Flavonoid glycosides are required for a number of crucial roles in planta and have the potential for development in a variety of agricultural, medicinal, and biotechnological applications. A number of recent advancements have been made in characterizing glycosyltransferases, the enzymes that are responsible for the synthesis of these important molecules. In this review, glycosyltransferases are considered with regard to biochemical properties, expression patterns, levels of enzyme activity during development, and structure/function relationships. This is presented with historical context to highlight critical findings, particularly with regard to the innovative work that has come from research on citrus species. The plant glycosyltransferase crystal structures that have been solved over the past decade, either alone or in complex with sugar donor and/or acceptor molecules, are discussed. The application of results from these structures to inform current structure/function work as well as implications and goals for future crystallography and tertiary modeling studies are considered. A thorough understanding of the properties of glycosyltransferases will be a critical step in any future biotechnological application of these enzymes in areas such as crop improvement and custom design of enzymes to produce desired compounds for nutritional and/or medicinal usage.

Yes

The exponential development of biotechnology has lead to a quasi unlimited number of potential products going from biopolymers to vaccines. Cell culture has therefore evolved from the simple cell growth outside its natural environment to its use to produce molecules that they do not naturally produce. This rapid development could not be continued without new control and supervising tools as well as a good process understanding. This requirement involves however a large diversity and a better accessibility of process measurements. In this framework, software sensors show numerous potentialities. The objective of a software sensor is indeed to provide an estimation of the system state variables and particularly those which are not obtained through in situ hardware sensors or laborious and expensive analysis. In this context, This work attempts to join the knowledge of increasing bioprocess complexity and diversity and the time scale of process developments and favours systematic modelling methodology, its flexibility and the speed of development. In the field of state observation, an important modelling constraint is the one induced by the selection of the state to estimate and the available measurements. Another important constraint is the model quality. The central axe of this work is to provide solutions in order to reduce the weight of these constraints to software sensors development. On this purpose, we propose four solutions to four main questions that may arise. The first two ones concern modelling uncertainties.<p><p>1."How to develop a software sensor using measurements easily available on pilot scale bioreactor?" The proposed solution is a static software sensor using an artificial neural network. Following this modelling methodology we developed static software sensors for the biomass and ethanol concentrations in a pilot scale S. cerevisae cell culture using the measurement of titrating base quantity, agitation rate and CO& / Doctorat en sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

Thesis (PhD)--University of Stellenbosch, 2004. / 271 Leaves printed single pages, preliminary pages i-xviii and 253 numberd pages. Includes bibliography. List of figures, List of tables. / ENGLISH ABSTRACT: A Neurocontrol Paradigm for Intelligent Process Control using Evolutionary Reinforcement Learning Balancing multiple business and operational objectives within a comprehensive control strategy is a complex configuration task. Non-linearities and complex multiple process interactions combine as formidable cause-effect interrelationships. A clear understanding of these relationships is often instrumental to meeting the process control objectives. However, such control system configurations are generally conceived in a qualitative manner and with pronounced reliance on past effective configurations (Foss, 1973). Thirty years after Foss' critique, control system configuration remains a largely heuristic affair. Biological methods of processing information are fundamentally different from the methods used in conventional control techniques. Biological neural mechanisms (i.e., intelligent systems) are based on partial models, largely devoid of the system's underlying natural laws. Neural control strategies are carried out without a pure mathematical formulation of the task or the environment. Rather, biological systems rely on knowledge of cause-effect interactions, creating robust control strategies from ill-defined dynamic systems. Dynamic modelling may be either phenomenological or empirical. Phenomenological models are derived from first principles and typically consist of algebraic and differential equations. First principles modelling is both time consuming and expensive. Vast data warehouses of historical plant data make empirical modelling attractive. Singular spectrum analysis (SSA) is a rapid model development technique for identifying dominant state variables from historical plant time series data. Since time series data invariably covers a limited region of the state space, SSA models are almost necessarily partial models. Interpreting and learning causal relationships from dynamic models requires sufficient feedback of the environment's state. Systemisation of the learning task is imperative. Reinforcement learning is a computational approach to understanding and automating goal-directed learning. This thesis aimed to establish a neurocontrol paradigm for non-linear, high dimensional processes within an evolutionary reinforcement learning (ERL) framework. Symbiotic memetic neuro-evolution (SMNE) is an ERL algorithm developed for global tuning of neurocontroller weights. SMNE is comprised of a symbiotic evolutionary algorithm and local particle swarm optimisation. Implicit fitness sharing ensures a global search and the synergy between global and local search speeds convergence.Several simulation studies have been undertaken, viz. a highly non-linear bioreactor, a rigorous ball mill grinding circuit and the Tennessee Eastman control challenge. Pseudo-empirical modelling of an industrial fed-batch fermentation shows the application of SSA for developing partial models. Using SSA, state estimation is forthcoming without resorting to fundamental models. A dynamic model of a multieffect batch distillation (MEBAD) pilot plant was fashioned using SSA. Thereafter, SMNE developed a neurocontroller