Η πρωτεΐνη La περιγράφηκε για πρώτη φορά το 1974 ως αυτοαντιγόνο στον ορό των ασθενών που έπασχαν από συστηματικό ερυθηματώδη λύκο και σύνδρομο Sjögren. Απαντάται σε μεγάλες ποσότητες μέσα στα κύτταρα, όπου υπό φυσιολογικές συνθήκες δεσμεύει τα πρόδρομα μετάγραφα της RNA πολυμεράσης ΙΙΙ προστατεύοντάς τα από την εξωνουκλεολυτική αποικοδόμηση. Επιπλέον, έχει βρεθεί ότι μπορεί να δεσμεύεται σε μία πληθώρα κυτταρικών και ιικών mRNA ρυθμίζοντας τη μετάφρασή τους. Πρόσφατες έρευνες έχουν επιβεβαιώσει το ρόλο της ανθρώπινης πρωτεΐνης ως RNA συνοδό (RNA chaperone) που συμβάλλει στην ορθή αναδίπλωση των μορίων RNA που δεσμεύει. Παρά το γεγονός ότι η La περιγράφηκε για πρώτη φορά στον άνθρωπο, ομόλογες πρωτεΐνες έχουν βρεθεί σε μία ποικιλία ευκαρυωτικών οργανισμών. Σε όλους αυτούς τους οργανισμούς η πρωτεΐνη παρουσιάζει μία κοινή οργάνωση επικρατειών. Στο Ν-τελικό άκρο υπάρχει το εξαιρετικά συντηρημένο μοτίβο La motif, το οποίο ακολουθείται από ένα τουλάχιστον μοτίβο RNA αναγνώρισης (RRM) και ένα αρκετά μεταβλητό C-τελικό άκρο. Παρότι ο λειτουργικός ρόλος της πρωτεΐνης έχει μελετηθεί επαρκώς, ελάχιστα είναι γνωστά για τη δομή της και τον τρόπο με τον οποίο δεσμεύεται στα φυσικά της υποστρώματα, όπως είναι τα πρόδρομα tRNA. Η παρούσα διατριβή εστιάζεται στη μελέτη τριών επιμέρους επικρατειών της πρωτεΐνης La του οργανισμού Dictyostelium discoideum, η οποία παρουσιάζει πολύ μεγάλη ομολογία και παρόμοια δομική οργάνωση με την ανθρώπινη πρωτεΐνη. Κάθε επικράτεια μελετάται ως προς τη δυνατότητα και τον τρόπο αλληλεπίδρασης με ομόλογα πρόδρομα tRNA του D. discoideum. Για το σκοπό αυτό, πραγματοποιήθηκε μοριακή κλωνοποίηση των επικρατειών La motif, RRM1 και La motif-RRM1 και υπερέκφρασή τους σε βακτηριακά κύτταρα. Οι ανασυνδυασμένες επικράτειες απομονώθηκαν σε δύο στάδια καθαρισμού και εξετάστηκαν ως προς την ικανότητα δέσμευσης στα πρόδρομα tRNA, με Electrophoresis Mobility Shift Assay και Micro Scale Thermophoresis. Επιπλέον, με ανάλυση αποτυπώματος (footprinting) αποκαλύφθηκαν τα σημεία της αλληλεπίδρασης του κάθε τομέα επάνω στο μόριο του pre-tRNAArg. Από τα αποτελέσματα προκύπτει ότι η La χρησιμοποιεί τις διαφορετικές επικρατειές της για να αναγνωρίζει ξεχωριστά σημεία επάνω στο μόριο του pre-tRNA. Τέλος, με τη χρήση φασματοσκοπίας NMR σε διάλυμα, επιβεβαιώθηκε ότι τόσο το La motif όσο και το RRM1 μπορούν να αναδιπλώνονται ανεξάρτητα σε μία διαμόρφωση που πιθανότατα διατηρούν και μέσα σε ολόκληρο το μόριο in vivo. / La is a highly abundant nuclear phosphoprotein. It was first described as an autoantigen found in the sera of patients suffering from systemic lupus erythematosus and Sjögren’s syndrome. Under normal conditions, La protein associates with the newly synthesized RNA polymerase III transcripts and protects them from exonucleases. It has also been reported that La associates with a variety of cellular and viral mRNAs and regulates their translation. Recent research has established the role of human La as an RNA chaperone that contributes to the proper folding of the associated RNA molecules. Although La was first described in human, homologs have been identified in a wide variety of eukaryotes. All these proteins share a common domain organization. The N-terminus of all known La proteins contains the highly conserved La motif. La motif is followed by a less conserved RNA Recognition motif (RRM) and a notably variable C-terminus. While the functional properties of La have been adequately investigated, information regarding the exact mode of binding and the structure remains elusive. This study focusses on the interaction between distinct domains of Dictyostelium discoideum La and homologous pre-tRNAs. La motif, RRM1 and La motif-RRM1 were cloned and overexpressed in E. coli. The recombinant domains were purified by affinity chromatography and subsequent FPLC. Each domain was assayed regarding its capacity to bind the pre-tRNA using Electrophoresis Mobility Shift Assay and Micro Scale Thermophoresis. Foot-printing analysis revealed the specific pre-tRNA recognition patterns for each domain. The results suggest that La uses its distinct domains to bind different sites of the pre-tRNA molecule. Finally, structural analysis based on solution NMR spectroscopy, confirmed the independent folding of the domains. Probably each domain preserves its tertiary structure in the wild type protein.

