A utilização de produtos naturais para a terapêutica do câncer foi iniciada com a actinomycina D, obtida a partir de culturas de Streptomyces e desde então, a busca por compostos bioativos de origem natural constitui uma importante linha de pesquisa. Estima-se que aproximadamente 60% dos agentes antineoplásicos, introduzidos para a terapia do câncer nas últimas décadas, tem origem vegetal ou microbiana. Dentre os micro-organismos proeminentes para produção de compostos ativos, as actinobactérias se destacam pela versatilidade metabólica, praticidade para cultivo in vitro e eficiência para produção de compostos com atividade anticâncer. Em seu último relatório, a Organização Mundial da Saúde reportou 8,8 milhões de mortes em decorrência de câncer, no ano de 2017. O índice representa um em cada seis óbitos em todo o mundo, sendo mais expressivo em países de média e baixa renda. Vale ressaltar que avanços significativos foram alcançados nos últimos anos para o tratamento de leucemia aguda infantil e tumores derivados de células germinais. Contudo, tumores sólidos de pulmão, próstata, mama e cólon ainda representam altos índices de mortalidade. Frente a isso, torna-se evidente a necessidade de identificar e desenvolver estratégias para o tratamento da doença. Com intuito de acessar novos recursos microbianos com potencial biotecnológico, a prospecção avança para áreas pouco exploradas, como por exemplo, o Continente Antártico. A Antártica foi o último dos continentes a ser acessado pelo homem e apresenta características edafoclimáticas favoráveis ao endemismo. Em vista da problemática e da potencialidade do Continente Antártico, os recursos microbiológicos associados à rizosfera de Deschampsia antarctica Desv. foram acessados e avaliados para a produção de compostos com propriedade antitumoral. Em resultado foram obtidos 42.528 clones metagenômicos e 72 linhagens de actinobactérias, dentre as quais Streptomyces sp. CMAA 1527, que apresentou pronunciada atividade antiproliferativa in vitro, para tumores de mama, pulmão, rim e sistema nervoso central, através da produção de cinerubina B. A análise taxonômica das actinobactérias isoladas revelou a presença de linhagens com baixo índice de similaridade, com as linhagens tipo conhecidas, o que pode significar a presença de novas espécies para os gêneros Nocardia, Rhodococcus e Streptomyces, reconhecidos pela capacidade de produzir metabólitos ativos e enzimas de interesse biotecnológico. A análise taxonômica polifásica da linhagem CMAA 1533 possibilitou a descrição da espécie Rhodococcus psychrotolerans sp. nov. (TaxoNumber TA00191; NRRL B-65465T = DSM 104532T), grupo bacteriano promissor como agente de biorremediação e produção de compostos bioativos. Com isso, o Continente Antártico foi considerado um ambiente promissor para a busca de novos micro-organismos, dentre eles actinobactérias, eficientes na produção de compostos antitumorais e outras substâncias com potencial biotecnológico. / The use of natural products for cancer therapy was initiated with the actinomycin D, obtained from Streptomyces. Since then, the search of bioactive from natural sources represent an essential line of research. It is estimated that approximately 60% of the antineoplasic agents inserted for the cancer therapy in recent decades have vegetal and microbial origin. Among the prominent microorganisms used to produce active compounds, actinobacterias are known by their metabolical versatility, convenience related to in vitro culture, and efficiency on the production of anticancer compounds. The Health World Organization, on its last review, reported 8.8 million of deaths in 2017, caused by cancer. Those numbers represent one out of six deaths worldwide, being more expressive in middle and low income countries. It is worth pointing out that meaningful advances were established in recent years for the treatment of childhood acute leukemia and germ cell-derived tumors. However, solid tumors of the lung, prostate, breast and colon still represent high mortality rates. For this reason, it is necessary to identify and develop strategies for the treatment of the disease. With the aim of accessing new microbial resources that contain biological potential, the prospection advance to areas barely explored, such as the Antarctic Continent. Antarctica was the last of the continents to be accessed by man and presents edaphoclimatic characteristics favorable to endemism. In light of the problematic and the potentiality of the Antarctic Continent, the microbiological resources associated with the rhizosphere of Deschampsia antarctica Desv. were accessed and evaluated for the production of compounds with antitumor properties. The results obtained had shown 42,528 metagenomic clones and 72 strains of actinobacteria, among them Streptomyces sp. CMAA 1527, which had presented anti-proliferative activity in vitro to breast, lung, kidney and central nervous system tumors, through the production of cinerubin B. The taxonomic analysis of the actinobacteria isolated revealed the presence of strains with low rate of similarity, with known type strains, which may mean the presence of new species for the genera Nocardia, Rhodococcus and Streptomyces, recognized for the ability to produce active metabolites and enzymes of biotechnological interest. The polyphasic approach of the CMAA 1533 strain made possible the description of the species Rhodococcus psychrotolerans sp. nov. (TaxoNumber TA00191; NRRL B-65465T = DSM 104532T), promising bacterial group as a bioremediation agent and production of bioactive compounds. As a result, the Antarctic Continent was considered a promising environment to search new microorganisms, among them, the actinobacteria, which is efficient on the production of antitumor compounds and other substances with biotechnological potential.

