This thesis describes the design and development of the SmartBadge; an electronic replacement for the standard paper name badge worn at conferences and similar events. Both hardware and software have been designed for the SmartBadge; the hardware has been developed around a CC1010 microcontroller and RF transceiver. Attached to this are an infrared transceiver, an LCD display, some LEDs, buttons and a piezoelectric buzzer. There is also an antenna for the RF transceiver whose design is the result of SuperNEC [1] simulations. Protocol software development has focussed on the communication between a SmartBadge and other badges and base stations, yet there is still space available in the CC1010s flash memory to develop applications beyond the business card exchange example developed to demonstrate the communication software. The SmartBadge communicates with other badges by using the infrared transceiver. In the business card application a SmartBadge is worn by a person and is collecting the ID and a time counter from SmartBadges worn by other facing people as this person mingles through a conference or similar event. This data is then collected in real time using the RF transceiver to communicate with base stations which would be scattered around the venue. The RF network has been designed as a single hop network and a new Medium Access Control (MAC) protocol has been designed to allow the SmartBadges to share the links to the base stations while conserving as much energy as possible. This protocol is called Uplink MAC (or U-MAC) and is described in section 6.2.

The construction industry is characterised by a number of problems in crucial fields such as health, safety and logistics. Since these problems affect the progress of construction projects, the construction industry has attempted to introduce the use of innovative information and communication technologies on the construction site. Specific technologies which find applicability on the construction site are wireless sensors, and especially radio-frequency identification (RFID) technology. RFID tagging is a technology capable of tracking items. The technology has been applied on the construction site for various applications, such as asset tracking. There are many problems related to health, safety and logistics on the construction site which could be resolved using RFID technology. In the health and safety field, the problems which exist are the monitoring of dangerous areas on the construction site, such as large excavation areas, the collisions between workers and vehicles, between vehicles and equipment and between vehicles, the detection of hazardous substances on the construction site when the construction work has been completed and the collection of hazard notifications from specific areas of the construction site as feedback for the prevention of future accidents. In the logistics field, the tracking of a material during its delivery on the construction site, its transportation to specific subcontractors and its future utilisation as well as the monitoring of the rate of use of materials on the construction site, the checking of the sequence of steel members and the monitoring of the temperature of porous materials are issues which can be realised using RFID technology. In order to facilitate the use of RFID technology for the specific health, safety and logistics problems, a system has been developed. The operation of this system is based on the combined use of hardware and software elements. The hardware elements of the developed system are a wireless local area network, RFID readers and tags. Its software elements are a software development kit based on which, a number of graphical user interfaces have been created for the interaction of the users with the REID tags, and Notepad files which store data collected from REID tags through the graphical user interfaces. Each of the graphical user interfaces is designed in such a way so that it corresponds to the requirements of the health, safety or logistics situation in which it is used. The proposed system has been tested on a simulated construction site by a group of experts and a number of findings have been produced. Specifically, the testing of the proposed system showed that RFID technology can connect the different stages which characterise the construction supply chain. In addition, it showed the capability of the technology to be integrated with construction processes. The testing of the system also revealed the barriers and the enablers to the use of RFID technology in the construction industry. An example of such a barrier is the unwillingness of the people of the construction industry to quit traditional techniques in favour of a new technology. Enablers which enhance the use of RFID technology in the construction industry are the lack of complexity which characterises the operation of RFID tagging and the relatively low cost of RFID tags. In general, RFID technology is an innovative sensor technology which can help the construction industry through its asset tracking ability. However, further research should be done on the improvement of RFID technology on specific characteristics, such as its inability to provide location coordinates and the resilience of the electromagnetic signal emitted by the RFID reader when there are metallic objects around the reader.