The message that climate change response should be central to municipal planning is clearly communicated in policy, science, and practice; and given that municipal planning is the core function of local government, the task of climate change response mainstreaming lies with them. There is however limited guidance offered to municipalities on how to go about mainstreaming climate response into planning. This study explores how climate change response, with a specific focus on adaptation, can be mainstreamed into South African local government planning instruments and processes. The study is largely framed in critical pragmatism in that it looks into real-world situations and appreciates context-specific complexity to make recommendations relevant to practice. Using a comparative case study design, two cases where mainstreaming has been undertaken were explored. Cape Town Metropolitan Municipality in the Western Cape Province and Thulamela Local Municipality in the Limpopo Province were selected as atypical cases with core similarities and contextual differences, which are able to offer information on the phenomenon of mainstreaming. A qualitative content analysis was undertaken of their latest Integrated Development Plans and Spatial Development Frameworks for both the cases, and the Built Environment Performance Plan in the case of Cape Town. Individual interviews were done in one case and a group interview in the other. Participants included spatial planners, environmental practitioners, and an infrastructure planner, all of whom have been involved in planning, climate change response and resilience. Similar themes with different findings emerged from the two cases, indicating that planning processes and experiences are very context-specific. The themes or factors that emerged can contribute to success in one case and cause significant challenges in another. These factors are a) champions, leadership and momentum, b) networks, mobilisation and organisation, c) information gathering, use and sharing, d) capacity, resources and skills, e) institutional support and coordination, and f) intergovernmental relations and mandate. The study contributes to the fields of local government, municipal planning, climate change adaptation mainstreaming, and the intersection between these fields. Insights are provided into the factors and conditions that can either support or hinder effective mainstreaming of climate change adaptation into local municipal planning instruments and processes, and recommendations are provided to support more effective mainstreaming in local government. / Dissertation (MTRP)--University of Pretoria, 2020. / Council for Scientific and Industrial Research (CSIR), International Development Research Centre (IDRC) / Town and Regional Planning / MTRP / Unrestricted

No description available.

Diverse research into the model organism, Escherichia coli, has added substantial depth to our understanding of genome editing of bacteria. Recombineering using the λ Red system is the most disruptive molecular technology discovered thus far, and improved our ability to introduce targeted single nucleotide variants by ~1E4 fold. This discovery has catalyzed incredible progress and enabled ambitious genome/organism engineering projects such as high throughput metabolic engineering to genome-wide codon reassignment. While efforts in E. coli have since accelerated further, work in other bacterial model organisms has lacked this catalyst and continues to fall behind E. coli. To facilitate development of disruptive technologies for non-standard model organisms, we produced a library of homologs to the λ Red recombinase, λ β (NP_040617.1), to generate a toolbox for recombinase discovery in organisms with minimal tools. We demonstrated the recombinase discovery workflow, called Serial Evolutionary Enrichment for Recombinases (SEER), in E. coli and present a number of alternatives to using λ Red for genome editing. We then moved on to explore λ β-mediated recombination in vitro where we able to show that bet specifically unloads E. coli Ssb from Ssb-coated oligos to facilitate annealing. We hypothesized that ssb represents the minimal host interaction node that a recombinase must achieve to facilitate recombination in vivo, and demonstrated a gain-of-function phenotype when species-matched recombinase/ssb pairs are ported into foreign organisms, potentially opening up poorly understood organisms to recombineering using well understood recombinase/ssb pairs.

