briq.) Kudô) have been studied in detail (Papers B, D and E). Comparative analyses by applying distillation sampling and SPME have been carried out in order to study the advantages and disadvantages of both techniques (Papers B and E). It could be shown, that SPME offers a fast and reliable method for detecting quality-impact compounds from the p-menthane group (menthol, menthone, neomenthol, isomenthone and menthyl acetate). A distinct increase in the menthol/menthone ratio in the basipetal direction could be detected for peppermint and sachalinmint by applying SPME, thus revealing within-plant quality differences according to pharmacopeial requirements. Taking the increase of EO production from the vegetative to the generative growth stage into account, the harvest of mint plants in bloom will result in better EO yield and quality with regard to higher amounts of menthol.

When applying HS-SPME on complex EO volatile matrices such as known for yarrow (Achillea millefolium L.; Paper C), one might deal with fibre-partitioning effects of the different mono- and sesquiterpenes due to their physical and chemical properties. Despite these disadvantages, HS-SPME appears to be a sensitive extraction method for the screening of EO volatiles from complex sample matrices. Comparative analyses of volatiles from rose root rhizomes (Rhodiola rosea L.) have been carried out in order to characterize the rose-like odour compounds (Paper F). A total of 75 and 59 compounds have been identified by distillation sampling and HS-SPME, respectively, thus underscoring the excellent extraction properties and applicability of the SPME fibre.

Paper A gives a brief overview of EO biosynthesis and chemical structures, plant sources and methods of EO production. Before leading over to the main topic of HS-SPME applications by referring to numerous examples from the research work at The Plant Biocenter in the past 5 years, an introduction of solid-phase microextraction with regard to devices, procedures and extraction parameters is given.

The advantages and disadvantages of distillation vs. SPME are outlined on the background of comparative analyses of peppermint, chamomile, basil and dill. Furthermore, the utilization of HS-SPME for quantitative studies with regard to extraction time and analyte concentration is being highlighted. Examples for the screening of chemotypes (hops −Humulus lupulus L.) and cultivars (dill – Anethum graveolens L.) and ontogenetic studies are given (Mentha species; arnica −Arnica montana L.). Finally, the applicability of HS-SPME for the quality assessment of processed herbs (sweet basil −Ocimum basilicum L.) and phytomedicinal preparations (red coneflower – Echinacea purpurea L.) is being discussed.

The advantages of HS-SPME over classical distillation and headspace applications are impressive due to drastically reduced analysis time and will introduce new frontiers in plant volatile research with regard to secondary metabolism, plant-insect interactions and in vivo studies. The user-friendliness of operating SPME will initiate the development of future applications and equipment for the monitoring of volatiles for plant biological and environmental studies, extraction automation, on-site sampling and on-fibre storage of analytes.

Paper VI reprinted with kind permission of Elsevier, Sciencedirect, www.sciencedirect.com

The aims of this study were to investigate the effects of known environmental contaminants on defined behavioural variables in fish, and to discuss properties of these behavioural traits that make them useful as potential indicators of pollution.

In studying the effects of pollution, the resulting biochemical and physiological alterations are more commonly measured. However, effects of pollution can manifest itself at all levels of biological organisation, including behaviour. In this respect, behaviour can be considered a valid biomarker of pollution in that it is expected to be both susceptible to pollution and of high ecological significance, as it influences the fitness of the affected individuals.

This thesis is based on four individual studies, in which the threespine stickleback Gasterosteus aculeatus was used as a model species. Results from these studies show that antipredator behaviour, feeding behaviour, shoaling behaviour, bottom-dwelling behaviour and reproductive behaviour are all sensitive to exposure to sublethal concentrations of defined environmentally relevant chemicals.

The results showed that antipredator behaviour and fright response in threespine stickleback were impaired following exposure to sublethal concentrations of bis(tributyltin)oxide (TBTO). However, for some of the tested antipredator variables the effects were reversed after the ending of exposure. Further, it was shown that feeding motivation in fish exposed to butyl benzyl phthalate (BBP) and/or 2,2-bis(p-chlorophenyl)-1,1-dichloroethylene (DDE) was increased in that exposed fish initiated feeding more often than the controls. Exposure to BBP also caused sticklebacks to aggregate into tight shoals and to spend more time at the bottom of the aquarium compared to the control fish.

The reported significant differences between the controls and BBPexposed fish with respect to feeding and shoaling behaviour were shown even though the levels of BBP were below the analytical detection limit. Different suggested explanations, for example, too high detection limit, or degradation to its BBP metabolites are given to this result.

17β-Oestradiol (E₂) exposed male sticklebacks started nest building later than non-exposed males, but there were no differences between exposed and control males with respect to the number of males that built nests. Further, the exposed males spent less time displaying paternal care compared to the control males, although there were no differences between the two groups in the number of performed courtship displays. Because of the significant effect upon some but not all reproductive behavioural traits, it was suggested that the different variables might vary in sensitivity, implying that a variety of variables should be studied in order to obtain a more reliable evaluation of the effects of pollution.

Chemicals can cause deleterious effects at one or more levels of biological organisation, from biochemical, physiological, individual, population and through to the ecosystem levels. In contrast to the established hypothesis that a pollutant affects the different biological levels in an escalating timedependent pattern, starting at the biochemical level, it is here suggested that biomarkers at the biochemical, physiological and behavioural levels often will respond early and simultaneously in the same individual.

Whereas some biochemical responses are specifically related to one class of exposure agents and thus may act as specific indicators of pollution, most behavioural traits may be altered in response to a variety of chemicals. One exception may be alterations in reproductive behaviour caused by endocrine disrupting chemicals, due to effects of the chemicals on hormones that result in immediate reproductive behavioural effects. In spite of the specific action of some biochemical biomarkers, they are often considered to be of little ecological relevance since many of them are not related to individual fitness.

In this thesis, it is argued that behavioural variables can be employed as useful and reliable biomarkers of environmental contamination. It is also important to focus on behaviour to map and quantify the resposes. However, to reliably evaluate the effects of pollution, behavioural variables should be used in association with biochemical and physiological traits. Moreover, optimal combination of results from laboratory and field experiments would enhance the ecological relevance of the study.

All papers reprinted with kind permission of Elsevier, sciencedirect.com

This thesis deals with effects of climate on tree growth of the dominating conifer species, Picea abies (L) Karst. (Norway spruce) and Pinus sylvestris L (Scots pine), in central Norway and Fennoscandia. Both species are sampled along major environmental gradients, i.e. altitude and oceanicity, and growth responses to climate, i.e. temperature and precipitation, are examined along these gradients. Additionally, time is considered as an environmental gradient and temporal responses are carefully deciphered. Special attention is given to large-scale climate oscillation and their effect on tree growth. In the individual papers the specific aims have been to:

1. identify climate variables (all seasons) with significant influence on radial tree growth of P. abies and P. sylvestris along major environmental gradients (Paper I-IV)

2. identify if and how the growth response to climate has changed through time along these gradients (Paper I-IV)

3. make interregional comparisons of P. sylvestris growth pattern across Fennoscandia from oceanic western Norway to continental eastern Finland (Paper III)

4. analyse to what degree large-scale circulation patterns of air masses are registered in regional tree growth of both P. abies and P. sylvestris (Paper I, III and IV)

5. discuss possible effects on radial tree growth of a predicted warmer climate (Paper I, III and IV)

When the study of this thesis was initiated, hardly any work had been carried out on how plant odour information was encoded by the olfactory RNs in heliothine moths. The method of gas chromatography linked to single cell recordings (GC-SCR) was employed and improved for identifying naturally occurring plant odorants that are detected by single RNs and can be considered as biologically relevant. Three species of the subfamily Heliothinae were included in this work, the two polyphagous H. virescens and H. armigera and the oligophagous H. assulta. The American H. virescens is geographically separated from the other two species. H. armigera and H. assulta are partly sympatric in Asia and Australia.

The aims of the thesis elucidated in Papers I-IV were as follows:

1. To identify plant produced volatiles detected by antennal RNs in the three species of the subfamily Heliothinae.

2. To elucidate whether the single RNs can be classified into distinct types according to their specificity.

3. To characterise the plant odour RN types by their molecular receptive ranges, sensitivity and specificity.

4. To compare the specificity of plant odour RN types across the three related species of Heliothinae, with the aim to reveal any differences in the peripheral olfactory system that may have evolved through evolution.