for on-line implementation using the SSA model of the MEBAD pilot plant. Both simulated and experimental studies confirmed the robust performance of ERL neurocontrollers. Coordinated flow sheet design, steady state optimisation and nonlinear controller development encompass a comprehensive methodology. Effective selection of controlled variables and pairing of process and manipulated variables were implicit to the SMNE methodology. High economic performance was attained in highly non-linear regions of the state space. SMNE imparted significant generalisation in the face of process uncertainty. Nevertheless, changing process conditions may necessitate neurocontroller adaptation. Adaptive neural swarming (ANS) allows for adaptation to drifting process conditions and tracking of the economic optimum online. Additionally, SMNE allows for control strategy design beyond single unit operations. SMNE is equally applicable to processes with high dimensionality, developing plant-wide control strategies. Many of the difficulties in conventional plant-wide control may be circumvented in the biologically motivated approach of the SMNE algorithm. Future work will focus on refinements to both SMNE and SSA. SMNE and SSA thus offer a non-heuristic, quantitative approach that requires minimal engineering judgement or knowledge, making the methodology free of subjective design input. Evolutionary reinforcement learning offers significant advantages for developing high performance control strategies for the chemical, mineral and metallurgical industries. Symbiotic memetic neuro-evolution (SMNE), adaptive neural swarming (ANS) and singular spectrum analysis (SSA) present a response to Foss' critique. / AFRIKAANSE OPSOMMING: 'n Neurobeheer paradigma vir intelligente prosesbeheer deur die gebruik van evolusionêre versterkingsleer Dit is 'n komplekse ontwikkelingstaak om menigte besigheids- en operasionele doelwitte in 'n omvattende beheerstrategie te vereenselwig. Nie-lineêriteite en vele komplekse prosesinteraksies kombineer om ingewikkelde aksie-reaksie verwantskappe te vorm. Dit is dikwels noodsaaklik om hierdie interaksies omvattend te verstaan, voordat prosesbeheer doelwitte doeltreffend gedoen kan word. Tog word sulke beheerstelsels dikwels saamgestel op grond van kwalitatiewe kriteria en word ook dikwels staatgemaak op historiese benaderings wat voorheen effektief was (Foss, 1973). Dertig jaar na Foss se kritiek, bly prosesbeheerstelsel ontwerp 'n heuristiese saak. Die biologiese prosessering van informasie is fundamenteel verskillend van metodes wat gebruik word in konvensionele beheertegnieke. Biologiese neurale meganismes (d.w.s., intelligente stelsels) word gebaseer op gedeeltelike modelle, wat grotendeels verwyderd is van die onderskrywende natuurwette. Neurobeheerstrategieë word toegepas sonder suiwer wiskundige formulering van die taak of die omgewing. Biologiese stelsels maak eerder staat op kennis van aksie-reaksie verhoudings en skep robuuste beheerstrategieë van swak gedefineerde dinamiese stelsels. Dinamiese modelle is of fundamenteel of empiries. Fundamentele modelle word ontwikkel vanaf eerste beginsels en word tipies uit algebraïese en differensiële vergelykings saamgestel. Modellering vanaf eerste beginsels is beide tydrowend en duur. Groot databasisse van historiese aanlegdata maak empiriese modellering aantreklik. Singuliere spektrumanalise (SSA) maak die vinnige ontwerp van empiriese modelle moontlik, waardeur dominante veranderlikes vanaf historiese tydreekse onttrek kan word. Aangesien tydreeksdata slegs 'n gedeelte van die prosesomgewing verteenwoordig, is SSA modelle noodwendig gedeeltelike modelle. Die interpretasie en aanleer van kousale verhoudings vanaf dinamiese modelle vereis voldoende terugvoer van omgewingstoestande. Die leertaak moet sistematies uitgevoer word. Versterkingsleer is 'n ramingsbenadering tot 'n doelwit-gedrewe leerproses. Hierdie tesis bewerkstellig 'n neurobeheerparadigme vir nie-lineêre prosesse met hoë dimensies binne 'n evolusionêre versterkingsleer (EVL) raamwerk. Simbiotiese, memetiese neuro-evolusie (SMNE) is 'n EVL algoritme wat ontwikkel is vir globale verstelling van die gewigte van ‘n neurobeheerder. SMNE is saamgestel uit 'n simbiotiese evolusionêre algoritme en 'n lokale partikelswerm-algoritme. Implisiete fiksheidsdeling verseker 'n globale soektog en die sinergie tussen globale en lokale soektogte bespoedig konvergensie.Verskeie simulasie studies is onderneem, o.a. die van 'n hoogs nie-lineêre bioreaktor, 'n balmeulaanleg en die Tennessee Eastman beheer probleem. Empiriese modellering van 'n industriële enkelladingsfermentasie demonstreer die aanwending van SSA vir die ontwikkeling van gedeeltelike modelle. SSA benader die toestand van 'n dinamiese stelsel sonder die aanwending van fundamentele modellering. 'n Dinamiese model van 'n multi-effek-enkelladingsdistillasie (MEBAD) proefaanleg is bewerkstellig deur die gebruik van SSA. Daarna is SMNE gebruik om 'n neurobeheerder te skep vanaf die SSA model vir die beheer van die MEBAD proefaanleg. Beide simulasie en eksperimentele studies het die robuuste aanwending van EVL neurobeheerders bevestig. Die gekoördineerde ontwerp van vloeidiagramme, gestadigde toestand-optimering en nie-lineêre beheerderontwikkeling vereis 'n omvattende metodologie. Beheerveranderlikes en die koppeling van proses- en uitvoerveranderlikes is implisiet en effektief. Maksimale ekonomiese aanwins was moontlik in hoogs nie-lineêre dele van die toestandsruimte. SMNE het besondere veralgemening toegevoeg tot neurobeheerderstrategieë ten spyte van prosesonsekerhede. Nietemin, veranderende prosestoestande mag neurobeheerderaanpassing genoodsaak. Aanpasbare neurale swerm (ANS) algoritmes pas neurobeheerders aan tydens veranderende proseskondisies en volg die ekonomiese optimum, terwyl die beheerder die proses beheer. SMNE bewerkstellig ook die ontwikkeling van beheerstrategieë vir prosesse met meer as een eenheidsoperasie. SMNE skaal na prosesse met hoë dimensionaliteit vir die ontwikkeling van aanlegwye beheerstrategieë. Talle kwelvrae in konvensionele aanleg-wye prosesbeheer word deur die biologies gemotiveerde benadering van die SMNE algoritme uit die weg geruim. Toekomstige werk sal fokus op die verfyning van beide SMNE en SSA. SMNE en SSA bied 'n nie-heuristiese, kwantitatiewe benadering wat minimale ingenieurskennis of oordeel vereis. Die metodologie is dus vry van subjektiewe ontwerpsoordeel. Evolusionêre versterkingsleer bied talle voordele vir 'n ontwikkeling van effektiewe beheerstrategieë vir die chemiese, mineraal en metallurgiese industrieë. Simbiotiese memetiese neuro-evolusie (SMNE), aanpasbare neurale swerm metodes (ANS) en singulêre spektrum analise (SSA) gee antwoord op Foss se kritiek.

