Submitted by Victor Alexandre Zonaro (victor.zonaro@gmail.com) on 2017-01-10T14:22:50Z No. of bitstreams: 1 Dissertação - Victor Alexadre Zonaro.pdf: 4719611 bytes, checksum: 075493217f07f41d58c2e71196b60eea (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-01-13T17:16:22Z (GMT) No. of bitstreams: 1 zonaro_va_me_araiq.pdf: 4719611 bytes, checksum: 075493217f07f41d58c2e71196b60eea (MD5) / Made available in DSpace on 2017-01-13T17:16:22Z (GMT). No. of bitstreams: 1 zonaro_va_me_araiq.pdf: 4719611 bytes, checksum: 075493217f07f41d58c2e71196b60eea (MD5) Previous issue date: 2016-12-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As 5,6-diidro-2-pironas 6-substituídas, também conhecidas como estirilpironas, são uma importante classe de metabólitos secundários presentes em plantas. São substâncias biologicamente ativas, apresentando como, por exemplo, atividade anticâncer, antioxidante, antifúngica e antiviral. O gênero Cryptocarya, pertence à família Lauraceae e apresenta diversas estirilpironas em suas mais diversas partes: folhas, cascas, sementes e raízes. Foram selecionadas para análise as espécies C. mandioccana, C. moschata e C. botelhensis. Foram utilizadas apenas as folhas, por apresentarem facilidade para coleta e abundância. Este trabalho tem como objetivo a identificação das estirilpironas presentes em três espécies brasileiras de Cryptocarya com o uso das técnicas HPLC-DAD-MS. Foi desenvolvido método cromatográfico para análise do extrato hidroalcoólico das folhas de espécies de Cryptocarya por HPLC-DAD-MS utilizando etanol como fase orgânica na fase móvel. Com os espectros no UV foi possível identificar que as classes de metabólitos presentes nas amostras eram alcaloides, flavonoides e estirilpironas. Utilizando os dados de MS e MS/MS, foi possível a caracterização de estirilpironas presentes nos extratos, assim como a sugestão de suas fragmentações por espectrometria de massas, além da identificação dos íons m/z 163 e m/z 189 característicos da fragmentação das estirilpironas. Foi detectado o íon m/z 423 no extrato de C. moschata, referente a um possível dímero da goniotalamina, sem relato anterior para as espécies de Cryptocarya. Com o auxilio de pironas purificadas obtidas pelo grupo, foi possível a comparação de seus tempos de retenção com os dados obtidos a partir dos extratos de folhas das espécies de Cryptocarya ajudando na identificação das substâncias presentes. Além das estirilpironas, foram identificados os alcalóides menisperina e xantoplanina nas três espécies estudadas. Nota-se uma grande diferença na quantidade de metabólitos entre as espécies analisadas, sendo que a C. mandioccana é a mais rica em estirilpironas. / The 6-substituted 5,6-dihydro-2-pyrones, also known as styrylpyrones, are an important class of secondary metabolites present in plants. They are biologically active substances, presenting, for example, anticancer, antioxidant, antifungal and antiviral activity. The genus Cryptocarya belongs to the family Lauraceae and presents several styrylpyrones in its most diverse parts: leaves, barks, seeds and roots. The species C. mandioccana, C. moschata and C. botelhensiswere selected for analysis. We chose to use only leaves, because they are easy to collect and abundant. The objective of this work is to identify the styrylpyrones present in three Brazilian Cryptocarya species using the HPLC-DAD-MS techniques. A chromatographic method was developed to analyze the hydroalcoholic extract from leavesCryptocarya species by HPLC-DAD-MS using ethanol as the organic solvent in the mobile phase. With the UV spectra were possible to identify the classes of metabolites present in the samples as alkaloids, flavonoids and styrylpyrones. Addtitionaly, with the MS and MS2 data, was possible to characterize the styrylpyrones present in the extracts, as well as the suggestion of their fragments by mass spectrometry, in addition to the identification of íons m/z 163 and m/z 189 characteristics of the fragmentation of styrylpirones. The m/z 423 ion detected in C. moschata extract was tentatively attributed to a goniotalamine dimer, with no previuous reports for Cryptocarya species. With the help of purified pirones obtained by the group, it was possible to compare their retention times with the data obtained from leaf extracts of the Cryptocarya species, helping to identify the substances present. Besides the styrylpyrones, the alkaloids menisperin and xantoplanin were also identified in the three species studied. There is a great difference in the amount of metabolites among the analyzed species, with C. mandioccana being the richest in styrylpyrones.

