Characterization of the Staphylococcus aureus bone sialoprotein-binding protein SdrE and the serine protease EpiP

In an attempt to develop a Staphylococcus aureus vaccine, we have applied reverse vaccinology approach, mainly based on in silico screening and proteomics. By using this approach SdrE, a protein belonging to serine-aspartate repeat protein family was identified as potential vaccine antigen against S. aureus. We have investigated the biochemical properties as well as the vaccine potential of SdrE and its highly conserved CnaBE3 domain. We found the protein SdrE to be resistant to trypsin. Further analysis of the resistant fragment revealed that it comprises a CnaBE3 domain, which also showed partial trypsin resistant behavior. Furthermore, intact mass spectrometry of rCnaBE3 suggested the possible presence of isopeptide bond or some other post-translational modification in the protein.However, this observation needs further investigation. Differential Scanning Fluorimetry study reveals that calcium play role in protein folding and provides stability to SdrE. At the end we have demonstrated that SdrE is immunogenic against clinical strain of S. aureus in murine abscess model. In the second part, I characterized a protein, annotated as epidermin leader peptide processing serine protease (EpiP), as a novel S. aureus vaccine candidate. The crystal structure of the rEpiP was solved at 2.05 Å resolution by x-ray crystallography . The structure showed that rEpiP was cleaved somewhere between residues 95 and 100 and cleavage occurs through an autocatalytic intra-molecular mechanism. In addition, the protein expressed by S. aureus cells also appeared to undergo a similar processing event. To determine if the protein acts as a serine protease, we mutated the catalytic serine 393 residue to alanine, generating rEpiP-S393A and solved its crystal structure at a resolution of 1.95 Å. rEpiP-S393A was impaired in its protease activity, as expected. Protective efficacy of rEpiP and the non-cleaving mutant protein was comparable, implying that the two forms are interchangeable for vaccination purposes.

Modelling and analysis of networked control strategies in smart power distribution grids: development of co-simulation tools

This thesis is focused on Smart Grid applications in medium voltage distribution networks. For the development of new applications it appears useful the availability of simulation tools able to model dynamic behavior of both the power system and the communication network. Such a co-simulation environment would allow the assessment of the feasibility of using a given network technology to support communication-based Smart Grid control schemes on an existing segment of the electrical grid and to determine the range of control schemes that different communications technologies can support. For this reason, is presented a co-simulation platform that has been built by linking the Electromagnetic Transients Program Simulator (EMTP v3.0) with a Telecommunication Network Simulator (OPNET-Riverbed v18.0). The simulator is used to design and analyze a coordinate use of Distributed Energy Resources (DERs) for the voltage/var control (VVC) in distribution network. This thesis is focused control structure based on the use of phase measurement units (PMUs). In order to limit the required reinforcements of the communication infrastructures currently adopted by Distribution Network Operators (DNOs), the study is focused on leader-less MAS schemes that do not assign special coordinating rules to specific agents. Leader-less MAS are expected to produce more uniform communication traffic than centralized approaches that include a moderator agent. Moreover, leader-less MAS are expected to be less affected by limitations and constraint of some communication links. The developed co-simulator has allowed the definition of specific countermeasures against the limitations of the communication network, with particular reference to the latency and loss and information, for both the case of wired and wireless communication networks. Moreover, the co-simulation platform has bee also coupled with a mobility simulator in order to study specific countermeasures against the negative effects on the medium voltage/current distribution network caused by the concurrent connection of electric vehicles.

Engineering the synthesis of lantibiotics in e. Coli by combining the cynnamicin and nisin modification systems.

