Ultra high molecular weight polyethylene (UHMWPE) is a polymer commonly used in various applications, particularly in hip joint replacements. Despite its good performance, it is susceptible to oxidation degradation, which can be mitigated with the addition of vitamin E, and to excessive wear against metal countersurfaces, which can potentially be improved with the addition of nanoparticles. In this work, sixteen newly developed UHMWPE nanocomposites with different nanofillers (multi-walled carbon nanotubes, graphene oxide and nanodiamonds), blended with and without vitamin E, and both irradiation crosslinked and non-crosslinked were studied. Thermomechanical characterisation (dynamic mechanical analysis and thermogravimetric analysis) and tribological pin-on-disc testing showed that both nanofillers and the addition of vitamin E ensured improved the performance of the nanocomposites compared to the virgin UHMWPE. Vitamin E improved the oxidative and thermal stability of UHMWPE, decreased the wear and increased the coefficient of friction. The nanoparticle reinforcements contributed to improved thermal stability to some degree and decreased the wear. Crosslinking was also shown to result in improved thermomechanical performance, although it appears that the addition of vitamin E inhibits the crosslinking process.

A Programação Orientada a Aspectos (POA) é uma técnica contemporânea de desenvolvimento de software fortemente baseada no princípio da separação de interesses. Ela tem como objetivo tratar de problemas de modularização de software por meio da introdução do aspecto como uma nova unidade de implementação que encapsula comportamento relacionado aos interesses transversais do software. A despeito dos benefícios que podem ser alcançados com o uso da POA, seus mecanismos de implementação representam novas potenciais fontes de defeitos que devem ser tratados durante a fase de teste de software. Nesse contexto, o teste de mutação consiste em um critério de seleção de testes baseado em defeitos que tem sido bastante investigado para demonstrar a ausência de defeitos pré-especifiados no software. Acredita-se que o teste de mutação seja uma ferramenta adequada para lidar com as particularidades de técnicas de programação contemporâneas como a POA. Entretanto, até o presente momento, as poucas iniciativas para adaptar o teste de mutação para o contexto de programas orientados a aspectos (OA) apresentam cobertura limitada em relação aos tipos de defeitos simulados, ou ainda requerem adequado apoio automatizado e avaliações. Esta tese visa a mitigar essas limitações por meio da definição de uma abordagem abrangente de teste de mutação para programas OA escritos na linguagem AspectJ. A tese inicia como uma investigação da propensão a defeitos de programas OA e define uma taxonomia de defeitos para tais programas. A taxonomia inclui uma variedade de tipos de defeitos e serviu como base para a definição de um conjunto de operadores de mutação para programas OA. Suporte automatizado para a aplicação dos operadores também foi disponibilizado. Uma série de estudos quantitativos mostra que a taxonomia de defeitos proposta é suficiente para classificar defeitos encontrados em vários sistemas OA. Os estudos também mostram que os operadores de mutação propostos são capazes de simular defeitos que podem não ser relevados por conjuntos de teste pré-existentes, não derivados para cobrir mutantes. Além disso, observou-se que o esforço requerido para evoluir tais conjuntos de teste de forma a torná-los adequados para os requisitos gerados pelos operadores / Aspect-Oriented Programming (AOP) is a contemporary software development technique that strongly relies on the Separation of Concerns principle. It aims to tackle software modularisation problems by introducing the aspect as a new implementation unit to encapsulate behaviour required to realise the so-called crosscutting concerns. Despite the benefits that may be achieved with AOP, its implementation mechanisms represent new potential sources of faults that should be handled during the testing phase. In this context, mutation testing is a widely investigated fault-based test selection criterion that can help to demonstrate the absence of prespecified faults in the software. It is believed to be an adequate tool to deal with testing-related specificities of contemporary programming techniques such as AOP. However, to date, the few initiatives for customising the mutation testing for aspect-oriented (AO) programs show either limited coverage with respect to the types of simulated faults, or a need for both adequate tool support and proper evaluation. This thesis tackles these limitations by defining a comprehensive mutation-based testing approach for AO programs written in the AspectJ language. It starts with a fault-proneness investigation in order to define a fault taxonomy for AO software. Such taxonomy encompasses a range of fault types and underlay the definition of a set of mutation operators for AO programs. Automated tool support is also provided. A series of quantitative studies show that the proposed fault taxonomy is able to categorise faults identified from several available AO systems. Moreover, the studies show that the mutation operators are able to simulate faults that may not be revealed by pre-existing, non-mutation-based test suites. Furthermore, the effort required to augment the test suites to provide adequate coverage of mutants does not tend to overwhelm the testers. This provides evidence of the feasibility of the proposed approach and represents a step towards the practical fault-based testing of AO programs

