Dissertations / Theses on the topic 'Production de mutants de trypanosomes'

Trypanosoma brucei, un parasite extracellulaire responsable de la trypanosomiase africaine, doit s’adapter à différents environnements dans ses hôtes mammifères et l’insecte vecteur (la mouche tsétsé). Dans le sang des mammifères, le glucose est la principale source de carbone soutenant une croissance rapide du parasite, en alimentant son métabolisme et la production d'ATP. Lorsque les formes sanguines prolifératives « slender » atteignent des densités cellulaires élevées, un quorum sensing induit leur différenciation en formes non réplicatives « stumpy » (stumpy-QS). Ces formes stumpy-QS protègent l’hôte d’une parasitémie létale et sont également compétentes pour la transmission à la mouche tsétsé. Cependant, le glucose peut être remplacé par le glycérol pour alimenter le métabolisme central du parasite, suggérant un rôle significatif in vivo. Cela s'aligne avec la démonstration récente que les trypanosomes résident principalement dans les espaces extravasculaires de la peau et du tissu adipeux, où le glycérol interstitiel est 5 à 20 fois plus concentré que dans le plasma. Le glycérol est produit par les adipocytes via la glycolyse et la lipolyse, cette dernière induite par les trypanosomes, pourrait protéger l'hôte contre l'infection. Ces données suggèrent que les interactions entre les adipocytes et les trypanosomes, potentiellement médiées par le glycérol, jouent un rôle dans le cycle de vie du parasite.Cette thèse explore l’impact du glycérol sur les formes sanguines slender de Trypanosoma brucei. Nos résultats ont démontré que le glycérol induit la différenciation des formes prolifératives en formes non réplicatives (stumpy-Glyc) ressemblant aux formes stumpy-QS, mais avec une survie allongée. Des conditions similaires à celles des tissus (4 mM glucose, 0,2-0,5 mM glycérol) induisent la production de formes intermédiaires prolifératives (intermédiaire-Glyc) capables de se différencier in vitro en formes procycliques (parasites de l’intestin de l'insecte vecteur) et d’infecter les mouches tsétsé. De plus, le glycérol prolonge considérablement la durée de vie des formes stumpy-QS induites par le quorum sensing. Ces données nous ont conduit à proposer un modèle révisé de la transmission du parasite de l’hôte mammifère à l’insecte vecteur, où les formes stumpy-QS protègent l'hôte contre une parasitémie élevée, tandis que les formes intermédiaires-Glyc prolifératives et les formes stumpy à longue durée de vie, induites par le glycérol des adipocytes, assurent la transmission à l’insecte vecteur.Un autre aspect de ma thèse concerne la dissection de la voie de signalisation impliquée dans la différenciation induite par le glycérol. En exploitant la durée de vie prolongée des cellules stumpy-Glyc en culture, nous avons sélectionné des mutants résistants à la différenciation induite par le glycérol. L'analyse génomique comparative entre ces mutants a permis d'identifier des mutations candidates associées au phénotype de résistance. Une mutation a notamment été trouvée dans le gène de la sous-unité régulatrice de la protéine kinase A (PKAR), dont le rôle dans la voie de signalisation a été ensuite validé.Enfin, nous avons exploré la capacité de T. brucei à métaboliser le glycérol sécrété par les adipocytes, même en présence d'un excès de glucose. Pour ce faire, nous avons utilisé un système de co-culture in vitro permettant d'analyser les interactions entre les trypanosomes parentaux ou mutants et les adipocytes. Le profilage métabolique par résonance magnétique nucléaire (RMN) couplé à des approches de marquage au 13C a permis de tracer les métabolites produits par les adipocytes et les trypanosomes. Nos données ont montré que T. brucei utilise efficacement le glycérol sécrété par les adipocytes pour alimenter son métabolisme central, même en présence de grandes quantités de glucose.En conclusion, ces données ont démontré que le glycérol est un acteur clé dans la biologie de Trypanosoma brucei
Trypanosoma brucei, an extracellular parasite responsible for African trypanosomiasis, must adapt to distinct environments in its mammalian hosts and the tsetse fly vector. In the mammalian bloodstream, glucose serves as the primary carbon source, fueling the parasite's central carbon metabolism and ATP production, which supports its rapid growth. Once the parasites reach high cell densities, a quorum-sensing mechanism induces a transition from proliferative slender forms to growth-arrested stumpy forms (stumpy-QS). These stumpy forms help prevent host mortality by limiting parasitaemia and are primed for transmission to the tsetse fly. However, it has been demonstrated that glycerol can effectively replace glucose in feeding the parasite’s central carbon metabolism, suggesting a significant role in vivo. This aligns with findings that trypanosomes predominantly reside in the extravascular spaces of tissues such as the skin and adipose tissue, where interstitial glycerol concentrations are 5 to 20 times higher than in plasma. Glycerol is released from adipocytes through both lipolysis and lipolysis-independent processes such as glycolysis, and it has been suggested that trypanosome-induced adipocyte lipolysis may even protect the host against trypanosome infection. Together, these data suggest that interactions between adipocytes and trypanosomes, potentially mediated by glycerol, play a critical role in the parasite’s life cycle.This thesis explores the impact of glycerol on bloodstream form (BSF) Trypanosoma brucei. Our findings demonstrated that glycerol induces the differentiation of slender BSF into growth-arrested forms that resemble stumpy-QS, but with enhanced survival. Furthermore, under tissue-like conditions, characterized by glycerol levels between 0.2-0.5 mM and glucose at 4 mM, proliferative intermediate forms were generated, which were capable of differentiating into the insect vector stage (procyclics) and sustaining infections in tsetse flies. Additionally, glycerol extended the lifespan of quorum-sensing-induced stumpy forms, which normally have a limited lifespan of a few days. All these data led us to propose a revised model for transmission, in which quorum sensing-induced stumpy-QS forms protect the host from high parasitaemia, while glycerol from adipocytes induces intermediate-Glyc or long-lived stumpy forms that facilitate transmission to the fly.Another key aspect of my thesis concerns the dissection of the signalling pathway involved in glycerol-induced differentiation. By exploiting the extended lifespan of stumpy-Glyc cells in culture, we selected mutants resistant to glycerol-induced differentiation through extended in vitro culturing in a glycerol-containing medium. Comparative genomic analyses between these mutants and cells grown in glucose, which are sensitive to glycerol-induced differentiation, identified candidate mutations associated with the resistance phenotype. Notably, these mutations were found to affect the protein kinase A regulatory subunit (PKAR), whose role in the signalling pathway was validated.Finally, we explored whether T. brucei can metabolize glycerol secreted by adipocytes even in the presence of excess glucose. To investigate this, we used an in vitro co-culture system using a transwell assay, which allowed us to analyse the interactions between parental and mutant trypanosomes and adipocytes. We examined growth and exometabolome profiles using nuclear magnetic resonance (NMR)-based metabolite profiling, coupled with 13C-labeling to trace specific metabolites. Our data showed that T. brucei efficiently utilized glycerol secreted by adipocytes to support its central carbon metabolism, even when glucose was abundant.Together, these data demonstrated that glycerol is a key player in the biology of Trypanosoma brucei

