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Using molecular tools to differentiate closely related blackfly species of the genus Simulium
(2008)
Biodiversity data are the foundation for conservation and managemet and taxonomy provides the reference system, skills and tools used to identify organisms. Species level data such as species richness, composition and diversity are common metrics. However, species level identification of organisms tends to be neglected within ecological work, especially within monitoring programmes, but also in conservation biology (Giangrande, 2003). This is because collection of species level data is time consuming, with identification of species-specific characteristics traditionally involving lengthy examination of samples using microscopy. In addition it is costly and species level data is almost impossible to collect if the taxa involved are species rich and difficult to identify (Báldi 1999). Other reasons why species level identification is neglected include the fact that sample collection can damage organisms, so diagnostic morphological features are lost, or that individuals may be in a life history stage or of a sex that does not have diagnostic morphological characteristics. Furthermore, the numbers of available expert taxonomists needed for species identification are in decline and have been for several decades. Species identification using molecular taxonomy where DNA is used as a marker is championed as a tool for resolving a range of morphological problems, such as the association of all life history stages, correlating male and female specimens to the same species and identifying partial specimens. Traditional taxonomy is built around morphological variations between species, with systematic inferences based upon shared physical characters. In molecular taxonomy on the other hand, proteins and genes are used to determine evolutionary relationships. ’DNA barcoding’ aims to provide an efficient method for species-level identification and it is thought that it will provide a powerful tool for taxonomic and biodiversity research (Hajibabaei et al. 2007). Cited strengths of a molecular based approach to species identification include the potential universality and objective nature of DNA data as taxonomic information, the usefulness of molecular data in animal groups characterized by morphological cryptic characters and the use of DNA sequence information to determine otherwise ‘unidentifiable’ biological material (such as incomplete specimens or immature specimens). Its aim is to increase the speed, precision and efficiency of field studies involving diverse and difficult to identify taxa and it has the potential to be automated to provide a rapid and consistently accurate supplementary identification system to traditional taxonomy. This project was a proof-of-concept study that investigated the feasibility of using DNA barcodes to differentiate closely related blackfly species of the genus Simulium. The longer term objective would be to apply such molecular approaches to organisms used in water quality monitoring and to biodiversity studies to provide a quick, robust but practical and cost effective tool for species identification. Great Britain is currently home to 33 morphospecies of blackfly many of which are morphologically close to other species and have been the cause of much systematic revision. In addition to evaluating the use of DNA barcodes in species identification, a non-destructive DNA extraction method was developed to preserve voucher pecimens that will allow a complete morphological classification to be carried after DNA extraction. Using molecular tools to differentiate closely related blackfly species of the genus Simulium v Finding an effective DNA barcode for an individual species involves accurate taxonomic identification and the retention of voucher specimens for future morphological studies. A rapid non-destructive method for DNA extraction from small insects was developed where no clean-up step was required prior to amplification and it was possible to extract DNA of sufficient quality in minutes retaining diagnostic morphological characteristics. For any molecular tool used for species discrimination, an important consideration is defining the specific genetic loci (e.g. the position of genes on a chromosome) to be monitored. All blackfly species in this study were successfully amplified with the standard barcoding coxI gene primer pair LCO1490 5'-GGT CAA CAA ATC ATA AAG ATA TTG G-3' and HCO2198 5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3' (Folmer et al. 1994) and we did not need to optimise or redesign the primer sequence.