The bacterial origin of mitochondria from an ancient endosymbiosis is now widely accepted and the mitochondrial ancestor is generally believed to belong to the bacterial subdivision α-proteobacteria. The high fraction of mitochondrial proteins encoded in the nucleus has commonly been explained with a massive transfer of genes from the genome of the ancestral mitochondrion.

The aim of this work was to get a better understanding of the mitochondrial origin and evolution by comparative genomics and phylogenetic analyses on mitochondria and α-proteobacteria. To this end, we sequenced the genomes of the intracellular parasites Bartonella henselae and Bartonella quintana, the causative agents of cat-scratch disease and trench fever, and compared them with other α-proteobacteria as well as mitochondrial eukaryotes.

Our results suggest that the adaptation to an intracellular life-style is coupled to an increased rate of genome degradation and a reduced ability to accommodate environmental changes. Reconstruction of the α-proteobacterial ancestor and phylogenetic analyses of the mitochondrial proteome in yeast revealed that only a small fraction of the proteins used for mitochondrial functions could be traced to the α-proteobacteria. Furthermore, a substantial fraction of the mitochondrial proteins was of eukaryotic origin and while most of the genes of the α-proteobacterial ancestor have been lost, many of those that have been transferred to the nuclear genome seem to encode non-mitochondrial proteins.

Symbiosis is a widely common phenomenon in nature and has undoubtedly contributed to the evolution of all organisms on earth. Symbiotic associations can be of varying character, such as parasitic or mutualistic, but all imply a close relationship. To study the evolution of genomes of insect endosymbionts, we have sequenced the genomes of the mutualist Buchnera aphidicola from the aphid Schizaphis graminum (Sg) and the reproductive manipulator Wolbachia pipientis strain wRi from Drosophila simulans that show strikingly different evolutionary patterns.

The comparison between the genome of B.aphidicola (Sg) and the genome of B.aphidicola from the aphid Acyrthosiphon pisum (Ap), that are believed to have diverged 50 million years ago, revealed a perfect gene order conservation and loss of only 14 genes in either of the lineages. In contrast, the rate of nucleotide turnover is very fast probably due to relaxed selection and loss of DNA repair genes. The genomic stasis observed in Buchnera was attributed to the loss of repeats and of the gene recA.

In striking contrast to the genomes of B.aphidicola, a vast amount of repeats were found in the genome sequence of W.pipientis strain wMel. The comparison between the genomes of W.pipientis strain wRi and W.pipientis strain wMel shows that a lot of rearrangements have occurred since their divergence. The massive amount of repeats might stem from relaxed selection pressure but possibly also from selection to create variability via recombination.

Comparisons between pairs of genomes from closely related bacteria showed that the stability of gene order and content is connected to an intracellular lifestyle and indicated that homologous recombination between repeats is an important mechanisms for causing intrachromosomal rearrangements. Our studies show that the lifestyle of a bacterium to a great extent shapes the evolution of their genetic material and future capabilities to adapt to new environments.

The tRNALeu (UAA) intron has been recorded in the plastid genome of many algae and land plants and was the first intron to be discovered in cyanobacteria. In all known cases it interrupts the tRNALeu anticodon loop at a conserved position (U-intron-AA). Cyanobacteria are a diverse group of photosynthetic prokaryotes, some involved in symbiotic associations with a wide range of organisms. The most studied associations are those with plants, where strains of Nostoc are the common cyanobacterial partner. In this thesis two aspects of the biology of the cyanobacterial tRNALeu (UAA) intron are focused: first, the use of the intron as a genetic marker for studying the diversity and specificity of two cyanobacterial symbiosis (bryophytes and cycads) and second, the evolutionary patterns of the intron by using the unique data set generated from the diversity analysis. From the studies, many different Nostoc strains are involved in the two symbiotic associations, although no variation was observed within a single bryophyte cavity or cycad coralloid root. Furthermore, a certain level of temporal stability in the cyanobiont composition of the bryophyte population was found and, in the cycad association different coralloid roots from a single specimen may harbor different cyanobacteria. That a minor cyanobiont could have avoided detection is still possible but unlikely. The sequence alignment of the Nostoc tRNALeu (UAA) introns reveals great sequence similarity with size variation only found in the structural element P6b. This element was found to consist of heptanucleotide repeats and of other non-repetitive genetic elements (NIS elements). The sporadic occurrence of the NIS elements indicates recent origins and a mechanism for its dispersal is proposed. In this thesis new insights are given concerning cyanobacterial symbioses and also on the mechanisms involved in the evolution of an old genetic element: the tRNALeu (UAA) intron in cyanobacteria.

Small GTP binding proteins function as molecular switches which cycles between GTP-bound ON and GDP-bound OFF states, and regulate a wide variety of cellular processes as biological timers. The first characterized member of the small GTPase family, the mutated oncogene p21 src, later known as Harvey-Ras, was identified in the early 1980s (Shih, T. Y. et al. 1980). In the following years small Ras-lik GTPases were found in several organisms and it was soon discovered that they took part in processes, such as signal transduction, gene expression, cytoskeleton reorganisation, microtubule organisation, and vesicular and nuclear transport. The first Rho (Ras homology) gene was cloned in 1985 from the sea slug Aplysia (Madaule, P. et al. 1985) and because of their homology to Ras it was first suspected that they could act as oncogenes. Later studies have shown that even though they participate in processes such as cell migration and motility they are not mutated in cancers.

The first indications that Rho was a signaling protein regulating the actin cytoskeleton, came from experiments where activated forms of human RhoA was microinjected into 3T3 cells (Paterson, H. F. et al. 1990). Another Rho-like GTPase Rac1 (named after Ras-related C3 botulinum toxin substrate) was later shown to regulate actin cytoskeletal dynamics as well, suggesting that Rho-family members cooperate in controlling these processes (Ridley, A. J. et al. 1992). The Rac GTPase was also implicated in regulating the phagocytic NADPH oxidase, which produce superoxide for killing phagocytized microorganisms (Abo, A. et al. 1991). Thus, it soon became clear that Rac/Rho and the related GTPase Cdc42 (cell division cycle 42) had central functions in many important cellular processes.

There are at least three types of regulators for Rho-like proteins. The GDP/GTP exchange factors (GEFs) which stimulates conversion from the GDPbound form to the GTP-bound form. GDP dissociation inhibitors (GDIs) decrease the nucleotide dissociation from the GTPase and retrieve them from membranes to the cytosol. GTPase activating proteins (GAPs) stimulates the intrinsic GTPase activity and GTP hydrolysis. In addition there are probably regulators that dissociate GDI from the GTPase leaving it open for activation by the RhoGEFs.

Ras and Rho-family proteins participate in a coordinated regulation of cellular processes such as cell motility, cell growth and division. The Ral GTPase is closely related to Ras and recent studies have shown that this GTPase is involved in crosstalk between both Ras and Rho proteins (Feig, L. A. et al. 1996; Oshiro, T. et al. 2002). Ral proteins are not found in plants and they appear to be restricted to animalia and probably yeast. During a screen for small GTPases in Drosophila melanogaster I discovered in 1993 several new members of the Ras-family, such as Drosophila Ral (DRal), Ric1 and Rap2. The functions of Ral GTPases in Drosophila have until recently been poorly known, but in paper 2 we present some of the new findings.

Rho-like GTPases have been identified in several eukaryotic organisms such as, yeast (Bender, A. et al. 1989), Dictyostelium discoideum (Bush, J. et al. 1993), plants (Yang, Z. et al. 1993), Entamoeba histolytica (Lohia, A. et al. 1993) and Trypanosoma cruzi (Nepomuceno-Silva, J. L. et al. 2001). In our first publication, (Winge, P. et al. 1997), we describe the cloning of cDNAs from RAC-like GTPases in Arabidopsis thaliana and show mRNA expressions pattern for five of the genes. The five genes analyzed were expressed in most plant tissues with the exception of AtRAC2 (named Arac2 in the paper), which has an expression restricted to vascular tissues. We also discuss the evolution and development of RAC genes in plants. The third publication, (Winge, P. et al. 2000), describe the genetic structure and the genomic sequence of 11 RAC genes from Arabidopsis thaliana. As most genomic sequences of the AtRACs we analyzed came from the Landsberg erecta ecotype and the Arabidopsis thaliana genome was sequenced from the Columbia ecotype, it was possible to compare the sequences and identify new polymorphisms. The genomic location of the AtRAC genes plus the revelation of large genomic duplications provided additional information regarding the evolution of the gene family in plants. A summary and discussion of these new findings are presented together with a general study of small Ras-like GTPases and their evolution in cellular organisms. This study suggests that the small GTPases in eukaryots evolved from two bacterial ancestors, a Rab-like and a MglA/Arp-like (Arf-like) protein. The MglA proteins (after the mgl locus in Myxococcus xanthus) are required for gliding motility, which is a type of movement that take place without help of flagella.