The innovation of DNA analysis techniques has enabled arevolution in the field of molecular biology. In the 70’s,first technologies for sequence determination of DNA wereinvented and these techniques enormously increased thepossibilities of genetic research. A large proportion ofmethods for DNA sequencing is based on enzymatic DNA synthesiswith chain termination followed by electrophoretic separationand detection. However, alternative approaches have beendeveloped and one example of this is the pyrosequencingtechnology, which a four-enzyme DNA sequencing method based onreal-time monitoring of DNA synthesis. Currently, the method is limited to analysis of short DNAsequences and therefore it has primarily been used for mutationdetection and single-nucleotide polymorphism analysis. In orderto expand the use of the pyrosequencing technology, the readlength obtained in the methods needs to be improved. However,it was previously shown that the data quality in pyrosequencingtechnology could be significantly increased by addition ofEscherichia coli single-stranded DNAbinding protein, SSB, tothe sequencing reaction. Since little was known about themechanism of this enhancement, we performed a systematic effortto analyse the effect of SSB on 103 clones randomly selectedfrom a cDNA library. We investigated the effect of SSB on theobtained read length in pyrosequencing and identified thecauses of low quality sequences. Moreover, the effciency ofprimer annealing and SSB binding for individual cDNA clones wasinvestigated by use of real-time biosensor analysis. Resultsfrom these experiments show that templates with highperformance in pyrosequencing without SSB possess effcientprimer annealing and low SSB affnity. To minimise the cost of the pyrosequencing system, effcientand scaleable procedures for production and isolation of theprotein components are required. Therefore, protocol foreffcient expression in E.coliand rapid isolation of native SSB was developed.Moreover, by use of a gene fusion strategy, Klenow polymerasewas produced in fusion with the Zbasic domain at high levels inE. coli. This highly charged protein handle enables selectiveand effcient ion exchange purification at physiological pH.Furthermore, active Apyrase was expressed in Methyltropic yeastPichia pastoris and purified by two chromatographic steps. Since pyrosequencing analysis mainly is performed in a96-sample plate format, an increase in sample capacity would bevery beneficial. One approach to achieve this would be to usemicromachined filter chamber arrays where nano-liter samplescan be monitored in real-time. However, to enable accuratepyrosequencing analysis of parallel samples, the produced lightshould preferable be docked to the correct DNA template.Therefore, two different gene fusion strategies were utilisedbased on directed immobilisation of the light-harvesting enzymeLuciferase on the DNA molecules. The thermostable variant ofthe enzyme was genetically fused to a DNA binding protein(either SSB or Klenow) and the Zbasic purification handle, which could beselectively removed by protease cleavage. A protocol wasdeveloped for effcient expression in E.coliand purification by Ion Exchange Chromatography.The proteins were analysed by complete extension of DNAtemplates immobilised on magnetic beadspyrosequencing monitoredby pyrosequencing chemistry. Results from these experimentsshow that the proteins bound selectively to the immobilised DNAand that their enzymatic domains were active. In summary, the work presented in this thesis pinpointsfeatures in the pyrosequencing technology that needs to befurther developed. Moreover, various protein-based strategiesare presented in order to overcome these limitations. <b>Keywords:</b>pyrosequencing, SSB, Zbasic, Klenow, Apyrase, expression, purification,Biacore, DNA template length, Luciferase, affnity, gene fusion,immobilisation.