Submitted by Maicon Juliano Schmidt (maicons) on 2015-05-25T17:09:42Z No. of bitstreams: 1 Elis Cristina Uhry Lauxen.pdf: 1169503 bytes, checksum: 4cb8a48fdda3cfb749632df05c6c2924 (MD5) / Made available in DSpace on 2015-05-25T17:09:42Z (GMT). No. of bitstreams: 1 Elis Cristina Uhry Lauxen.pdf: 1169503 bytes, checksum: 4cb8a48fdda3cfb749632df05c6c2924 (MD5) Previous issue date: 2014-12-10 / Nenhuma / As indagações com relação ao termo inicial da vida humana perpassam a sociedade desde a antiguidade. No mundo pós-moderno, os avanços da ciência e da técnica potencializam as discussões acerca do tema, de modo que os debates que anteriormente se concentravam em torno do aborto, também passaram a ter como objeto as novas tecnologias. Desse modo, as questões contemporâneas sobre o marco inicial da vida humana referem-se, principalmente, a dois aspectos: o primeiro, relacionado à interrupção da gestação e o segundo, no que diz respeito às intervenções das novas tecnologias no embrião e sua ingerência na natureza humana. A presente Dissertação trata do segundo aspecto e, nesse contexto, busca refletir em que medida a dignidade humana pode ser considerada um referencial (hermenêutico) na construção de parâmetros ético-jurídicos para os avanços biotecnológicos na definição do marco inicial da vida humana. Para desenvolver a pesquisa, utiliza-se como método de abordagem a ética hermenêutica crítica, mediante a qual a ética encontra-se no centro do processo de compreensão e interpretação, observando-se os contornos da faticidade. O assunto será tratado com uma perspectiva transdisciplinar, que transpassa diferentes áreas do conhecimento, como a Bioética, o Biodireito, o Direito, a Filosofia, a Antropologia, a Sociologia, a Biologia e a Medicina. A Dissertação utiliza os métodos de procedimento histórico, comparativo e monográfico, a partir do referencial teórico, da legislação e da jurisprudência relevantes acerca do assunto. Como resultado, constatou-se que não há consenso no que diz respeito ao termo inicial da vida humana. Diante desse contexto, é importante dialogar com a nova realidade decorrente dos avanços biotecnológicos, no processo de construção de parâmetros éticos e jurídicos para a proteção do embrião e da natureza humana, tendo como referencial hermenêutico a dignidade humana. / Inquiries with respect to the beginning of human life pervade society since antiquity. In the postmodern world, the advances of science and technology enhance the discussions on the subject in such a way that the debates that earlier focused around abortion also started having as object the new technologies. Thus, the contemporary questions about the beginning of human life relate mainly to two aspects: the first one is related to the termination of pregnancy and the second concerns the interventions of new technologies in the embryo and as interference in human nature. This dissertation deals with the second aspect and, in this context, aims to consider to what extent human dignity can be regarded as a (hermeneutic) reference in the construction of ethical and legal parameters for the biotechnological advances in defining the beginning of human life. In order to develop the research, the critical hermeneutic ethics is used as method of approach, whereby ethics is at the center of the process of understanding and interpretation, observing the contours of facticity. The matter will be treated with a transdisciplinary perspective, which pervades different areas of knowledge, such as bioethics, biolaw, law, philosophy, anthropology, sociology, biology and medicine. As research methods, this dissertation uses historical, comparative and monographic research, from the theoretical framework of relevant legislation and case law on the subject. As a result, it was found that there is no consensus regarding the beginning of human life. In this context, it is important dealing with the new reality which results from biotechnological advances, in order to create parameters of protection for the embryo and for the human nature, in which the human dignity is a hermeneutic reference.