The second publication describes experiments done with the Drosophila melanogaster DRal gene and its effects on cell shape and development. Ectopic expression of dominant negative forms of DRal reveals developmental defects in eye facets and hairs, while constitutive activated forms affects dorsal closure, leaving embryos with an open dorsal phenotype. Results presented in this publication suggest that DRal act through the Jun N-terminal kinase (JNK) pathway to regulate dorsal closure, but recent findings may point to additional explanations as well. The results also indicate a close association between processes regulated by Rac/Rho and Ral proteins in Drosophila.

In the post-"omic" era the analysis of high-throughput data is regarded as one of the major challenges faced by researchers. One focus of this data analysis is uncovering biological network topologies and dynamics. It is believed that this kind of research will allow the development of new mathematical models of biological systems as well as aid in the improvement of already existing ones. The work that is presented in this dissertation addresses the problem of the analysis of highly complex data sets with the aim of developing a methodology that will enable the reconstruction of a biological network from time series data through an iterative process. The first part of this dissertation relates to the analysis of existing methodologies that aim at inferring network structures from experimental data. This spans the use of statistical tools such as correlations analysis (presented in Chapter 2) to more complex mathematical frameworks (presented in Chapter 3). A novel methodology that focuses on the inference of biological networks from time series data by least squares fitting will then be introduced. Using a set of carefully designed inference rules one can gain important information about the system which can aid in the inference process. The application of the method to a data set from the response of the yeast Saccharomyces cerevisiae to cumene hydroperoxide is explored in Chapter 5. The results show that this method can be used to generate a coarse-level mathematical model of the biological system at hand. Possible developments of this method are discussed in Chapter 6.
Ph. D.

Hvordan lærer og husker vi dufter? Hvordan skiller vi dem fra hverandre?

Det finnes liten kunnskap om hvilke duftsubstanser ulike lukteceller er utviklet for. I dag er gener for luktereseptorer identifisert i mange organismer, men kunnskapen om relevante duftstimuli mangler. Denne kunnskapen kan kun komme fra fysiologiske studier. I laboratoriet ble det brukt beslektede skadeinsekter som modeller for å studere innkoding av luktinformasjon i sanseceller og generelle prinsipper for luktesansens mekanismer.

Dette ble utført ved bruk av elektrofysiologiske målinger av nerveimpulser fra enkelte celler på insektantennene, med direkte kopling til kjemiske analyser av substansene avgitt av plantene. Resultatene har gitt ny kunnskap om hvilke plantesubstanser disse insektene detekterer i naturen. I tillegg har resultatene vist at luktecellene kan klassifiseres i distinkte typer, og samme type finnes hos de tre beslektede artene. Hver type er spesialisert for en enkelt substans, men de svarer også på høye konsentrasjoner av noen få andre substanser med lignende molekylstrukturer. Det er ingen overlapping av substanser mellom de ulike celletypene, noe som viser hvordan luktinformasjonen formidles via spesifikke nerveceller til hjernen hos disse insektene. Atferdsreaksjoner på en spesielt interessant substans viste at substansen økte tiltrekningen av gravide hunninsekter og økte antallet egg lagt på planter med substansen.

Å kjenne de biologiske luktesubstansene er viktig for å studere luktesansen generelt. Informasjon om hvilke plantedufter de studerte insektene lukter, kan brukes for å bekjempe skadene de påfører avlinger over hele verden.

The aim of this study was to investigate the effects of water temperature and salinity on lipid nutrition of farmed Atlantic salmon (Salmo salar L.) parr and smolt. Salmon parr were held at low water temperature (2°C) for six months while being fed feeds that differed in oil source (i.e. marine fish oil or vegetable oil blend) and concentration (low, 21% and high, 34%). The responses at low temperature were compared with those of fish held at 8°C using full-factorial design. Feeding and growth were maintained at 2°C, although at lower rates than at 8°C. Growth and feed utilisation improved over time, suggestive for a long-term acclimation response in fish held at low temperature. Overall feed efficiency was better at the lower temperature. A gradual decrease in growth rate and feed utilisation was seen at the higher temperature as the fish grew larger. The fish compensated for reduced energy density by increasing feed intake. At the higher temperature, better growth was found for fish fed the low-fat feeds, and there was also a tendency for improved growth when vegetable oil was used. Thus, there were no signs that vegetable oils are inferior to marine fish oil in promoting growth of Atlantic salmon parr in fresh water. Fish fed high fat feed were fatter than fish fed low fat feed, suggestive of lipostatic regulation of feed intake. Fat and protein digestibility were high at both 2°C and 8°C, although both fat and protein digestibility were lower at 2°C. At the lower temperature, increased dietary fat level increased the fat digestibility, and improved protein digestibility were seen when vegetable oil was included in the feed. Protein retention was higher at the higher temperature irrespective of feed treatment, indicating that proteins were both readily digested and converted into new tissues. The effects of feed treatment on low temperature acclimation responses were assessed from deposition of dietary fatty acids in fish tissues and from n-3 and n-6 essential fatty acid (EFA) budgets. Fatty acid composition of polar (membrane) and non-polar (storage) lipids in muscle, viscera and carcass were markedly influenced by the dietary oil, and non-polar lipids were more influenced than polar lipids. The retention n-6 EFAs was lower than for n- 3 EFAs, and was independent of temperature. The retention of n-3 EFAs retention was higher at the 2°C, especially amongst fish given the fish oil based diets. This may be a reflection of the importance of n-3 HUFAs during low temperature acclimation. However, the unsaturation (UFA:SFA ratio) of polar lipids was higher in fish fed the vegetable oils than for fish fed fish oil based feed. This may imply that vegetable oils produced fish that were better able to withstand exposure to low temperature, while having membrane lipids less susceptible to oxidative damage, due to the lower contents of n-3 HUFAs (mainly EPA and DHA). The six months feeding period in freshwater was followed by parr-smolt transformation, and a subsequent 42-days on-growing in seawater. Feed history during freshwater rearing influenced on-growth of smolts. A positive effect of using a vegetable oil was indicated, but this effect was only seen when there was a shift to a high-lipid fish oil based feed at the time of transfer to seawater. As such, it was evident that use of vegetable oils in freshwater feed did not interfere with low temperature acclimation or parr-smolt transformation of juvenile salmon, and subsequent on-growing in seawater was better when vegetable oil had been used in the feed. This indicates that fatty acid (lipid) requirement of Atlantic salmon are probably different in fresh water and seawater, and that these changes are linked to parr-smolt transformation. It could be speculated that that salinity may be more important than temperature as an environmental influence on the fatty acid requirements of Atlantic salmon.
Målet med dette studiet har vært å undersøke vanntemperaturens og saltholdighetens innvirkning på lipidernæringen hos parr og smolt av oppdrettet atlantisk laks (Salmo salar L.). Lakseparr ble holdt ved lav vanntemperatur (2ºC) i seks måneder mens de ble fôret med en av fire fôrtyper med ulike fettkilde (dvs. marin fiskeolje eller vegetabilsk olje) og ulik konsentrasjon (lav, 21% og høy, 34%). Responsene ved den lave temperaturen ble sammenlignet med responsene en fikk hos fisk holdt ved 8ºC i et full-faktorielt forsøksdesign. Fôrinntak og vekst ble opprettholdt ved 2ºC, men var lavere enn ved 8ºC. Over tid ble vekst og fôrutnyttelse forbedret, noe som indikerer en langtids akklimeringsrespons hos fisken ved den lave temperaturen. Totalt sett var utnyttelsen av fôret bedre ved den laveste temperaturen. En kunne observere en gradvis reduksjon i veksthastighet og fôrutnyttelse ved den høyeste temperaturen ettersom fisken ble større. Fisken kompenserte for lavere energitetthet i fôret ved å øke fôrinntaket. Ved den høyeste temperaturen var veksten bedre hos fisk fôret med lav-fett-fôrene. Det var også en tendens til forbedret tilvekst når vegetabilsk olje ble brukt. Det var ingen tegn til at vegetabilsk olje var dårligere enn marin fiskeolje til å fremme vekst hos lakseparr i ferskvann. Fisken som ble fôret med høy-fett-fôr ble fetere enn den som fikk lav-fett-fôr. Det indikerer lipostatisk regulering av fôrinntak. Fett- og proteinfordøyeligheten var høy både ved 2ºC og 8ºC, selv om både fett- og proteinfordøyeligheten var lavest ved 2ºC. Ved den laveste temperaturen, ga økt fettinnhold en forbedret fettfordøyelighet, og bruk av vegetabilsk olje i fôret ga bedre proteinfordøyelighet. Proteinretensjonen var høyere ved den høyeste temperaturen uavhengig av fôrtype, noe som indikerer at proteinet ble både lett fordøyd og omdannet til nytt vev. Effektene av fôrtype på akklimeringen til lav temperatur ble bestemt fra deponeringen av fettsyrer fra fôret i ulike vev og fra budsjetter for n-3 og n-6 essensielle fettsyrer (EFS). Fettsyresammensetningen i polare (membran) lipider og upolare (lagrings) lipider i muskel, innvoller og ’rest’ ble tydelig påvirket av oljene i fôret, og de upolare lipidene ble mer påvirket enn de polare lipidene. Retensjonen av n-6 EFS var lavere enn for n-3 EFS, og var uavhengig av temperatur. Retensjonen av n-3 EFS var høyere ved 2ºC, spesielt hos fisk som fikk et fiskeoljebasert fôr. Dette kan reflektere betydningen av n-3 HUFA fettsyrer i akklimeringen til lav temperatur. Imidlertid var de polare lipider hos fisk som ble gitt fôr med vegetabils olje, mer umettet (UFA:SFA forhold) enn hos fisk gitt fôr med marine fiskeoljer. Dette kan bety at vegetabilske oljer produserte fisk som var bedre i stand til å tåle eksponering til lav temperatur, samtidig som membranlipidene var mindre utsatt for oksidering som følge av et lavere innhold av n-3 HUFA fettsyrer (hovedsaklig EPA og DHA). Etter seks måneder i ferskvann ble fisken smoltifisert, etterfulgt av en 42-dagers periode i sjøvann. Fôrhistorie i ferskvannsfasen påvirket påvekst hos smolt. En positiv effekt av vegetabilsk olje ble funnet, men denne effekten ble bare funnet i grupper som hadde et skifte til et høy-fett-fiskeoljefôr ved overføring til sjøvann. Det var derfor tydelig at vegetabilsk olje ikke hadde negative konsekvenser for akklimering til lav temperatur eller for smoltifiseringen hos unglaks, og påfølgende tilvekst i sjøvann var bedre når vegetabilske oljer hadde blitt brukt. Dette indikerer at fettsyre (fett) behovet til atlantisk laks er forskjellig mellom ferskvann og sjøvann, og at forskjellene er knyttet til smoltifiseringen. Det kan derfor spekuleres i om saltholdigheten i miljøet er viktigere enn temperaturen i å bestemme fettsyrebehovet hos atlantisk laks.
Papers II and V reprinted with kind permission of Elsevier, sciencedirect.com.

