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.

[EN] Plant viruses are obligate intracellular parasites which were used to develop recombinant plant virus vectors to express heterologous proteins and to modify endogenous metabolic pathways of natural products in plants. The main limitation of many plant virus-based systems is the difficulty to co-express various heterologous proteins in the same cell with proper subcellular localization, which is a crucial question in metabolic engineering. This work provides a solution to overcome this problem by using a potyvirus-based vector system. Potyviruses (genus Potyvirus, family Potyviridae) are plus-strand single-stranded RNA viruses, which have a genome expression strategy that allows the equimolar production of most viral proteins. On the basis of an infectious clone of Tobacco etch virus (TEV), Bedoya et al. (2010) developed an expression system in which the RNA-dependent RNA polymerase (NIb) gene was replaced by an expression cassette, harboring several heterologous proteins. This viral vector was able to express three fluorescent proteins with nucleocytoplasmic localization in equimolar amounts in transgenic tobacco plants in which NIb was supplemented in trans. Despite of the apparent simplicity of potyvirus genome expression strategy, foreign cDNA insertion is a complicated task. Thus, our first goal was to analyze the effect of gene insertion on TEV genome stability. As a result of this work, a novel insertion position was discovered at the amino-terminal end of the potyvirus polyprotein, which opened the possibility to explore new questions of recombinant protein expression. Since metabolic pathways are highly compartmentalized, proper subcellular targeting of enzymes is an essential task. Thus, our second objective centralized on the subcellular targeting of expressed proteins from the TEV-based viral vector. cDNAs coding for the green fluorescent protein (GFP) fused to chloroplast, nucleus and mitochondria targeting signal sequences were inserted into the newly described amino-terminal insertion position or into an internal site, replacing the NIb cistron. Our results showed that for protein delivery to chloroplasts and mitochondria, foreign genes have to be inserted at the amino-terminal site of the viral vector, but for nuclear delivery, both insertion positions are suitable. The last objective of this work was to investigate whether the potyvirus-based vector was able to express an entire heterologous multistep biosynthetic pathway in plant cells. For this aim we purposed to produce lycopene, a plant pigment with health promoting properties. To do so, we inserted cDNAs coding for the enzymes of a three-step metabolic pathway of bacterial origin into the potyvirus-based vector. Infected tobacco plants developed orange symptoms indicating lycopene accumulation, which was confirmed by high-performance liquid chromatography analysis and microscopy observations. Our results also illustrated that the sole expression of Pantoea ananatis phytoene synthase, crtB, is enough to induce carotenoid accumulation, conferring yellow coloration to the infected tissue and serves as reporter system to visually track viral infection in several plant species. / [ES] Los virus de plantas son parásitos intracelulares obligados que han sido utilizados para desarrollar vectores virales y expresar proteínas heterólogas y modificar rutas metabólicas endógenas de productos naturales. La principal limitación de muchos sistemas basados en virus de plantas es la dificultad de coexpresar diversas proteínas heterólogas en la misma célula con la localización subcelular apropiada, lo cual es una cuestión crucial en ingeniería metabólica. Este trabajo presenta una solución para superar este problema mediante el uso de un vector viral basado en un potyvirus. Los potyvirus (género Potyvirus, familia Potyviridae) son virus de RNA de cadena positiva simple que tienen una estrategia de expresión génica que permite la producción de la mayoría de las proteínas virales en cantidades equimolares. Basado en un clon infeccioso del virus del grabado del tabaco (Tobacco etch virus, TEV) Bedoya et al. (2010) desarrollaron un sistema de expresión en el que el gen de la RNA polimerasa dependiente de RNA (NIb) fue sustituido por un casete de expresión, que albergaba varias proteínas heterólogas. Este vector viral fue capaz de expresar tres proteínas fluorescentes con localización nucleocitoplásmica en cantidades equimolares en plantas de tabaco transgénicas que complementaban el cistron NIb en