The purpose of this study was to investigate the impact of popular culture, especially prime-time television, on women learner-viewers’ identity development. More specifically, this study explores one specific television show, the 1962-64 Cathy Gale episodes of The Avengers as a portal to adult learning. It further explores the ways in which television, as a form of public pedagogy, can help facilitate the formation of a critical or feminist identity among adult learner viewers. The research questions guiding this study were: 1) How and what did women learn from watching The Avengers? 2) How did women incorporate that learning into their lives and into their identities? and 3) How did women interpret and accommodate the feminist example of Cathy Gale? Data for this study was collected over a two-and-a-half year period. Data consisted of interviews with contemporaneous viewers of the Cathy Gale Avengers episodes, interviews with scriptwriters and the actor who played Cathy Gale, Honor Blackman, numerous documents from statistics obtained at the British Film Institute, fanzines, and newspaper articles of the period. Analysis revealed that in particular historical times and situations television viewing can become a form of public pedagogy, facilitating transformational learning in adult viewers that produces lasting, life-changing effects. The investigation revealed that not only did biologically-born women incorporate Cathy Gale’s feminist example into their identities and actions, but biologically born males whose core gender identity was female did also. This dissertation is written in article format. Each of the six sections has been designed as stand-alone pieces to aid accessibility and enhance readers’ engagement with the study.

Die von mir vorgelegte Arbeit widmet sich insgesamt der Fragestellung, wie große gelenkte Projekte an einer ostdeutschen Schule in der Wendezeit durchgeführt wurden, was aus ihnen folgte und welche Konsequenzen für die Theorie der schulischen Projektarbeit daraus gezogen werden können. Insbesondere zeigt sie auf inwieweit die Arbeit mit Projekten die Forderungen der Gesellschaft, Schule als Lern-, Arbeits-, Lebens- und Freizeitort für Schüler/innen zu gestalten, erfüllt werden kann. Unterschiedlichste Schülerbeispiele (für Begabte, (Hoch)begabte, "schwierig Begabte", Verhaltensschwierige, überraschende Entwicklungen, Lernunwillige u.a.) werden vorgestellt und daran aufgezeigt, was Projektarbeit leisten kann, um die individuellen Leistungsmöglichkeiten von allen Schüler/innen gerecht zu werden, um diese auszuschöpfen, zu fördern und weiter zu entwickeln.

In this thesis, the aim was to attain a deeper understanding according to the students meaning of learning related to their own responsibility and independence within a framework of an educa¬tional programme. An empirical study with an ethnographic approach was carried out during one term of a nursing programme that uses the PBL-approach. Based on observations, conversations, inter¬views and documents, three narratives were formulated. One describes the phases students experience in the planned curricula. Periods characterised of chaos, uncertainty and a heavy workload, alternate with periods of ‘cosmos’, optimism, curiosity and satisfaction. The second narrative is about students' learning objectives while realising that they have to make choices and decisions on their own to succeed. A dialectic driving force emerging from frustration and stimulation, chaos and cosmos, results in questions about what to learn and how to act. The third narrative describes how students handle these questions. The narratives reveal two dimensions. One concerns how the students form attitudes about the relevance of learning objectives and how they go about learning a knowledge base necessary for their future profession. The other dimension takes as its starting point the fact that students try to manage their learning situation taking into consideration the framework of the educa¬tional programme. These dimensions were further analysed using theoretical references. The first dimension was analysed from the perspective of teacher/learner control and a didactic analysis of the meaning of an educational setting. The second dimension was analysed on the basis of phenomenographic learning theory. As regards responsibility and independence there seems to be a point in abandoning the concept of self-directed learning. Instead of emphasising self, the interaction between people, the individual and the educational framework and the interaction with content, are found to be fundamental. This interaction includes communication, dialogue and active participation in all the parts of a learning situation. Based on the results of this study, I claim that the driving force in student-centred learning is the dialectic relation between frustration and stimulation, chaos and cosmos. This stimulates the students to engage in the teachers' traditional didactic ques¬tions concerning an educational programme: what are we going to study, how and why, and what are the objectives? The students' conduct as regards independence, vis à vis dependence, are related to a dialectic relationship between the prerequisites provided by the educational frame¬work and the students' interpretation and ability to use them. Expressions of responsibility and independence emerge as choices and decisions concerning the didactic questions, initiative, activity, search for opportunities to reflect and co-operate and self-confidence. The opposite, dependence, is characterised by strategies for “survival”. The students plan their learning situa¬tion so that the examinations and assignments can be successfully tackled, and the learning situation takes on features of a surface approach. I believe that further insights into learning in student-centred education can be found in the two dialectic relationships described above.

