<p> </p><p> </p><p>According to Faircloth, Capella & Alford, (2001), a brand is one of the most important assets a company can possess. A brand is what the consumers relate to when differentiating one com-pany from another and therefore plays a vital role for determining competitive advantage. How-ever, in the modern world, with the increasing technology advances, companies are losing more and more control of what is said and spread about their brands. What takes companies years to build can nowadays be destroyed in just a short amount of time. When dealing with negative publicity, a company‟s actions have a crucial role in determining the outcome of the negative publicity. The theoretical literature suggests that strong respective weak corporate response, will decide whether the consumers‟ brand attitude will be improved or wors-ened. Furthermore, it is also argued that consumers‟ commitment level can temper the effects of negative publicity in the sense that the more committed a consumer is, the more he/she will re-sist a change in brand attitude. Therefore, the purpose of this study is <em>"to examine if consumers’ atti-tude towards a brand is changed depending on strong or weak corporate response to the negative publicity. A significant aspect is to investigate and further associate the commitment variable to the outcome of change in attitudes as a result of the negative publicity." </em></p><p>In order to determine the cause-and-effect relationship between corporate response and brand attitude, an experiment was conducted where corporate response was the independent variable and brand attitude was the dependent variable. Furthermore, the commitment variable was in-cluded as a covariate; an independent variable not manipulated by the experimenter but still ex-pected to affect the outcome. Three different questionnaires were created: 1) Negative publicity with weak corporate response, 2) Negative publicity with strong corporate response, and 3) Negative publicity only. The experiment was conducted on consumers in Jönköping. The results indicate that whether a company decides to reply with a strong or weak corporate re-sponse to negative publicity, it will in the end have an effect on the consumers‟ brand attitude. Furthermore, the results also reveal that a consumer‟s level of commitment reinforces the effect of corporate response on his/her attitude towards a brand. In other words, the degree of the consumers‟ commitment towards a brand can temper the effect of negative publicity, ultimately saving companies from the consequences of negative publicity.</p><p> </p><p> </p> / <p>Ett varumärke är enligt Faircloth, Capella & Alford (2001), företagets viktigaste tillgång. Varu-märket spelar en viktig roll i att avgöra ett företags konkurrensfördel, då konsumenter relaterar till ett varumärke för att kunna differentiera mellan olika företag. I takt med de ökande teknolo-giska framryckningar i den moderna världen, har företag däremot börjat förlora alltmer kontroll över det som sägs och sprids om företagets varumärke. Det som tar företag åratal att bygga upp kan numera förgöras under en kort tidsperiod. När det gäller att handskas med negativ publicitet har företagets handlingar en stor inverkan på konsekvensen av den negativa publicitet som företaget har utsatts för. Den teoretiska litteraturen föreslår att stark respektive svag företagsrespons kommer att avgöra om konsumenternas attityd gentemot varumärket kommer förbättras eller försämras. Dessutom menar man att konsumen-tens lojalitetsnivå har en dämpande effekt på negativ publicitet. Ju lojalare en konsument är, des-to mer kommer han/hon att motstå en ändring i attityd gentemot varumärket i fråga trots den negativa publiciteten. Därmed är syftet med denna uppsats att ”undersöka om konsumenters attityd gentemot ett varumärke ändras beroende på stark eller svag företagsrespons i förhållande till den negativa publici-teten. En betydelsefull aspekt är att utreda och associera den kompletterande variabeln, lojalitet, med de utfallande ändringarna i attityd till följd av negativ publicitet”. I avsikt att utröna orsak-och-verkan relationen mellan företagsrespons och varumärkesattityd, ut-fördes ett experiment där företagsrespons var den oberoende variabeln och varumärkesattityd var den beroende variabeln. Därutöver, var lojalitetsvariabeln inkluderad som en covariate, dvs. en oberoende variabel som inte var manipulerad av forskarna men som ändå förväntades påver-ka resultatet. Tre olika enkäter var utformade: 1) Negativ publicitet med svag företagsrespons, 2) Negativ publicitet med stark företagsrespons, och 3) Endast negativ publicitet. Experimentet ut-fördes på konsumenter i Jönköping. Resultaten påvisar att vare sig ett företag väljer att hantera negativ publicitet genom stark eller svag företagsrespons, kommer resultatet att ha en inverkan på konsumenternas varumärkesatti-tyd. För övrigt visar resultaten att nivån av konsumentens lojalitet gentemot ett varumärk kom-mer att förstärka effekten av företagsresponsen på kundens varumärkesattityd. Med andra ord, graden av konsumenters lojalitet mot ett varumärke kan dämpa effekten av negativ publicitet och därmed rädda företag från degeneration av varumärket till följd av negativ publicitet.</p> / Grade: VG, (ECTS) A