This practice-led, project-based research charts, simultaneously, my disenchantment and re-engagement with graphic design. By it's dissemination I hope to articulate: 1. How an evolving understanding of my sense disenchantment emerged from the research, and enabled the process of re-engagement. 2. The role, and importance, of provocation and doubt in creative practice generally, but specifically in practice-led research. The difficulty of provoking one's self, and the strategies through which I have tried to enact a disruptive reframing of my practice. 3. That through the highly reflective nature of practice-led research and the greater sense of self-awareness that inevitable comes from that I have been able to re-engage with graphic design. That this re-engagement has, for me specifically, had much to do with my ability to begin to negotiate my own personal terms of reference, so as to be able to locate myself within a community of practice, and to begin to take part in a discourse that has a certain resonance for me. Central to this research are questions about professional practice, dislocation/disinterest, research, resonance and reinvention. As disenchantment is common, perhaps pervasive, within professional practice, my account of this research will propose that a more general understanding of practice-led research-highly reflective, self-initiated work-is essential if graphic design is to support and sustain imaginative, innovative, and inventive practitioners. Rather than target graphic design's inability to support provocative practices (the studio, or the industry), my research focuses on the potential of the individual practitioner to motivate and design a more generative and engaged practice. As such any observations and/or discoveries are not presented as quantitative 'findings', but should be seen rather as generative understandings that promote future possibility and potential for the practice.

Biology is often characterized as an historical science. Why and to what extent does history matter in biology? Overall, this analysis shows that biology is deeply historical. Using examples from various fields (evolution, ecology and development), I develop a theory of biological historicity starting from the idea that the most fundamental property of historical processes is their capacity to retain information from the past, which ultimately depends on the existence of divergent outcomes and a causal dependence between a process’ outcome and its past. Yet the conditions generating divergent outcomes and information preserving processes are numerous and can be illustrated in various ways. These are developed and clarified through the notions of historical contingency, path dependence, irreversibility, and generative entrenchment.