O exame de resultados de pesquisas anteriores realizadas pelo Nomads.usp - Núcleo de Estudos de Habitares Interativos - em que foram utilizados vários instrumentos metodológicos clássicos de pesquisa qualitativa a fim de entender contextos urbano e social, revelou limites desses instrumentos quando se desejava abarcar nuances referentes a modos de vida, visões de mundo e pontos de vistas de indivíduos pesquisados. O objetivo dessa pesquisa é a verificação dos limites e potencialidades do emprego de três atividades, concebidas para uso em eventos culturais com presença de público - Rádio de Rua, Graffiti Digital e Projeção de Comentários -, no sentido de utilizá-las também como procedimentos de obtenção de informação sobre seus públicos, estimulando processos comunicacionais e interlocuções com auxílio de meios digitais. Essas três atividades foram definidas em conjunto com os interesses do Projeto de Políticas Públicas \"Territórios Híbridos: meios digitais, comunidades e ações culturais\", o qual, por outro lado, tornou possível a realização dos experimentos aqui estudados. / The evaluation of previous research carried out by Nomads.usp - Center of Interactive Living Studies - which used several classical methodological instruments of qualitative research in order to understand urban and social contexts, found limits of these tools when they were employed to encompass nuances related to ways of life, worldviews and viewpoints of surveyed individuals. The aim of this reseach is to verify the limits and the possibilities of using three activities conceived for usage in cultural events with audience\'s presence - Street Radio, Digital Graffiti and Comment\'s Projection -, in a matter of utilising it as well as procedures for gathering informations about its audiences by proposing communication processes and dialogs, with the aid of digital media. These three activities were established according to the interests of the Public Policy Project \"Hybrid Territories: digital media, communities and cultural activities\", which, on the other hand, made possible the accomplishment of these studied experiments.

O exame de resultados de pesquisas anteriores realizadas pelo Nomads.usp - Núcleo de Estudos de Habitares Interativos - em que foram utilizados vários instrumentos metodológicos clássicos de pesquisa qualitativa a fim de entender contextos urbano e social, revelou limites desses instrumentos quando se desejava abarcar nuances referentes a modos de vida, visões de mundo e pontos de vistas de indivíduos pesquisados. O objetivo dessa pesquisa é a verificação dos limites e potencialidades do emprego de três atividades, concebidas para uso em eventos culturais com presença de público - Rádio de Rua, Graffiti Digital e Projeção de Comentários -, no sentido de utilizá-las também como procedimentos de obtenção de informação sobre seus públicos, estimulando processos comunicacionais e interlocuções com auxílio de meios digitais. Essas três atividades foram definidas em conjunto com os interesses do Projeto de Políticas Públicas \"Territórios Híbridos: meios digitais, comunidades e ações culturais\", o qual, por outro lado, tornou possível a realização dos experimentos aqui estudados. / The evaluation of previous research carried out by Nomads.usp - Center of Interactive Living Studies - which used several classical methodological instruments of qualitative research in order to understand urban and social contexts, found limits of these tools when they were employed to encompass nuances related to ways of life, worldviews and viewpoints of surveyed individuals. The aim of this reseach is to verify the limits and the possibilities of using three activities conceived for usage in cultural events with audience\'s presence - Street Radio, Digital Graffiti and Comment\'s Projection -, in a matter of utilising it as well as procedures for gathering informations about its audiences by proposing communication processes and dialogs, with the aid of digital media. These three activities were established according to the interests of the Public Policy Project \"Hybrid Territories: digital media, communities and cultural activities\", which, on the other hand, made possible the accomplishment of these studied experiments.