There are many applications of small organic compounds, e.g. as drugs or as tools to study biological systems. Once a compound with interesting biological activity has been found, medicinal chemists typically synthesize small libraries of compounds with systematic differences to the initial “hit” compound. By screening the new ensemble of compounds for their ability to perturb the biological system, insights about the system can be gained. In the work presented here, various ways to synthesize small libraries of ring-fused 2‑pyridones have been developed. Members of this class of peptidomimetic compounds have previously been found to have a variety of biological activities, e.g. as antibacterial agents targeting virulence, and as inhibitors of the aggregation of Alzheimer b‑peptides. The focus in this work has been to alter the core skeleton, the central fragment, of the previously discovered biologically active 2‑pyridones and evaluate the biological effects of these changes. Several new classes of compounds have been constructed and their preparations have included the development of multi-component reactions and a method inspired by diversity-oriented synthesis. Some of the new compounds have been evaluated for their effect on the fibrillation of different amyloid proteins. Both the Parkinson-associated amyloid protein a-synuclein and the bacterial protein CsgA that is involved in bacterial biofilm formation are affected by subtle changes of the compounds’ central fragments. This is an example of the usefulness of central-fragment alterations as a strategy to probe structure-activity relationships, and the derived compounds may be used as tools in further study of the aggregation of amyloid proteins.

Preterm birth (&lt; 37 complete gestation weeks, GWs) is the single most prominent risk factor for the development of cerebral palsy (CP). This is due to the immature physiological state of the preterm born infant which increases the risk of brain lesions. In CP, prominent sensorimotor, cognitive, and perceptual deviations are manifested with varying degrees of functional impairment. Although most children born preterm (PT) do not develop CP, sensorimotor and cognitive deficits are frequently reported in the absence of major disability. To date, few studies have focused on detailed kinematic analysis of upper-body goal-directed movements and how aspects of movement organization and control are related to hand preference, intellectual function, gestational age, and sex within groups of prematurely born children. Further, studies evaluating effects of sensorimotor training in persons with CP is needed and of importance as positive effects on movement performance may increase individual autonomy as a consequence.To investigate the prevalence of non-right hand preference within children born preterm (PT) in comparison to children born fullterm (FT), a meta-analysis and literature review were performed (study I). It was shown that children born PT had increased rates of non-right handedness (NRH) corresponding to 22% compared to 12% in the FT group (odds ratio 2.12). In study II, hand preference and side specific movement performance in children born PT (. 35 GWs) compared to an age matched group of children born FT were investigated. All included participants were 4-8 years of age without major disability. Movement performance was studied through detailed kinematic registrations of the head, arm, and hand during a goal-directed task and hand preference through observations of the hand used when manipulating different objects. On a group level, the children born PT, specifically those born &lt; 33 GWs, showed an increased rate of NRH, weaker strength of hand preference, and a lack of side specificity in terms of movement kinematics. In general, the children born &lt; 33 GWs also displayed poorer movement organization and control as expressed by longer durations, less smooth and longer distances of the movement trajectories. These findings imply that preterm birth has long term effects on sensorimoror organization and function, possibly reflecting a developmental delay and/or a persistent effect that may be associated with the increased risk of deviations in brain development.In study III, associations between movement performance, assessed with the same task as in study II, and intellectual function (Wechsler Intelligence Scale for Children, 4th ed; WISC-IV) were studied. This study included a subsample of the children from study II at 6-8 years of age. The results showed a link between movement organization and general intellectual functioning (IQ), when