<p style="text-align: justify;">Jedno od najvećih dostignuća u dvadesetom veku je bio pronalazak antibiotika i njihova primena u humanoj medicini. Međutim, vremenom se pokazalo da su izazivači bolesti mikroorganizmi koji &ldquo;uče&rdquo; i imaju sposobnost da se &ldquo;menjaju&rdquo;, &scaron;to neminovno dovodi do pojave antibiotske rezistencije, a čemu ide u prilog &scaron;iroko rasprostranjena upotreba antibiotika u lečenju pacijenata i primena humanih antimikrobnih lekova u veterini i fitofarmaciji. Razvoj novih i unapređenje postojećih farmaceutika zahteva ogromna ulaganja koja se mogu ali i ne moraju vratiti godinama. Prvi korak u razvoju novog farmaceutika je identifikacija ciljanog antibiotskog delovanja metabolita izabranog proizvodnog mikroorganizma, nakon čega sledi optimizacija uslova biosinteze u smislu sastava medijuma za proizvodnju.<br />Osnovni cilj ove doktorske disertacije je optimizacija formulacije medijuma za kultivaciju prirodnog izolata Streptomyces hygroscopicus u pogledu izvora makronutrijenata i njihovih količina, kako bi se metabolička aktivnost primenjenog proizvodnog mikroorganizma, u definisanim proizvodnim uslovima, usmerila ka sintezi antibiotika sa ciljanim delovanjem.<br />Optimizacijom formulacije medijuma za kultivaciju prirodnog izolata <em>Streptomyces hygroscopicus</em>, u primenjenim eksperimentalnim uslovima, odabrani su najpogodniji izvori makronutrijenata i definisane su njihove optimalne koncentracije za proizvodnju antibiotika sa ciljanim delovanjem. Utvrđeno je da je biosinteza baktericida efikasnih protiv <em>B. cereus</em> ATCC 10876, <em>S. aureus</em> ATCC 11632 i P. <em>aeruginosa</em> ATCC 27853 najizraženija u medijumu sa fruktozom, sojinim bra&scaron;nom i fosfatnim solima, dok je za dobijanje fungicida koji deluju na <em>C. albicans</em> ATCC 10231 i <em>A. niger</em> ATCC 16404 najadekvatnije primeniti medijum sa glukozom kao izvorom ugljenika i već pomenutim izvorima azota i fosfora pri čemu je odnos navedenih sastojaka medijuma specifičan za svaki test mikroorganizam. Sa tehnolo&scaron;kog aspekta, rezultati ovih istraživanja predstavljaju pouzdan izvor informacija za unapređenje proizvodnih karakteristika primenjenog biokatalizatora, izbor tehnike kultivacije, definisanje toka i optimizaciju postupka proizvodnje baktericida i fungicida sa krajnjim ciljem uvećanja razmera posmatranog bioprocesa.</p> / <p>One of the greatest achievements in the twentieth century was the invention of antibiotics and their application in human medicine. However, over time, it has been proven that disease susceptors are &quot;learning&quot; microorganisms and have the ability to &quot;change&quot;, which inevitably leads to the emergence of antibiotic resistance, which is in favor of widespread use of antibiotics in the treatment of patients and the application of human antibiotics in veterinary medicine and phytopharmacy. The development of new and the advancement of existing pharmaceuticals requires huge investments that may or may not be back for years. The first step in the development of a new pharmaceutical is the identification of the target antibiotic activity of the metabolite of the selected production microorganism, followed by the optimization of the conditions of biosynthesis in terms of composition of the production medium.<br />The main goal of this PhD thesis is the optimization of the formulation of the medium for the cultivation of the natural isolate Streptomyces hygroscopicus in terms of the source of macronutrients and their amounts, in order to direct the metabolic activity of the applied production microorganism, in the defined production conditions, towards the synthesis of antibiotics with targeted action. By optimizing the formulation of the medium for the culture of the natural isolate Streptomyces hygroscopicus, in the applied experimental conditions, the most suitable sources of macronutrients were selected and their optimal concentrations for the production of antibiotics with targeted action were defined. The biosynthesis of bactericides was found to be effective against B. cereus ATCC 10876, S. aureus ATCC 11632 and P. aeruginosa ATCC 27853 most pronounced in the medium with fructose, soy flour and phosphate salts, while for the production of fungicides acting on C. albicans ATCC 10231 and A. niger ATCC 16404 most appropriately apply a glucose medium as carbon source and already mentioned sources of nitrogen and phosphorus wherein the ratio of said ingredients to the medium is specific to each test microorganism. From a technological point of view, the results of these studies represent a reliable source of information for improving the production characteristics of the applied biocatalyst, a selection of cultivation techniques, defining the flow and optimizing the production of bactericides and fungicides with the ultimate goal of increasing the size of the observed bioprocess.</p>

Infecções fúngicas em humanos vem aumentando nos últimos anos e acometem principalmente pacientes imunocomprometidos, portadores do vírus HIV, transplantados ou com câncer. Os antifúngicos empregados no tratamento pertencem a poucos grupos de fármacos e o aparecimento de resistência antifúngica em muitos patógenos leva à necessidade de desenvolvimento de novos agentes antifúngicos. As cepas Trichosporon japonicum QU139 e Candida catenulata LV102 apresentam atividade killer sobre diversas leveduras patogênicas, apresentando bom potencial para desenvolvimento de novos agentes antimicrobianos. O objetivo do trabalho foi a otimização das condições para produção e detecção dos compostos antimicrobianos, para seu futuro uso terapêutico, e sua purificação. O efeito killer da cepa T. japonicum QU139 foi avaliado pelo método dos poços contra células sensíveis de Cryptococcus gattii C20 nos meios GYP, YM e Queijo em diferentes pH e temperaturas. A máxima atividade killer foi encontrada no meio GYP, pH 4,5 à 25°C após 24 horas de incubação para T.japonicum QU139 e C. catenulata LV102. Não foi possível isolar o composto antimicrobiano produzido pela levedura T.japonicum QU139 pelos métodos de isolamento de proteína/glicoproteína, corroborando a hipótese de que a toxina seja um glicolipídeo. / Human fungal infections have increased in the last years and affect mainly immunocompromised patients, carriers of HIV vírus, transplanted or with cancer. The antifungal agents used in treatment belong to a few groups of drugs and the increase of antifungal resistance in many pathogens leads to the necessity of developing new antifungal agents. Strains Trichosporon japonicum QU139 and Candida catenulata LV102 showed killer activity against several pathogenic yeasts, having a good potential for the development of new antimicrobial agents. The objective of the work was the optimization of conditions for production and detection of the antimicrobial compounds, aiming their future terapeutic use, and their purification. The killer effect of T. japonicum QU139 strain was evaluated by the well method against sensitive cells of Cryptococcus gattii C20 in media GYP, YM and Cheese in different pH and temperatures. The maximum killer activity was found in media GYP, pH 4.5, 25°C after 24 hours of incubation for T.japonicum QU139 and C. catenulata LV102. The isolation of the antimicrobial compound produced by the yeast T.japonicum QU139 was not possible by the methods for isolation of proteins/glicoproteins, corroborating the hypothesis that the toxin is a glycolipid.