Estudamos a ação das proteases bothropasina e Bothrops protease A, do veneno da serpente Bothrops jararaca, sobre o fibrinogênio (FBG), fibronectina (FN) e cininogênio (HK), como ferramenta para geração de peptídeos bioativos. As sequências primárias dos produtos de digestão foram identificadas por espectrometria de massas, com as buscas direcionadas por peptídeos em comum gerados pelas duas proteases. Foram encontradas oito sequências em comum provenientes do FBG e onze, da FN. Apenas a bothropasina clivou o HK, liberando desArg9BK. Foram sintetizados peptídeos derivados do FBG (FBG1-6) e da FN (FN1-4), além de des-Arg9-BK. Oito peptídeos apresentam potencial atividade antiangiogênica predita in silico. Observamos a inibição da elastase (28-20%) causada por FBG1-2-5-6. A melhor inibição da trombina foi de 17%, por FBG1. Contudo, a maioria dos peptídeos intensificou sua atividade. Por fim, este trabalho sugere que as proteases de veneno de serpentes podem ser usadas como ferramentas para processar componentes do plasma, visando à busca por peptídeos bioativos. / We studied the action of the proteases bothropasin and Bothrops protease A purified from the venom of snake Bothrops jararaca upon fibrinogen (FBG), fibronectin (FN) and kininogen (HK), as a tool to generate bioactive peptides. The primary sequences of the digestion products were identified by mass spectrometry and we focused the search for common peptides released by both proteases simultaneously. Sequences in common released by both proteases were found, being eight peptides from FBG, and 11 from FN. Only bothropasin was able to cleave HK releasing des-Arg9-BK. Peptides from fibrinogen (FBG1-6) and from fibronectin (FN1-4), as well as the des-Arg9-BK were synthetized. Eight peptides have potential antiangiogenic predicted in silico. We observed the inhibition of elastase (28-20%) caused by FBG1-2-5-6. The best inhibition of thrombin was 17% by FBG1. However, most of the peptides intensified its activity. Finally, this work suggests that the snake venom protease can be used as tools to process plasma components in order to search for bioactive peptides.

Rhodococci are notable for their ability to degrade a variety of natural and xenobiotic compounds. Recently, interest in Rhodococcus has increased due to the discovery of a large number of genes for secondary metabolism. Only a few secondary metabolites have been characterized from the rhodococci (including 3 recently described antibiotics). Twenty-four new Rhodococcus strains were isolated from soils in East Tennessee using acetonitrile enrichment culturing and identified using 16S rRNA analysis. Forty-seven Rhodococcus strains were screened for antibiotic production using a growth inhibition assay. One strain, MTM3W5.2, had 90% similarity to the Rhodococcus opacus 16S rRNA gene sequence and produced a large zone of inhibition against R. erythropolis and a large number of closely related species. The antimicrobial compound produced by MTM3W5.2 had a large MW of 911.5452 Da and acts much like a bacteriocin but no amino acids were detected in this molecule based on TLC analysis.