controlling for effects of GA and sex, for the PT born but not the FT born children. These findings suggest shared neural underpinnings and interrelated development of motor and intellectual functions.In study IV, kinematic analysis of upper-body movements and subjective experience of change in upper-body function were applied before and after a period of synchronized metronome training (SMT) in adolescents with varying degree of CP to study the effects of this type of training. The SMT method applied entailed elements of movement timing and rhythmic activation coupled with feedback and was hypothesized to train sensorimotor integration. It was found that SMT did improve, to varying degrees, the organization and control of movements in adolescents with CP. The participants with more severe forms of CP reported substantial effects in daily living activities. The observed effects of SMT warrant further study of specific effects on movement planning, biomechanical constraints, and attention.The relation between hand preference and movement performance, movement performance and intellectual function, and aspects related to the SMT method applied in study IV are discussed. Specifically, the lack of side specific movement organization within the group of children born PT is discussed from perspectives of motor learning, plasticity, and genetics. The relations between movement performance and intellectual functions are considered and ideas for how this association could be tested are given. With relation to study IV, the functions trained by the specific SMT method applied and the accessibility of individuals with different degree of CP is discussed. Methodological considerations and ideas for future research approaches within these areas are presented. / Varje år föds det drygt 100 000 barn i Sverige. Cirka 5% av dessa barn föds för tidigt, det vill säga före 37 kompletta graviditetsveckor. Av de barn som föds för tidigt har majoriteten en gestationsålder (antalet graviditetsveckor) över 33 veckor (Medicinska födelseregistret, 2009). Omkring 8% av barn som föds för tidigt diagnosticeras senare med cerebral pares (CP) (Ancel, et al., 2006) och omkring hälften av personer med diagnosen CP har en för tidig födelsehistorik (Beaino et al., 2010). CP är ett paraplybegrepp som innefattar ett flertal icke-progressiva motoriska utvecklingsavvikelser av olika karaktär och funktionsnedsättningsgrad, orsakade av hjärnskador som inträffar under fosterlivet eller vid förlossningen. Vanligtvis så påverkas även sensorik, perception, kognition, och beteende negativt (Rosenbaum, Paneth, Leviton, Goldstein, &amp; Bax, 2008). En prematur födsel utgör den största riskfaktorn för utvecklingen av CP där risken är störst för de barn som föds allra tidigast (Murphy, Sellers, MacKenzie, Yudkin, &amp; Johnson, 1995). De allra flesta barn som föds för tidigt drabbas dock inte av grav sensomotorisk funktionsnedsättning som till exempel CP trots att det finns en ökad risk för hjärnskador hos denna grupp. Ett flertal studier har ändå visat att barn som fötts för tidigt och ej diagnosticerats med CP i högre grad än fullgångna barn generellt har både motoriska (e.g., Williams et al., 2010) och kognitiva (e.g., Bhutta, 2002) funktionsnedsättningar.I dagsläget så har endast ett fåtal studier fokuserat på att i detalj studera organiseringen av målinriktade hand-armrörelser hos barn som fötts för tidigt. Av dessa har merparten fokuserat på spädbarn i syfte att finna tidiga markörer för sensomotoriska avvikelser. Då de sensomotoriska problemen hos för tidigt födda barn kvarstår i skolåldern och i vissa fall är så diskreta att de upptäcks först när barnet börjar skolan så kan rörelseanalys vara en metod som kan bidra till ökad förståelse och kunskap om hur den sensomotoriska påverkan uttrycks hos äldre barn. Ett av syftena med denna avhandling var således att noggrant studera organiseringen av huvud-, arm-, och handrörelser under en målinriktad handling hos barn som fötts för tidigt jämfört med en grupp barn i samma åldrar (4-8 år) som fötts efter fullgången graviditet. Av intresse är att studera relationen mellan aspekter associerade med rörelseorganisation och handpreferens, intellektuell förmåga, gestationsålder, samt kön. Då barn och ungdomar med CP utgör den största diagnosgruppen som behandlas inom barn- och ungdomshabiliteringar (Odding, Roebroeck, &amp; Stam, 2006) finns det ett stort behov av att utvärdera effekten av redan existerande träningsmetoder. Ett ytterligare syfte med detta avhandlingsarbete var att utvärdera både kort- och långtidseffekter av en etablerad träningsmetod (synchronized