<p style="text-align: justify;">Plodovi jabuke su tokom čitave godine prisutni u ishrani beba, dece i odraslih ljudi zbog čega je kvalitet i zdravstevena bezbednost ovih namirnica od izuzetnog značaja. Veliki broj fitopatogenih gljiva su prouzrokovači bolesti jabuka međutim, među značajnim prouzrokovačima bolesti jabuka u skladi&scaron;tima spominju se gljive iz rodova Alternaria i Fusarium. Smatra se da su zdravstveno bezbedni plodovi jabuke pre svega zdravi plodovi, bez prisustva fitopatogenih gljiva i bez ostataka fungicida. Sve veća briga oko primene sintetičkih fungicida zbog njihove toksičnosti, razvoja rezistentnosti patogena na fungicide i &scaron;tetnih efekata na životnu sredinu i zdravlje ljudi, doveli su do potrebe za pronalaženjem alternative hemijskoj za&scaron;titi, a jedno od mogućih se ogleda upravo u primeni biotehnolo&scaron;kim postupkom proizvedenih agenasa. Budući da su visoki tro&scaron;kovi biotehnolo&scaron;ke proizvodnje osnovna prepreka za &scaron;iru primenu ovih agenasa, optimizacija uslova izvođenja biosinteze predstavlja najvažniji korak ka njenom prevazilaženju. Stoga je kao cilj istraživanja u okviru ove doktorske disertacije definisana optimizacija biosinteze antagonista skladi&scaron;nih patogena jabuke primenom Streptomyces hygroscopicus na nivou laboratorijskog bioreaktora u pogledu odabira izvora ugljenika i azota i količina izvora ugljenika, azota i fosfora u hranljivoj podlozi za biosintezu željenih antagonističkih agenasa, kao i parametara biosinteze sa posebnim osvrtom na intenzitet primenjene aeracije i me&scaron;anja. Analizom dobijenih rezultata zaključeno je da se podloga za kultivaciju S. hygroscopicus koja je sadržala glicerol i pored već prisutnog azota u podlozi nije sadržala dodatni izvor azota, pokazala kao najpogodnija za produkciju željenih antagonističkih agenasa efikasnih protiv izolata vrsta A. alternata i F. avenaceum. Rezultati optimizacije sastava hranljive podloge ukazuju da se primenom definisanih optimalnih vrednosti ispitivanih faktora, koje su: sadržaj glicerola od 20 g/l, sadržaj (NH₄)₂SO₄ od 0,25 g/l i sadržaj K₂HPO₄ od 1,41 g/l u podlozi za biosintezu antagonista skladi&scaron;nih patogena jabuke primenom S. hygroscopicus, na osnovu definisanih matematičkih modela predviđaju sledeće vrednosti parametara toka i parametara uspe&scaron;nosti biosinteze antagonista skladi&scaron;nih patogena jabuke: rezidualni glicerol od 5,3418 g/l; rezidualni azot od 0,193 g/l; rezidualni fosfati od 0,8601 g/l; prečnik zone inhibicije rasta micelije izolata A. alternata KA10 od 28,22 mm; prečnik zone inhibicije rasta micelije izolata A. alternata T1Jg3 od 36,86 mm; prečnik zone inhibicije rasta micelije izolata F. avenaceum KA12 od 25,54 mm i prečnik zone inhibicije rasta micelije izolata F. avenaceum KA13 od 26,78 mm. Rezultati dobijeni ispitivanjem uticaja različitog intenziteta aeracije i brzine me&scaron;anja na biosintezu antagonista skladi&scaron;nih patogena jabuke primenom S. hygroscopicus u laboratorijskom bioreaktoru standardnih geometrijskih odnosa ukupne zapremine 3 l ukazuju da se primenom definisanih optimalnih vrednosti ispitivanih faktora, brzine me&scaron;anja od 100 o/min, intenziteta aeracije od 1,2 l/l/min i pri trajanju kultivacije od 96 h na osnovu definisanih matematičkih modela predviđaju sledeće vrednosti parametara toka i parametara uspe&scaron;nosti biosinteze antagonista skladi&scaron;nih patogena jabuke: rezidualni glicerol od 8,2645 g/l; rezidualni azot od 0,1723 g/l; rezidualni fosfati od 0,5167 g/l; suva materija 1,5023 g/l; prečnik zone inhibicije rasta micelije izolata A. alternata KA10 od 62,62 mm; prečnik zone inhibicije rasta micelije izolata A. alternata T1Jg3 od 61,09 mm; prečnik zone inhibicije rasta micelije izolata F. avenaceum KA12 od 45,55 mm i prečnik zone inhibicije rasta micelije izolata F. avenaceum KA13 od 43,02 mm. Validnost dobijenih rezultata potvrđena je izvođenjem biosinteze antagonista skladi&scaron;nih patogena jabuke na podlozi optimalnog sastava i primenom optimalnih procesnih parametara u laboratorijskom bioreaktoru standardnih geometrijskih odnosa ukupne zapremine 7l, a efikasnost produkovanih antagonističkih agenasa efikasnih na izolate A. alternata (KA10 i T1Jg3) i F. avenaceum (KA12 i KA13) potvrđena je i u in planta ogledima. Ostavreni rezultati optimizacije biosinteze antagonista skladi&scaron;nih patogena jabuke primenom Streptomyces hygroscopicus na nivou laboratorijskog bioreaktora predstavljaju osnovu za dalje uvećanje razmera do industrijskih, &scaron;to je ključni korak ka potencijalnoj industrijalizaciji predloženog proizvodnog postupka.