For bioactive chemical classes, it is often assumed that environmental transformation eliminates associated ecosystem risks. However, for endocrine-active steroid hormones, modest changes in structure can have a significant influence on biological activity and thus, subtle environmental transformations can yield products with conserved, enhanced, or activity across different biological endpoints. The aim of this work was to explore the environmental fate of high potency, endocrine-active steroid hormones during natural or engineered water processes in order to test the hypothesis that steroid transformation products generated during these processes are likely to contribute to residual bioactivity often reported in water resources. Specifically, laboratory experiments were used to simulate chemical disinfection (e.g., chlorination) or natural processes (e.g., photolysis) to: (i) determine the rate and extent of steroid transformation, (ii) isolate and identify products that are formed, and (iii) evaluate products or product mixtures for biological activity. These experimental results can be used to help guide occurrence studies for any products of concern in the environment and also guide computational predictions or rationalizations of chemical reactivity. Ultimately, the goal is to expand upon our awareness and understanding of how these potent endocrine ligands behave in the environment and how they potentially affect ecosystem health. Chapter 2 discusses the reaction of glucocorticoids (GC)s with aqueous chlorine (effectively, HOCl) to simulate their fate during engineered drinking water and wastewater chemical disinfection. Numerous transformation pathways were unveiled, including interconversion of GCs (e.g., endogenous cortisol to synthetic prednisolone), production of known androgens in the adrenosterone class, and chlorination of GCs (e.g., formation of 9-chloro-prednisone). We also showed that other advanced processes (e.g., oxidation via ozonation) result in more complete degradation of such pollutants, and may be better alternatives to chemical disinfection at eliminating bioactive steroidal product formation. In Chapter 3, results of the direct photolysis of dienogest (DNG), a widely prescribed oral contraceptive agent, are presented to simulate its fate in natural sunlit surface waters and engineered photochemical treatment systems (e.g., UV disinfection systems). The major products (~ 80% of the converted mass in neutral aqueous solutions) were identified to be photohydrates resulting from photochemical-induced incorporation of water into parent DNG. These products were found to be prone to dehydration in the dark, and thus, a source of substantial DNG regeneration (~ 65% after 72 h in neutral solutions). Other minor, non-revertible products were also identified, including two known estrogens. Although minor in initial yield, these estrogens are likely to accumulate over time through repeated cycling between DNG and its photohydrates, and thus, dominate DNG long-term fate. It was also found that DNG undergoes an unusual photochemical rearrangement to produce a minor product with a novel tetracyclic ring system--the subject of Chapter 4. Further, the generality of this unique photorearrangement process was explored through extension to the photolysis of two other dienone pharmaceutical steroids (e.g., the androgens methyldienolone and dienedione). Surprisingly, despite the significant change in core steroidal structure, the rearrangement products retain some progesterone receptor (PR) and androgen receptor (AR) bioactivity (i.e., low-µM to sub-nm EC50 values). Again, these represent other non-revertible, minor products that are likely to accumulate over time, with likely adverse ecological consequences. Chapter 5 covers results arising from the direct photolysis of trenbolone acetate (TBA) metabolites in the presence of model nucleophiles (e.g., sodium azide, sodium thiosulfate, ammonium hydroxide, hydroxylamine, and humic acid), some of which would be expected to be present, along with TBA metabolites, in agriculturally-impacted water resources. Previous studies by our group revealed that TBA metabolites undergo photohydration-thermal dehydration cycling, like that described above for DNG photolysis. The objective of this study was to determine if other nucleophiles would outcompete water for photochemical incorporation across the TBA metabolite extended conjugation system. It was found that TBA metabolite photolysis results in photochemical (and at times thermal) addition of the nucleophile to the TBA metabolites. It was also found that the addition products undergo thermal elimination in the dark and contribute to TBA metabolite regeneration, and therefore, are expected to increase TBA metabolite persistence in water resources. Finally, Chapter 6 discusses the reactions of various trienone and dienone steroids with aqueous chlorine to simulate their fate during engineered drinking water and wastewater chemical disinfection. Single-step transformation pathways were unveiled for each steroid class, including 4-chlorination (trienones) and 9,10-epoxidation (dienones). Chlorination at position C-4 is known to enhance anabolic potency of androgenic steroids and the 9,10-epoxy products were found to undergo acid- or base-catalyzed ring-opening and aromatization to yield known estrogenic products. In addition, Chapter 7 provides conclusions and future directions, while Chapter 8 details the experimental methods and procedures used throughout this thesis. Collectively, the results presented herein confirm our overall hypothesis that steroid transformation products would be expected to contribute to residual biological activity often detected in water resources. Furthermore, the results indicate that the transformation of high potency pharmaceuticals does not automatically equate with reduction or elimination of hazards to exposed organisms, especially in cases where such compounds have potential to form products exhibiting diverse biological endpoints. More holistic approaches to risk assessment of such high potency environmental contaminants are needed in order to accurately assess the fate and effects of such emerging pollutant classes and their bioactive transformation products.