I lantibiotici sono molecole peptidiche prodotte da un gran numero di batteri Gram-positivi, posseggono attività antibatterica contro un ampio spettro di germi, e rappresentano una potenziale soluzione alla crescente problematica dei patogeni multi-resistenti. La loro attività consiste nel legame alla membrana del bersaglio, che viene quindi destabilizzata mediante l’induzione di pori che determinano la morte del patogeno. Tipicamente i lantibiotici sono formati da un “leader-peptide” e da un “core-peptide”. Il primo è necessario per il riconoscimento della molecola da parte di enzimi che effettuano modifiche post-traduzionali del secondo - che sarà la regione con attività battericida una volta scissa dal “leader-peptide”. Le modifiche post-traduzionali anticipate determinano il contenuto di amminoacidi lantionina (Lan) e metil-lantionina (MeLan), caratterizzati dalla presenza di ponti-tioetere che conferiscono maggior resistenza contro le proteasi, e permettono di aggirare la principale limitazione all’uso dei peptidi in ambito terapeutico. La nisina è il lantibiotico più studiato e caratterizzato, prodotto dal batterio L. lactis che è stato utilizzato per oltre venti anni nell’industria alimentare. La nisina è un peptide lungo 34 amminoacidi, che contiene anelli di lantionina e metil-lantionina, introdotti dall’azione degli enzimi nisB e nisC, mentre il taglio del “leader-peptide” è svolto dall’enzima nisP. Questo elaborato affronta l’ingegnerizzazione della sintesi e della modifica di lantibiotici nel batterio E.coli. In particolare si affronta l’implementazione dell’espressione eterologa in E.coli del lantibiotico cinnamicina, prodotto in natura dal batterio Streptomyces cinnamoneus. Questo particolare lantibiotico, lungo diciannove amminoacidi dopo il taglio del leader, subisce modifiche da parte dell’enzima CinM, responsabile dell’introduzione degli aminoacidi Lan e MeLan, dell’enzima CinX responsabile dell’idrossilazione dell’acido aspartico (Asp), e infine dell’enzima cinorf7 deputato all’introduzione del ponte di lisinoalanina (Lal). Una volta confermata l’attività della cinnamicina e di conseguenza quella dell’enzima CinM, si è deciso di tentare la modifica della nisina da parte di CinM. A tal proposito è stato necessario progettare un gene sintetico che codifica nisina con un leader chimerico, formato cioè dalla fusione del leader della cinnamicina e del leader della nisina. Il prodotto finale, dopo il taglio del leader da parte di nisP, è una nisina completamente modificata. Questo risultato ne permette però la modifica utilizzando un solo enzima invece di due, riducendo il carico metabolico sul batterio che la produce, e inoltre apre la strada all’utilizzo di CinM per la modifica di altri lantibiotici seguendo lo stesso approccio, nonché all’introduzione del ponte di lisinoalanina, in quanto l’enzima cinorf7 necessita della presenza di CinM per svolgere la sua funzione.

Noninvasive assessment of exercise-related intramyocellular acetylcarnitine in euglycemia and hyperglycemia in patients with type 1 diabetes using ¹H magnetic resonance spectroscopy: a randomized single-blind crossover study

Intramyocellular acetylcarnitine (IMAC) is involved in exercise-related fuel metabolism. It is not known whether levels of systemic glucose influence IMAC levels in type 1 diabetes.

Skeletal muscle (1) H MRSI before and after prolonged exercise. I. muscle specific depletion of intramyocellular lipids

Aim of the study was to determine distribution and depletion patterns of intramyocellular lipids (IMCL) in leg muscles before and after two types of standardized endurance exercise. ¹H-magnetic resonance spectroscopic imaging was performed (1) in the thigh of eight-trained cyclists after exercising on an ergometer for 3 h at 52 ± 8% of maximal speed and (2) in the lower leg of eight-trained runners after exercising on a treadmill for 3 h at 49 ± 3% of maximal workload. Pre-exercise IMCL contents were reduced postexercise in 11 out of 13 investigated upper and lower leg muscles (P < 0.015 for all). A strong linear correlation with a slope of ∼0.5 between pre-exercise IMCL content and IMCL depletion was found. IMCL depletion differed strongly between muscles. Absolute and also relative IMCL reduction was significantly higher in muscles with predominantly slow fibers compared to those with fast fibers. Creatine levels and fiber orientation were stable and unchanged after exercise, while trimethyl-ammonium groups increased. This is presented in the accompanying paper. In conclusion, a systematic comparison of metabolic changes in cross sections of the upper and lower leg was performed. The results imply that pre-exercise IMCL levels determine the degree of IMCL depletion after exercise.

Skeletal muscle (1) H MRSI before and after prolonged exercise. II. visibility of free carnitine