</p> / <p>Apple fruits are available on the market all year round and their safety is of great importance for the consumers. Unfortunately, many phytopathogenic fungi can cause apple diseases but fungi from the genera Alternaria and Fusarium are listed among important storage pathogens of apple fruits. It is considered that health-safe apple fruits are primarily healthy fruits, without the presence of phytopathogenic fungi and without the remains of fungicides. Excessive use of pesticides in modern agriculture, including those used for control of fungal phytopathogens, has led to several problems related to pollution, environmental degradation and emergence of resistant strains. Biological<br />control of plant pathogens by means of microorganisms is considered as an attractive alternative to chemical-based treatments, with minimal impact on the environment. The main lack of biotechnology production of the antifungal agents are the high costs, so the optimization of the biosynthesis conditions is the most important step towards its overcoming. Therefore, the aim of this research is to optimize the medium composition and process conditions (agitation and aeration rate) of the antagonists of storage apple pathogens biosinthesis by Streptomyces hygroscopicus in laboratory-scale bioreactor. Based on the obtained results, it can be concluded that the S. hygroscopicus cultivation medium containing glycerol as a carbon source, with no additional nitrogen source, proved to be the most suitable for the production of the A. alternate and F. avenaceum antagonistic agents. The results of the optimization of medium for biosynthesis of the antagonists of storage apple pathogens using S. hygroscopicus indicated the following optimal values of the examined factors: the content of glycerol of 20 g/l, (NH₄)₂SO₄ of 0.25 g/l and K₂HPO₄ of 1.41 g/l. By applying the defined optimal values and using the appropriate mathematical models, the following responses are predicted: residual glycerol of 5.3418 g/l; residual nitrogen of 0.193 g/l; residual phosphates of 0.8601 g/l; inhibition zone diameters of A. alternata KA10 of 28.22 mm; inhibition zone diameters of A. alternata T1Jg3 of 36.86 mm; inhibition zone diameters of F. avenaceum KA12 of 25.54 mm and inhibition zone diameters of F. avenaceum KA13 of 26.78 mm. Examining the effect of different agitation and aeration rate on the biosynthesis of antagonists of storage apple pathogens using S. hygroscopicus in a laboratory-scale bioreactor with total volume of 3 l, the following optimal values of the investigated factors are obtained: the agitation rate of 100 rpm, the aeration of 1.2 l/l/min and 96-hour as optimal time of cultivation. Bringing together the appropriate mathematical models and defined optimal values, the following responses are predicted: residual glycerol of 8.2645 g/l; residual nitrogen of 0.1723 g/l; residual phosphates of 0.5167 g/l; biomass content of 1.5023 g/l; inhibition zone diameters of A. alternata KA10 of 62.62 mm; inhibition zone diameters of A. alternata T1Jg3 of 61.09 mm; inhibition zone diameters of F. avenaceum KA12 of 45.55 mm and inhibition zone diameters of F. avenaceum KA13 of 43.02 mm. The validity of the results was confirmed by performing the biosynthesis of the antagonists of storage apple pathogens in the medium with optimal composition and the optimal process parameters in a laboratory-scale bioreactor with the total volume of 7l. The efficacy of produced antagonistic agents effective against A. alternata (KA10 and T1Jg3) and F. avenaceum (KA12 and KA13) isolates was confirmed in vitro and in planta. The results obtained within this research represents the basis for the further enlargement of the scale, which is a key step towards the potential industrialization of the proposed production process.</p>

<p>Sve veća potreba za hranom kao posledica povećanja broja ljudi &scaron;irom sveta dovela je do intenzifikacije poljoprivrednih aktivnosti usmerenih na direktnu proizvodnju hrane i hrane za životinje. U poslednjih nekoliko decenija sve vi&scaron;e se govori o problemu prekomerne upotrebe hemijskih pesticida, &scaron;to je je rezultovalo degradacijom kvaliteta zemlji&scaron;ta, kao i sve većim zagađenjem podzemnih i povr&scaron;inskih voda. Osim toga, nedovoljno naučnih podataka o uticaju hemijskih jedinjenja koja ulaze u sastav pesticida na zdravlje ljudi izaziva jo&scaron; veći stepen zabrinutosti. Stoga se iznalaženje novih načina za kontrolu biljnih &scaron;tetočina i prevenciju i tretman bolesti biljaka postavlja kao imperativ održive poljoprivredne proizvodnje, a samim tim i održivog razvoja. Jedna od alternativa koje su pokazale najveći potencijal jeste primena biopesticida, koji po svojoj naj&scaron;iroj definiciji predstavljaju žive organizme ili njihove proizvode koji pokazuju sposobnost suzbijanja &scaron;tetočina. Kada su u pitanju mikrobiolo&scaron;ki biopesticidi, bakterije roda <em>Bacillus</em> predstavljaju najzastupljenije mikroorganizme koji se mogu sresti u komercijalno dostupnim proizvodima. Proizvodnja mikrobiolońkih biopesticida, iako u konstantnom porastu, i dalje čini samo mali deo ukupnog svetskog trži&scaron;ta sredstava za za&scaron;titu bilja, najvi&scaron;e zbog visokih tro&scaron;kova i komplikovanog procesa proizvodnje ovog tipa biokontrolnih agenasa. Stoga je kao osnovni cilj istraživanja ove doktorske disertacije postavljena optimizacija uslova umnožavanja i primarnog izdvajanja biomase soja <em>Bacillus velezensis</em> sa primenom za sprečavanje pojave i &scaron;irenja bakterioza povrtarskih useva koje izazivaju fitopatogene vrste roda<em> Xanthomonas</em>, koji u Republici Srbiji izazivaju velike ekonomske gubitke u proizvodnji kupusa, paprike i paradajza. Prvi korak optimizacije bioprocesa podrazumevao je odabir optimalnog