HIV-1 is the cause of the majority of global HIV infections. Not only being more virulent, and relatively easily transmitted than HIV-2, HIV-1 is also more extensively studied. HIV-1 is known for its highly recombinogenic nature, together with an extreme genetic variety, both attributable to an error-prone reverse transcriptase which gives rise to heterozygous virion. Sequence diversity of HIV-1 has resulted in identification of 9 subtypes of HIV-1 M group, as well as 43 circulating and a number of other unique recombinant forms of HIV-1. The extensive heterogeneity of HIV-1 has become the main consideration in vaccine development, mainly due to the inherent variability of HIV-1 and the frequent generation of new recombinant forms, which subsequently makes the effort to control the HIV-1 pandemic more challenging. The inter-subtype recombination event is a common phenomenon observed in Malaysia whereby there is a co-circulation of multiple HIV-1 subtypes; CRF01_AE and subtype B. Therefore, it becomes crucial to widen the knowledge of currently emerging CRF01_AE/B inter-subtype recombinants, in order to assist the future regional vaccine design and also to prevent wider spread of these strains. Concurrently, with a better understanding on the characteristics of HIV-1 CRF01_AE/B recombinant forms, further diversification of these strains can possibly be thwarted. The objectives of this study included, firstly to study the molecular epidemiology pattern of different HIV-1 strains, as well as to observe their frequency and distribution. Our second aim was to identify possible derivative from CRF33_01B, and also other new CRF01_AE/B inter-subtype recombinant forms in Malaysia. Thirdly, we aimed to identify possible biological advantages of the CRF33_01B isolates over its parental strains; CRF01_AE and subtype B. Currently, the HIV-1 epidemic in Malaysia is in a concentrated phase with evidence of predominance of both CRF01_AE and subtype B found among heterosexuals and injecting drug users, respectively. There is urgent necessity to apply a more detailed and continuous molecular characterization and epidemiological monitoring of these recombinant forms in Malaysia. We obtained plasma samples from 115 HIV-1-infected patients who attended HIV clinic at the University Malaya Medical Centre in Kuala Lumpur, Malaysia. The HIV-1 PR-RT, gp120-env and gp41-env genes were amplified and sequenced from 50 samples, while the remaining 65 samples were successfully studied at either one or two HIV-1 specific genomic regions. Cloning, phylogenetic analyses, together with bootscanning methods were employed to assign subtypes and to identify inter-subtype recombination based on all three genomic regions. From the plasma-derived sequences of 50 patients, 46% were found to harbour CRF01_AE, 10% and 6% had subtype B and B’, and a total of 18% of the patients were infected with CRF33_01B, while the remaining 18% of patients was found to have unique recombinant forms. As for the other 65 patients, majority of them harboured CRF01_AE and subtype B. This study shows that co-circulation of multiple HIV-1 subtypes and their recombinant strains are frequent in the Malaysian population, while capable of spreading to different HIV-1 risk groups. Possible recombination hotspots in CRF01_AE/B recombinants are suggested to be within the HIV-1 PR-RT gene region. Further, this study highlights the need to characterize and monitor the molecular epidemiology of these recombinant forms. The ideal environment for the inter-subtype recombination event to take place is created by the co-circulation and dual infections of both CRF01_AE and subtype B. With more HIV-1 CRF01_AE/B recombinant forms emerging and shaping the nature of HIV epidemic in Malaysia, certainly it will complicate the timely diagnosis of these molecularly altered HIV-1 forms. The recent identification of the novel CRF33_01B suggests the emergence of other new CRF01_AE/B inter-subtype recombinant forms in Malaysia, as preliminarily demonstrated in some HIV-1 patients identified in the first part of this study. The peripheral blood mononuclear cells (PBMCs) of these HIV-1 patients were co-cultured with those of healthy donors, which we then isolated the proviral genomic DNA. The nested long-range PCR was performed to obtain seven overlapping viral genome fragments that made up the whole viral genome. The detailed phylogenetic, as well as bootscan analyses confirmed the mosaic compositions and recombinant structures of the newly emerging CRF01_AE/B recombinant forms derived from CRF01_AE and subtype B. One of them in particular; HIV-1 isolate 06MYKLD46 is structurally similar to CRF33_01B, except for an extra subtype B fragment within the env region. It also has close phylogenetic relationship and similar breakpoints with CRF33_01B, mainly at the PR-RT region. Furthermore, the other three distinct HIV-1 recombinants; isolates 07MYKLD47, 07MYKLD48 and 07MYKLD49 also display near full-length genomes composed of the backbone of CRF01_AE, with insertions of subtype B fragments at different gene regions. These results indicate the high possibility of second generation of minor recombinant forms derived from CRF33_01B, as well as the continuous evolution and rapid dispersal of CRF01_AE/B recombinants in Malaysia. The high prevalence of newly emerging CRF33_01B (CRF01_AE/B inter-subtype recombinant) may cause a possible epidemiologic shift, attributable to its altered virologic characteristics and possible transmission advantages compared to its parental strains. Two major determinants; the viral factor and host factor have influenced the progress of a productive HIV-1 infection upon virus entry into the host cells. We have assessed the two main viral factors; the in vitro viral replication capacity and the viral fitness of the circulating HIV-1 strains in Malaysia. We have determined that CRF33_01B primary isolate (07MYKLVik) replicates better in activated whole PBMCs and CD4+ T-lymphocytes and is ‘fitter’ than one of its parental strain; CRF01_AE (07MYKLNBL) but not subtype B (07MYKLAfik). Subtype B has more advanced ability to produce a progressive infection in all cell types, including MDMs, and has a comparable viral fitness to that of CRF33_01B. We also investigated the role of host factors in a productive HIV-1 infection, by determining the viral effect on the host cell morphological features. We found that CRF33_01B (07MYKLVik) culture displayed more large syncytia (multinucleated giant cells) with multiple nuclei compared to subtype B (07MYKLAfik) culture, while no snycytia was observed in CRF01_AE (07MYKLNBL) culture. Generally, the cells within CRF33_01B and subtype B cultures appeared to be morphologically distinct from CRF01_AE cultures. This may indicate a more productive HIV-1 infection of CRF33_01B and subtype B, similar to our finding from the in vitro viral replicative capacity and viral fitness assays of these HIV-1 strains. We also studied the effect of different HIV-1 strain infections on host differential gene expression profiles, by using the PCR Array, which detects a total of 84 genes known to be involved in the host response to HIV-1 infection. It was observed that the in vitro infection with CRF33_01B isolates resulted in a more damaging effect on host cells and caused more apoptotic death within the infected cultures, compared to the isolates of its parental subtypes. Moreover, subtype B isolates resulted in a poorer cell response upon viral infection, compared to CRF01_AE/B isolate. Concurrently, it also gave less productive spread of viral infection within the infected cultures, in comparison to CRF01_AE/B isolate. We speculate that if the same scenario is reflected in vivo, CRF01_AE/B inter-subtype recombinant including CRF33_01B would have a better survival rate within the host upon their infection, in comparison to their parental strains. This again strengthens our presumption that CRF33_01B has potential ability to disseminate widely in the Malaysian population and gives a progressive change of the current molecular epidemiological trend by gradually replacing the current predominance of CRF01_AE in the country.

PhD
HIV-1 is the cause of the majority of global HIV infections. Not only being more virulent, and relatively easily transmitted than HIV-2, HIV-1 is also more extensively studied. HIV-1 is known for its highly recombinogenic nature, together with an extreme genetic variety, both attributable to an error-prone reverse transcriptase which gives rise to heterozygous virion. Sequence diversity of HIV-1 has resulted in identification of 9 subtypes of HIV-1 M group, as well as 43 circulating and a number of other unique recombinant forms of HIV-1. The extensive heterogeneity of HIV-1 has become the main consideration in vaccine development, mainly due to the inherent variability of HIV-1 and the frequent generation of new recombinant forms, which subsequently makes the effort to control the HIV-1 pandemic more challenging. The inter-subtype recombination event is a common phenomenon observed in Malaysia whereby there is a co-circulation of multiple HIV-1 subtypes; CRF01_AE and subtype B. Therefore, it becomes crucial to widen the knowledge of currently emerging CRF01_AE/B inter-subtype recombinants, in order to assist the future regional vaccine design and also to prevent wider spread of these strains. Concurrently, with a better understanding on the characteristics of HIV-1 CRF01_AE/B recombinant forms, further diversification of these strains can possibly be thwarted. The objectives of this study included, firstly to study the molecular epidemiology pattern of different HIV-1 strains, as well as to observe their frequency and distribution. Our second aim was to identify possible derivative from CRF33_01B, and also other new CRF01_AE/B inter-subtype recombinant forms in Malaysia. Thirdly, we aimed to identify possible biological advantages of the CRF33_01B isolates over its parental strains; CRF01_AE and subtype B. Currently, the HIV-1 epidemic in Malaysia is in a concentrated phase with evidence of predominance of both CRF01_AE and subtype B found among heterosexuals and injecting drug users, respectively. There is urgent necessity to apply a more detailed and continuous molecular characterization and epidemiological monitoring of these recombinant forms in Malaysia. We obtained plasma samples from 115 HIV-1-infected patients who attended HIV clinic at the University Malaya Medical Centre in Kuala Lumpur, Malaysia. The HIV-1 PR-RT, gp120-env and gp41-env genes were amplified and sequenced from 50 samples, while the remaining 65 samples were successfully studied at either one or two HIV-1 specific genomic regions. Cloning, phylogenetic analyses, together with bootscanning methods were employed to assign subtypes and to identify inter-subtype recombination based on all three genomic regions. From the plasma-derived sequences of 50 patients, 46% were found to harbour CRF01_AE, 10% and 6% had subtype B and B’, and a total of 18% of the patients were infected with CRF33_01B, while the remaining 18% of patients was found to have unique recombinant forms. As for the other 65 patients, majority of them harboured CRF01_AE and subtype B. This study shows that co-circulation of multiple HIV-1 subtypes and their recombinant strains are frequent in the Malaysian population, while capable of spreading to different HIV-1 risk groups. Possible recombination hotspots in CRF01_AE/B recombinants are suggested to be within the HIV-1 PR-RT gene region. Further, this study highlights the need to characterize and monitor the molecular epidemiology of these recombinant forms. The ideal environment for the inter-subtype recombination event to take place is created by the co-circulation and dual infections of both CRF01_AE and subtype B. With more HIV-1 CRF01_AE/B recombinant forms emerging and shaping the nature of HIV epidemic in Malaysia, certainly it will complicate the timely diagnosis of these molecularly altered HIV-1 forms. The recent identification of the novel CRF33_01B suggests the emergence of other new CRF01_AE/B inter-subtype recombinant forms in Malaysia, as preliminarily demonstrated in some HIV-1 patients identified in the first part of this study. The peripheral blood mononuclear cells (PBMCs) of these HIV-1 patients were co-cultured with those of healthy donors, which we then isolated the proviral genomic DNA. The nested long-range PCR was performed to obtain seven overlapping viral genome fragments that made up the whole viral genome. The detailed phylogenetic, as well as bootscan analyses confirmed the mosaic compositions and recombinant structures of the newly emerging CRF01_AE/B recombinant forms derived from CRF01_AE and subtype B. One of them in particular; HIV-1 isolate 06MYKLD46 is structurally similar to CRF33_01B, except for an extra subtype B fragment within the env region. It also has close phylogenetic relationship and similar breakpoints with CRF33_01B, mainly at the PR-RT region. Furthermore, the other three distinct HIV-1 recombinants; isolates 07MYKLD47, 07MYKLD48 and 07MYKLD49 also display near full-length genomes composed of the backbone of CRF01_AE, with insertions of subtype B fragments at different gene regions. These results indicate the high possibility of second generation of minor recombinant forms derived from CRF33_01B, as well as the continuous evolution and rapid dispersal of CRF01_AE/B recombinants in Malaysia. The high prevalence of newly emerging CRF33_01B (CRF01_AE/B inter-subtype recombinant) may cause a possible epidemiologic shift, attributable to its altered virologic characteristics and possible transmission advantages compared to its parental strains. Two major determinants; the viral factor and host factor have influenced the progress of a productive HIV-1 infection upon virus entry into the host cells. We have assessed the two main viral factors; the in vitro viral replication capacity and the viral fitness of the circulating HIV-1 strains in Malaysia. We have determined that CRF33_01B primary isolate (07MYKLVik) replicates better in activated whole PBMCs and CD4+ T-lymphocytes and is ‘fitter’ than one of its parental strain; CRF01_AE (07MYKLNBL) but not subtype B (07MYKLAfik). Subtype B has more advanced ability to produce a progressive infection in all cell types, including MDMs, and has a comparable viral fitness to that of CRF33_01B. We also investigated the role of host factors in a productive HIV-1 infection, by determining the viral effect on the host cell morphological features. We found that CRF33_01B (07MYKLVik) culture displayed more large syncytia (multinucleated giant cells) with multiple nuclei compared to subtype B (07MYKLAfik) culture, while no snycytia was observed in CRF01_AE (07MYKLNBL) culture. Generally, the cells within CRF33_01B and subtype B cultures appeared to be morphologically distinct from CRF01_AE cultures. This may indicate a more productive HIV-1 infection of CRF33_01B and subtype B, similar to our finding from the in vitro viral replicative capacity and viral fitness assays of these HIV-1 strains. We also studied the effect of different HIV-1 strain infections on host differential gene expression profiles, by using the PCR Array, which detects a total of 84 genes known to be involved in the host response to HIV-1 infection. It was observed that the in vitro infection with CRF33_01B isolates resulted in a more damaging effect on host cells and caused more apoptotic death within the infected cultures, compared to the isolates of its parental subtypes. Moreover, subtype B isolates resulted in a poorer cell response upon viral infection, compared to CRF01_AE/B isolate. Concurrently, it also gave less productive spread of viral infection within the infected cultures, in comparison to CRF01_AE/B isolate. We speculate that if the same scenario is reflected in vivo, CRF01_AE/B inter-subtype recombinant including CRF33_01B would have a better survival rate within the host upon their infection, in comparison to their parental strains. This again strengthens our presumption that CRF33_01B has potential ability to disseminate widely in the Malaysian population and gives a progressive change of the current molecular epidemiological trend by gradually replacing the current predominance of CRF01_AE in the country.