Dans le cadre de la recherche pharmaceutique, les propriétés relatives à l’Absorption, la Distribution, le Métabolisme, l’Elimination (ADME) et la Toxicité (Tox) sont cruciales pour le succès des phases cliniques lors de la conception de nouveaux médicaments. Durant ce processus, la chémoinformatique est régulièrement utilisée afin de prédire le profil ADME-Tox des molécules bioactives et d’améliorer leurs propriétés pharmacocinétiques. Ces modèles de prédiction, basés sur la quantification des relations structure-activité (QSAR), ne sont pas toujours efficaces à cause du faible nombre de données ADME-Tox disponibles et de leur hétérogénéité induite par des différences dans les protocoles expérimentaux, ou encore de certaines erreurs expérimentales. Au cours de cette thèse, nous avons d’abord constitué une base de données contenant 150 000 mesures pour une cinquantaine de propriétés ADME-Tox. Afin de valoriser l’ensemble de ces données, nous avons dans un deuxième temps proposé une plateforme automatique de création de modèles de prédiction QSAR. Cette plateforme, nommée MetaPredict, a été conçue afin d’optimiser chacune des étapes de création d’un modèle statistique, dans le but d’améliorer leur qualité et leur robustesse. Nous avons dans un troisième temps valorisé les modèles obtenus grâce à la plateforme MetaPredict en proposant une application en ligne. Cette application a été développée pour faciliter l’utilisation des modèles, apporter une interprétation simplifiée des résultats et moduler les observations obtenues en fonction des spécificités d’un projet de recherche. Finalement, MetaPredict permet de rendre les modèles ADME-Tox accessibles à l’ensemble des chercheurs. / Absorption, Distribution, Metabolism, Elimination (ADME) and Toxicity (Tox) properties are crucial for the success of clinical trials of a drug candidate. During this process, chemoinformatics is regularly used to predict the ADME-Tox profile of bioactive compounds and to improve their pharmacokinetic properties. In silico approaches have already been developed to improve poor pharmacokinetics and toxicity of lead compounds. These predictive models, based on the quantification of structure-activity relationships (QSAR), were not always efficient enough due to the low number of accessible biological data and their heterogeneity induced by the differences in experimental assays or the significant experimental error. In this thesis, we first built a database containing 150,000 data points for about 50 ADME-Tox properties. In order to valorize all this data, we then proposed an automatic platform for creating predictive models. This platform, called MetaPredict, has been designed to optimize each step of model development, in order to improve their quality and robustness. Third,, we promoted the statistical models using the online application of MetaPredict platform. This application has been developed to facilitate the use of newly built models, to provide a simplified interpretation of the results and to modulate the obtained observations according to the needs of the researchers. Finally, this platform provides an easy access to the ADME-Tox models for the scientific community.