izvora ugljenika i organskog izvora azota za biotehnolo&scaron;ku proizvodnju biokontrolnih agenasa, pri čemu su najbolji rezultati dobijeni primenom glicerola i ekstrakta kvasca, &scaron;to je otvorilo mogućnost ispitivanja primene sirovog glicerola iz proizvodnje biodizela kao izvora ugljenika. Nakon toga je usledilo modelovanje sastava hranljive podloge na bazi komercijalnog i sirovog glicerola u pogledu ispitivanja uticaja komponenata podloge (glicerola, ekstrakta kvasca, (NH₄)₂SO₄ i K₂HPO₄) na odabrane odzive (prečnik zona inhibicije i rezidualne koncentracije nutrijenata &ndash; glicerola, ukupnog azota i ukupnog fosfora). Dobijeni matematički modeli posmatranih odziva, za koje je dokazano da su statistički značajni na nivou značajnosti od 95%, dalje su kori&scaron;ćeni za optimizaciju sastava hranljivih podloga. Optimizovan sastav hranljive podloge na bazi komercijalnog glicerola je sledeći: glicerol 10 g/l, ekstrakt kvasca 2,83 g/l, (NH₄)₂SO₄ 3 g/l, K₂HPO₄ 1,07 g/l i MgSO₄∙7H₂O 0,3 g/l. Sa druge strane, optimizovan sastav hranljive podloge na bazi sirovog glicerola dobija se primenom sledećih koncentracija nutrijenata: glicerol 10 g/l, K₂HPO₄ 4,66 g/l i MgSO₄∙7H₂O 0,3 g/l, &scaron;to ukazuje na mogućnost smanjenja tro&scaron;kova pripreme ove hranljive podloge zbog izostavljanja izvora azota. Validacija dobijenih matematičkih modela izvr&scaron;ena je eksperimentima kultivacije proizvodnog mikroorganizma u uvećanim razmerama - u Vulfovim bocama (2 l) i laboratorijskom bioreaktoru (3 l), čiji su rezultati pokazali zadovoljavajuću korelaciju sa modelima predviđenim vrednostima odziva bioprocesa, pri čemu su bolji rezultati dobijeni primenom hranljive podloge na bazi komercijalnog glicerola i kultivacijom proizvodnog mikroorganizma u laboratorijskom bioreaktoru. Unapređenje procesa unakrsne mikrofiltracije kultivacionih tečnosti dobijenih nakon kultivacije proizvodnog mikroorganizma <em>Bacillus velezensis</em> na hranljivoj podlozi na bazi komercijalnog glicerola primenom različitih hidrodinamiĉkih metoda pokazalo je značajan potencijal primene Kenics statičkog me&scaron;ača, uduvavanja vazduha i kombinacije ovih metoda za pobolj&scaron;anje fluksa permeata u stacionarnom stanju.<em> In planta</em> ispitivanje antimikrobne aktivnosti preparata na bazi <em>Bacillus velezensis</em> protiv fitopatogenih sojeva vrste <em>Xanthomonas euvesicatoria</em>, koji izazivaju bakterioznu pegavost paprike, pokazalo je značajan potencijal primene bikontrolnih agenasa na bazi <em>Bacillus velezensis</em> za suzbijanje ovog oboljenja paprike. Ispitivanje &scaron;ireg spektra delovanja preparata na bazi<em> Bacillus velezensis </em>pokazalo je značajan potencijal za suzbijanje oboljenja izazvanih fitopatogenim sojevima roda Fusarium, koji izazivaju truljenje jabuka u toku skladi&scaron;tenja. Rezultati proistekli iz okvira ove doktorske disertacije predstavljaju osnovu za dalje unapređenje i povećanje razmera biotehnolońkog postupka proizvodnje biokontrolnih agenasa na bazi proizvodnog mikroorganizma <em>Bacillus velezensis</em>, uz predlog re&scaron;enja za iskori&scaron;ćenje sirovog glicerola iz proizvodnje biodizela u biotehnolońkom postupku dobijanja proizvoda sa dodatom vredno&scaron;ću.</p> / <p>The raise of human population worldwide requires higher amount of available groceries, which has led to intensification of agricultural activities directed at production of food and feed. In the last few decades the problem of chemical pesticides‟ overuse has resulted in soil deterioration and contamination of surface and underground water reservoirs. Furthermore, insufficient scientific data concerning the chronic effect of chemicals used in pesticides on human health contribute to the global concern when it comes to pesticides‟ usage. Therefore finding new ways to control plant pests and to prevent or suppress plant diseases has become an imperative for sustainable agricultural production and consequently for sustainable development. One of the alternatives showing great potential is usage of biopesticides, comprising of living organisms or their products able to suppress plant pathogens. When it comes to microbial biopesticides, bacteria of the genus Bacillus are the most often used in commercial biocontrol products. Microbial biopesticides, although being a raising alternative, still represent only small share of the global pesticides‟ market, mostly due to expensive and complicated production process. Hence the main goal of the research in the framework of this PhD thesis was to optimize conditions of cultivation and separation processes for production of Bacillus velezensis biomass intended for application in prevention and suppression of plant diseases caused by phytopathogenic species of the genus Xanthomonas, which cause significant economic losses during agricultural production of cabbage, pepper and tomato in the Republic of Serbia. The first step of bioprocess optimization was to choose optimal carbon and organic nitrogen sources for biocontrol agents‟ production, where the best results have been achieved by using glycerol and yeast extract, which opened a new chapter of possibilities to utilize raw glycerol from biodiesel production as carbon