Abstract

The sediment requirements for freshwater pearl mussel (Margaritifera margaritifera) recruitment, in 18 rivers in the counties of Västra Götaland, Örebro, Värmland and Västmanland in Sweden, were investigated. The top 4 cm of sediment in the rivers was analysed in terms of size, distribution and organic compound within the fine sediment. The aims of the study were to determine whether there is a relation between sediment particle size compound and freshwater pearl mussel recruitment as well as between organic compound in fine sediment and recruitment of mussels. The study shows that there is a significant difference in the amount of organic silt between non-recruitment and recruitment sites with a higher percentage of organic silt in recruitment sites. There is also a legible difference between the amounts of silt per sample between non-recruitment sites and recruitment sites where there was significantly more silt in sediment samples of non-recruitment sites. With the exception of fine sediment, no significant difference was found between non-recruitment and recruitment sites regarding size class distribution.

Sammanfattning

Sedimentkraven för rekrytering hos flodpärlmussla (Margaritifera maragaritifera) i 18 svenska vattendrag belägna i Västra Götalands, Örebro, Värmlands och Västmanlands län undersöktes. Vattendragens översta 4 cm sediment analyserades gällande storlek, fördelning och organisk sammansättning i finsedimentet. Målen med studien var att fastställa huruvida det finns förhållanden mellan sedimentets partikelstorleksammansättning och rekrytering av flodpärlmussla samt mellan finsedimentets organiska sammansättning och rekrytering av musslor. Studien visar en signifikant skillnad i organiskt finsediment mellan icke-rekryteringsplatser och platser med rekrytering där rekryteringsplatser hade högre procentuell andel organiskt finsediment. En signifikant skillnad påvisades också mellan andel finsediment per sedimentprov där sedimentprov från icke-rekryteringsplatser innehöll en högre procentuell andel finsediment. Förutom gällande finsediment påvisades ingen signifikant skillnad i sedimentets storleksdistribution mellan rekryterings- och icke-rekryteringsplatser.

O objetivo deste trabalho foi caracterizar aspectos biológicos e ecológicos de Austinixa aidae, um caranguejo simbionte em galerias construídas pelo Thalassinidea Callichirus major, na Praia do Perequê-Açu, Ubatuba, litoral Norte do Estado de São Paulo. As coletas foram realizadas por meio de bombas de sucção manual na região intermareal da praia arenosa, de Maio/2005 a Setembro/2006. Nesta dissertação foram apresentados, em três capítulos distintos, a Estrutura Populacional, o Crescimento Relativo e as Estratégias Reprodutivas deste caranguejo. No Capítulo I, sua população foi estudada com ênfase em aspectos estruturais como abundância sazonal, freqüência de indivíduos em classes de tamanho, razão sexual, densidade e incidência nas galerias de seus hospedeiros, os períodos reprodutivo e de recrutamento. Além disso, foram avaliadas as correlações entre sua densidade e seu tamanho com o tamanho de seus respectivos hospedeiros. Foi constatado que as espécies do gênero Austinixa possuem aspectos ecológicos e biológicos semelhantes, mesmo em casos de localidades muito distantes geograficamente, corroborando o que foi postulado por outros autores quanto à similaridade destes parâmetros entre os membros do grupo. No Capítulo II, devido ao forte dimorfismo sexual e uma série de adaptações morfológicas atribuídas ao estilo de vida simbionte destes caranguejos, foram estudadas as características corporais, o crescimento relativo, a maturidade e o dimorfismo sexual destes indivíduos. Os adultos de A. aidae possuem a largura da carapaça em média 2,4 vezes o seu comprimento, essas características foram sugeridas como sendo adaptações ao estilo de vida simbionte. O crescimento relativo evidenciou que alterações morfológicas processadas ao longo da ontogenia dos animais estão relacionadas à maturidade sexual da espécie. No Capítulo III, o objetivo foi conhecer a estratégia reprodutiva relacionada à fecundidade, determinando-se o número de ovos produzidos pelas fêmeas ovígeras de distintos tamanhos e em diferentes períodos, além do desenvolvimento dos ovos, seu diâmetro e volume. A. aidae apresentou uma estratégia reprodutiva com padrões característicos de outros crustáceos Decapoda, com evidências de eficiência reprodutiva no local onde esta população está instalada. Considerando a ausência de estudos populacionais e reprodutivos para esta espécie, esta dissertação visou suprir esta demanda na literatura científica.
The biological and ecological aspects of Austinixa aidae, a symbiotic crab inhabiting burrows of the Thalassinidea ghost shrimp Callichirus major, from Perequê-Açu beach, Ubatuba, North shore of State of São Paulo, Brazil, were studied. The samples were conducted bimonthly with manual suction pumps in the intertidal region of the sand beach, from May/2005 to September/2006. This project was subdivided into three chapters: Population Structure, Relative growth and Reproductive Strategies. In Chapter I, the population of A. aidae was determined in terms of seasonal abundance, size frequency distribution, sex ratio, density and incidence in the burrows of the thalassinids, reproductive and recruitment periods. In addition, were evaluated the correlations between its density and size with size of their respective hosts. Was found that Austinixa species posses similar biological and ecological aspects, even in distant localities. In Chapter II, due to sexual dimorphism and the morphological adaptations assigned to symbiotic life style, body features, relative growth, maturity and sexual dimorphism were studied. Adults of A. aidae have the carapace width on average 2.4 times the carapace length. Relative growth revealed that morphological changes are related to sexual maturity of this species. In Chapter III, the goal was to know the reproductive strategy related to production of eggs, to determine the fecundity of ovigerous females of different sizes and different periods, conditions of the development of eggs, and their parameters (diameter and volume). A. aidae presented patterns of reproductive strategy similar to other Decapoda crustaceans, with evidence of reproductive efficiency where this population is established. Due to a lack of population and reproductive studies for this species, this study aimed to supply this demand in the scientific literature.