This study developed a GC-MS method for the simultaneous detection of piperazines and congeners in street samples of amphetamine type stimulants. This research investigated the clandestine routes of synthesis and chemical profiles of phenylpiperazines, represented by 1- (4-fluorophenyl)piperazine (4-FPP) and 1-(3-trifluoromethylphenyl)piperazine (3-TFMPP). These drugs are part of the increasingly prevalent illicit new psychoactive substances. The presence of (2, 3, 4) FPP and (2, 3, 4) TFMPP positional isomers has been identified by other researchers as a limitation due to their similar chemical profiles. The method was optimized and confirmed as compliant with the International Conference on Harmonisation and the Center for Drug Evaluation and Research guidelines on validation. 4- FPP and 3-TFMPP were synthesised using potential routes for clandestine laboratories. Simple extraction and analysis of 11 street samples was conducted using the method developed. Furthermore, the stability of 22 drugs during analysis was investigated. Limits of detection were in the range 5 – 1.95ng/mL free base on column. The synthesised samples were identified as 4-FPP and 3-TFMPP. Several impurities were observed in the synthesised samples, which were identified and categorised as residual reactants, isomers of 4-FPP and of 3-TFMPP and by-products of synthesis. The percentage yields of the synthesised samples obtained were up to 82.4% 4-FPP and 78.7% 3-TFMPP. The street samples were found to contain MDMA, 3-TFMPP, BZP, caffeine, ephedrine and other impurities. The analytical method simultaneously separates 19 of the most common drugs found in piperazine samples and achieves for the first time the GC-MS separation of (i) 2-FPP, 3-FPP and 4-FPP and (ii) 2-TFMPP, 3-TFMPP and 4-TFMPP at the same time from a sample matrix containing all the 19 compounds. This method provides operational laboratories with a more effective method for the chemical characterisation of street samples of piperazines and also provides novel stability data.

<p>U disertaciji su predstavljena istraživanja uticajnih parametara u procesu izrade alata za utiskivanje tehnikom aditivne proizvodnje, tačnije tehnikom deponovanja istopljenog materijala (FDM). Izrađena je kontrolna grupa alata za utiskivanje konvencionalnom tehnikom hemijskog nagrizanja i SLA tehnikom 3D štampe. Cilj istraživanja je definisanje optimalnih procesnih parametara izrade alata za<br />utiskivanje FDM tehnikom štampe. Ustanovljena je metodologija za karakterizaciju proizvodnih parametara koja se može primeniti na ostale tehnike aditivne proizvodnje. Analizom dobijenih rezultata i zaključaka istraživanja ustanovljena su ograničenja i mogućnosti zamene konvencionalnih alata za utiskivanje alatima izrađenim tehnikama 3D štampe.</p> / <p>The study of the influencing parameters in the production process of embossing dies using Fused Deposition Modelling (FDM) additive manufacturing technique, was investigated in this dissertation. Embossing dies, produced using conventional chemical etching and vat photopolymerization technique, were developed as a control group. This research aims to define the optimal process parameters of Fused Deposition Modelling (FDM) in embossing dies manufacturing. A new methodology for the characterisation of production parameters, which can be applied to other additive production techniques, has been established. By analysing the results and the conclusions of this research, the possibility of replacing<br />conventional embossing dies produced using 3D printing techniques has been established as well as its limitations.</p>