The dissertation, ”Coping with emergent hearing loss”, is written in English and is based on the diversity of problems connected to hearing loss and the adaptation of hearing aids. The research was carried out in Denmark and involves adults with an emergent hearing loss, who have decided to become hearing aid users. The data is analyzed through methods of cultural anthropology with focus on the following topics: How do the new users cope with the status passage towards being hearing aid users, how do they integrate the hearing aids into their lives, and what are the involved learning processes. What are the consequences of the provision by the state of free hearing aids, their free adaptation as well as free batteries, and does the state take part in the construction of the social group of new hearing aid users? The method is based on qualitative field work. Two public hospitals were helpful in identifying 24 new users, who acquired a free hearing aid through the hearing health care system. Through ENTs and private dispensers 17 further respondents joined the study – these acquired their hearing aids with a considerable state subsidy but mostly they themselves contributed financially as well. The 41 respondents between the ages of 42 to 92 years of age came from a wide range of professions and were followed throughout 2003 und 2004. After an in-depth qualitative interview, the contact to the respondents was maintained in order to follow the process of integration into their lives. When possible, the respondents were accompanied to their appointments in the private or public clinics. Moreover, interviews with experts from the public hearing health care system, politicians and user organisations were carried out, and the general public debate on the hard of hearing and hearing loss was followed and recorded. The second chapter gives an overview of the position of audiology in Denmark, of epidemiological information on hearing loss in the Danish society and statistics to the use of hearing aids. Moreover, basic information is given about the functioning of the human ear, the auditive perception and diagnosis and classification of hearing loss as well as a short introduction to the hearing aid technology. The structure of the further thesis divides the material into three pillars that make the discussion of the interaction processes possible. (1) The user’s interaction with the lifeworld concerns the meaning of hearing in relation to social participation. For some of the users, a good sense of hearing was essential to communicate freely and uphold their position in relation to others, whereas other respondents paid less attention to the information they acquired through their sense of hearing. A number of the respondents were selective and only used their hearing aids in specific situations, whereas another group discontinued the use of their hearing aids for various reasons. Status passages that hold specific challenges like a new work place or a new marriage motivate the continued everyday use. On the whole, the thesis illustrates that hearing loss is a socially dividing factor that complicates the interaction with others. In comparison to other bodily impairments or diseases, the hearing loss is rarely used as occasion to unite with fellow sufferers, join patient organisations or form self help groups. (2) The users’ interaction with the institutions The medical anthropologist Arthur Kleinman conceptualises health care as a moral process in which essential issues are at stake for the users. Different factors interact in the process: the training of the experts, allocation of funds, the quality of the technology, the dispensing procedures and the motivation and individual characteristics of the new users. The integration of the hearing aid into the lifeworld can be compared to a learning process, for which reason the learning theory of the anthropologist Gregory Bateson is outlined. Susanne Bisgaard’s own theory lists the meaning creating elements that serve as motivation for the users to counteract contingency (occurrences that influence the adaptation negatively). In the interaction between individual and society, the individual can apply strategies in order to eliminate stumbling blocks. (3) The users’ interaction with the technology A number of theorists from Anthropology as well as Science and Technology Studies are discussed in order to question their validity with regard to human action autonomy vs. technological determination and test the theoretical models with regard to their usability for the thesis. Hearing aids have a supporting function in everyday life and have the capability of moderating the user’s perception of sound. The alienating experience of hearing one’s own voice amplified, of wearing a foreign body in the ear and the different strategies that emerge from the more or less successful handling of the technology is reported by way of case stories and quotes from the interviews.
We study the effect of randomness in the adversarial queueing model. All proofs of instability for deterministic queueing strategies exploit a finespun strategy of insertions by an adversary. If the local queueing decisions in the network are subject to randomness, it is far from obvious, that an adversary can still trick the network into instability. We show that uniform queueing is unstable even against an oblivious adversary. Consequently, randomizing the queueing decisions made to operate a network is not in itself a suitable fix for poor network performances due to packet pileups.
This work presents the study on the suitability of single-crystal CVD diamond for particle-detection systems in present and future hadron physics experiments. Different characterization methods of the electrical and the structural properties were applied to gain a deeper understanding of the crystal quality and the charge transport properties of this novel semiconductor material. First measurements regarding the radiation tolerance of diamond were performed with sensors heavily irradiated with protons and neutrons. Finally, detector prototypes were fabricated and successfully tested in various experiments as time detectors for minimum ionizing particles as well as for spectroscopy of heavy ions at the energy ranges available at the SIS and the UNILAC facilities of GSI. ...