Submitted by (edna.saturno@ufrpe.br) on 2016-06-29T14:39:18Z No. of bitstreams: 1 Milena Dutra da Silva.pdf: 584735 bytes, checksum: 4ed04e843193f8526a523d7cedcbdb96 (MD5) / Made available in DSpace on 2016-06-29T14:39:18Z (GMT). No. of bitstreams: 1 Milena Dutra da Silva.pdf: 584735 bytes, checksum: 4ed04e843193f8526a523d7cedcbdb96 (MD5) Previous issue date: 2008-02-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Myracrodruon urundeuva Allemão (Anacardiaceae), Sideroxylon obtusifolium (Roem. & Schult.) T. D. Penn. (Sapotaceae) and Zizyphus joazeiro Mart. (Rhamnaceae) are native species of the caatinga, widely used as medicinal, specially parts from the stem bark of adult individuals. In general, it is collected hazardously and can let the plant to die and, consequently, reduce the biodiversity. This study aimed to characterize the anatomical, histochemical and phytochemical profile of these species, to the botanical certification, to identify and to localize the accumulation of the metabolites, comparing young stem and mature leaves in young and adult individuals. Usual methods in plant anatomy, phytochemical tests to detect the metabolites classes and specific phytochemical tests to phenolic compounds, iridoids and starch were made. M. urundeuva shows amphystomatic leaves with anomocytic stomata; unicellular simple and glandular trichomes, more abundant over the veins; idioblasts with prismatic crystals in the spongy parenchyma; secretory ducts associated with the phloem in the main vein of the leaf vein, in the petiole and in the stem. S. obtusifolium shows hypostomatic leaves with actinocytic stomata; tector trichomes in the leaf lamina and in the petiole; unistratified hypodermis, with idioblasts with druses; sclereids in the mesophyll; secretory ducts in the medulla of the petiole and the stem. Z. joazeiro shows anomocytic and tetracytic stomata, unistratified hypodermis, with idioblasts with druses, braciform cells in the spongy parenchyma, prismatic crystals in the main vein of the leaf, cap of gelatinous fibers in the stem. Z. joazeiro shows cumarin molecules and cinamic derivates in the stem, only in adult individuals. The other species show similar characteristics related to the metabolites in the stem and in the leaves, in young and adult individuals with different stadium of development. / Myracrodruon urundeuva Allemão (Anacardiaceae), Sideroxylon obtusifolium (Roem. & Schult.) T. D. Penn. (Sapotaceae) e Zizyphus joazeiro Mart. (Rhamnaceae) são espécies nativas da caatinga, amplamente utilizadas como medicinais, especialmente de partes do caule de plantas adultas. Em sua grande maioria, eles são coletados de forma danosa, podendo ocasionar a morte do vegetal e, por conseguinte, a perda da biodiversidade. Este estudo objetivou caracterizar o perfil anatômico, histoquímico e fitoquímico dessas espécies, para certificação botânica, identificação e localização de acúmulo de metabólitos, comparando partes jovens de caule e folhas maduras em indivíduos jovens e adultos. Foram utilizados métodos usuais em anatomia vegetal, testes fitoquímicos para detecção das classes de metabólitos e testes histoquímicos específicos para compostos fenólicos, triterpenos e amido. M. urundeuva apresenta folhas anfiestomáticas com estômatos anomocíticos; tricomas unicelulares simples e glandulares, em maior quantidade sobre as nervuras; idioblastos com cristais prismáticos no parênquima esponjoso; ductos secretores associados ao floema na nervura principal da lâmina foliar, no pecíolo e no caule. S. obtusifolium apresenta folhas hipoestomáticas com estômatos actinocíticos; tricomas tectores na lâmina foliar e no pecíolo; hipoderme uniestratificada, com idioblastos contendo drusas; esclereídeos no mesofilo; ductos de secreção na região medular do pecíolo e do caule. Z. joazeiro apresenta estômatos anomocíticos e tetracíticos, hipoderme uniestratificada, com idioblastos contendo drusas, células do esponjosobraciformes, cristais prismáticos na nervura principal da folha, calotas de fibras gelatinosas no caule. Z. joazeiro apresentou moléculas de cumarinas e derivados cinâmicos no caule, apenas na planta adulta, as demais espécies apresentaram características comuns quanto à presença dos metabólitos e sua localização no caule e nas folhas, em indivíduos jovens e adultos. Isto indica que os metabólitos ocorrem em indivíduos com graus de desenvolvimento variado.