The twin arginine translocase pathway : a functional and structural study in Escherichia coli and Synechocystis sp. PCC6803

The bacterial twin-arginine translocase (Tat) pathway is able to export large folded proteins across the cytoplasmic membrane. It derives its name from the almost invariant twin-arginine motif present in the signal peptide of Tat substrates. Components of the Tat pathway have been identified in many bacteria, and in Escherichia coli one of these essential proteins, TatA, forms large complexes of variable size. The research presented in this thesis sought to gain insight into the Tat pathway in E. coli and the cyanobacterium Synechocystis sp. PCC6803, mainlyfocusing on the TatA component of the E. coli Tat pathway and how its various domains contribute to function and complex formation. Green fluorescent protein tagged with a Tat signal peptide was used to compare the Tat pathway in E. coli and Synechocystis, using biochemical and bioimaging techniques. Substitution of the twin-arginine motif with lysine residues highlighted differences between the specificity of the two systems. The transmembrane (TM) domain and amphipathic helix (APH) of E. coli TatA were analysed using synthetic peptides and the TOXCAT assay. Investigation of the TM domain demonstrated it is α-helical and spontaneously inserts into membranes. It interacts relatively weakly and substitution of a glutamine does not affect these interactions or function of the protein at high expression levels. In contrast, the APH only forms an α-helical secondary structure in high detergent concentration or in the presence of negatively charged lipids, and does not insert into lipid bilayers. Analysis of the unstructured C-terminus of TatA led to the identification of an acidic motif just after the APH. Removal of this motif has a severe effect on function and complex formation of TatA. Removal of the C-terminus does not affect the proofreading abilities of TatA. The research presented here has led to the proposal of a new model for TatA complex formation in E. coli – the weak interactions of the TM domains bring the monomers together, whilst interactions between the acidic motif and other regions stabilise these complexes

Unseen and unheard? Women managers and organizational learning

This paper aims to use (in)visibility as a lens to understand the lived experience of six women managers in the headquarters of a large multinational organization in the UK to identify how “gender” is expressed in the context of organizational learning. Design/methodology/approach: The researchers take a phenomenological approach via qualitative data collection with a purposeful sample – the six female managers in a group of 24. Data were collected through quarterly semi-structured interviews over 12 months with the themes – knowledge, interaction and gender. Findings: Organizations seek to build advantage to gain and retain competitive leadership. Their resilience in a changing task environment depends on their ability to recognize, gain and use knowledge likely to deliver these capabilities. Here, gender was a barrier to effective organizational learning with women’s knowledge and experience often unseen and unheard. Research limitations/implications: This is a piece of research limited to exploration of gender as other, but ethnicity, age, social class, disability and sexual preference, alone or in combination, may be equally subject to invisibility in knowledge terms; further research would be needed to test this however. Practical implications: Practical applications relate to the need for organizations to examine and address their operations for exclusion based on perceived “otherness”. Gendered organizations cause problems for their female members, but they also exclude the experience and knowledge of key individuals as seen here, where gender impacted on effective knowledge sharing and cocreation of knowledge. Social implications: The study offers further evidence of gendered organizations and their impacts on organizational effectiveness, but it also offers insights into the continues social acceptance of a masculinized normative model for socio-economic practice. Originality/value: This exploration of gender and organizational learning offers new insights to help explain the way in which organizational learning occurs – or fails to occur – with visibility/invisibility of one group shaped by gendered attitudes and processes. It shows that organizational learning is not gender neutral (as it appears in mainstream organizational learning research) and calls for researchers to include this as a factor in future research

The relationship between psychopathy and indirect aggression in a community sample

This thesis sought to investigate the relationship between psychopathy and indirect aggression use. Psychopathy has been strongly linked with increased levels of aggressive behaviour and in particular violence (Porter ;Woodworth, 2006). However, thus far research has predominantly focused on direct forms of aggression with minimal research considering indirect forms of aggression. On the basis of previous research, it was hypothesised that not only would psychopathy be significantly related to indirect aggression use, but that this relationship would remain after controlling for the shared variance with direct aggression. It was also hypothesised that this relationship would be mediated by deficits in affective empathy and moderated by both gender and levels of social skills. A series of quasi-experimental studies were conducted to test this hypothesis using regression analysis and structural equation modelling. Study 1 sought to test the basic relationship between psychopathy and indirect aggression using the Psychopathic Personality Inventory - Revised and the Indirect Aggression Scale respectively on a sample of 103 university students. Study 2 ;3 then expanded this and sought to investigate both the role of empathy, using the Empathy Quotient (Study 2), and gender (Study 3) using a sample of 201 university students, 83 males and 118 females. Study 4 used the Social Skills Inventory in a sample of 107 students to test the hypothesised social skill moderation of this relationship. Finally Study 5 and 6 sought to redress issues of both the limited samples and use of self-report measures in the previous studies by replicating these findings in a general community population of 204 (Study 5) and using behavioural measures of empathy on a sample of 117 (Study 6).The results indicate that psychopathy is significantly related to the use of indirect aggression, even after controlling for direct aggression, and that this was driven predominantly by the impulsive antisociality and coldheartedness factors. This relationship was found to be significantly mediated by affective, but not cognitive, empathy deficits although only for males, not for females, which may arguably point towards differences in the function of indirect aggression for male compared to female psychopaths. Non-verbal social skills were found to significantly moderate this relationship among students, however this finding could not be replicated. These findings would appear to imply that psychopathy is related to a general increase in aggression, rather than a specific increase in violence. This supports the theorisation of non-criminal psychopathy as a moderated behavioural manifestation of the underlying personality traits rather than a sub-clinical version of the disorder. The sex differences in the relationship would seem to imply that the different types of aggression use may have different underlying meanings for males and females high on psychopathic traits.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