Après s’être interrogée sur la notion de sens, cette thèse se fixe pour objectif de dégager un certain nombre d’éléments linguistiques allant dans le sens d’une caractérisation abstraite à même de rendre compte des différents emplois du verbe anglais want. Il est proposé que ce verbe conserve aujourd’hui quelque chose de son emploi diachronique initial en tant que « verbe de manque » à partir d’une première opération de localisation par laquelle le sujet grammatical est mis en relation avec un objet ou une propriété non-immédiatement disponible pour lui dans la situation d’énonciation. Cette mise en relation particulière accompagne un processus de subjectivisation culminant dans une tendance forte à la modalisation de l’énoncé en want, en lien avec des effets déontiques et épistémiques. Par ailleurs, les interprétations mixtes associées à l’énoncé en want poussent à proposer d’en rendre compte par une « concentricité notionnelle » centrée sur un état stable p donné comme absent du domaine du sujet et à partir duquel s’élaborent les notions de manque, de désir et de volonté qui se convoquent et s’activent mutuellement à partir du second argument de want. Il semble intéressant de le traiter comme un domaine notionnel organisé autour de p, lequel sert de repère permettant d’envisager une seconde opération de localisation qui autorise le repérage d’une relation permettant d’atteindre p. Ainsi, la distinction désir / volonté est pensable en termes de degrés dans les déterminations que l’on peut construire à partir du manque. Ce travail met donc en relation les constructions syntaxiques possibles du second argument de want et ces déterminations. / After an overview of what underpins the notion of meaning, this thesis intends to identify a number of linguistic features that could serve as an abstract characterisation informing the uses of the English verb want. It is claimed that a vestige of its original use as a verb expressing lack has been kept in its modern uses as a verb expressing desire in that it marks a prime operation of localisation by which the grammatical subject connects to an object or a property p that is not immediately available to him in the speaking situation. It is shown that this relation relies on a subjectification process which culminates in a strong tendency towards the modalisation of the want sentence resulting in both epistemic and deontic effects. Given that want is used in utterances which convey mixed interpretations, it is suggested that those partially overlapping notions are best described in terms of a « notional concentricity » organised around a! n object or stable property which is absent from the dominion of the grammatical subject. It serves as the conceptual basis from which the mutually evocative and mutually activating notions of lack, desire and volition arise. It is argued that this organisation can be described as a notional domain centring on the missing object p, acting as a starting point for a second localisation operation whose aim is to locate a relation to palliate the lack of p. It is thus possible to account for the difference between desire and volition as degrees in the determinations that can be constructed from the missing object. This thesis then correlates the syntactic constructions in the internal argument of want with those types of determination.