metronome training; SMT) som antas främja den sensomotoriska integreringen hos ungdomar (12-17 år) med diagnosen CP. Effekterna av denna träningsmetod utvärderades med hjälp av detaljerad rörelseanalys samt ett frågeformulär gällande individens upplevelse av förändring.Då barn som fötts för tidigt i vissa studier har visat sig ha en ökad prevalens av icke-högerhänthet, det vill säga fler som är vänsterhänta eller tvåhänta (ambidextra), så utfördes en systematisk litteraturgenomgång och en metaanalys för att undersöka detta fenomen närmare (studie I). I denna studie visade det sig att barn som fötts för tidigt i större utsträckning var icke-högerhänta jämfört med barn som fötts efter en fullgången graviditet (odds ratio 2.12). Hos de för tidigt födda barnen var 22% icke högerhänta jämfört med 12% av de som fötts efter fullgången graviditet. Dock säger handpreferensstudier inget om hur kvaliteten på rörelser utförda med den föredragna eller icke-föredragna handen ser ut. För att undersöka sambandet mellan handpreferens och kvalitet på rörelser kopplade till den föredragna och icke-föredragna handen hos barn som fötts för tidigt, jämfört med fullgångna åldersmatchade kontroller, utfördes kinematiska rörelseanalyser samt ett testbatteri som undersöker handpreferens (studie II). Barnen utförde målinriktade rörelser, med en hand i taget, i en uppgift som ställer höga krav på öga-hand-koordination och precision. Uppgiften bestod i att plocka upp och trä så många pärlor som möjligt på en smal pinne under 30 sekunder. Denna studie visade att barnen med en för tidig födelsehistorik, speciellt de mycket för tidigt födda (före graviditetsvecka 33), i större utsträckning var klassificerade som icke-högerhänta och hade en svagare handpreferens i jämförelse med barnen som inte fötts för tidigt. De mycket för tidigt födda barnen visade även, som grupp, en sämre organisering av huvud-, arm-, och handrörelser vilket manifesterades i en ökad längd, tidsåtgång och ojämnhet av rörelsebanorna. Oavsett gestationsålder så visade den för tidigt födda gruppen barn inga sidoskillnader i kvaliteten på rörelserna vilket däremot var tydligt hos gruppen barn som fötts vid fullgången graviditet. Dock så skiljde sig gruppen barn som fötts moderat för tidigt (mellan graviditetsvecka 33 och 35) inte statistiskt signifikant åt från barnen i den fullgånget födda gruppen. Dessa resultat indikerar en långvarig effekt av för tidig födsel på sensomotorisk funktion. Den markanta påverkan som barnen som fötts före graviditetsvecka 33 uppvisade, kan tolkas som en effekt av den ökade risken för avvikelser i hjärnans utveckling.I studie III undersöktes sambanden mellan intellektuell förmåga och organiseringen av rörelser hos barn som fötts för tidigt samt hos barn som fötts efter en fullgången graviditet i 6-8 års ålder (en subgrupp ur studie II). Den intellektuella förmågan undersöktes med hjälp av Wechsler Intelligence Scale for Children, 4:e upplagan, och den kinematiska uppgiften var den samma som beskrivits ovan. Denna studie visade på skillnader i intellektuell förmåga, specifikt gällande generell intelligens samt verbal och perceptuell förmåga, där de för tidigt födda barnen hade lägre poäng än de fullgångna barnen. Viktigt att poängtera är dock att medelvärdena för gruppen för tidigt födda barn låg inom det normala spannet. Vidare så visade sig gestationsålder och generell intellektuell förmåga ha betydelse för det motoriska utförandet hos de för tidigt födda barnen, speciellt på den föredragna sidan. Kön bidrog inte till att förklara skillnader i organiseringen av rörelser. Intellektuell förmåga hade ingen relation till den sensomotoriska funktionen hos gruppen barn som fötts efter en fullgången graviditet. Detta belyser vikten av att studera kognitiva aspekter av motorisk funktion för att bättre förstå de skillnader eller avvikelser som ofta påvisas hos barn med en för tidig födelsehistorik. Dessutom skulle en sådan ansats öka kunskapen om den interrelaterade utvecklingen av motorisk och intellektuell förmåga, både ur ett beteendeperspektiv och ur ett hjärnutvecklingsperspektiv.Kinematisk rörelseanalys användes även i studie IV för att beskriva eventuella förändringar i rörelseorganisation efter SMT-träning hos ungdomar med olika typer av CP och varierande grad av funktionsnedsättning (studie IV). Ungdomarna utförde en målinriktad uppgift som bestod i att tända små lampor i sekventiell ordning. Testerna genomfördes vid tre tillfällen, före, i anslutning till avslutad SMT-träning samt 6 månader efter avslutad SMT-träning. Träningsperioden varade i 4 veckor och innefattade 12 individanpassade