Pseudevernia furfuracea is a shrubby lichen commonly found around the Baltic Sea. The lichen appears to reproduce mainly by producing isidia, propagules of the lichen cortex. It is very morphologically and chemically diverse and can be found on different substrates, such as nutrient poor bark trees and rocks. The main objective of this study was to describe genetic variation in the ITS region and link this to variations in morphology, substrate ecology and secondary chemistry. In total, 36 specimens of P. furfuracea were collected from 21 sites in Sweden, Finland and Estonia. Seven haplotypes were distinguished in the ITS region. A statistically significant connection between haplotype and colouration of the lichen was found. Lighter coloured specimens of P. furfuracea are more likely to be of the ancestral haplotype. No other significant correlations between the different characteristics studied were found, suggesting that P. furfuracea should be regarded as a single species within the Baltic Sea area. This is also supported by the haplotype network.

More so than any other science in the past several decades, Biology has seen an explosion of new information and monumental discoveries that have had a profound impact on much more than the science itself. Much of this has occurred at the molecular level. Many of these modern concepts, ideas, and technologies, as well as their historical context, can be easily understood and appreciated at the high school level. Moreover, it is argued here that the integration of this is critical for making biology relevant as a modern science. A contemporary high school biology curriculum should adequately reflect this newly acquired knowledge and how it has already has already begun to revolutionize medicine, agriculture, and the study of biology itself. This curriculum provides teachers with a detailed framework for integrating molecular biology into a high school biology curriculum. It is not intended to represent the curriculum for an entire academic year, but should be considered a significant component. In addition to examining key concepts and discoveries, it examines modern molecular techniques, their applications, and their relevance to science and beyond. It also provides several recommended labs and helpful protocols.

Thesis (Ph. D.)--University of Sydney, 2009.
Title from title screen (viewed 31 March 2009). Submitted in fulfilment of the of the requirements for the degree of Doctor of Philosophy to the Discipline of Medicine, Faculty of Medicine. Degree awarded 2009; thesis submitted 2008. Includes bibliographical references. Also available in print form.

The Cerulean Warbler (Dendroica cerulea) is a Neotropical migrant bird species which is experiencing severe population declines. This study fills in gaps in the information available concerning Cerulean Warbler territoriality and breeding in a site in southeastern Indiana. During the summers of 2002 and 2003, 51 territories were mapped, with an average territory size of 0.21 hectares. Differences existed between territories and random sites for canopy cover, slope, canopy height, number of trees, diameter at breast height (DBH), the number of trees between 3 - 7.9 cm DBH, the number of trees between 8 - 14.9 cm DBH, the number of trees between 15 - 22.9 cm DBH, and the number of trees > 38 cm DBH. Nest productivity was very low in the study area, suggesting that Big Oaks National Wildlife Refuge was a sink population of Cerulean Warblers during the two years of the study.
Department of Biology

When the Biological Weapons Convention opened for signature in 1972, the battle against infectious disease appeared to have been won and the biological warfare programs of the United States and Soviet Union seemed to be irrelevant in the global conflict d

In Chapter one I introduce the subject of geographic profiling, its use in criminology and its previous application to biology. I go on in Chapter two to examine the original model and develop a likelihood-based approach to fit the parameters to data from 53 UK invasive species. GP performs well on this novel problem, and outperforms other simple spatial modelling techniques. Using simulations I show that GP is particularly efficient at locating sources when there is more than a single source. Chapter three develops a Bayesian approach using Dirichlet Processes to account for the problem of multiple sources. This model was developed in collaboration with Robert Verity. This new Bayesian model outperforms the original model used in criminology and offers a range of additional information from the data. The Bayesian GP model is then used to determine the sources of malaria outbreaks in Cairo. These developments significantly improve and extend the theory and application of GP. In Chapter four I discuss the possible shapes of dispersal functions. I conduct a review of the literature and find a geometric mistake in the way linear distributions have been extracted from two-dimensional data. The correct back-transformation allows these dispersal distributions to be properly generated. Using this information; ecologists, conservationists and resources managers can now apply GP to real world problems and effectively allocate limited resources to locate sources of species invasions and disease outbreaks. I go on in Chapter five to develop a method for fitting the primary parameter sigma from the point pattern data and run simulations to show the effectiveness of this new approach. In Chapter six I illustrate the application of GP to three problems, one in criminology, one in ecology and one in epidemiology. I finish by summarising the work in this thesis and discussing the potential future developments and applications of GP.

PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO DE JANEIRO
Os livros didáticos modernos constituem-se num objeto de estudo valioso para o lingüista por serem elaborados a partir de múltiplas modalidades semióticas e porque, em geral, são gêneros disciplina-específicos. Entretanto, no que tange à disciplina de Biologia, ainda há poucos estudos que exploram a interação entre as modalidades verbal e visual nos livros de Ensino Médio e, até o momento, nenhum trabalho de natureza contrastiva foi realizado no Brasil sobre esse aspecto. Na presente análise, investigam-se os tipos de representação visual e o modo de estabelecimento da relação entre figuras e o texto verbal associado em dois livros didáticos de Biologia do Ensino Médio, um brasileiro e outro norteamericano. Este estudo baseia-se principalmente nos pressupostos teóricos de Kress e van Leeuwen (1996, 2001) e inspira-se nos trabalhos de Myers (1997) e Nascimento (2002). Implícita nesses pressupostos está a abordagem sistêmicofuncional de Halliday e Hasan (1976) e de Halliday (1994). Os resultados desta pesquisa revelam que, dentre outros mecanismos, a coesão lexical exerce importante papel na interação entre o componente verbal das figuras, o texto principal e as legendas em ambos os livros analisados. Dentre as diferenças encontradas nos dois livros, destacam-se as representações visuais distintas referentes a um mesmo tópico, o modo de referenciação às figuras no texto, a função das legendas e a proporção de utilização de termos técnicos na coesão intermodal. Esses resultados mostram que existem variações no gênero livro didático de Biologia quanto ao modo de utilização dos mecanismos de interação entre figuras e texto verbal, que podem estar relacionadas com os propósitos dos autores. Isso pode ter importantes implicações para o ensino da Biologia e de Inglês para fins específicos, pois auxilia na conscientização de educadores e alunos sobre a necessidade de se levar em consideração os meios de interação entre as linguagens visual e verbal na interpretação e produção dos textos multimodais próprios de cada comunidade discursiva.
Modern textbooks are valuable as a research object in applied linguistics because they usually present their content through multiple semiotic modalities and because in general they are discipline-specific genres. However, concerning the discipline of Biology, there are still few studies that exploit the interaction between verbal and visual languages in highschool textbooks and, up to the moment, no contrastive works on this topic have been published in Brazil. For this reason, the goal of the present study is to investigate the types of visual representations and the strategies that establish the interaction between pictures and verbal text used in two highschool-level Biology textbooks, a Brazilian and a North-American one. This study is based mainly on Kress and van Leeuwen s (1996, 2001) theory of multimodality and is inspired in Myers (1997) and Nascimento s (2002) previous works. This theoretical basis presupposes Halliday and Hasan s (1976) and Halliday s (1997) systemic- functional approach to text. The results of this investigation reveal that, among other mechanisms, lexical cohesion has an important role in the interaction between the verbal element in images and the verbal language of the corresponding texts and captions in both books. Among the differences found between these two books, there are the different visual representations concerning the same topic, the way pictures are referred to in the text, the role of the captions and the proportion of technical terms used in intermodal cohesion. These results show that there are intrageneric variations in the use of such resources that are probably due to the authors purposes. The findings may have important implications for the teaching of Biology and of English for specific purposes. They may enhance the awareness of educators and students about the need of taking the interaction between visual and verbal languages into consideration for the appropriate interpretation of texts and for the production of multimodal texts specific to each discursive community.