Acid proteases, commonly known as aspartic proteases are degredative enzymes which catalyze the cleavage reaction of peptide bonds in proteins with a pH optimum in the acidic range (pH 3-4). Acid proteases have crucial roles in metabolism. Moreover, they are used in different fields of industry. Thermophilic microorganisms, especially archaea, gain special interest because of their thermal stability for both fundamental and industrial researches. Thermopsin is an extracellular acid protease and a member of A5 family of proteases. This thermophilic enzyme has no characteristic active aspartyl residue, is insensitive to pepstatin and no apparent sequence homology to other acid proteases and therefore represents a new class of acid proteases. Thermophilic archaeal strain Thermoplasma volcanium GSS1 (optimum temperature 550C and pH 2.7) in the genome has a putative thermopsin gene encoding 998 amino acid enzyme. In this study thermopsin gene from Thermoplasma volcanium was expressed in E. coli as fusion with 6xHis tag under the control of T5 transcription/translation system. Putative thermopsin gene from Thermoplasma volcanium was amplified by PCR method using two primer sets and cloned. A 3080 bp and a 3070bp PCR products were obtained by using TP1/TP2 primer set (thermopsin gene with the start codon) and TP1&rsquo / /TP2 primer set (thermopsin gene missing start codon) respectively. PCR amplified thermopsin genes pDrive and pUC18 vectors in E. coli TG1 were cloned using and then cloned genes were sub-cloned directionally into pQE triple vector set for expression. In these expression vectors, cloned genes are placed downstream of a 6XHis tag to produce an expression fusion. E.coli strains (M15[pREP4], SG13009[pREP4], and TG1) used as hosts. Recombinant colonies screened by colony blot/hybridization method based on immunological detection of the expressed 6XHis tag fusion by Anti-His HRP conjugates which are specific for 6xHis tag, and DAB chromogenic substrate was used for colony blot procedure. PCR amplified thermopsin gene containing 3080bp could not expressed in pQE30 and 31 vectors in TG1 strains. It is thought that pQE32 open reading frame can be true for thermopsin gene (3080bp). Three expression constructs, pQE31-1, pQE31-4 and pQE31-6 plasmids containing PCR amplified 3070bp thermopsin gene were confirmed as true recombinant plasmids according to both colony blot hybridization result and restriction digestion profile the agarose gel.

Ribosomal stalk proteins are known to play important role in protein synthesis. The ‘stalk’, an extended structure on the large subunit of the ribosome is composed mainly of two to three dimers of L12 and one L10 protein, which forms the base of the stalk. In E. coli, four copies of L12 molecules exist as dimer of dimers forming the pentameric L8 complex together with L10. This thesis is a collection of four interlinked studies on the structure, function and application of the ribosomal stalk protein L12. In the first study, we have mapped the interaction sites of the four major translation GTPase factors (IF2, EF-Tu, EF-G & RF3) on L12 molecule using heteronuclear NMR spectroscopy. Surprisingly, all these factors produced an overlapping interaction map spanning two α-helices on the C terminal domain of L12, thereby suggesting a general nature of the interaction between L12 and the GTPase factors. L12 is known to stimulate GTPase activity of the elongation factors EF-Tu and EF-G. Here, we have clarified the role of L12 in IF2 mediated initiation of protein synthesis. Our data suggest that rapid subunit association requires a specific interaction between the L12 protein on the 50S and IF2·GTP on the 30S preinitiation complex. We have also shown that L12 is not a GAP for IF2 and GTP hydrolysis triggers IF2 release from the 70S initiation complex. The next question we have addressed is why multiple copies of L12 dimer are needed on the ribosome. For this purpose, we created a pure E. coli strain JE105, where the terminal part of rplJ gene coding for the binding site of one L12 dimer on protein L10 was deleted in the chromosomal locus. Using ribosomes with single L12 dimer we have observed that the rate of the initiation and elongation involving IF2 and EF-G gets most compromised, which in turn decreases the growth rate of the bacteria.  This study also indicates that L12 can interact with different GTPase factors in a specialized manner. Lastly, we have developed an application making advantage of the multiple L12 dimers on the ribosome. By inserting a (His)6-tag at the C-terminus of the L12 protein we have created a novel E. coli strain (JE28), where all ribosomes are tetra-(His)6-tagged. Further, we have developed a single step method for purification of the active (His)6-tagged ribosomes from JE28.

Thesis (Ph. D.)--Utrecht University, 2007. / Afterword and vita in both English and Dutch. Includes bibliographical references (p. 115-120).