source. After that, modeling of cultivation medium composition based on commercial and raw glycerol was performed in order to investigate the effect of cultivation medium components (glycerol, yeast extract, (NH4)2SO4 and K2HPO4) on the selected responses (inhibition zone diameter and residual concentration of nutrients &ndash; glycerol, total nitrogen and total phosphorus). The obtained mathematical models for the selected responses, proved to be statistically significant at the significance level of 95%, were further used for optimization of cultivation media composition. Optimized composition of the cultivation medium based on commercial glycerol was: glycerol 10 g/L, yeast extract 2.83 g/L, (NH4)2SO4 3 g/L, K2HPO4 1.07 g/L and MgSO4∙7H2O 0.3 g/L. On the other hand, optimized composition of the cultivation medium based on raw glycerol was: glycerol 10 g/L, K2HPO4 4.66 g/L and MgSO4∙7H2O 0.3 g/L, which has opened a possibility to reduce cost of cultivation medium preparation by excluding nitrogen sources. Validation of the obtained mathematical models was carried out by cultivating the producing microorganism at a larger scale &ndash; in Woulff bottles (2 L) and in a laboratory-scale bioreactor (3 L). Validation results were in good correlation with the model predicted values of the selected responses, where better results were achieved by using cultivation medium based on commercial glycerol and cultivation of the producing microorganism in the laboratory-scale bioreactor. Application of different hydrodynamic methods, such as Kenics static mixer, air sparging and their combination, has showed a significant potential for improvement of cross flow microfiltration of Bacillus velezensis cultivation broth based on commercial glycerol in terms of permeate flux enhancement. Also, significant potential of the Bаcillus velezensis cultivation broth for suppression of phytopathogenic Xanthomonas euvesicatoria strains, which cause bacterial spot of pepper, has been proven during in planta experiments. Investigation of wider spectra of antimicrobial activity expressed by preparation based on Bаcillus velezensis cultivation broth has revealed a significant potential for suppression of Fusarium phytopathogenic species responsible for apple rot during storage. The results arising from the research performed in the framework of this PhD thesis represent solid basis for further improvement and scale-up of biotechnological process for production of biocontrol agents based on Bacillus velezensis, with a proposal for utilization of raw glycerol from biodiesel production in biotechnological production of value-added products.</p>

Infecções fúngicas em humanos vem aumentando nos últimos anos e acometem principalmente pacientes imunocomprometidos, portadores do vírus HIV, transplantados ou com câncer. Os antifúngicos empregados no tratamento pertencem a poucos grupos de fármacos e o aparecimento de resistência antifúngica em muitos patógenos leva à necessidade de desenvolvimento de novos agentes antifúngicos. As cepas Trichosporon japonicum QU139 e Candida catenulata LV102 apresentam atividade killer sobre diversas leveduras patogênicas, apresentando bom potencial para desenvolvimento de novos agentes antimicrobianos. O objetivo do trabalho foi a otimização das condições para produção e detecção dos compostos antimicrobianos, para seu futuro uso terapêutico, e sua purificação. O efeito killer da cepa T. japonicum QU139 foi avaliado pelo método dos poços contra células sensíveis de Cryptococcus gattii C20 nos meios GYP, YM e Queijo em diferentes pH e temperaturas. A máxima atividade killer foi encontrada no meio GYP, pH 4,5 à 25°C após 24 horas de incubação para T.japonicum QU139 e C. catenulata LV102. Não foi possível isolar o composto antimicrobiano produzido pela levedura T.japonicum QU139 pelos métodos de isolamento de proteína/glicoproteína, corroborando a hipótese de que a toxina seja um glicolipídeo. / Human fungal infections have increased in the last years and affect mainly immunocompromised patients, carriers of HIV vírus, transplanted or with cancer. The antifungal agents used in treatment belong to a few groups of drugs and the increase of antifungal resistance in many pathogens leads to the necessity of developing new antifungal agents. Strains Trichosporon japonicum QU139 and Candida catenulata LV102 showed killer activity against several pathogenic yeasts, having a good potential for the development of new antimicrobial agents. The objective of the work was the optimization of conditions for production and detection of the antimicrobial compounds, aiming their future terapeutic use, and their purification. The killer effect of T. japonicum QU139 strain was evaluated by the well method against sensitive cells of Cryptococcus gattii C20 in media GYP, YM and Cheese in different pH and temperatures. The maximum killer activity was found in media GYP, pH 4.5, 25°C after 24 hours of incubation for T.japonicum QU139 and C. catenulata LV102. The isolation of the antimicrobial compound produced by the yeast T.japonicum QU139 was not possible by the methods for isolation of proteins/glicoproteins, corroborating the hypothesis that the toxin is a glycolipid.