Milk is a highly nutritious natural product and research over the last 10 years has proven that these milk proteins not only provide a rich source of amino acids to the consumer but also contains many bioactive proteins and peptides known to exert biological activity benefitting human health. In this research, proteomic methods were first used to characterize the low abundance proteome within the skim milk fraction produced by Holstein and Jersey dairy cows maintained under the same diet, management and environmental conditions. Milk samples were collected over a seven day period from six Holstein and six Jersey dairy cows. Samples were depleted of casein (CN) by acidification and ultracentrifugation followed by ProteoMiner treatment. Extracts were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation followed by liquid chromatography mass spectrometry (LC-MS). Over 930 low abundance proteins were identified and label-free proteomic analysis allowed for semi-quantification of proteins. Gene ontology (GO) classified proteins into various cellular localization and function categories. Forty-three low abundance proteins were differentially expressed between the two dairy breeds. Some bioactive proteins with immunomodulatory activities were present at significantly different abundance between breeds such as lactotransferrin (P <0.01) and Complement C2 (P <0.01), whereas others like osteopontin (P = 0.17) and lactoperoxidase (P = 0.29) were present at similar levels. This work has identified the highest number of low abundance proteins within the whey fraction in bovine skim milk, providing a foundation for future research exploring the bovine milk proteome. Nutrition is a significant animal factor that has potential to alter milk protein composition. Therefore in the second phase of this work, nutritional perturbances were used to alter the bovine milk proteome by feeding Holstein dairy cows different proportions of rumen degradable (RDP) and rumen undegradable protein (RUP) to alter whole-body nitrogen (N) metabolism. Six multiparous Holstein cows in mid-lactation were randomly assigned to one of two treatment groups. The experiment was conducted as a double-crossover design consisting of three 21-day periods. Within each period, treatment groups received diets with either 1) a high RDP:RUP ratio (control: 62.4:37.6 % of CP) or 2) a low RDP:RUP ratio (RUP: 51.3:48.7 % of CP). Both diets were isonitrogenous (CP = 18.5%) and isoenergetic (NEL = 0.8 Mcal lbs-1). Feeding a diet high in RUP decreased β-casein (P = 0.06), κ-casein (P =0.04) and total milk casein concentrations in milk (P <0.001). Milk urea nitrogen (MUN) and plasma urea nitrogen (PUN) were significantly higher in the RDP group (P = 0.04; P < 0.01, respectively). Over 590 low abundance proteins were identified and only three proteins were found to be differentially expressed between the two dietary groups. The high dietary crude protein (CP) inclusion may explain the lack of treatment effect since protein synthesis within the mammary gland (MG) may not be responsive to dietary changes when total CP levels is offered in excess. Additional feeding trials are needed to alter N utilization patterns within a dairy cow while maintaining isonitrogenous and isoenergetic diets and offering normal CP levels. Nutritional perturbances offer opportunities to selectively alter the bovine proteome, providing a tool to enhance the healthfulness of milk.

Thesis (M.Sc. (Microbiology)) --University of Limpopo, 2011 / The leaves of fifteen plant species were collected from the Lowveld Botanical Garden in Nelspruit, Mpumalanga Province, South Africa. The collection was based on a list of plants and their ethnopharmacological information provided by the Phytomedicine Programme at the University of Pretoria. The dried leaves of the plants were powdered and extracted using hexane, dichloromethane, acetone and methanol. The extracts were screened for antibacterial activity against Mycobacterium smegmatis and Rhodococcus erythropolis. The acetone extract of Milletia stulhimannii was the most active, showing activity against Mycobacterium smegmatis and Rhodococcus erythropolis with MIC values 0.13 and 0.08 mg/ml, respectively. Acetone extracts for all plants had the lowest MIC values ranging between 0.11-1.25 mg/ml and 0.08-1.25 mg/ml for M. smegmatis and R. erythropolis, respectively. Milletia stulhimannii, Albizia gummifera, Xanthocercis zambesiaca and Barringtonia racemosa extracts have shown the greatest potential for anti-tubercolosis agents. These were all active