A multimodal network (MMN) is a novel mathematical construct that captures the structure of biological networks, computational network models, and relationships from biological databases. An MMN subsumes the structure of graphs and hypergraphs, either undirected or directed. Formally, an MMN is a triple (V,E,M) where V is a set of vertices, E is a set of modal hyperedges, and M is a set of modes. A modal hyperedge e=(T,H,A,m) in E is an ordered 4-tuple, in which T,H,A are subsets of V and m is an element of M. The sets T, H, and A are the tail, head, and associate of e, while m is its mode. In the context of biology, each vertex is a biological entity, each hyperedge is a relationship, and each mode is a type of relationship (e.g., 'forms complex' and 'is a'). Within the space of multimodal networks, structural operations such as union, intersection, hyperedge contraction, subnetwork selection, and graph or hypergraph projections can be performed. A denotational semantics approach is used to specify the semantics of each hyperedge in MMN in terms of interaction among its vertices. This is done by mapping each hyperedge e to a hyperedge code algo:V(e), an algorithm that details how the vertices in V(e) get used and updated. A semantic MMN-based model is a function of a given schedule of evaluation of hyperedge codes and the current state of the model, a set of vertex-value pairs. An MMN-based computational system is implemented as a proof of concept to determine empirically the benefits of having it. This system consists of an MMN database populated by data from various biological databases, MMN operators implemented as database functions, graph operations implemented in C++ using LEDA, and mmnsh, a shell scripting language that provides a consistent interface to both data and operators. It is demonstrated that computational network models may enrich the MMN database and MMN data may be used as input to other computational tools and environments. A simulator is developed to compute from an initial state and a schedule of hyperedge codes the resulting state of a semantic MMN model.
Ph. D.

The thesis is that contrary to the received popular wisdom, the combination of David Hume's sceptical enquiry and Charles Darwin's provision of an alternative theoretical framework to the then current paradigm of natural theology did not succeed in defeating the design argument. I argue that William Paley's work best represented the status quo in the philosophy of biology circa 1800 and that with the logical mechanisms provided us by William Dembski in his seminal work on probability, there is a strong argument for thr work of Michael Behe to stand in a similar position today to that of Paley two centuries ago. The argument runs as follows: In Sections 1 and 2 of Chapter 1 I introduce the issues. In Section 3 I argue that William Paley's exposition of the design argument was archetypical of the natural theology school and that given Hume's already published criticism of the argument, Paley for one did not feel the design argument to be done for. I further argue in Section 4 that Hume in fact did no such thing and that neither did he see himself as having done so, but that the design argument was weak rather than fallacious. In Section 5 I outline the demise of natural theology as the dominant school of thought in the philosophy of biology, ascribing this to the rise of Darwinism and subsequently neo-Darwinism. I argue that design arguments were again not defeated but went into abeyance with the rise of a new paradigm associated with Darwinism, namely methodological naturalism. In Chapter 2 I advance the project by a discussion of William Dembski's formulation of design inferences, demonstrating their value in both everyday and technical usage. This is stated in Section 1. In Sections 2 and 3 I discuss Dembski's treatment of probability, whilst in Section 4 I examine Dembski's tying of different levels of probability to different mechanisms of explanation used in explicating the world. Section 5 is my analysis of the logic of the formal statement of the design argument according to Dembski. In Section 6 I encapsulate objections to Dembski. I conclude the chapter (with Section 7) by claiming that Dembski forwards a coherent model of design inferences that can be used in demonstrating that there is little difference between the way that Paley came to his conclusions two centuries ago and how modem philosophers of biology (such as I take Michael Behe to be, albeit that by profession he is a scientist) come to theirs when offering design explanations. Inference to the best explanation is demonstrated as lying at the crux of design arguments. In Chapter 3 I draw together the work of Michael Behe and Paley, showing through the mechanism of Dembski's work that they are closely related in many respects and that neither position is to be lightly dismissed. Section 1 introduces this. In Section 2 I introduce Behe's concept of irreducible complexity in the light of (functional) explanation. Section 3 is a detailed analysis of irreducible complexity. Section 4 raises and covers objections to Behe with the general theme being that (neo-) Darwinians beg the question against him. In Section 4 I apply the Dembskian mechanic directly to Behe's work. I argue that Behe does not quite meet the Dembskian criteria he needs to in order for his argument to stand as anything other than defeasible. However, in Section 5 I conclude by arguing that this is exactly what we are to expect from Behe's and similar theories, even within competing paradigms, in the philosophy of biology, given that inference to the best explanation is the logical lever therein at work.
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This thesis describes a path from a model of a biological system to a biologically-inspired algorithm. The thesis commences with a discussion of the principled design of biologically-inspired algorithms. It is argued that modelling a biological system can be tremendously helpful in eventual algorithm construction. A proposal is made that it is possible to reduce modelling biases by modelling the biological system without any regard to algorithm development, that is, with only concern of understanding the biological mechanisms. As a consequence the thesis investigates a detailed model of T cell signalling process. The model is subjected to stochastic analysis which results in a hypothesis for T cell activation. This hypothesis is abstracted to form a simplified model which retains key mechanisms. The abstracted model is shown to have connections to Kernel Density Estimation, through developing these connections the Receptor Density Algorithm is developed. By design, the algorithm has application in tracking probability distributions. Finally, the thesis demonstrates the algorithm on a related but different problem of detecting anomalies in spectrometer data.

Machine learning has been a source for continuous methodological advances in the field of computational learning from data. Systems biology has profited in various ways from machine learning techniques but in particular from network inference, i.e. the learning of interactions given observed quantities of the involved components or data that stem from interventional experiments. Originally this domain of system biology was confined to the inference of gene regulation networks but recently expanded to other levels of organization of biological and ecological systems. Especially the application to species interaction networks in a varying environment is of mounting importance in order to improve our understanding of the dynamics of species extinctions, invasions, and population behaviour in general. The aim of this thesis is to demonstrate an extensive study of various state-of-art machine learning techniques applied to a genetic regulation system in plants and to expand and modify some of these methods to infer species interaction networks in an ecological setting. The first study attempts to improve the knowledge about circadian regulation in the plant Arabidopsis thaliana from the view point of machine learning and gives suggestions on what methods are best suited for inference, how the data should be processed and modelled mathematically, and what quality of network learning can be expected by doing so. To achieve this, I generate a rich and realistic synthetic data set that is used for various studies under consideration of different effects and method setups. The best method and setup is applied to real transcriptional data, which leads to a new hypothesis about the circadian clock network structure. The ecological study is focused on the development of two novel inference methods that exploit a common principle from transcriptional time-series, which states that expression profiles over time can be temporally heterogeneous. A corresponding concept in a spatial domain of 2 dimensions is that species interaction dynamics can be spatially heterogeneous, i.e. can change in space dependent on the environment and other factors. I will demonstrate the expansion from the 1-dimensional time domain to the 2-dimensional spatial domain, introduce two distinct space segmentation schemes, and consider species dispersion effects with spatial autocorrelation. The two novel methods display a significant improvement in species interaction inference compared to competing methods and display a high confidence in learning the spatial structure of different species neighbourhoods or environments.

The study examined interpretational frameworks of six experienced biology teachers related to their roles in assessing students’ written answers to test questions. Data comprised in-depth interviews, sample tests, marking keys and student answers. Teachers’ interpretational frameworks depend on their expectations and are complex, three-dimensional, relational, predominantly visual and incorporate dynamic decision-making processes in forming judgments about students’ answers. Pedagogical Content assessment Knowledge (PCaK) is proposed as a major organising feature.