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-07-22T12:40:38Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação - Nataliane Marques de Vasconcelos.pdf: 1186432 bytes, checksum: 7aaaefe15061c83ecc86656db0d731f1 (MD5) / Made available in DSpace on 2016-07-22T12:40:38Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação - Nataliane Marques de Vasconcelos.pdf: 1186432 bytes, checksum: 7aaaefe15061c83ecc86656db0d731f1 (MD5) Previous issue date: 2016-02-26 / CNPq / A resistência microbiológica aos antibióticos constitui uma série problemas de saúde pública por dificultar o tratamento das infecções. As actinobactérias são fontes importantes para a descobertas de novas moléculas com atividades biológicas. A este grupo, o gênero Streptomyces possuem dentre outros, dois grupos de enzimas multimoduladoras conhecidas como policetídeo sintase (PKS) e peptídeo sintase não ribossomal (NRPS), genes relacionados com a produção de metabólitos secundários. O presente trabalho teve como objetivo investigar o potencial in vitro de metabólitos bioativos produzidos por Actinobactérias isoladas do bioma Caatinga com atividade contra diferentes isolados clínicos de Candida spp. Após ensaio primário das 45 actinobactérias apenas a linhagem PR- 32 apresentou atividade contra Candida spp, com halos de até 20 mm no meio ISP2. Posteriormente, essa linhagem foi cultivada em seis diferentes meios de cultura sendo observada melhor produção do metabólito secundário no meio 400 em 48 horas (h) de fermentação. A determinação da concentração mínima inibitória (CMI) foi determinada a partir do extrato etanólico da biomassa de PR- 32 em pH 7.0 e foi evidenciada uma CMI entre 31,25 μg/mL a 3,9 μg/mL para as leveduras testadas. A cinética de morte reforçou o resultado da CMI e mostrou que no período de 4-8 h o extrato inibiu as cepas de Candida spp. A caracterização da actinobactéria foi identificada por metodologias clássicas e pela pesquisa do gene 16S rRNA como Streptomyces sp. PR- 32. Os resultados desta caracterização sugerem uma possível espécie nova, contudo outras análises ainda precisam ser realizadas. Foi evidenciada a presença do gene nrps com aproximadamente 750 kb. Diante destes resultados podemos concluir que Streptomyces sp. PR- 32 é um isolado promissor para produção de compostos antifúngicos, sendo possível sugerir que a atividade biológica deste metabólito secundário é regulado por peptídeo sintase não ribossomal (NRPS). / The microbial resistance to antibiotics is a series of public health problems for hindering the treatment of infections. The actinomycetes are important sources for new molecules with biological activities discovered. In this group, the genus Streptomyces have among others, multimoduladoras two groups of enzymes known as polyketide synthase (PKS) and non-ribosomal peptide synthase (NRPS), genes involved in production of secondary metabolites. This study aimed to investigate the potential in vitro bioactive metabolites produced by isolated Actinobacteria biome Caatinga with activity against different clinical isolates of Candida spp. After screening of actinomycetes in 45 primary test only the PR- 32 strain showed activity against Candida spp, with halos of up to 20 mm in the middle ISP2. Subsequently, this strain was grown in six different culture media is best seen in secondary metabolite production means 400 at 48 h of fermentation. The determination of the minimum inhibitory concentration (MIC) was determined from the ethanolic extract of the biomass of PR- 32 at pH 7.0 and one MIC was observed between 31.25 mg / mL 3.9 mg / mL for yeast tested. The kinetics of death reinforced the result of CMI and showed that in the 4-8 hour period the extract inhibited the strains of Candida spp. The characterization of actinobacteria was identified by classical methods and research 16S rRNA gene as Streptomyces sp. PR- 32. The results of this characterization suggests a possible new species, but other tests that must be performed. the presence of the NRPS gene of approximately 750 kb was observed. From these results we conclude that Streptomyces sp. PR- 32 is a promising isolated to produce antifungal compounds, it is possible to suggest that the biological activity of this secondary metabolite is regulated by peptide synthase not ribosomal (NRPS).