<div>Additive manufacturing (AM) or 3D printing has gained significant advancement in recent years. However the potential of 3D printed metals still has not been fully explored. A main reason is the lack of accurate knowledge of the load capacity of 3D printed metals, such as fatigue behavior under cyclic load conditions, which is still poorly understood as compared with the conventional wrought counterpart.</div><div><br></div><div>The goal of the thesis is to advance the knowledge of fatigue behavior of 15-5 PH stainless steel manufactured through laser powder bed fusion process. To achieve the goal, a combined numerical and experimental study is carried out. First, using a rotary fatigue testing experiment, the fatigue life of the 15-5 PH stainless steel is measured. The strain life curve shows that the numbers of the reversals to failure increase from 13,403 to 46,760 as the applied strain magnitudes decrease from 0.214\% from 0.132\%, respectively. The micro-structure analysis shows that predominantly brittle fracture is presented on the fractured surface. Second, a finite element model based on cyclic plasticity including the damage model is developed to predict the fatigue life. The model is calibrated with two cases: one is the fatigue life of 3D printed 17-4 stainless steel under constant amplitude strain load using the direct cyclic method, and the other one is the cyclic behavior of Alloy 617 under multi-amplitude strain loads using the static analysis method. Both validation models show a good correlation with the literature experimental data. Finally, after the validation, the finite element model is applied to the 15-5 PH stainless steel. Using the direct cyclic method, the model predicts the fatigue life of 15-5 PH stainless steel under constant amplitude strain. The extension of the prediction curve matches well with the previously measured experimental results, following the combined Coffin-Manson Basquin Law. Under multi-amplitude strain, the kinematic hardening evolution parameter is incorporated into the model. The model is capable to capture the stresses at varied strain amplitudes. Higher stresses are predicted when strain amplitudes are increased. The model presented in the work can be used to design reliable 3D printed metals under cyclic loading conditions.</div>

<p>Proteini imaju osnovnu ulogu u&nbsp; životnim&nbsp;procesima, usled čega je isptivanje raznovrsnosti&nbsp;njihove strukture jedna od ključnih potreba&nbsp;modernih biolo&scaron;kih istraživanja. Cilj ove&nbsp;doktorske disertacije je predviđanje&nbsp;trodimenzionalne strukture beta-galaktozidaza&nbsp;odabranih bakterijskih sojeva (<em>Streptococcus&nbsp;thermophillus,&nbsp; Lactobacillus acidophilus</em>,&nbsp; i&nbsp;<em>Bifidobacterium animalis</em>&nbsp; ssp.&nbsp; <em>lactis</em>) kao i&nbsp;karakterizacija &nbsp;njihovih aktivnih mesta i&nbsp;određivanje aktivnih rezdua. Takođe, cilj&nbsp;disertacije obuhvata i ispitivanje beta-galaktozidaze&nbsp;<em> Bacteroides thetaiotaomicron</em>-a,&nbsp;kao i ispitivanje aktivnosti beta-galaktozidaze&nbsp;Kluyveromyces lactis-a. Na osnovu sličnosti&nbsp;<br />analizirane sekvence se mogu podeliti u dva&nbsp;klastera. Članovi klastera 1 pripadaju GH-2&nbsp;subfamiliji, dok članovi klastera 2 pripadaju GH-42 subfamiliji. Na osnovu klaster analize i poravnanja sekvenci&nbsp; predložene su&nbsp; katalitički&nbsp;aktivne rezidue beta-galaktozidaza ispitivanih&nbsp;sojeva: GLU458/GLU546 kod <em>S. thermophillus</em>-a,&nbsp;GLU148/GLU307 kod&nbsp;<em> L. Acidophilus-</em>a i&nbsp;GLU164/GLU324 kod<em> B. animalis</em> ssp.<em> lactis</em>-a.&nbsp;Konstruisani su homologi modeli beta-galaktozidaza<em> S. thermophilus, L. &nbsp;acidophilus, B.&nbsp;&nbsp; animalis</em>&nbsp; ssp.&nbsp;<em> lactis</em>.&nbsp; Iz rezultata simulacije&nbsp;molekularnog dokinga svih beta-galaktozidaza&nbsp;uočljivo je da se supstrat veže za aktivno mesto&nbsp;koje se nalazi u regionu visoke&nbsp;elektronegativnosti. Simulacije dokinga su&nbsp;potvrdile aktivna mesta predložena poravnanjem&nbsp;sekvenci i otkrile rezidue koje učestvuju u&nbsp;vezivanju laktoze i galaktoze kao supstrata.