SMT-sessioner. Den typ av SMT-träning som användes i denna studie antas främja sensomotorisk integrering genom rytmisk aktivering och timing av rörelser mot en auditiv och visuell signal. Feedback på avvikelsen i synkronisering, och en instruktion om hur deltagaren skall uppnå bättre rörelsetiming, ges direkt både auditivt och visuellt. Denna studie visade på förändringar i rörelseorganisation och motorisk kontroll hos flertalet av deltagarna. Dessa effekter kvarstod till viss del vid uppföljningen efter 6 månader. Deltagarna med gravare funktionsnedsättning rapporterade substantiella förändringar i förmågan att delta i dagliga aktiviteter både direkt efter avslutad träning samt 6 månader senare. De rapporterade effekterna gällde ökad snabbhet, minskad muskeltonus, bättre arm-, hand- och fingerkontroll, samt ökad motivation att använda armen och handen både i träning och vardagliga aktiviteter. Dessa subjektivt upplevda förändringar tillsammans med de vi observerat i de kinematiska parametrarna utgör en grund för fortsatta studier av effekter av SMT hos denna grupp ungdomar och även hos andra grupper barn och ungdomar med motoriska problem. En ansats där effekter av SMT-metoder studeras utifrån rörelseplanering, biomekaniska restriktioner, samt uppmärksamhet skulle vara av betydelse.I avhandlingen diskuteras relationen mellan handpreferens och kvalitet på rörelser i relation till den föredragna och icke-föredragna handen, samband mellan organisering av rörelser och intellektuell förmåga, samt aspekter relaterade till SMT-metoden som applicerats i studie IV. Specifikt diskuteras avsaknaden av en sidospecifik rörelseorganisering hos gruppen barn som fötts för tidigt i relation till motorisk inlärning, hjärnans förmåga att förändra sig, och genetik. Relationen mellan organisering av rörelser och intellektuell förmåga betänks och förslag på hur detta samband kan undersökas i kommande studier ges. Vidare så diskuteras vilka funktioner som SMT-metoden tränar samt dess tillgänglighet för barn och ungdomar med olika grad av funktionsnedsättning relaterad till typ av CP diagnos. Metodologiska överväganden och idéer för framtida forskningsansatser inom dessa områden presenteras. / <p>En viktig annan finansiär är Norrbacka-Eugenia Stiftelsen som bekostade hela min forskarutbildning.</p>

The members of the mammalian neuropeptide Y family, i.e. the peptides neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP), are all involved in regulation of food intake. In human and most other mammals they act via receptors Y1, Y2, Y4 and Y5. NPY is released in the hypothalamus and is one of the strongest appetite-stimulating neurotransmitters whereas PP and PYY are secreted from gut endocrine cells after meals and function as appetite-reducing hormones. This thesis describes studies of the NPY system at both the molecular and the physiological level. The first part describes two investigations of receptor-ligand interactions with the human Y1 and Y2 receptors. The results clarify the importance of several amino-acid residues of the human Y1 receptor. Three amino acids previously suggested by others to form a binding pocket for the carboxy-terminus of the peptide were confirmed to be crucial for interaction with peptide ligands. However, they were found to be too distantly located from each other to be able to form a binding pocket. Further investigation of the three corresponding positions in the human Y2 receptor showed that only one of the positions was important for interaction with full-length peptides. The results indicate overlapping but, surprisingly, non-identical binding of the different peptides to human Y1 and Y2 receptors, despite the fact that the two receptors share a common ancestor. The second part of the thesis describes an investigation of the effect of PP on food intake in six beagle dogs and a test for personality characteristics in dogs (TFPC). Treatment with physiological doses of PP decreased both the appetitive and the consummatory drive but had no effect on the amount food consumed. The TFPC protocol was used to map individual behavioral differences in a population of sixteen beagle dogs. The test, which included several situations that may appear in an experimental study, revealed considerable inter-individual differences in behavioral responses despite the fact that the dogs were born and housed in the same animal facility in constant controlled conditions. These results demonstrate that PP can influence food intake in distantly related mammals and emphasize the importance of considering differences in personality in experimental animals.