trans. A pesar de la aparente simplicidad de la estrategia de expresión génica de los potyvirus, la inserción de un cDNA foráneo es una tarea complicada. Por lo tanto, nuestro primer objetivo fue analizar el efecto de la inserción en la estabilidad del genoma de TEV. Como resultado de este trabajo, descubrimos una nueva posición de inserción en el extremo amino-terminal de la poliproteína viral que nos permitió explorar otras cuestiones sobre la expresión de proteínas recombinantes. Dado que las vías metabólicas son muy compartimentalizadas, la adecuada localización subcelular de enzimas es una tarea esencial en ingeniería metabólica. Por eso, nuestro segundo objetivo se centró en la distribución de las proteínas heterológas expresadas con el vector viral a diferentes orgánulos subcelulares. cDNAs que codificaban la proteína fluorescente verde (green fluorescent protein, GFP) fusionada a péptidos señal se insertaron en la nueva posición amino-terminal y en un sitio interno, sustituyendo el cistrón NIb, para enviarla al cloroplasto, núcleo y a la mitocondria. Nuestros resultados mostraron que para la distribución de proteínas al cloroplasto y mitocondria, los genes foráneos deben ser insertados en el sitio amino-terminal del vector viral, pero para la distribución nuclear, ambas posiciones son adecuadas. El último objetivo de este trabajo fue estudiar si el vector viral basado en potyvirus es capaz de expresar una ruta biosíntética de múltiples pasos en células vegetales. Para ello nos propusimos producir licopeno, un pigmento vegetal con propiedades beneficiosas para la salud humana. Para ello, insertamos un cDNA que codificaba las enzimas de una ruta metabólica de tres pasos de origen bacteriano en el vector viral. Las plantas de tabaco infectadas con el vector viral desarrollaron síntomas de color naranja indicando la acumulación de licopeno, que fue confirmado por análisis de cromatografía líquida de alta eficacia y observaciones de microscopía. Nuestros resultados también ilustraron que la sola expresión de la fitoeno sintasa de Pantonea ananatis, crtB, es suficiente para inducir la acumulación de carotenoides que confieren una coloración amarilla al tejido infectado y sirve como sistema reportero visual en varias especies de plantas. / [CAT] Els virus de plantes són paràsits intracel·lulars obligats que han estat utilitzats per a desenvolupar vectors virals i expressar proteïnes heteròlogues y modificar rutes metabòliques endògenes de productes naturals silenciant certs gens o expressant factors de transcripció i enzims metabòlics. La principal limitació de molts sistemes basats en virus de plantes és la dificultat de coexpressar diverses proteïnes heteròlogues en la mateixa cèl·lula amb la localització subcel·lular apropiada, cosa que és una qüestió crucial en enginyeria metabòlica. Aquest treball presenta una solució per a superar aquest problema mitjançant l'ús d'un vector viral basat en un potyvirus. Els potyvirus (gènere Potyvirus, família Potyviridae) són virus d'RNA de cadena positiva simple que tenen una estratègia d'expressió gènica que permet la producció de la majoria de les proteïnes virals en quantitats equimolars. Basat en un clon infecciós del virus del gravat del tabac (Tobacco etch virus, TEV) Bedoya et al. (2010) van desenvolupar un sistema d'expressió en el qual el gen de l'RNA polimerasa depenent d'RNA (NIb) va ser substituït per un casset d'expressió, que albergava diverses proteïnes heteròlogues. Aquest vector viral va ser capaç d'expressar tres proteïnes fluorescents amb localització nucleocitoplàsmica en quantitats equimolars en plantes de tabac transgèniques que complementaven el cistró NIb en trans. Malgrat l'aparent simplicitat de l'estratègia d'expressió gènica dels potyvirus, la inserció d'un cDNA forà és una tasca complicada. Per tant, el nostre primer objectiu va ser analitzar l'efecte de la inserció en l'estabilitat del genoma de TEV. Com a resultat d'aquest treball, hem descobert una nova posició d'inserció en l'extrem amino terminal de la poliproteïna viral que ens va permetre explorar altres qüestions sobre l'expressió de proteïnes recombinants. Atès que les vies metabòliques són molt compartimentalitzades, l'adequada localització subcel·lular d'enzims és una tasca essencial en enginyeria metabòlica. Per açò, el nostre segon objectiu es va centrar en la distribució de les proteïnes heteròlogues expressades amb el vector viral a diferents orgànuls subcelul·lars. cDNAs que codificaven la proteïna fluorescent verda (green fluorescent protein, GFP) fusionada a pèptids senyal es