Esponjas do gênero Dysidea (Ordem: Dictyoceratida) caracterizam-se por apresentarem grande diversidade de metabólitos secundários, muitos dos quais apresentam potentes atividades biológicas. Este trabalho descreve o estudo de duas amostras de esponjas da espécie Dysidea robusta, DR1 e DR2, coletadas no litoral da Bahia em 1999. Tal estudo consistiu no fracionamento das amostras, nas análises de seus extratos brutos por LC-MS e técnicas de RMN- mono e bidimensionais. Dentre os extratos de DR1, a fração DR1-EP-5A obtida do extrato éter de petróleo apresentou uma mistura de três ceramidas saturadas (22, 23 e 24). Já da amostra DR2, as frações do extrato aquoso DR2-AQ-6B e DR2-AQ-6D mostraram ser constituídas por derivados do ácido pirodisinóico (18, 19, 20 e 21). Com exceção do ácido pirodisinóico (18), os demais compostos isolados ainda não foram relatados na literatura. / Sponges of the genus Dysidea (Order: Dyctioceratida) are characterized as sources of several biologically active secondary metabolites. This work describes the study of two samples of D.robusta, DR1 and DR2, both collected at the Bahia state coastline, in 1999. The investigation aimed the crude extract fractionation and analysis by LC-MS and by 1D and 2D NMR techniques. Among the extracts DR1, the fraction DR1-EP-5A obtained from the petroleum ether extract showed a mixture of three saturated ceramides, represented by 22, 23 and 24. From the DR2 sample, the fractions obtained from the aqueous extract DR2-AQ-6B and -6D presented pirodisinoic acid derivates 18, 19, 20 and 21. Except for pyrodisinoic acid (18), all other isolated compounds haven´t been reported in the literature yet.

Recreational abuse of the drugs cocaine, methamphetamine, and morphine continues to be prevalent in the United States of America and around the world. While numerous methods of detection exist for each drug, they are generally limited by the lifetime of the parent drug and its metabolites in the body. However, the covalent modification of endogenous proteins by these drugs of abuse may act as biomarkers of exposure and allow for extension of detection windows for these drugs beyond the lifetime of parent molecules or metabolites in the free fraction. Additionally, existence of covalently bound molecules arising from drug ingestion can offer insight into downstream toxicities associated with each of these drugs. This research investigated the metabolism of cocaine, methamphetamine, and morphine in common in vitro assay systems, specifically focusing on the generation of reactive intermediates and metabolites that have the potential to form covalent protein adducts. Results demonstrated the formation of covalent adduction products between biological cysteine thiols and reactive moieties on cocaine and morphine metabolites. Rigorous mass spectrometric analysis in conjunction with in vitro metabolic activation, pharmacogenetic reaction phenotyping, and computational modeling were utilized to characterize structures and mechanisms of formation for each resultant thiol adduction product. For cocaine, data collected demonstrated the formation of adduction products from a reactive arene epoxide intermediate, designating a novel metabolic pathway for cocaine. In the case of morphine, data expanded on known adduct-forming pathways using sensitive and selective analysis techniques, following the known reactive metabolite, morphinone, and a proposed novel metabolite, morphine quinone methide. Data collected in this study describe novel metabolic events for multiple important drugs of abuse, culminating in detection methods and mechanistic descriptors useful to both medical and forensic investigators when examining the toxicology associated with cocaine, methamphetamine, and morphine.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第22754号 / 薬科博第128号 / 新制||薬科||14(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 馬見塚 拓, 教授 緒方 博之, 教授 石濱 泰 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Les bactéries doivent s'adapter rapidement aux modifications de leur environnement en ajustant leur modèle d'expression génétique et leurs activités enzymatiques. Dans la plupart des cas, les variations de leur habitat impliquent de petites molécules que les bactéries peuvent détecter et auxquelles elles peuvent réagir. Le ribosome, la machinerie de la cellule qui catalyse la formation de la liaison peptidique, est capable de détecter les métabolites ou les antibiotiques afin de réguler l'expression des gènes, où le peptide naissant au sein du ribosome est capable d’induire