against M. smegmatis with an average MIC value of acetone extracts of 0.13 mg/ml. Apodytes dimidiata was selected for the isolation of active compounds since its activity on qualitative antibacterial activity assays was highly prominent on TLC plates in comparison to the other plant extracts. Two compounds were isolated from A. dimidiata but after purification, their MICs were above 2.5 mg/ml indicating a possible loss of activity during purification. The preliminary NMR spectra analysis suggested that the compounds were a long fatty acid and a triterpene. Future work is required to elucidate the chemical structures of the latter compounds and to test the activity of these compounds against Mycobacterium tuberculosis. / Department of Water Affairs, and University of Limpopo Research Development and Administration Office

<p>U okviru ove doktorske disertacije prikazan je postupak izolacije i karakterizacije denitrifikatora uz optimizaciju biotehnolo&scaron;kih uslova proizvodnje biomase i ispitivanje efikasnosti proizvedene biomase u pogledu bioči&scaron;ćenja različitih građevinskih materijala.<br />U okviru istraživanja koja su obuhvaćena ovom doktorskom disertacijom, izolovan je nov soj denitrifikatora koji je pomoću metode MALDI TOF identifikovan kao Pseudomonas stutzeri. U svim eksperimentima karakteristike i performanse novoizolovanog soja P. stutzeri, interno označenog kao D1, su poređene sa referentnom kulturom P. stutzeri ATCC 17588, koji je model mikroorganizam na kome se izučava proces denitrifikacije. Primenom PCR metode utvrđeno je postojanje četiri ključna denitrifikujuća gena (napA, nirS, norB i nosZ) u genomu P. stutzeri D1 i referentnog soja, čime je pokazano da ispitivani sojevi imaju genetsku predispoziciju za izvođenje potpune aerobne denitrifikacije. Takođe, uz primenu Boks-Benkenovog eksperimentalnog dizajna i metode odzivne povr&scaron;ine, definisan je sastav hranljive podloge za dobijanje maksimalnog sadržaja biomase P. stutzeri D1 (1 g/L glukoze, 3 g/l KNO₃ i 4 g/L peptone) i P. stutzeri ATCC 17588 (2 g/L KNO₃ i 4 g/L peptona). Osim toga, optimizovani su i odabrani bioprocesni parametri za proizvodnju biomase oba denitrifikatora &ndash; temperatura 30&deg;C, pH vrednost kultivacionog medijuma 7 jedinica i veličina inokuluma 5% (v/v). Validacija optimizovanih parametara kultivacije izvr&scaron;ena je izvođenjem nove serije ogleda pri definisanim uslovima u laboratorijskim bioreaktorima ukupnih zapremina 3 L i 7 L, čime je pokazana stabilnost bioprocesa prilikom uvećanja zapremine kultivacione tečnosti uz ostvarenje maksimalnog sadržaja biomase od 9 log CFU/ml za oba ispitivana soja uz minimalno opterećenje otpadnog toka. Rezultati primene denitrifikatora u vidu bioaktivnog sistema (pulpa sa suspenzijom denitrifikatora) koji je nano&scaron;en na povr&scaron;inu ve&scaron;tački kontaminiranih model supstrata opeke ukazuju na visoku efikasnost uklanjanja nitrata i to 95 % u slučaju P. stutzeri D1 i 72% u slučaju referentnog soja. Ispitivanjem bioaktivnih sistema primenjenih na kontaminirane uzorke stenskog materijala konstatovano je da se najveća efikasnost postiže tokom prvih 24 h, kada je efikasnost bioaktivnog sistema sa P. stutzeri ATCC 17588 47,7%, a bioaktivnog sistema sa P. stutzeri D1 čak 89,2%.<br />U okviru doktorske disertacije izolovanjem i karakterizacijom visoko efikasnog denitrifikatora P. stutzeri D1 i detaljnim prikazom postupka optimizacije bioprocesnih parametara i validacije optimizovanih bioprocesnih parametara u laboratorijskim bioreaktorima načinjeni su prvi koraci ka definisanju idejnog re&scaron;enja i uvećanju razmera predloženog bioprocesa. Takođe, visoka efikasnost odabranih denitrifikatora u postupcima bioči&scaron;ćenja različitih građevinskih materijala, koja je postignuta u laboratorijskim uslovima predstavlja dobar osnov za nastavak istraživanja u realnim uslovima, kako na građevinskim materijalima, tako i u tretmanima zemlji&scaron;ta i otpadnih voda.</p> / <p>The main goal of this PhD thesis is to establish the procedure of isolation and characterization of denitrifiers along with the optimization of biotechnological conditions for the biomass production and testing the efficiency of the produced biomass in terms of biocleaning of various building materials.