van inserir en la nova posició amino terminal i en un lloc intern, substituint el cistró NIb, per a enviar-la al cloroplast, nucli i al mitocondri. Els nostres resultats van mostrar que per a la distribució de proteïnes al cloroplast i mitocondri, els gens forans han de ser inserits en el lloc amino terminal del vector viral, però per a la distribució nuclear, ambdues posicions són adequades. El lloc amino terminal va resultar ser més adequat per a produir quantitats més grans de proteïnes recombinants, però el lloc d'inserció intern va demostrar ser més estable. Sobre la base d'aquests resultats, hem sigut capaços de distribuir dues proteïnes fluorescents diferents als cloroplasts i nuclis des d'un únic vector viral. L'últim objectiu d'aquest treball va ser estudiar si el vector viral basat en potyvirus és capaç d'expressar una ruta biosintètica de múltiples passos en cèl·lules vegetals. Per açò ens vam proposar produir licopè, un pigment vegetal amb propietats beneficioses per a la salut humana. Per això inserírem un cDNA que codificaba els tres enzims de una ruta metabòlica de tres passos d'origen bacterià en el vector viral. Les plantes de tabac infectades amb el vector viral van desenvolupar símptomes de color taronja indicant l'acumulació de licopè, que va ser confirmat per anàlisi de cromatografia líquida d'alta eficàcia i observacions de microscòpia. Els nostres resultats també van il·lustrar que la sola expressió de fitoè sintasa de Pantonea ananatis, crtB, és suficient per a induir l'acumulació de carotenoides que confereixen una colora / Majer, E. (2016). Metabolic engineering of plants using a disarmed potyvirus vector [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/68477 / TESIS

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.

Plant cell culture is an attractive platform technology for production and supply of important plant derived medicinals. A unique characteristic of plant cells is the ability to grow as multicellular aggregates in suspension. The presence of these non-uniform aggregates results in creation of distinct microenvironments, which can induce variations in cellular metabolism (e.g., growth, oxygen consumption and secondary metabolite synthesis). This heterogeneity can lead to unpredictable and suboptimal performance in large scale bioprocesses. One example is the Taxus cell culture system, which produces a widely used chemotherapeutic drug - paclitaxel (Taxol ®). Despite extensive process engineering efforts which have led to increased yields of paclitaxel, Taxus cells exhibit variability in productivity that is poorly understood. Elicitation of Taxus cultures with methyl jasmonate (MeJA) induces the accumulation of paclitaxel, but to varying extents in culture. A significant negative correlation was observed between paclitaxel level and mean aggregate size of the culture, demonstrating the relevance of measuring, and potentially controlling aggregate size during long term subculture. Understanding the regulation of gene expression can provide rational engineering strategies to control variability and optimize performance of Taxus cell cultures. Biosynthetic pathway gene analyses revealed upregulation of genes upon elicitation with MeJA; results also suggested additional molecular regulatory points outside of the biosynthetic pathway. In order to fully understand Taxus molecular regulation and the relationship to paclitaxel production variability, a transcriptome-wide analysis using next generation sequencing (454 and Illumina) methods was performed. Several pathways outside of paclitaxel biosynthesis were found active upon MeJA elicitation. Global comparison of gene expression amongst cultures accumulating different levels of paclitaxel is being performed to completely understand the interactions amongst the paclitaxel biosynthetic pathway and other complimentary and competing pathways to suggest effective targets for metabolic engineering. This work collectively represents the first molecular studies to understand metabolic regulation in Taxus cell cultures. Apart from inducing paclitaxel biosynthesis, MeJA decreases cell growth in Taxus cell cultures. The MeJA-mediated repression of cell growth was shown to correlate with inhibition of cell cycle progression as evident both at the culture level through flow cytometric analyses and at the transcriptional level by repression of key cell cycle-associated genes. Results from this study provide valuable insight into the mechanisms governing MeJA perception and subsequent events leading to repression of Taxus cell growth.