&nbsp;Obzirom da je objavljena samo jedna kristalna&nbsp;struktura beta-galaktozidaze sa laktozom u&nbsp;aktivnom mestu, analiza molekularnog dokinga&nbsp;laktoze kao supstrata je sprovedena i sa beta-alaktozidazama koje imaju određenu&nbsp;trodimenzionalnu strukturu. Na osnovu slučnosti&nbsp;sekvenci može se zaključiti da je nukleofilna&nbsp;rezidua beta-galaktozidaze <em>B. thetaiotaomicron</em>-a&nbsp;GLU259. Analizom konzervativnih regiona, kao i&nbsp;doking simulacije, može se zaključiti da ulogu&nbsp;proton donora ima rezidua GLU182. Hemijski&nbsp;sastav mleka za ispitivanje aktivnosti beta-galaktozidaze <em>Kluyveromyces lactis-</em>a je u skladu sa pravilnikom o kvalitetu proizvoda od mleka i&nbsp;starter kultura&nbsp; koji izdaje Ministarstvo&nbsp;<br />poljoprivrede i za&scaron;tite životne sredine Republike&nbsp;Srbije.&nbsp; Celokupna laktoza se razgradila u&nbsp;permeatu na temperaturi od 40&deg;C i pri&nbsp;koncentraciji enzima od 0,1% nakon 60 minuta od&nbsp;trenutka dodatka enzima. Maksimalna brzina&nbsp;postiže se toku prvih 10 minuta. Iz dobijenih&nbsp;rezultata može se zaključiti da beta-galaktozidaza&nbsp;<br /><em>K. lactis</em>-a ima visoku efikasnost prilikom&nbsp;razgradnje laktoze &scaron;to je čini pogodnom za&nbsp;<br />komercijalnu upotrebu u industriji mleka. U ovoj&nbsp;disertaciji predstavljen je protokol za&nbsp;<br />modelovanje komercijalno važnih beta-galaktozidaza i analiziranje njihovih &nbsp;interakcija sa laktozom kao supstratom. Dobijeni rezultati treba&nbsp;da pruže okvir za molekularna istraživanja&nbsp;vezivanja i razgradnje laktoze, kao i za buduće&nbsp;<br />dizajniranje komercijalno interesantnih beta-galaktzidaza.&nbsp;</p> / <p>Proteins have an essential role in all life&nbsp;processes. Therefore, investigation of their&nbsp;<br />structure diversity is one of the key focuses of&nbsp;modern biological researches. The aim of this&nbsp;PhD thesis is prediction of three-dimensional&nbsp;structure of&nbsp; beta-galactosidases of&nbsp; selected&nbsp;bacterial strains (<em>Streptococcus thermophillus,&nbsp;Lactobacillus acidophilus </em>and <em>Bifidobacterium&nbsp;animalis</em> ssp. <em>lactis</em>), as well as characterization&nbsp;of their active sites and active residues.&nbsp;Furthermore, this thesis include investigation&nbsp;<em>Bacteroides thetaiotaomicron</em>&nbsp; and&nbsp;<em>Kluyveromyces lactis</em> beta-galactosidases. Based&nbsp;on sequence &nbsp;similarity analyzed sequences can&nbsp;be clustered in two clusters. Members of cluster&nbsp;<br />1 belong to GH-2 subfamily, while members of&nbsp;cluster 2 belong to GH-42 subfamily. Cluster&nbsp;analysis and sequence alignment revealed&nbsp;potential active residues: GLU458/GLU546 - S.&nbsp;thermophillus,&nbsp; GLU148/GLU307&nbsp; -&nbsp; L.&nbsp;Acidophilus&nbsp; and&nbsp; GLU164/GLU324&nbsp; -&nbsp; B.&nbsp;animalis ssp. lactis.&nbsp; Homologous models of&nbsp;beta-galactosidases from&nbsp; <em>S. thermophilus,&nbsp; L.&nbsp;acidophilus,&nbsp; B. animalis&nbsp;</em> ssp.&nbsp; <em>lactis</em>&nbsp; are&nbsp;<br />constructed as a result of this PhD thesis.&nbsp;Molecular docking simulations of all beta-galactosidases revealed that substrate binds in&nbsp;region with high electronegativity. &nbsp;Furthermore,&nbsp;docking simulations confirmed active sites&nbsp;suggested by sequence alignment. As only one&nbsp;crystal structure with lactose in its active site&nbsp;has been published, docking simulations with&nbsp;lactose as a ligand were also performed with&nbsp;<br />experimentally determined beta-galactosidases&nbsp;structures. Also, lactose and &nbsp;alactose &nbsp;binding&nbsp;residues are predicted. Based on sequence&nbsp;similarity&nbsp; GLU259&nbsp; can