The main role of roadway lighting is to produce quick, accurate and comfortable visibility during nighttime conditions. It is commonly known that good lighting levels enable motorists, pedestrians and bicyclists to obtain necessary visual information in an effective and efficient manner. Many previous studies also proved that roadway lighting minimizes the likelihood of crashes by providing better visibility for roadway users. Appropriate and adequate roadway lighting illuminance levels for each roadway classification and pedestrian areas are essential to provide safe and comfortable usage. These levels are usually provided by national, or local standards and guidelines. The Florida Department of Transportation (FDOT) Plan Preparation Manual recommends a roadway lighting illuminance level average standard of 1.0 horizontal foot candle (fc) for all the roadway segments used in this research. The FDOT Plan Preparation Manual also states that this value should be considered standard, but should be increased if necessary to maintain an acceptable uniformity illuminance ratio. This study aimed to find the relationship between nighttime crash injury severity and roadway lighting illuminance. To accomplish this, the research team analyzed crash data and roadway lighting illuminance measured in roadway segments within the West Central Florida Region. An Ordered Probit Model was developed to understand the relationship between roadway lighting illuminance levels and crash injury severity. Additionally, a Negative Binomial Model was used to determine which roadway lighting illuminance levels can be more beneficial in reducing the counts of crashes resulting in injuries. A comprehensive literature review was conducted using longitudinal studies with and without roadway lighting. Results showed that on the same roadways there was a significant decrease in the number of nighttime crashes with the presence of roadway lighting. In this research, roadway lighting illuminance was measured every 40 feet using an Advanced Lighting Measurement System (ALMS) on a total of 245 centerline miles of roadway segments within the West Central Florida Region. The data were mapped and then analyzed using the existing mile post. During the process of crash data analysis, it was observed that rear-end collisions were the most common first harmful event observed in all crashes, regardless of the lighting conditions. Meanwhile, the average injury severity for all crashes, was found to be possible injury regardless of the lighting conditions (day, dark, dusk, and dawn). Finally, this research presented an Ordered Probit Model, developed to understand the existing relationship between roadway lighting illuminance levels and injury severity within the West Central Florida Region. It was observed that having a roadway lighting average moving illuminance range between 0.4 to 0.6 foot candles (fc) was more likely to have a positive effect in reducing the probability of injury severity during a nighttime crash. A Negative Binomial Model was conducted to determine if the roadway lighting average moving illuminance level, found on the Ordered Probit Model was beneficial in reducing crash injury severity during nighttime, would also be beneficial in reducing the counts of crashes resulting in injuries. It was observed that a roadway lighting average moving illuminance, range between 0.4 to 0.6 fc, was more likely to reduce the count of crashes resulting in injuries during nighttime conditions, thus increasing roadway safety. It was also observed that other factors such as pavement condition, site location (intersection or no intersection), number of lanes, and traffic volume can affect the severity and counts of nighttime crashes. The results of this study suggest that simply adding more roadway lighting does not make the roadway safer. The fact is that a reduction in the amount of roadway lighting illuminance can produce savings in energy consumption and help the environment by reducing light pollution. Moreover, these results show that designing roadway lighting systems go beyond the initial design process, it also requires continuous maintenance. Furthermore, regulations for new developments and the introduction of additional lighting sources near roadway facilities (that are not created with the intent of being used for roadway users) need to be created.