Univ., Magisterarb., 2000--Kassel

Thesis (MSc (Genetics))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The South African abalone, Haliotis midae, is the only endemic species of commercial value. Aquaculture remains the only avenue for expanding the industry, since the closure of the fishery. The current focus is on implementing a molecular breeding programme; thus the development of molecular markers for linkage mapping and QTL analysis is a priority. Various markers, mainly anonymous, have been developed for H. midae; however emphasis is being placed on the development of gene-linked type I molecular markers. The present study investigates and demonstrates the use of public sequence collections to develop type I markers for a species with limited genomic resources, via three strategies: Surveying anonymous H. midae microsatellite markers’ flanking regions to find homology to gene sequences in public databases, cross-species marker transfer of anonymous markers from H. rubra and H. discus hannai demonstrating putative gene associations and lastly EST marker mining (SNP and microsatellites) from various Haliotids and testing transfer to the target species. Approximately 17% of H. midae anonymous markers showed significant similarity to genes. The current study also reports higher cross-species transferability from both H. rubra and H. discus hannai to H. midae (39% and 20.5%, respectively) than previously demonstrated and 15 EST-microsatellites and 16 EST-SNPs were successfully mined. Furthermore, the non-random distribution of microsatellites and high nucleotide diversity in the H. midae genome was confirmed. This is a low cost and time effective method for marker development and presents a continuous and dynamic resource that could be used for future marker development and characterisation as sequence information in public databases grow exponentially. / AFRIKAANSE OPSOMMING: Die Suid-Afrikaanse perlemoen, Haliotis midae, is die enigste van vyf inheemse spesies van kommersiële waarde. Na die noodgedwonge sluiting van die vissery, is akwakultuur die mees praktiese oplossing om die perlemoen industrie uit te brei. Die huidige fokus is gerig op die implementering van ‘n molekulêre teel-program en dus is die ontwikkeling van molekulêre merkers vir genetiese kartering en kwantitatiewe kenmerk lokus analise, van uiterste belang. Tipe II merkers is voorheen vir die perlemoen ontwikkel, maar huidige tendense lê klem op die ontwikkeling van geen-gekoppelde tipe I merkers. Die huidige studie ondersoek die gebruik van publieke databasisse vir die ontwikkeling van tipe I molekulêre merkers vir ‘n spesie met beperkte genomiese bronne. Drie strategieë is geïmplementeer: Eerstens is ‘n opname gemaak van die homologie van perlemoen tipe II merker-vleuelende volgordes met geen volgordes in databasisse. Verder is die oordraagbaarheid van tipe II merkers vanaf H. rubra en H. discus hannai wat assosiasie met gene toon ondersoek. Laastens is ‘n Uitgedrukte Volgorde Merk (UVM) (Expressed Sequence Tag, EST) merker-ontginnings metode vanaf verskeie Haliotis spesies en toetsing van oordraagbaarheid na die teiken spesie uitgevoer. Ongeveer 17% van die tipe II H. midae merkers het geniese assosiasie getoon. ‘n Hoër tussen-spesie oordraagbaarheid vanaf beide H. rubra en H. discus hannai na H. midae (39% en 20.5%, onderskeidelik) word gerapporteer in vergelyking met vorige studies en 15 UVM-mikrosatelliete en 16 UVM-enkel nukleotied polimorfismes (single nucleotide polimorphism, SNP) is ontwikkel. Verder bevestig die studie die nie-lukrake verspreiding van mikrosatelliete en hoë nukleotied diversiteit in die perlemoen genoom. Die gebruik van publieke databasise vir die ontwikkeling en karakterisering van tipe I molekulêre merkers is tyd- en koste-besparend en bied ‘n volgehoue en dinamiese bron vir toekomstige gebruik.