be recognized as&nbsp;nucleophile of&nbsp;<em> B. thetaiotaomicron&nbsp;</em> beta-galactosidase. By analysis of conserved regions&nbsp;and docking simulation, GLU182 is predicted as&nbsp;a proton donor residue. Chemical composition&nbsp;of milk used for Kluyveromyces lactis&nbsp; beta-galactosidase research had appropriate quality&nbsp;according to directions of Ministry of&nbsp;<br />agriculture and environmental protection of&nbsp;Republic of Serbia. Total lactose content was&nbsp;degraded in permeate after 60 min using&nbsp;enzyme concentration of 0,1% at&nbsp; 40&deg;C.&nbsp;Maximal degradation was reached in the first 10&nbsp;min. Based on the results, it can be concluded&nbsp;that <em>Kluyveromyces lactis</em> beta-galactosidase has&nbsp;high efficiency for &nbsp;lactose degradation, which&nbsp;makes it commercially interesting for milk&nbsp;industry. Protocol for modeling of commercially&nbsp;important beta-galactosidases and their&nbsp;<br />interactions with lactose is developed as a result&nbsp;of this PhD thesis. Obtained results should&nbsp;provide the frame for molecular investigation of&nbsp;lactose binding and degradation, as well as for&nbsp;designing of commercially suitable beta-galactozidases.&nbsp;</p>

Clavicipitaceous fungal endophytes of the genera Epichloë and Neotyphodium form symbioses with grasses of the family Pooideae in which they can synthesise an array of bioprotective alkaloids. Some strains produce the ergot alkaloid ergovaline, which is implicated in livestock toxicoses caused by ingestion of endophyteinfected grasses. Cloning and analysis of a plant-induced non-ribosomal peptide synthetase (NRPS) gene from Neotyphodium lolii and analysis of the E. festucae E2368 genome sequence revealed a complex gene cluster for ergot alkaloid biosynthesis. The EAS cluster contained a single-module NRPS gene, lpsB, and other genes orthologous to genes in the ergopeptine gene cluster of Claviceps purpurea and the clavine cluster of Aspergillus fumigatus. Functional analysis of lpsB confirmed its role in ergovaline synthesis and bioassays with the lpsB mutant unexpectedly suggested that ergovaline was not required for black beetle (Heteronychus arator) feeding deterrence from epichloë-infected grasses. Southern analysis showed the cluster was linked with previously identified ergot alkaloid biosynthetic genes, dmaW and lpsA, at a subtelomeric location. The ergovaline genes are closely associated with transposon relics, including retrotransposons, autonomous DNA transposons and miniature inverted-repeat transposable elements (MITEs), which are very rare in other fungi. All genes in the cluster were highly expressed in planta but expression was very low or undetectable in mycelia from axenic culture, including under nitrogen-, carbonor phosphate-limited conditions. Comparative analysis of the EAS gene cluster in four different epichloë strains showed marked differences in gene expression and ergot alkaloid synthesis. Gene order is conserved in each strain although evidence for recombination between two MITEs and expansion or reduction of a simple sequence repeat (SSR) at a single intergenic region was observed. Heterologous expression of a candidate regulatory gene, laeA, from Aspergillus nidulans, which is a global regulator of secondary metabolism in aspergilli, did not affect eas gene expression. This, along with phylogeny and microsynteny analysis, suggests there is not an orthologue of this gene in epichloë. This work provides a genetic foundation for elucidating biochemical steps in the ergovaline pathway, the ecological role of individual ergot alkaloid compounds, and the regulation of their synthesis in planta.