Carnitine (Car) buffers excess acetyl-CoA through the formation of acetylCar (AcCar). AcCar's acetyl group (AG-AcCar) gives rise to a peak at 2.13 ppm in ¹H MR spectra of skeletal muscle, whereas the trimethylammonium (TMA) groups of both, AcCar and Car, are thought to contribute to the TMA peak at 3.23 ppm. Surprisingly, in previous studies both resonances, AG-AcCar and TMA, increased after exercise. The aim of this study was to assess if the exercise-related TMA increase correlated with AcCar production. Magnetic resonance spectroscopic imaging (pulse repetition time/echo time = 1200/35 ms) was performed before and after prolonged exercise in the lower leg and thigh of eight runners and eight cyclists, respectively. TMA and AG-AcCar increased after exercise (P < 0.001). TMA's increase correlated with the AG-AcCar increase (R² = 0.73, P < 0.001, lower leg; R² = 0.28, P < 0.001, thigh). The correlation of ΔTMA with ΔAG-AcCar suggests that the TMA increase is due to AcCar formation. As total Car (Car + AcCar) remains unchanged with exercise, these findings suggest that the contribution of free Car to the TMA peak is limited and, therefore, is partly invisible in muscle ¹H MR spectra. This indicates that the biochemically relevant cytosolic content of free Car is considerably lower than the overall concentration determined by radioisotopic assays, a potentially important result with respect to regulation of substrate oxidation.

Streptococcus spp. and related bacteria: Their identification and their pathogenic potential for chronic mastitis - A molecular approach

Streptococcus spp. and related bacteria form a large group of organisms which are associated with bovine intramammary Infections (IMI). Some of them are the well-known mastitis pathogens Streptococcus uberis and Streptococcus agalactiae. In addition, there are a considerable number of these gram-positive, catalase-negative cocci (PNC) with unclear mastitic pathogenicity such as Aerococcus viridans which make the conventional diagnostics of PNC difficult. One diagnostic, API 20 Strep (API, Biomerieux) is recommended which, as a phenotypic assay, involves a series of miniaturized biochemical tests. Recently, preference is given to genotypic identification methods. In particular, sequencing of the 16S rRNA gene allows highly reproducible and accurate identification of bacteria and permits discovery of novel, clinically relevant bacteria. As a consequence, the aim of the present study was to compare identification of IMI-associated PNC by the API method as well as by sequencing of their 16S rRNA gene (16S). Furthermore, the correlation of these bacteria to bovine chronic mastitis and their phylogeny was investigated. 102 PNC isolated from single quarter milk samples were identified by API and 16S sequencing. Considering Streptococcus uberis, Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus agalactiae, both methods generated fully concordant results. In contrast, a very high disconcordance was observed for most of the other PNC, in particular Enterococcus spp., Aerococcus viridans and the viridans streptococci were shown as apathogenic. Lactococcus garvieae was found to be an opportunistic pathogen causing IMI during late lactation. In addition, PNC isolated from milk were frequently observed together with other bacteria, in particular with Staphylococcus spp. In these cases, the levels of somatic cell counts (SCC) were determined by the specific PNC present in the sample. Considering PNC phylogeny based on 16S sequencing, 3 major clusters were observed. They included all the common mastitis pathogens (cluster I), the Lactococcus spp., Enterococcus spp. and Aerococcus spp. (cluster II) and all the viridans streptococci (cluster III).

Effects of fructose and glucose overfeeding on hepatic insulin sensitivity and intrahepatic lipids in healthy humans

To assess how intrahepatic fat and insulin resistance relate to daily fructose and energy intake during short-term overfeeding in healthy subjects.

Development of Socio-Economic Behaviour and Attitudes of Baltic Nationalities: Perspectives for Integration with Europe

This project is the third stage of a comparative research project, The New Baltic Barometer, which was carried out simultaneously with the "New Democracies Barometer" of the Paul Lazerfeld Society (Vienna) and The Russian Barometer. It studied the opinion and behaviour of the largest Baltic ethnic groups (Estonians, Latvians, Lithuanians). The main focus was on the attitudes of Baltic residents towards the changes in the economic and political system, attitudes towards political values, political trust, and attitudes to the Baltic countries joining the European Union. An analysis of macroeconomic indicators of the Baltic states made it possible to deduce the link between the country's economic development, and satisfaction with the political regime and attitudes towards democratic values. The study analysed the conditions for the democratisation of society, i.e. the development of culture and public opinion in the Baltic states. Attention was also paid to the development of a social network of individuals, showing the transition from informal networks to impersonal institutions. The group concluded that the participation of residents in formal organisations, NGOs in particular, considerably fosters political trust and also increases political efficacy. Participation in formal organisations also reduces the importance of esteem for an authoritarian leader.