l’arrêt de la traduction. Dans ce mécanisme, le peptide en cours de traduction (peptide d'arrêt) bloque le ribosome en interagissant avec les parois du tunnel ribosomal correspondant à la cavité par laquelle le peptide atteint le cytoplasme. L'arrêt peut dépendre uniquement de la séquence du peptide ou bien nécessiter la liaison d’une petite molécule. L’arrêt du ribosome en cours de traduction contrôle à son tour l'expression sur le même ARNm d'un gène situé en aval. Malgré plusieurs études biochimiques et structurales antérieures, le mécanisme exact de détection de ces petits métabolites par le peptide d’arrêt est encore inconnu. Mon travail de doctorat a porté sur : (1) comprendre comment de petites molécules sont détectées par les peptides d'arrêt ribosomaux, et (2) un cas particulier d'arrêt de la traduction dépendant du ligand : la détection des antibiotiques par des peptides d'arrêt courts.Pour répondre au premier problème, j'ai étudié biochimiquement et structurellement un nouveau peptide d'arrêt (appelé SpeFL) qui détecte l’ornithine (un petit métabolite) et qui est codé en amont de l'opéron speF chez Escherichia coli. La structure cryo-EM que j'ai résolue a révélé comment l’ornithine est détectée de manière très spécifique par un complexe ribosomal en cours de traduction. De plus, j'ai montré que le mécanisme d'induction du gène en aval speF implique un arrêt du ribosome au niveau de speFL empêchant ainsi une terminaison prématurée de la transcription Rho-dépendante.Dans la deuxième partie de ma thèse, je me suis concentrée sur la façon dont un antibiotique ciblant les ribosomes, l'érythromycine, est détecté par un peptide d'arrêt court. L'érythromycine est capable de bloquer la traduction de manière séquence-dépendante, où le motif (+)X(+) est le motif principal de blocage. Des données biochimiques publiées antérieurement suggèrent que l'encombrement stérique et électrostatique causé par le premier acide aminé chargé positivement (+) empêche l'addition du second, arrêtant ainsi le ribosome en cours de traduction. La résolution de la structure cryo-EM d'un ribosome arrêté par un peptide MKFR en présence d'érythromycine suggère le contraire, ce qui ouvre la voie à d'autres recherches sur le sujet. / Bacteria need to rapidly adapt to the changing environment by adjusting their gene expression patterns and enzymatic activities. In most cases, the variations in their habitat involve small molecules that bacteria are able to sense and respond to. The ribosome, the machinery of the cell that catalyzes peptide bond formation, is able to detect metabolites or antibiotics to regulate gene expression via nascent-chain mediated translational arrest. In this mechanism, the peptide that is being translated (arrest peptide) stalls the ribosome by interacting with the walls of the ribosomal tunnel, the cavity through which it reaches the cytoplasm. The arrest may depend solely on the sequence of the peptide or need a small molecule to be triggered. Ribosomal stalling in turn, controls the expression of a gene that is located downstream on the same mRNA. Despite previous biochemical and structural studies, the exact mechanism of sensing of small metabolites by the nascent chain is still unknown. My PhD work focused on: (1) understanding how small molecules are sensed by ribosomal arrest peptides, and (2) a special case of ligand-dependent translational arrest: drug sensing by short arrest peptides.To address the first issue, I studied biochemically and structurally a novel L-ornithine sensing arrest peptide (SpeFL) encoded upstream the speF operon in Escherichia coli. The cryo-EM structure that I solved revealed how a small molecule is sensed by a ribosome nascent chain complex in a highly specific manner. Besides, I showed that the mechanism of induction of the downstream gene speF involves ribosomal arrest at speFL preventing premature Rho-dependent transcriptional termination.On the second part of my thesis, I focused on how a ribosome-targeting antibiotic, erythromycin, is sensed by a short arrest peptide. Erythromycin is able to block translation in a sequence dependent manner, with the (+)X(+) motif being the main stalling motif. Previously published biochemical data suggest that steric and static hindrance caused by the first positively charged amino acid prevents the addition of the second one arresting the ribosome. I solved the cryo-EM structure of a ribosome arrested by an MKFR peptide in the presence of erythromycin that shows otherwise and opens up further investigation on the matter.