Although work-family conflict (WFC) has been of particular interest to work-family researchers, little attention has been paid to the consequences of WFC that reside in the family domain. Research on WFC and child outcomes is especially scant. The current study addresses the gap in the literature by investigating the relationship between work-interfere-with-family (WIF) and three forms of parent-child interaction behavior (PB): physical and recreational PB (PRPB), cognitive and academic-oriented PB (CAPB), and passive and maintenance-oriented PB (PMPB). The mechanism by which WIF relates to PB was further investigated by examining negative emotion as a mediator and trait guilt as a moderator of the relationship. Employed parents of early school-aged children (n = 201) participated in the survey. Results indicated that both time- and strain-based WIF were negatively related to two types of active PB, PRPB and CAPB. However, negative emotion did not mediate the relationship between WIF and PB. With regard to the moderating role of trait guilt, support was found for PRPB. Theoretical and practical implications, as well as future directions, are discussed.

A persistent challenge in the design of composite materials is the ability to fabricate materials that simultaneously display high stiffness and high loss factors for the creation of structural elements capable of passively suppressing vibro-acoustic energy. Relevant recent research has shown that it is possible to produce composite materials whose macroscopic mechanical stiffness and loss properties surpass those of conventional composites through the addition of trace amounts of materials displaying negative stiffness (NS) induced by phase transformation [R. S. Lakes, et al., Nature, 410, pp. 565-567, (2001)]. The present work investigates the ability to elicit NS behavior without employing physical phenomena such as inherent nonlinear material behavior (e.g., phase change or plastic deformation) or dynamic effects, but rather the controlled buckling of small-scale structural elements, metamaterials, embedded in a continuous viscoelastic matrix. To illustrate the effect of these buckled elements, a nonlinear hierarchical multiscale material model is derived which estimates the macroscopic stiffness and loss of a composite material containing pre-strained microscale structured inclusions. The nonlinear multiscale model is then utilized in a set-based hierarchical design approach to explore the design space over a wide range of inclusion geometries. Finally, prototype NS inclusions are fabricated using an additive manufacturing technique and tested to determine quasi-static inclusion stiffness which is compared with analytical predictions. / text

在這網路發達的世界，電子口碑(electronic word of mouth)一直以來都是學術界研究的重點。電子口碑分成正面跟負面，尤其以負面電子口碑備受關注，因為這會帶給企業難以估計的傷害。而在2014年，開始有學者提出了網路風暴(online firestorm)的新名詞，描述在現代社群網路下，負面口碑在網路上突然大量流傳的現象。 關於網路風暴的文獻相當少，但在台灣2014年舉辦的九合一選舉中，可以看出網路風暴對選情的影響之大，我們觀察到某些候選人可以藉由應對來有效控制情勢，但有些回應卻會讓負面口碑風暴越趨惡化，因此我們想要探究這之間的問題所在。所以本研究首先透過文獻了解口碑風暴的定義，從而利用文獻推斷出口碑風暴形成過程。並以Google Trend設計出衡量口碑風暴的方式，以此找出選舉中符合口碑風暴標準的負面口碑案例。之後把案例分成三種情境，每種情境以兩個不同候選人的個案為例，把從Opview收集到的負面口碑資料套用在設計出的公式裡來判斷選舉人的應對有效與否。選舉人的應對則會利用Benoit印象修復理論來做進一步說明。最後，由於此篇研究是以選舉中的口碑風暴為主，因此我們會把選舉中的口碑風暴與傳統商業中的口碑風暴做比較，來看兩者間有何不同或相同之處。 / During the political election period in Taiwan in Nov. 2014, this study observed significant effects caused by online firestorms: some candidates used appropriate responses to mitigate their effects or minimize the harm, some turned negative into positive support of their brand, but some candidates were unable to manage the crisis and lost their brand value. To date, few studies have noted the importance of negative WOM (NWOM) management in political election campaigns. Nor have studies noted the effects of online firestorms on brand value. This study undertakes research to seek answers to the following research questions: What is the uniqueness of online firestorms in a political election? And, how do candidates respond the online firestorm to retain brand value? This study initially reviewed the literature on negative WOM and online firestorm, and consolidated these studies to formulate the forming process of an online firestorm. Then the study classified online political election firestorm cases into three categories including fault, defect, and counterforce slander, and analyzed the best responses in each situation. Next, the study applied Google Trend to measure the online firestorm and referred image restoration theory to form an understanding of the response of a brand manager. The study results show that for fault situation, the most useful strategies seem to be mortification, minimization the offensive feeling, and ignoring to respond to most offensive accusations. For defect situation, the most used strategies are insisting the main opinion and using attack strategy to evade the most offensive accusations. Last, for counterforce slander situation, most candidates used full denial, blame shifting and accuser attack strategies to prove their innocence. Finally, the study further distinguished the difference between conventional business online firestorm and political elections online firestorm from several angles which include: online firestorm targets, causes, consumer reactions, initiatives, effects and dissemination.