<p>The innovation of DNA analysis techniques has enabled arevolution in the field of molecular biology. In the 70’s,first technologies for sequence determination of DNA wereinvented and these techniques enormously increased thepossibilities of genetic research. A large proportion ofmethods for DNA sequencing is based on enzymatic DNA synthesiswith chain termination followed by electrophoretic separationand detection. However, alternative approaches have beendeveloped and one example of this is the pyrosequencingtechnology, which a four-enzyme DNA sequencing method based onreal-time monitoring of DNA synthesis.</p><p>Currently, the method is limited to analysis of short DNAsequences and therefore it has primarily been used for mutationdetection and single-nucleotide polymorphism analysis. In orderto expand the use of the pyrosequencing technology, the readlength obtained in the methods needs to be improved. However,it was previously shown that the data quality in pyrosequencingtechnology could be significantly increased by addition ofEscherichia coli single-stranded DNAbinding protein, SSB, tothe sequencing reaction. Since little was known about themechanism of this enhancement, we performed a systematic effortto analyse the effect of SSB on 103 clones randomly selectedfrom a cDNA library. We investigated the effect of SSB on theobtained read length in pyrosequencing and identified thecauses of low quality sequences. Moreover, the effciency ofprimer annealing and SSB binding for individual cDNA clones wasinvestigated by use of real-time biosensor analysis. Resultsfrom these experiments show that templates with highperformance in pyrosequencing without SSB possess effcientprimer annealing and low SSB affnity.</p><p>To minimise the cost of the pyrosequencing system, effcientand scaleable procedures for production and isolation of theprotein components are required. Therefore, protocol foreffcient expression in E.<i>coli</i>and rapid isolation of native SSB was developed.Moreover, by use of a gene fusion strategy, Klenow polymerasewas produced in fusion with the Zbasic domain at high levels inE. coli. This highly charged protein handle enables selectiveand effcient ion exchange purification at physiological pH.Furthermore, active Apyrase was expressed in Methyltropic yeastPichia pastoris and purified by two chromatographic steps.</p><p>Since pyrosequencing analysis mainly is performed in a96-sample plate format, an increase in sample capacity would bevery beneficial. One approach to achieve this would be to usemicromachined filter chamber arrays where nano-liter samplescan be monitored in real-time. However, to enable accuratepyrosequencing analysis of parallel samples, the produced lightshould preferable be docked to the correct DNA template.Therefore, two different gene fusion strategies were utilisedbased on directed immobilisation of the light-harvesting enzymeLuciferase on the DNA molecules. The thermostable variant ofthe enzyme was genetically fused to a DNA binding protein(either SSB or Klenow) and the Z_basic purification handle, which could beselectively removed by protease cleavage. A protocol wasdeveloped for effcient expression in E.<i>coli</i>and purification by Ion Exchange Chromatography.The proteins were analysed by complete extension of DNAtemplates immobilised on magnetic beadspyrosequencing monitoredby pyrosequencing chemistry. Results from these experimentsshow that the proteins bound selectively to the immobilised DNAand that their enzymatic domains were active.</p><p>In summary, the work presented in this thesis pinpointsfeatures in the pyrosequencing technology that needs to befurther developed. Moreover, various protein-based strategiesare presented in order to overcome these limitations.</p><p><b>Keywords:</b>pyrosequencing, SSB, Z_basic, Klenow, Apyrase, expression, purification,Biacore, DNA template length, Luciferase, affnity, gene fusion,immobilisation.</p>

No description available.

This thesis explores the conversation surrounding the recent attempts by the Vermont Legislature to pass a Sugar-Sweetened Beverage tax in the years 2014-2016. We explore the common perceptions expressed by a sample of Vermont residents and also look at how Vermont media outlets portrayed the tax through frames of reference. Framing is a method of emphasizing certain points of an issue. This thesis reports the common opinions of Vermonters, the media framing of the issue, and if there is any relationship between them in two academic journal articles. The first article looks at the common frames used in Vermont media during the 2014-2016 period. Classifying 10 pro- and anti-tax frames from 30 common arguments, the article analyzes the use of these frames, their prevalence in different news outlets, and their frequency during time periods. The article also looks at sponsors of these frames and measures which frames individuals and organizations are sponsoring. The study finds that anti-tax advocates most often cite economic hindrances as a reason to oppose the tax and pro-tax advocates predominately cite health benefits and economic tax benefits as a reason to support the tax. In the final year, pro-tax advocates sponsored economic benefits more than any other frames and this argument coincided with the statewide discussion of a budget shortfall. The second article measures the relationship between the media portrayal of the Sugar-Sweetened Beverage tax and the opinions of Vermont citizens regarding the tax. By looking at the prevalence of pro- and anti-tax frames usage in each year, a logistic regression model was built to measure the odds of people favoring tax based off of independent variables, including frames. Vermont residents fluctuated in their opinion of the tax over the years. It was found that in 2015, pro-tax frames made people more likely to support the tax. Democrats were also more likely to support the tax and Republicans were more likely to oppose the tax. This thesis provides insight into the conversation surrounding Sugar-Sweetened Beverage taxes in Vermont. It helps to shed light on the issue, how different groups feel about the issue, and how frames of thought presented through the media can relate to Vermonters' opinion of the tax.