Commingling of horses from various populations, together with physiological stress associated with transport and confinement at a sales complex, may be associated with detection and transmission of equine herpesvirus type-1 (EHV-1) and -4 (EHV-4). This prospective cohort study aimed to investigate the currently undefined prevalence of EHV-1 and -4 in young Thoroughbreds at an auction sale in South Africa, and associations between clinical signs, physiological stress and viral detection. Ninety, two-year old Thoroughbreds (51 colts, 39 fillies) were consigned from eight farms and sampled at a South African auction sale. The horses were monitored for pyrexia and nasal discharge. Nasal swabs were collected for quantitative polymerase chain reaction (qPCR) assay to detect EHV-1 and -4 and faecal samples were collected for enzyme immunoassay (EIA) to determine faecal glucocorticoid metabolite (FGM) concentrations. EHV-4 nucleic acid was detected in some and EHV-1 nucleic acid in none of the population. Pyrexia and nasal discharge were poor indicators of EHV-4 status. Variation in FGM concentrations was best explained by transportation and preparation for auction. Peaks in EHV-4 detection and increases in FGM concentrations were identified shortly post-arrival and on the first day of auction. Temporal changes in FGM concentrations of horses from individual farms showed two distinct patterns: Pattern A (biphasic peaks) and Pattern B (single peak). It was concluded that sales consignment was associated with some EHV-4 nucleic acid detection and distinctive physiological stress patterns in this population of young Thoroughbreds. / Dissertation (MSc)--University of Pretoria, 2014. / tm2015 / Companion Animal Clinical Studies / MSc / Unrestricted

Biofilms of the bovine rumen bacterium Actinobacillus succinogenes have demonstrated their exceptional capabilities as biocatalysts for high productivity, titre and yield production of succinic acid (SA). Succinic acid is set to become a significant building block chemical in the biobased economy. Although substantial progress has been made towards understanding the productive aspect of this microorganism with regard to its metabolic limits and performance on unrefined biorefinery stream substrates, more research is still required to address other challenges. One aspect is to understand how the biofilm biocatalyst is affected by bioreactor conditions, which would help in developing stable and highly active biofilms. For this reason the aim of this thesis was (i) to characterise how the accumulation of acid metabolites in continuous operation impacts A. succinogenes biofilms with respect to biofilm development, biofilm structure and cell activity within the biofilm, (ii) to show how shear conditions in the fermenter can be used to manipulate the biofilm structure and viable cell content of biofilms, leading to improved cell-based succinic acid productivities, and lastly (iii) to investigate the internal mass transfer effects on biofilm performance, further showing the role played by differences in shear and acid accumulation conditions in this respect. The first part of the study addressed the interaction between the biofilm and the accumulation of metabolites produced. The results showed that biofilms of A. succinogenes develop rapidly and with high activity when cultivated under low product accumulation (LPA) conditions (< 10 g L-1 SA). High product accumulation (HPA) conditions considerably slowed down biofilm development, and increased cell mortality. Under HPA conditions some cells exhibited severe elongation while maintaining a cross-sectional diameter like the rod/cocci-shaped cells predominantly found in LPA conditions. The elongated cells formed in HPA conditions were found to be more viable and thus more resistant than the clusters of rod-shaped or cocci-shaped cells. The global microscopic structure of the HPA biofilms also differed significantly from that of the LPA biofilms. Although both exhibited shedding after 4 days of growth, the LPA biofilms were more homogenous (less patchy), thicker and had high viability throughout the biofilm depth. In the second part of the study, two custom-designed bioreactors were used to evaluate the effect of shear on the biofilms. The first bioreactor allowed for in situ removal of small biofilm samples used for microscopic imaging. The second bioreactor allowed for complete removal of all biofilm and was used to analyse biofilm composition and productivity. Results clearly indicated that high shear biofilm cultivation in LPA conditions has beneficial morphological, viability and cell-based productivity characteristics. The smooth, low-porosity biofilms obtained under high shear and LPA conditions had an average cell viability of 79% (over a 3-day cultivation period) compared with the low shear value of 57%, also developed under LPA conditions. The EPS content of the high shear biofilm was 58% compared with 7% of the low shear