To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton. The PG synthases that build this structure are called penicillin-binding proteins (PBPs). Since they are the targets of penicillin and related antibiotics, the structures and in vitro biochemical functions of the PBPs have been extensively studied. However, the in vivo functions of the PBPs and the factors they work with to build the PG meshwork remain poorly understood. PBPs work in the context of multicomponent complexes organized by cytoskeletal elements. A major outstanding question has been whether or not these complexes contain factors required for PBP function. I addressed this using Escherichia coli as a model system by taking advantage of the synthetic lethal phenotype resulting from simultaneous inactivation of the major PG synthases: PBP1a and PBP1b. Using a screen for mutants synthetically lethal with the inactivation of PBP1b, I identified LpoA as a factor required for PBP1a function. A colleague in the lab performed the analogous screen for mutants synthetically lethal with the inactivation of PBP1a and identified LpoB as a factor required for PBP1b function. We showed that the Lpo factors are outer membrane lipoproteins that form specific trans-envelope complexes with their cognate PBPs in the inner membrane and that LpoB can stimulate the activity of PBP1b in vitro. Our results reveal unexpected complexity in the control of PBP activity and indicate that they likely receive regulatory input from the outer membrane in addition to cytoskeletal elements in the cytoplasm. To investigate the role of LpoB in morphogenesis further, I took a genetic approach that has identified PBP1b* variants capable of functioning in vivo in the absence of LpoB. Preliminary characterization of these variants indicates that LpoB has cellular functions in addition to PBP1b activation and that LpoB may be important for coordinating the two different catalytic activities of PBP1b. Future study of these mutants is likely to uncover important insights into PBP function and their control by the Lpo factors. These insights may open new avenues for the development of novel therapeutics that target the PBPs.

Background: Developing a well-adept social competence in preschool years is considered important and seems to play a pivotal role in later social functioning like school readiness and academic competence. Due to the individual development in children, establishing potential early markers of early social problems has been difficult. Although parent, peer, and contextual factors may be important to children’s development of social competence, the present study addressed the range of individual differences in children that may facilitate or impede social skills development. Method: The paper is based on data from the comprehensive longitudinal Trondheim Early Secure Study (TESS) of a screen-stratified community sample of 2475 children who were assessed at 4 year of age (T1) and followed up at the age of 6 (T2) (n=797). General linear modeling weighting data back to yield true population estimates of the predictive value of Social Competence, Gender, Negative Affectivity, Surgency, Effortful Control, Inattention, Hyperactivity, Impulsivity, Peer Problems, Disorganized Attachment and Callous-Unemotional traits assessed at T1 in predicting Social Competence at T2, when adjusting for Social Competence at T1. Results: Analysis indicates that Social Competence, Surgency, Inattention, Peer Problems high levels of Callous-Unemotional traits and Disorganization were unique predictors of Social Competence when adjusting for all variables. Negative Affectivity failed to predict Social Competence. Conclusions: Beyond a sizable continuity in social competence a range of child characteristics may enhance social skills development in young children . The identification of such child factors, when controlling for other potential factors, may inform health promotion efforts towards increasing young children’s social competence.