Genome stability has great importance in maintaining cell viability and optimal functionality of cellular processes. Loss of genome stability can lead to cell death in the simplest organisms and to deregulation of the cell proliferation machinery in higher organisms, potentially causing cancer or morbid states. The Single Stranded DNA Binding (SSB) protein of Escherichia coli is an essential protein that binds and stabilises ssDNA stretches. Its role is particularly crucial during DNA replication, recombination and repair processes and it has therefore been predicted to play a prominent role in the maintenance of genome stability. The role of SSB in genome instability was investigated using an E. coli strain in which, the expression of the ssb gene was placed under the control of the arabinose promoter. The level of SSB protein present in the cell could therefore be tuned by varying the arabinose concentration in the medium. A wide characterisation of the behaviour of the strain at low SSB level was carried out. Viability and growth tests showed that a threshold level of protein is required to allow normal growth. Microscopy analyses were carried out to follow cell division, nucleoid morphology and SOS response activation. Cells grown at low SSB level, showed a phenotype consistent with impaired cell division and altered nucleoid morphology. The SOS response was activated at low SSB levels and cell elongation was detected. Lowering the arabinose concentration in solid medium allowed the selection of suppressor strains that could form colonies under the new conditions. Sequencing of the entire genome of one such suppressor strain was carried out revealing a possible candidate for the phenotype change. The stability of a 105bp and of a 246bp DNA imperfect palindromes and the stability of CAG·CTG trinucleotide repeat arrays, inserted in the E. coli chromosome, were investigated in correlation to the SSB cellular level. Lowering the SSB level in cells grown on solid medium, increased the instability of the 105bp palindrome presumably by increasing the number of slippage events. On the other hand, SSB overexpression did not have an effect on the stability of the 246bp palindrome. The stability of a leading strand (CAG)75 repeat array was highly increased by overexpressing SSB, while the same effect was not observed for a leading strand (CTG)137 repeat array. Furthermore, excess SSB caused a change in the deletion size distribution profile for the leading strand (CAG)75 strain, lowering the bias towards big deletions. This is consistent with SSB being able to preferentially impede the formation of big DNA hairpins. Also, SbcCD nuclease was shown to have an effect on the deletion size distribution profile of the leading strand (CTG)137 strain. The lack of SbcCD led to a slight reduction of the number of big deletions.

There are four mammalian SSB proteins (SSB-1 to -4), and these are characterized by a C-terminal SOCS box and central SPRY domain. The C-terminal SOCS box was first observed in proteins that were found to act as Suppressors of Cytokine Signalling and function by virtue of their SH2 domain. Other families containing the SOCS box motif were defined by the domains N-terminal to this, such as the ASBs (Ankyrin repeats), WSBs (WD40 repeats) and of course the SSBs (SPRY domains). This thesis describes a very broad investigation of the SSBs, a protein family about which very little was known. To begin with, functional investigation into the evolution of this family and analysis of murine SSB expression patterns was performed. This highlighted that the family was highly conserved and had differential expression in the mouse, suggestive of important, unique functional roles for the individual family members. The majority of work in the thesis then proceeds in three directions; (i) analysis of the SSB proteins in vivo, with genetic deletion of SSB-2 in the mouse, (ii) biochemically, with analysis of SSB binding partners, and (iii) structurally, with functional analysis of the structure of SSB-2.