<br />Within the research involved in this PhD thesis, novel denitrifier strain was isolated and identified as Pseudomonas stutzeri by using MALDI TOF. In all experiments, the characteristics and performances of the newly isolated strain of P. stutzeri (internally denoted as D1) were compared with the reference strain P. stutzeri ATCC 17588, which is a model microorganism for studying the denitrification process. By using PCR method the presence of four key denitrification genes (napA, nirS, norB and nosZ) in genome of the both strains was established indicating their genetic predisposition to perform complete aerobic denitrification. Also, the composition of the cultivation medium for obtaining the maximum content of biomass of P. stutzeri D1 (1 g/L glucose, 3 g/l KNO₃ and 4 g/L peptone) and P. stutzeri ATCC 17588 (2 g/L KNO₃ and 4 g/L peptone) was detemined by employing Box-Bhenken experimental design and RSM. In addition, the selected bioprocess parameters were optimized for the production of biomass of the both denitrifiers &ndash; temperature 30 &deg;C, pH value of the cultivation medium of 7 units and inoculum size of 5% (v/v). Validation of the optimized bioprocess parameters was carried out by performing new series of experiments under previously define conditions in laboratory bioreactors with total volumes of 3 L and 7 L. The results of validation showed the stability of bioprocess while increasing the volume of the cultivation broth with the achievment of a maximum biomass content og 9 log CFU/ml for the both strains with minimum load of waste stream. The results of the application of the denitrifiers as a part of bioactive system (poultice with a suspension of denitrifiers) used onto the artificially contaminated brick model supstrates indicate a high efficiency of nitrate removal, namely 95% in the case of P. stutzeri D1 and 72% in the case of reference strain. By examing bioactive systems applied to contaminated stone material it was found that the highest efficiency in nitrate removal was achieved during the first 24 h (47.7% for the reference strian and 89.2% for the P. stutzeri D1).<br />The results obtained in this PhD thesis including the established procedure for isolation and characterization of denitrifiers and detailed presentation of the optimization and validation process of bioprocess parameters represent the first steps made towards defining the conceptual solution and scale up of the proposed bioprocess. Moreover, the high efficiency of selected denitrificators in the biological treatment of various building materials, achieved in laboratory conditions is a good basis for continuing research in real conditions on construction materials, as well as in soil and wastewater treatment.</p>

<p>U okviru ove doktorske disertacije prikazan je holistiĉki pristup proučavanju objekata kulturnog nasleđa, koji je podrazumevao postavljanje metodologije ispitivanja originalnih istorijskih materijala, simulacije procesa degradacije na laboratorijski pripremljenim model supstratima, kao i razvoj novih materijala koji imaju funkciju či&scaron;ćenja degradiranih struktura. Kroz multidisciplinaran istraživački rad je izvr&scaron;ena je detaljna dijagnostika stanja srednjevekovne tvrđave Bač, procesirani su (proizvedeni i ve&scaron;tački ostareni) model supstrati opeka tako da poseduju slične karakteristike kao i ispitivani istorijski materijali, razvijene su i primenjenje glinene pulpe i na bazi njih projektovani su bioaktivni sistemi za uklanjanje nitratnih soli. Ovi novorazvijeni sistemi predstavljaju kombinaciju glinenih pulpi srednje efikasnosti koje imaju ulogu nosača bakterijske kulture Pseudomonas stutzeri, koja povećava kapacitet či&scaron;ćenja razvijenih glinenih pulpi. Rezultati ispitivanja efikasnosti bioaktivnih sistema pokazali su da upotreba suspenzije P.stutzeri (direktno naneta ili ume&scaron;ana u projektovane pulpe) dovodi do značajnog povećanja kapaciteta či&scaron;ćenja polaznih pulpi, odnosno do formiranja sistema visoke efikasnosti za proces desalinacije nitratnih soli. Pored laboratorijske primene, novorazvijeni bioaktivni sistem primenjen je u realnim uslovima na severoistočnom bedemu Tvrđave Bač varijacijom procedura nano&scaron;enja (jednostepene, dvostepene i trostepene primene). Ovaj sistem pokazao je vi&scaron;e pozitivnih performansi u realnim uslovima primene u odnosu na očekivanja bazirana na laboratorijskim istraživanjima. Na osnovu svih eksperimentalnih podataka dobijenih u okviru laboratorijskih istraživanja razvijen je algoritam (Matematičko modelovanje ve&scaron;tačkih neuronskih mreža). Ovaj algoritam bi u budućnosti svoju primenu mogao naći u predviđanju pona&scaron;anja razvijenih sistema na drugim supstratima čija je poroznost poznata, čime se skraćuje vreme projektovanja desalinacionih sistema za druge objekte.