This thesis presents four studies investigating (1) whether incidentally primed control-related words can attenuate the impact of activated stereotypes on subsequent evaluation of a target person, (2) the impact of motivated forgetting on the recall of stereotypically congruent and incongruent information, and (3) the impact of a directed forgetting instruction on the false recall and recognition of nonpresented stereotypical information. In three experiments, Study I showed that participants initially primed with the social category, immigrant, and subsequently primed with words that were evocative of control or self-control made less negative impression of a target displaying ambiguous behaviors than participants not exposed to such words. Study II, using a directed-forgetting paradigm, demonstrated in two experiments that participants subliminally primed with Swedish facial photographs who later studied stereotypically incongruent words roughly recalled an equal number of items regardless of the forget or remember instructions. Study III showed that participants primed with the social category, immigrant and then studied a list of stereotypically related and unrelated words falsely recognized more nonpresented stereotypical words when they were furnished with a forget than a remember instruction. Similarly, Study IV (Experiment 2) demonstrated that participants primed with the social category, immigrant, but not with a neutral category, falsely recalled more nonpresented stereotypical words when their cognitive capacity was depleted through a concurrent memory load task. The thesis presents a review and a discussion of some of the theoretical underpinnings of the extant literature on stereotyping and intergroup relations and of the social implications of the present findings.

The sun provides us daily with large quantities of energy in the form of light. With the world’s increasing demand of electrical energy the prospect of converting this solar light into electricity is highly tempting. In the strive towards mass-production and low cost solar cells, new types of solar cells are being developed, e.g. solar cells completely based on organic molecules and polymers. These materials offer a promising potential of low cost and large scale manufacturing and have the additional advantage that they can be produced on flexible and light weight substrate which opens for new and innovating application areas, e.g. integration with paper or textiles, or as building materials. In polymer solar cells a combination of two materials are used, an electron donor and an electron acceptor. The three dimensional distribution of the donor and acceptor in the active layer of the device, i.e. the morphology, is known to have larger influence of the solar cell performance. For the optimal morphology there is a trade-off between sometimes conflicting criteria for the various steps of the energy conversion process. The dissociation of photogenerated excitons takes place at an interface between the donor and acceptor materials. Therefore an efficient generation of charges requires a large interface between the two components. However, for charge transport and collection at the electrodes, continuous pathways for the charges to the electrodes are required. In this thesis, results from morphology studies by atomic force microscopy (AFM) and dynamic secondary ion mass spectrometry (SIMS) of spin-coated blend and bilayer thin films of polyfluorene co-polymers, especially poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-5,5-(4´,7´-di-2-thienyl-2´,1´,3´-benzothiadiazole)] APFO-3, and the fullerene derivative [6,6]-phenyl-C61-butyric acid methyl ester (PCBM) are presented. It is shown that by varying the blend ratio, the spin.-coating solvent, and/or the substrate, different morphologies can be obtained, e.g. diffuse bilayer structures, spontaneously formed multilayer structures and homogeneous blends. The connection between these different morphologies and the performance of solar cells is also analysed. The results indicate that nano-scale engineering of the morphology in the active layer may be an important factor in the optimization of the performance of polymer solar cells.