5-LO is the key enzyme in the biosynthesis of proinflammatory leukotrienes. It catalyses the conversion of arachidonic acid to the hydroperoxy intermediate 5(S)-hydroperoxy-6- trans-8,11,14-cis-eicosatetraenoic acid (5-HpETE). In a second step 5-LO catalyses a dehydration reaction forming the unstable epoxide intermediate 5(S)-trans-5,6-oxido-7,9- trans-11,14-cis-eicosatetraenoic acid (leukotriene A4 , LTA4). The 5-LO gene is subjected to versatile regulation mechanisms. Apart from regulation by DNA-methylation and histone acetylation / deacetylation 5-LO gene expression can be regulated by the differentiation inducers calcitriol (1,25-dihydroxyvitamin D3) and transforming growth factor beta (TGFβ) 5-LO gene expression. In the myeloid cell lines Mono Mac 6 (MM6) and HL-60, differentiation with both agents caused a prominent upregulation of 5-LO mRNA level, of 5-LO protein expression and of 5-LO activity. Treatment with calcitriol alone already has an impact on 5-LO gene expression which is additionally potentiated by TGFβ treatment. Previous nuclear run-off analysis and reporter gene analysis could not associate the 5-LO promoter with the induction of 5-LO mRNA expression mediated by calcitriol and TGFβ. Inclusion of the 5-LO coding sequence (cds) and inclusion of the 5-LO cds plus the last four introns of the gene (J to M) in the 5-LO promoter construct pN10 led to an enhanced reporter gene activity. The inductions were dependent on vitamin D receptor (VDR) and retinoid x receptor (RXR) cotransfection. Therefore the work was concentrated on identifying elements outside the 5-LO promoter region which contribute to the calcitriol / TGFβ effect on 5-LO mRNA expression. Insertion of the LTA4 hydrolase coding sequence – a coding sequence of similar size - instead of the 5-LO cds led to a loss of the calcitriol / TGFβ effect (pN10LTA4Hcds 1-fold induction). Therewith, it was proven that the presence of the 5-LO cds is crucial for the upregulating effect of calcitriol / TGFβ on 5-LO mRNA level. Cloning of the SV40 promoter instead of pN10 upstream of the 5-LO cds still showed inducibility by treatment with the inducers which argues for a promoter unspecific effect. Insertion of the 5-LO cds in a promoterless basic vector (pGL3cds) displayed same inductions by calcitriol / TGFβ treatment as the 5-LO promoter 5-LO cds construct (pN10cds). Thus, the effect of the inducers is not dependent on the 5-LO promoter under the in vitro conditions of the reporter gene assay. Hence, further cloning was done with promoterless constructs. Through 5-LO cds deletion constructs a positive regulating region in exon 10 to 14 was discovered. To adapt the natural gene context the last four introns (J-M) of the 5-LO gene were inserted in a promoterless construct containing exon 10 to 14 (pGL3cdsΔABInJM). 5end deletion constructs of it revealed putative vitamin D responsive elements (VDREs) in exon 12 and intron M. Mutation of the putative VDREs led to a reduced calcitriol effect –more prominent when the putative VDRE in intron M was mutated (reduction of 40%). Moreover another putative VDRE in exon 10 with an adjacent SMAD binding element (SBE) was detected. SMAD proteins are effector proteins of TGFβ signalling. Gelshift experiments demonstrated in vitro binding of the VDR-RXR heterodimer to those three putative VDREs. By chromatin immunoprecipitation (ChIP) assay in vivo binding of VDR and RXR was shown to the VDRE in the region of exon 10, exon 12 and intron M. 8h and 24h incubation with calcitriol / TGFβ resulted in enhanced expression of VDR in each of the examined regions. The VDR is able to bind to the VDRE without its ligand, whereas this goes along with corepressor recruitment and thus the VDR has a repressive effect on transcription. Histone H4 acetylation was increased when MM6 cells were treated for 8h or 24h with calcitriol or the combination of calcitriol / TGFβ. This finding implies that at that point of time corepressors associated with the VDR are replaced by coactivators. It seems convincing that 5-LO transcription is mainly promoted by calcitriol alone which leads to a more accessible chromatin structure. Previous data indicated that calcitriol and TGFβ upregulate 5-LO RNA maturation and 5- LO transcript elongation. Thus several elongation markers were investigated by ChIP analysis: Histone H3 lysine 36 (H3K36) trimethylation and H4K20 monomethylation were detected in the analysed regions in exon 10, exon 12 and intron M. In region exon 10 the H3K36 trimethylation status was enhanced after 24h calcitriol or calcitriol / TGFβ treatment. An increased H4K20 monomethylation status in all regions was observed when MM6 cells were treated for 24h with calcitriol / TGFβ. 24h treatment with both agents also enhanced the recruitment of the elongation form of RNA polymerase II, which is phosphorylated at serine 2 of the carboxyterminal domain, to the investigated regions. These findings prove the positive regulating role for calcitriol and TGFβ on 5-LO transcript elongation. A putative mechanism of the effect of calcitriol and TGFβ on 5-LO RNA maturation might be the elevated phosphorylation of serine 2 of the RNA Polymerase II which is known to be followed by recruiting polyadenylating factors.
Owing to higher performance on the Raven’s Progressive Matrices (RPM) than on the Wechsler Intelligence Scales (WIS), it has recently been argued that intelligence is underestimated in autism. This study examined RPM and WIS IQs in 48 individuals with autism, a mixed clinical (n = 28) and a neurotypical (n = 25) control group. Average RPM IQ was higher than WIS IQ only in the autism group, albeit to a much lesser degree than previously reported and only for individuals with WIS IQs <85. Consequently, and given the importance of reliable multidimensional IQ estimates in autism, the WIS are recommended as first choice IQ measure in high functioning individuals. Additional testing with the RPM might be required in the lower end of the spectrum.
Purim and parodies
(2008)