equivalent. The cell-based (EPS excluded) succinic acid productivity for the high shear biofilm was 2.4 g g-1DCW h-1 compared with the 0.8 g g-1DCW h-1 for the low shear biofilm. This threefold increase in productivity obtained from the second bioreactor corresponded to the cell viability differences obtained from the first bioreactor. Clear evidence was provided for shear-induced shaping of the biofilm which resulted in improved volumetric glucose turnover attributes within the biofilm matrix. The last section of the study investigated internal mass transfer effects in biofilm fermentations of Actinobacillus succinogenes by performing batch fermentations using attached and resuspended biofilms as biocatalysts. In the latter, the biofilms were resuspended after initial development to simulate mass transfer-free fermentations. Intrinsic kinetics for succinic acid production obtained from resuspended fermentations predicted faster production rates than for the attached biofilm runs (biofilm thicknesses in the range of 120–200 µm), indicating internal mass transfer limitations. A developed biofilm reaction diffusion model gave good prediction of attached biofilm batch operation results by accounting for internal mass transfer in the biofilm. Biofilm effectiveness factors ranged from 75% to 97% for all batches at the inception of batch conditions, but increased with the progression of batch operation due to the increased succinic acid titres which inhibited the production rates. Analysis of pseudo-steady-state continuous fermentation data from the literature, as well as from the second part of the study, using the model developed, showed that active biofilm thickness and effectiveness factors were dependent on the shear conditions and succinic acid titres in the biofilm reactors. A simplified algorithm was developed to estimate the pseudo-steady-state glucose penetration and biofilm effectiveness of A. succinogenes biofilms without the requirement to solve the overall mass transfer model. The results clearly showed that internal mass transfer needs to be considered in biofilm fermentations involving A. succinogenes as high biomass concentrations may not always equate to increased productivities if mass transfer effects dominate. / Thesis (PhD)--University of Pretoria, 2020. / NRF / Chemical Engineering / PhD / Unrestricted

Coral reefs are under considerable decline. The framework builders in coral reefs are scleractinian corals, which comprise so-called holobionts, consisting of cnidarian host, algal symbionts (genus Symbiodinium), and other associated microbes. Corals are commonly considered stenohaline osmoconformers, possessing limited capability to adjust to salinity changes. However, corals differ in their ability to cope with different salinities. The underlying mechanisms have not yet been addressed. To further understand putative mechanisms involved, I examined coral holobiont osmoregulation conducting a range of experiments on the coral Fungia granulosa. In my research F. granulosa from the Red Sea exhibited pronounced physiological reactions (decreased photosynthesis, cessation of calcification) upon short-term incubations (4 h) to high salinity (55). However, during a 29-day in situ salinity transect experiment, coral holobiont photosynthesis was unimpaired under high salinity (49) indicating acclimatization. F. granulosa microbiome changes after the 29-day high salinity exposure aligned with a bacterial community restructuring that putatively supports the coral salinity acclimatization (osmolyte synthesis, nutrient fixation/cycling). Long-term incubations (7 d) of cultured Symbiodinium exhibited cell growth even at ‘extreme’ salinity levels of 25 and 55. Metabolic profiles of four Symbiodinium strains exposed to increased (55) and decreased (25) salinities for 4 h indicated distinct carbohydrates and amino acids to be putatively involved in the osmoadjustment. Importantly, under high salinity the osmolyte floridoside was consistently increased. This could be corroborated in the coral model Aiptasia and in corals from the Persian/Arabian Gulf, where floridoside was also markedly increased upon short- (15 h) and long-term (>24 months) exposure to high salinity, confirming an important role of floridoside in the osmoadjustment of cnidarian holobionts. This thesis demonstrates osmoacclimatization of F. granulosa and osmoadjustment of cultured Symbiodinium. All three main compartments (i.e. coral host, Symbiodinium, bacteria) seem to contribute to the coral holobionts salinity adjustment. However, the exact mechanisms of coral host and bacteria contribution remain to be determined. Floridoside likely constitutes a conserved osmolyte increasing the salinity resilience of Symbiodinium and also of the cnidarian/coral holobiont. Floridoside further possess’ antioxidative properties, possibly providing a protection from reactive oxygen species formation as a result of salinity stress or/and other environmental stressors.