The twin arginine translocase pathway : a functional and structural study in Escherichia coli and Synechocystis sp. PCC6803

The bacterial twin-arginine translocase (Tat) pathway is able to export large folded proteins across the cytoplasmic membrane. It derives its name from the almost invariant twin-arginine motif present in the signal peptide of Tat substrates. Components of the Tat pathway have been identified in many bacteria, and in Escherichia coli one of these essential proteins, TatA, forms large complexes of variable size. The research presented in this thesis sought to gain insight into the Tat pathway in E. coli and the cyanobacterium Synechocystis sp. PCC6803, mainlyfocusing on the TatA component of the E. coli Tat pathway and how its various domains contribute to function and complex formation. Green fluorescent protein tagged with a Tat signal peptide was used to compare the Tat pathway in E. coli and Synechocystis, using biochemical and bioimaging techniques. Substitution of the twin-arginine motif with lysine residues highlighted differences between the specificity of the two systems. The transmembrane (TM) domain and amphipathic helix (APH) of E. coli TatA were analysed using synthetic peptides and the TOXCAT assay. Investigation of the TM domain demonstrated it is α-helical and spontaneously inserts into membranes. It interacts relatively weakly and substitution of a glutamine does not affect these interactions or function of the protein at high expression levels. In contrast, the APH only forms an α-helical secondary structure in high detergent concentration or in the presence of negatively charged lipids, and does not insert into lipid bilayers. Analysis of the unstructured C-terminus of TatA led to the identification of an acidic motif just after the APH. Removal of this motif has a severe effect on function and complex formation of TatA. Removal of the C-terminus does not affect the proofreading abilities of TatA. The research presented here has led to the proposal of a new model for TatA complex formation in E. coli – the weak interactions of the TM domains bring the monomers together, whilst interactions between the acidic motif and other regions stabilise these complexes.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

The twin arginine translocase pathway : a functional and structural study in Escherichia coli and Synechocystis sp. PCC6803

The bacterial twin-arginine translocase (Tat) pathway is able to export large folded proteins across the cytoplasmic membrane. It derives its name from the almost invariant twin-arginine motif present in the signal peptide of Tat substrates. Components of the Tat pathway have been identified in many bacteria, and in Escherichia coli one of these essential proteins, TatA, forms large complexes of variable size. The research presented in this thesis sought to gain insight into the Tat pathway in E. coli and the cyanobacterium Synechocystis sp. PCC6803, mainlyfocusing on the TatA component of the E. coli Tat pathway and how its various domains contribute to function and complex formation. Green fluorescent protein tagged with a Tat signal peptide was used to compare the Tat pathway in E. coli and Synechocystis, using biochemical and bioimaging techniques. Substitution of the twin-arginine motif with lysine residues highlighted differences between the specificity of the two systems. The transmembrane (TM) domain and amphipathic helix (APH) of E. coli TatA were analysed using synthetic peptides and the TOXCAT assay. Investigation of the TM domain demonstrated it is α-helical and spontaneously inserts into membranes. It interacts relatively weakly and substitution of a glutamine does not affect these interactions or function of the protein at high expression levels. In contrast, the APH only forms an α-helical secondary structure in high detergent concentration or in the presence of negatively charged lipids, and does not insert into lipid bilayers. Analysis of the unstructured C-terminus of TatA led to the identification of an acidic motif just after the APH. Removal of this motif has a severe effect on function and complex formation of TatA. Removal of the C-terminus does not affect the proofreading abilities of TatA. The research presented here has led to the proposal of a new model for TatA complex formation in E. coli – the weak interactions of the TM domains bring the monomers together, whilst interactions between the acidic motif and other regions stabilise these complexes.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

The quorum sensing transcription factor AphA directly regulates natural competence in Vibrio cholerae

Many bacteria use population density to control gene expression via quorum sensing. In Vibrio cholerae, quorum sensing coordinates virulence, biofilm formation, and DNA uptake by natural competence. The transcription factors AphA and HapR, expressed at low and high cell density respectively, play a key role. In particular, AphA triggers the entire virulence cascade upon host colonisation. In this work we have mapped genome-wide DNA binding by AphA. We show that AphA is versatile, exhibiting distinct modes of DNA binding and promoter regulation. Unexpectedly, whilst HapR is known to induce natural competence, we demonstrate that AphA also intervenes. Most notably, AphA is a direct repressor of tfoX, the master activator of competence. Hence, production of AphA markedly suppressed DNA uptake; an effect largely circumvented by ectopic expression of tfoX. Our observations suggest dual regulation of competence. At low cell density AphA is a master repressor whilst HapR activates the process at high cell density. Thus, we provide deep mechanistic insight into the role of AphA and highlight how V. cholerae utilises this regulator for diverse purposes