</p> / <p>The research approach to cultural heritage degradation and protection, presented in this thesis is a holistic one. It involved establishment of methodology for the investigation of original historical materials, the simulation of the degradation processes on laboratory prepared model substrates, as well as the development of novel materials with cleaning function for degraded structures. The thesis encompasses a detailed diagnostics of the medieval Baĉ Fortress, processing (manufacturing and artificial weathering) of brick model substrates with the characteristics similar to the investigated historical materials, and finally, the development and application of clay poultices which were the basis for the design of bioactive systems for the reduction of nitrate salts. These newly developed systems present a combination of moderately efficient clay poultices that are used as a carrier for bacterial culture Pseudomonas stutzeri. These bacteria had a role of increasing the cleaning capacity of the developed clay poultices. The results of the investigation of the efficacy of the developed systems showed that the use of P. stutzeri suspension (directly applied or mixed in the designed poultices) leads to a significant increase of the cleaning capacity of the developed poultices and to the formation of highly efficient bioactive systems for desalination of nitrate salts. Apart from laboratory application, the most efficient newly developed bioactive system was applied in real environmental conditions on the north-eastern rampart of Baĉ fortress, by varying the application procedure (one-step, two-step and three-step application). This system showed more positive performances in real environmental conditions of application compared to the expectations based on laboratory investigations. Based on all the experimental data obtained within laboratory investigations, an algorithm (mathematical modelling of artificial neuron networks) was programed. The developed model could find its application in the assessment of the application parameters of the developed systems on other substrates with known porosity. This would greatly decrease the time needed for the design of desalination systems for other objects.</p>

Using network architecture to describe a biological system is an effective organizational method. The utility of this approach, which generally applies to qualitative models, is enhanced by the addition of quantitative models characterizing the interactions between network nodes. A chromatophore-based signal transduction network is developed, and the highly interconnected major nodes of the network, guanine trisphosphate, adenylate cyclase, and protein kinase A, are identified. These reference nodes serve to partition the network into functional modules, and mechanistic models describing these modules are derived. Three elicitor compounds, forskolin, melanocyte stimulating hormone (MSH), and clonidine, were selected due to their ability to access the signal transduction network at specific reference nodes, and the module configurations corresponding to their mechanisms of action are presented. The chromatophore responses to the three elicitors and to a negative control, L-15 cell medium, were recorded for two experimental blocks consisting of genetically different fish cells. Significant differences in cell responsiveness were evident between the two blocks, but this variability was controlled by the transformation and normalization of the data. The model parameters for each agent were estimated, and the resulting response curves were highly accurate predictors of the changes in apparent cell area, with R-squared values in the 0.88 to 0.96 range. Two examples were presented for the application of a model discovery algorithm, which selects modules from an existing library, generates model output for all valid module configurations, and selects the configurations which best satisfy a fitness function for a given set of target data. The algorithm proved robust to the introduction of different levels of random error in the simulated data sets when applied to a model of the desensitization of a cell membrane receptor, and continued to classify the stochastic data sets correctly even when the underlying rate constants differed significantly from those embedded in the modules. When challenged with the chromatophore data, the model discovery algorithm successfully matched the forskolin and MSH module configurations to the data within the top three models proposed, with less precise classification for the clonidine model. / Graduation date: 2005