AcrA is the periplasmic component of an efflux system AcrA-AcrB-TolC, which can expel different classes of antibiotics. AcrB is the inner membrane (IM) pump that utilizes proton-motive force for the active transport, TolC is the outer membrane (OM) channel, and AcrA coordinates the actions of AcrB and TolC, so that substrates are expelled across the two membranes, bypassing the periplasm. It has been proposed that AcrA either provides a static seamless link between AcrB and TolC, or acts like its analogous viral membrane fusion protein (MFP) and actively brings the IM and OM closer for substrate transfer. To better understand the role of AcrA in the efflux mechanism, site-directed spin labeling (SDSL)/EPR (electron paramagnetic resonance) spectroscopy is used to investigate the structure and dynamics of AcrA in solution. My results demonstrated that AcrA is a dynamic protein that undergoes pH-dependent and reversible conformational changes. AcrA contains an interrupted alpha-helical, coiled-coil domain flanked by a pair of beta-stranded lipoyl motifs, and my SDSL/EPR analysis revealed that the pH-induced conformation change mainly involves the coiled-coil and the lipoyl domains. In addition, I found that each AcrA monomer folds into an intra-molecular hairpin and AcrA monomers oligomerize with their coiled-coil hairpins aligned in parallel. Unlike the pH-induced conformational rearrangement of a viral MFP, change in pH alters both intra- and inter-molecular interaction along the coiled-coil of AcrA without rearranging the hairpin fold. The organization of AcrA protomers and its pH-induced conformational switching are, however, congruent with the TolC coiled-coil hairpins in the iris-like opening of the TolC channel. Together, my studies suggest that rather than being a passive structural linkage between AcrB and TolC, AcrA plays an active role mediating the drug efflux. The reported AcrA dynamics provides new insights into the AcrA-TolC interactions for the channel opening during the efflux process.

ABSTRACT Protein-protein interactions play a central role in most biological processes. One such biological process is the maintenance of a reducing environment inside the cell. To maintain an internal reducing environment, living cells have evolved two enzymatic systems (glutathione and thioredoxin (Trx) systems). The Trx system is composed of the enzyme TrxR and its substrate Trx. The two proteins constitute an important thiol-dependent redox system that catalyzes the reduction of many proteins that are responsible for a variety of cellular functions. The system relies on transient protein-protein interactions between Trx and TrxR for its function. Cross-reactivity of components of the Trx system between species has been shown to be medically relevant. For example, Helicobacter pylori Trx (HP Trx) is thought to mediate catalytic reduction of human immunoglobulins and thus facilitate immune evasion. It has also been proposed that Helicobacter pylori gains access to the impenetrable gastric mucous layer by using secreted HP Trx to reduce the disulfide bonds present in the cysteine-rich mucin regions that are responsible for cross-linking mucin monomers. Therefore, disruption of secreted HP Trx-host protein interaction may result in restoration of the viscoelastic and hydrophobic protective properties of mucus. Previous studies aimed at understanding the nature of cross-reactivity of Trx system components among various species have shown that Trxs have higher affinity for cognate TrxRs (same species), than for TrxRs from different species. However, the basis for this specificity is not known. A growing body of evidence suggests that most protein-protein interactions are mediated by a small number of protein-protein interface residues, referred to as hot spot residues or binding epitopes. Therefore, understanding the biochemical basis of the affinity of proteins for their partners usually begins by identifying the hot spot residues responsible for the protein complex interactions. In this study, the crystal structures of Deinococcus radiodurans thioredoxin reductase (DR TrxR) and Helicobacter pylori TrxR (HP TrxR) were determined at 1.9 Å and 2.4 Å respectively. Analysis of the Trx-binding sites of both structures suggests that the basis of affinity and specificity of Trx for TrxR is primarily due to the shape rather than the charge of the surface. In addition, the complex between Escherichia coli thioredoxin reductase (EC TrxR) and its substrate thioredoxin (EC Trx) was used to identify residues that are responsible for TrxR-Trx interface stability. Using computational alanine scanning mutagenesis and visual inspection of the EC TrxR-Trx interface, 22 EC TrxR side chains were shown to make contact across the TrxR-Trx interface. Although more than 20 EC TrxR side chains make contact across the TrxR-Trx interface, our results suggest that only 4 residues (F81, R130, F141, and F142) account for the majority of the EC TrxR-Trx interface stability. Individual replacement of equivalent DR TrxR residues (M84, K137, F148, F149) with alanine resulted in drastic changes in binding affinity, confirming that the four residues account for most of TrxR-Trx interface stability. These hot spot residues are surrounded by less important residues (hydrophobic and hydrophilic) that are also predicted to contribute to interface stability. F148 and F149 are invariant across bacterial TrxRs, however other residues that contact Trx are less conserved including M84 and K137. When M84 and K137 were changed to match equivalent E. coli TrxR residues (K137R, M84F); D. radiodurans TrxR substrate specificity was altered from its own Trx to that of E. coli Trx. The results suggest that a small subset of the TrxR-Trx interface residues are responsible for the majority of Trx binding affinity and specificity, a property that has been shown to general to protein-protein interfaces.