The Voltage-Dependent Anion Channel (VDAC) is a channel located in the outer mitochondrialmembrane of nearly all eukaryotic cells. It is responsible for the passage of numerous ions andmetabolites in and out of the mitochondria but is also involved in the regulation of the cell functionthrough interactions with other proteins. Its activity is known to be modulated by lipids. Experimentson Phaseolus coccineus VDAC32 (PcVDAC32) have notably suggested a direct interaction betweendioleoylphosphatidylethanolamine (DOPE) head group and the bean VDAC32. Moreover, moleculardynamic simulations have proposed that charged residues of the mouse VDAC1 could be involved indirect interaction with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) polar head.During this PhD thesis, we first constructed and optimised expression systems for production ofPcVDAC32 and of Saccharomyces cerevisiae VDAC1 (ScVDAC1) in yeast and assessed those forcomplementation in yeasts lacking their endogenous VDAC1. We then tested the effect of a polyhistidine-tag placed in C-terminal of the ScVDAC1 on its electrophysiological properties to validate itas a tool for VDAC study. We further analysed the effect of the lipid environment on the ScVDAC1and compared it with the results obtained for PcVDAC32. Finally, we assessed the effect of thesuppression of the Glu185 charge on the ScVDAC1 conductance, selectivity and voltage dependence.We found that expression of PcVDAC32 could complement the growth deficiency of the yeast lackingendogenous VDAC. We also showed that the presence of calcium ions in the experimental solutionsallowed the ScVDAC1 closed states to reach lower conductances. We observed that preincubation withergosterol greatly enhanced the reconstitution of ScVDAC1 in soy extract PLB. We demonstrated thatpH and salt concentration influence the ScVDAC1 functional characteristics and that, according tothose parameters, the presence of a 6xHis-tag can influence VDAC functions. Finally, we showed thatthe ScVDAC1 Glu185 located in the loop between β-strand 12 and 13 is involved in ScVDAC1selectivity and that its substitution by a glutamine residue decreases the ScVDAC1 sensitivity tomembrane curvature stress. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program / Timothy P. Durrett / Many Euonymus species express an acetyltransferase enzyme in their seeds which catalyzes the transfer of an acetyl group from acetyl-CoA to the sn-3 position of diacylglycerol (DAG) producing unusual acetyl-1,2-diacyl-sn-glycerols (acetyl-TAG). The presence of the sn-3 acetate group gives acetyl-TAG with unique physical properties over regular triacylglycerol (TAG) found in vegetable oils. The useful characteristics of acetyl-TAG oil offer advantages for its use as emulsifiers, lubricants, and 'drop-in' biofuels. One enzyme, Euonymus alatus diacylglycerol acetyltransferase (EaDAcT), responsible for acetyl-TAG synthesis in nature was previously isolated from the seeds of Euonymus alatus (burning bush) and expressed in the oilseed crop Camelina sativa. Expression of EaDAcT successfully led to production of high levels of acetyl-TAG in camelina seeds. To further increase acetyl-TAG accumulation in transgenic camelina seeds, multiple strategies were examined in this study. Expression of a new acetyltransferase enzyme (EfDAcT) isolated from the seeds of Euonymus fortunei, which was previously shown to possess higher in vitro activity and in vivo acetyl-TAG levels compared to EaDAcT, increased acetyl-TAG accumulation by 20 mol%. Suppression of the endogenous competing enzyme DGAT1 further enhanced acetyl-TAG accumulation to 90 mol% in selected transgenic line. Studying the regulation of EfDAcT transcript, protein, and acetyl-TAG levels during seed development further provided new insights on the factors limiting acetyl-TAG accumulation.

Metal ions play essential roles in biological functions including catalysis, protein stability, DNA-protein interactions and cell signaling. It is estimated that 30% of proteins utilize metals in some fashion. Additionally, methods by which metal ions can be visualized have been utilized to study metal concentrations and localizations in relation to disease. Understanding the roles metals play in biological systems has great potential in medicine and technology. Chapters 1 and 2 of this dissertation analyzes the structure and function of the Mn-dependent enzyme oxalate decarboxylase (OxDc) and Chapter 2 presents a bioinformatic analysis of the cupin superfamily that provides the structural scaffold of the decarboxylase. The X-ray crystal structure of the W132F variant was determined and utilized together with EPR data to develop a computational approach to determining EPR spectra of the enzyme’s two metal-binding centers. Furthermore, a variant in which the catalytic Glu162 was deleted revealed the binding mode of oxalate, the first substrate-bound structure of OxDc. OxDc is a member of the cupin superfamily, which comprises a wide variety of proteins and enzymes with great sequence and functional diversity. A bioinformatics analysis of the superfamily was performed to analyze how sequence variation determines function and metal utilization. Chapters 3 and 4 discuss the expansion of lanthanide-binding tags (LBTs) to in cellulo studies. Lanthanide-binding tags are short sequences of amino acids that have high affinity and selectivity for lanthanide ions. An EGF-LBT construct used to quantify EGF receptors on the surface of A431 and HeLa cells. The results from the LBT quantification are consistent with previous studies of EGFR receptors in these cell types, validating the use of this method for future studies. The potential of using LBTs for X-ray fluorescence microscopy (XFM) was also investigated. LBT-labeled constructs were utilized to investigate if membrane bound as well as cytosolic LBT-containing proteins could be visualized and localized to their cell compartments via XFM; both membrane-localized and cytosolic proteins were successfully visualized. With the high resolution (< 150 Å) obtainable with new synchrotron beamline configurations LBTs could be used to study nanoscale biological structures in their near-native state.