Syftet med föreliggande studie var att undersöka positiva och negativa känslors inverkan på gymnasieungdomars sociala-, äventyrliga-, hälsomässiga- och etiska riskbeteenden. Syftet var även att undersöka könsskillnader i gymnasieungdomars positiva och negativa känslor samt riskbeteenden. Deltagarna gick på gymnasieskolor i Örebro län och var 18-21 år. Totalt medverkade 240 personer i studien. För att mäta riskbeteenden och känslor användes en enkät som bestod av två skalor. Resultatet visade att positiva och negativa känslor kombinerat förklarade 5 % av variansen i gymnasieungdomars riskbeteenden. Positiva respektive negativa känslor predicerade två typer av riskbeteenden vardera. Resultatet visade att killar sannolikt tar mer risker än tjejer i tre av fyra risktyper. Det fanns ingen könsskillnad i känslor. Sammanfattningsvis visade resultatet att känslor påverkar gymnasieungdomars riskbeteenden. / The purpose of this study was to examine whether positive and negative emotions affected high school students social, recreational, ethical and health risk behaviors. The goal was also to investigate gender differences in specific risk behaviors and positive and negative emotions. The participants were high school students in Örebro County, between 18 and 21 years old. In total there were 240 participants. To measure emotions and risk behaviors, we used a questionnaire consisting of two scales. The results showed that emotions explained 5 % of the variance in high school students risk behaviors. Positive and negative emotions predicted two types of risk behaviors each. The results showed that boys were more likely to take risks than girls in three out of four types of risks. There were no gender differences in emotions. In summary, the results showed that emotions play a role in high school students risk behaviors.

Σκοπός της παρούσας ερευνητικής εργασίας ήταν η επιδημιολογική μελέτη των λοιμώξεων από πηκτάση-αρνητικούς σταφυλοκόκκους (CNS) σε ασθενείς με προσθετικά υλικά, όπως ενδαγγειακούς καθετήρες και η σύγκριση με στελέχη που προκαλούν βακτηριαιμία. Συνολικά μελετήθηκαν 168 Staphylococcus epidermidis και 58 S. haemolyticus από βακτηριαιμίες (BSIs, 100 στελέχη) και εντοπισμένες λοιμώξεις σχετιζόμενες με την εφαρμογή προσθετικών υλικών (PDAIs, 126 στελέχη) από ασθενείς του Πανεπιστημιακού Γενικού Νοσοκομείου Πατρών (ΠΓΝΠ) και του Νοσοκομείου Παίδων Πεντέλης (ΝΠΠ). Η πλειοψηφία των στελεχών (89.8%) ήταν ανθεκτικά στην methicillin (MR-CNS) και πολυανθεκτικά (90.7%). Βιομεμβράνη συνέθεταν τα 106/226 στελέχη, ενώ 208 παρήγαγαν β-λακταμάση. Τα γονίδια σύνθεσης προσκολλητινών aap, fnbA και bap βρέθηκαν σε συχνότητα 40.3%, 35.8% και 20.4% αντίστοιχα. Οι S. epidermidis έφεραν τα γονίδια atlE και fbe σε ποσοστά 88.1% και 81%, αντίστοιχα. Από τα γονίδια σύνθεσης τοξινών, συχνότερο ήταν το γονίδιο της τοξίνης τοξικής καταπληξίας tst (8.4%) ενώ τα γονίδια που κωδικοποιούν τις εντεροτοξίνες sea, sec βρέθηκαν μόνο σε μικρό ποσοστό στελεχών S. epidermidis και S. haemolyticus (5.3% και 3.1% του συνολικού πληθυσμού αντίστοιχα). Κανένα στέλεχος δεν έφερε τα γονίδια σύνθεσης των εντεροτοξινών seb και sed. Ο πληθυσμός των στελεχών S. epidermidis έδειξε μεγάλη γενετική ποικιλομορφία, με 67 PFGE τύπους, μεταξύ των οποίων δύο κύριοι τύποι (a, b) με 50 και 36 στελέχη αντίστοιχα. Έλεγχος με MLST ανέδειξε τρεις κύριους