Through the back door : Melungeon literacies and 21st century technologies

The Melungeons, a minority recognized in Southern Appalachia where they settled in the early 1800s, have mixed heritage—European, Mediterranean, Native American, and Sub-Saharan African. Their dark skin and distinctive features have marked them and been the cause of racial persecution both by custom and by law in Appalachia for two centuries. Their marginalization has led to an insider mentality, which I call a “literacy” of Melungeon-ness that affects every facet of their lives. Just a century ago, while specialized practices such as farming, preserving food, hunting, gathering, and distilling insured survival in the unforgiving mountain environment, few Melungeons could read or write. Required to pay property taxes and render military service, they were denied education, suffrage, and other legal rights. In the late 1890s visionary Melungeon leader Batey Collins invited Presbyterian homemissionaries to settle in one Tennessee Melungeon community where they established a church and built a school of unparalleled excellence. Educator-ministers Mary Rankin and Chester Leonard creatively reified the theories of Dewey, Montessori, and Rauschenbusch, but, despite their efforts, school literacy did not neutralize difference. Now, taking reading and writing for granted, Melungeons are exploring their identity by creating websites and participating in listserv discussions. These online expressions, which provide texts for rhetorical, semiotic, and socio-linguistic analysis, illustrate not solidarity but fragmentation on issues of origins and legitimacy. Armed with literacies of difference stemming from both nature and nurture, Melungeons are using literacy practices to embrace the difference they cannot escape.

The Dark Side of Leadership: Dealing with a Narcissistic Boss

Some of the defining characteristics of narcissists include a grandiose sense of self-importance, preoccupation with success and power, a sense of infallibility, and a supreme confidence in their ability and intelligence. Ironically, many of these characteristics are rewarded in business organizations, which may explain why there seems to be so many narcissists in management positions. Assuming we will be dealing with narcissists throughout our career, it makes sense for us to understand what makes them tick, and more importantly what we can do to work with them in a constructive manner.

Remembering Lee Metcalf - Natural Resources, Environmental and Conservation Excerpts (done for 75th Anniversary of Montana State Parks System - 2014)

Montana's Lee Metcalf was an extraordinary Montana leader with an unbelievable record of accomplishment fighting for the little people against the forces of economic and political power. The public memory is so short that this film will serve to help reacquaint Lee & Donna Metcalf to most of those who were around during their time. But it will also provide an opportunity for new generations to receive a perspective of an important leader from an important time. (Language from YouTube version of the film, written and provided by Executive Producer Evan Barrett) Lee Warren Metcalf (January 28, 1911 – January 12, 1978) was an American lawyer, judge, and politician. A member of the Democratic Party, he served as a U.S. Representative (1953–1961) and a U.S. Senator (1961–1978) from Montana. He was permanent acting President pro tempore of the Senate, the only person to hold that position, from 1963 until his death in 1978. Excerpts from main film for 75th Anniversary of Montana State Parks System

Social Work Education and Training in Europe and the Bologna Process

A short review of the last three decades shows that social work programmes have developed similarly in (almost) all European countries, both in terms of structural and content-related characteristics. Here I would like to focus on the following aspects: Increased academic focus of training, Generalist programme, International/European orientation. Increased academic focus means that social work programmes have been established at uni-versities or comparable higher education institutions, such as universities of applied sciences. The only exception is France where the approximately 150, generally fairly small colleges and the 14 larger instituts regionaux have a hybrid position between vocational colleges and universities and are roughly comparable to academies.

Neoliberalism, the Third Way and Social Work: the UK experience

For most of the past two decades, the notion that there is no alternative to the market as a basis for organising society has constituted a kind of global 'common sense', accepted not only by the neo-liberal Right but also by social democratic thinkers and politicians, in the form of 'the Third Way'. This paper will critically assess the central claims of neoliberalism in the light of experience in the UK and internationally, evaluate the ways in which Third Way policies are shaping social work in the UK, and in the final section, begin to explore some of the ways in which the anti-capitalist movement which has emerged in recent years might contribute to the development of a new, engaged social work, based on social justice.

Modernising Social Work and the Ethics of Care

Recent attempts to 'modernise' social work have emphasised the importance of collaboration, partnership, and participation with individual users of services and the wider community. However, technical-rational aspects of managerialism have proved dominant. Managerialist approaches to social service administration and delivery threaten important dimensions of social work; specifically its caring and democratic-transformative dimensions. However, social work theorists have only recently begun to re-engage with ideas of care. We argue that closer attention to feminist debates about the ethics of care can make a significant contribution to not only rehabilitating the ideal of care for social work but also to moving forward the modernisation agenda itself. We develop a feminist critique of managerialism, and argue that the discourse of the ethics of care offers useful ways of framing arguments to counter some damaging impacts of managerial reforms.