κλώνους (ST2, ST5 και ST16) που ανήκαν στο ίδιο κλωνικό σύμπλεγμα (Clonal Complex 2). Τα στελέχη του PFGE τύπου a παρουσίασαν υψηλότερα ποσοστά αντοχής στα αντιμικροβιακά clindamycin, ciprofloxacin, fusidic acid, SXT και στις αμινογλυκοσίδες, ενώ τα στελέχη του τύπου b έφεραν συχνότερα το γονίδιο aap (p=0.049). Τα στελέχη S. haemolyticus παρουσίασαν μικρότερη γενετική ποικιλομορφία, με έναν κύριο PFGE τύπο (h), που περιελάμβανε 44/58 στελέχη (75.9% του συνολικού πληθυσμού). Τα στελέχη CNS από BSIs ήταν συχνότερα ανθεκτικά στην methicillin (p<0.001) και στα υπόλοιπα αντιμικροβιακά (p<0.05), ενώ υπερείχαν και στην παραγωγή biofilm (p=0.003). Αντιθέτως, οι CNS από PDAIs έφεραν συχνότερα τα γονίδια των προσκολλητινών aap (p=0.006) και bap (p=0.045). Σε ένα δεύτερο σκέλος της παρούσας ερευνητικής εργασίας μελετήθηκε ένας πληθυσμός S. lugdunensis από το ΠΓΝΠ (37 στελέχη) και το ΝΠΠ (1 στέλεχος). Ο S. lugdunensis κατέχει ιδιαίτερη θέση μεταξύ των CNS, καθώς μπορεί να μιμηθεί την παθογόνο δράση του S. aureus και να προκαλέσει σοβαρές λοιμώξεις. Είκοσι δύο S. lugdunensis απομονώθηκαν από ασθενείς με λοιμώξεις δέρματος και μαλακών μορίων (Skin and Soft Tissue Infections: SSTIs), εννέα από εν τω βάθει λοιμώξεις (Deep Sited Infections: DSIs), συμπεριλαμβανομένων τριών στελεχών από ασθενείς με βακτηριαιμία, και επτά στελέχη από λοιμώξεις σχετιζόμενες με προσθετικά υλικά, κυρίως ενδαγγειακούς καθετήρες, (PDAIs). Όλα τα στελέχη ήταν ευαίσθητα στην methicillin (MS-CNS), στις αμινογλυκοσίδες (kanamycin, gentamicin), καθώς και στα: ciprofloxacin, rifampicin, teicoplanin, vancomycin, linezolid και daptomycin, ενώ μόνο τέσσερα στελέχη ήταν πολυανθεκτικά. Οι S. lugdunensis της συλλογής μας έδειξαν μικρή γενετική ποικιλομορφία. Τα 38 στελέχη ταξινομήθηκαν σε επτά κλώνους, με δύο κύριους PFGE τύπους (C και D), οι οποίοι περιελάμβαναν 22 και εννέα στελέχη αντίστοιχα. Τα 26 από τα 38 στελέχη έφεραν το οπερόνιο ica, ενώ συνολικά 14 ήταν biofilm-θετικά. Δεν παρατηρήθηκε συσχέτιση της παρουσίας του ica με κάποιο κλώνο, αλλά ούτε και με την παραγωγή βιομεμβράνης. Οι S. lugdunensis από PDAIs ήταν συχνότερα biofilm-θετικοί σε σχέση με τα στελέχη από SSTIs και DSIs, ενώ ο κύριος κλώνος C παρήγαγε biofilm σε μεγαλύτερο ποσοστό από τον D, δεύτερο σε συχνότητα κλώνο. Το γονίδιο fbl ανιχνεύθηκε σε όλα τα στελέχη S. lugdunensis που εξετάστηκαν, επιβεβαιώνοντας την φαινοτυπική ταυτοποίηση σε επίπεδο είδους. Ο επόμενος κατά σειρά συχνότητας παράγοντας παθογένειας που ανιχνεύθηκε ήταν το γονίδιο atlL, το οποίο βρέθηκε σε 36 από τα 38 στελέχη (94.7%). Ακολουθούν οι παράγοντες vwbl και slush, που βρέθηκαν σε 31 (81.6%) και 15 (39.5%) S. lugdunensis, αντίστοιχα. Τα στελέχη από εν τω βάθει λοιμώξεις (DSIs) έφεραν σε μεγαλύτερο ποσοστό τα γονίδια vwbl και slush σε σχέση με αυτά από PDAIs και SSTIs . Ο κλώνος C υπερείχε στην παρουσία του ermC, ενώ τα στελέχη που ανήκαν στον κλώνο D έφεραν σε μεγαλύτερο ποσοστό τα γονίδια vwbl και slush. / Coagulase-negative staphylococci (CNS), especially Staphylococcus epidermidis and S. haemolyticus, have emerged as opportunistic pathogens in patients with low immune response or indwelling medical devices. In the present study, bloodstream (BSIs) and prosthetic-device associated infections (PDAIs) CNS isolates were compared in terms of biofilm formation, antimicrobial resistance, clonal distribution, adhesin and toxin genes carriage. A collection of 226 CNS (168 S. epidermidis and 58 S. haemolyticus) recovered from BSIs (100) and PDAIs (126) of different patients in the Patras tertiary-care University General Hospital (UGHP) and Pentelis Paediatric Hospital in Athens (PPHA), was tested for biofilm formation, antimicrobial susceptibility, mecA, ica operon, adhesin (aap, bap, fnbA, atlE, fbe) and toxin (tst, sea, seb, sec, sed) genes carriage. All CNS were classified into pulsotypes by PFGE, whereas S. epidermidis strains were assigned to sequence types by MLST. In total, 106 isolates (46.9%) produced biofilm, whereas 150 (66.4%) carried ica operon. Most isolates carried mecA and were multidrug resistant (90.7%). The adhesin encoding genes aap, fnbA and bap were identified in 40.3%, 35.8% and 20.4% of the total population, respectively. Genes encoding AtlE and Fbe were found in 88.1% and 81% of S. epidermidis isolates, respectively. CNS recovered from BSIs prevailed in biofilm formation (P=0.003), resistance to antimicrobials and mecA carriage (P<0.001) as compared to isolates derived from PDAIs. CNS from PDAIs carried more frequently aap and bap genes (P=0.006 and P=0.045, respectively). No statistically significant difference in toxin genes carriage was identified (P>0.05). Even though PFGE showed genetic diversity, especially among S. epidermidis, analysis of representative strains from the main PFGE types by MLST, revealed three major clones (ST2, ST5, ST16). A clonal relationship was found concerning antimicrobial susceptibility, ica and aap gene carriage, reinforcing the aspect of clonal expansion in hospital settings. Pathogenesis of BSIs is associated with biofilm formation and high-level antimicrobial resistance, whereas PDAIs are related to the adhesion capability of CNS. In the second part of this study we analyzed a collection of S. lugdunensis isolates recovered from different inpatients hospitalized in UGHP (37 isolates) and PPHA (one isolate) during a six-year period (2008-2013). S. lugdunensis has emerged as a significant human pathogen with distinct clinical and microbiological characteristics. A collection of 38 S. lugdunensis was tested for biofilm formation, antimicrobial susceptibility, clonal distribution, virulence factors (ica operon, fbl, atlL, vwbl, slush) and antibiotic resistance genes (mecA, ermC) carriage. The majority (22) was isolated from skin and soft tissue infections (SSTIs), nine from deep-sited infections (DSIs), including three bacteraemias and seven from PDAIs. All isolates were oxacillin-susceptible, mecA-negative and fbl-positive. The higher resistance rate was detected for ampicillin (50%), followed by erythromycin and clindamycin (18.4%). Fourteen isolates (36.8%) produced biofilm, 26 carried ica operon, but no relation between ica carriage and biofilm formation was identified. Biofilm formation was more frequent in isolates recovered from PDAIs. Thirty six strains (94.7%) carried atlL, 31 (81.6%) vwbl, whereas, slush was detected in fifteen (39.5%). PFGE revealed low level of genetic diversity: strains were classified into seven pulsotypes, with two major clones C and D including 22 and nine strains, respectively. Type C strains, recovered from all infection sites, prevailed in biofilm formation and ermC carriage, whereas, type D strains, associated with SSTIs and DSIs, carried more frequently vwbl, slush or both genes. Despite susceptibility to antimicrobials, clonal expansion and carriage of virulence factors combined with biofilm-producing ability render this species an important pathogen that should not be ignored.

The membrane proteins signal peptide peptidase, signal peptide peptidase like and presenilin are intramembrane aspartyl proteases located in the endoplasmic reticulum, plasma membrane and organelle. These membrane proteins are able to catalyze a hydrolytic reaction in a hydrophobic space. The downstream consequences of these reactions impact a variety of cellular functions such as cytokine production, inflammatory responses, embryogenesis, and immune system regulation. Additionally, the aspartyl proteases such as signal peptide peptidase and presenilin, a part of the γ-secretase complex, hydrolyze peptides leading to pathogen maturation and Alzheimer’s disease, respectively. Electron crystallography offers the unique aspect of studying membrane proteins in a near native state. Determining the structures of Haloarcula morismortui and Methanoculleus marisnigri JR1 signal peptide peptidases by electron crystallography may provide insight into how a hydrolysis reaction occurs in a hydrophobic environment and how the protein determines which transmembrane signal peptides to cleave. Additionally, structure determination may help answer questions regarding why human presenilin, part of the γ-secretase complex, incorrectly processes amyloid precursor protein into amyloid-beta peptides leading to Alzheimer’s disease. Such structural data may not only shed light on how amyloid precursor protein is processed but how other proteins are processed by signal peptide peptidase leading to immune responses, cell signaling, and pathogen maturation. In addition, structure-function data may have an impact on pharmaceutical drug designs that targets signal peptide peptidase, signal peptide peptidase like, and/or presenilin. To determine the structure of aspartyl proteases, two archaeal signal peptide peptidases were used for two-dimensional crystallization trials to be able to study their structure by electron crystallography. Haloarcula morismortui and Methanoculleus marisnigri JR1 signal peptide peptidases, both human signal peptide peptidase homologues, were recombinantly over-expressed and purified. During dialysis trials, various lipid-to-protein ratios, sodium chloride concentrations, temperatures, detergents and a variety of other variables were tested. Methanoculleus marisnigri JR1 signal peptide peptidase showed the most promising results in terms of crystallinity. Optimizing dialysis conditions, specifically narrowing the lipid to protein ratio, resulted in two-dimensional crystals. Ordered arrays measuring up to 200 nm x 200 nm were observed. These ordered arrays have been shown to be reproducible amongst multiple batches of purified Methanoculleus marisnigri JR1 signal peptide peptidase. Preliminary projection maps of negatively stained ordered arrays show unit cell dimensions of a = 178 Å, b = 160 Å, γ = 92.0 Å and a = 175 Å, b = 167 Å, γ = 92.0 Å. The monomer measurements are approximately 70 Å by 80 Å. This is the first time a signal peptide peptidase homologue has been crystallized by two-dimensional crystallization.