UDP-glucose pyrophosphorylase (UGPase) is a key component of carbohydrate production in plants, especially with respect to sucrose synthesis/ metabolism, by producing UDP-glucose, a key precursor to sucrose and to many polysaccharides in cell walls. UDP-glucose is also utilized in the synthesis of carbohydrate moiety of glycolipids, glycoproteins and a variety of secondary metabolites, among other functions. The UGPase enzyme may have a rate-limiting function in sugar biosynthesis, and its activity is now known to increase upon variety of abiotic stresses, with possible effects on an overall carbohydrate budget in stressed plants. The enzyme has been proposed to be regulated by (de)oligomerization and it has been estabished that only monomeric form of the enzyme is active. Based on mutant studies, the deoligomerization step (formation of monomers) was found as rate-limiting. A structural model of barley UGPase was recently suggested, based on homology to a human Antigen-X (AGX) protein that has a 40% protein sequence similarity to eukaryotic UGPase. The 3D model shows a bowl-shaped protein with three different domains: (a) N-terminal, (b) central part which includes the nucleotide binding loop (NB-loop) at the active centre and (c) C-terminal which includes an insertion loop (I-loop) that is possibly involved in dimer formation and stabilization. In this study, the model was used as a testable blueprint to verify details of the barley enzyme catalysis and substrate binding, as well as oligomerization process.  In order to test the model, site-directed mutagenesis approaches and heterologous (E. coli) expression system were used to produce several UGPase mutants: Del-NB, lacking 4 amino acids (aa) at the NB region; Del-I-4 and Del-I-8, lacking respectively 4 and 8 aa of the I-loop; and Y192A, by replacing an active-site tyrosine into alanine. The Y192A mutant had about half the apparent activity of the wild-type (wt), whereas Del-I-8 and Del-I-4 had only 0.5 and 0.2 % activity, respectively, of the wt, and Del-NB showed no activity at all. Based on native-PAGE, both Y192A and Del-NB mutants had similar oligomerization status as the wt, i.e. existing as monomer only or a mixture of monomer, dimer and higher order oligomers, depending on incubation conditions. Both Del-I-8 and Del-I-4 were present in all conditions as higher order oligomers. Whereas Y192A mutant had similar Kms with both substrates as the wt protein, significant difference between the Del-I-4 and Del-I-8 mutants and wt could be detected. Both mutants had approximately 16-fold higher Kms for UDP-glucose, and the Kms with PPi were 735- and 1500-fold higher for Del-I-4 and Del-I-8, respectively, when compared to wt.The conclusion of those results: (A) Tyr-192 is not essential for activity and is not involved in substrate binding and/ or oligomerization of the enzyme. (B) The NB-loop is essential for catalysis, as evidenced by a complete lack of activity of the Del-NB mutant, and is not involved in oligomerization. On the other hand, (C) the region corresponding to central part of I-loop is located in the model far from active center, but deletion in this region does affect very strongly both catalysis and substrate binding parameters. This can be explained by the involvement of I-loop in formation of dimers (inactive) from monomers (active), as earlier proposed. Apparently, the Del-I-4 and Del-I-8 mutations lead to an enzyme form with a very high oligomerization ability. This affects both Kms and Vmaxs of the Del-I mutants. Taken together the results verify the essentiality of NB-loop for catalysis support the involvement of I-loop region in oligomerization and, overall, the importance of oligomerization status for enzymatic performance of UGPase.

It has been shown previously in our lab that mutations in the pyrimidine pathway reduced the ability of Pseudomonas aeruginosa to produce virulence factors. Knockout mutations in pyrB, pyrC and pyrD genes of the pyrimidine pathway showed that virulence factor production was decreased. Pyoverdin, pyocyanin, hemolysin, iron chelation, motility, and adherence are all considered virulence factors. Here I further investigate the effects of mutations in the pyrimidine pathway by studying a pyrE mutant. I studied the effect of the pyrE mutation on the production of the above virulence factors. Just like the effect of pyrB, pyrC and pyrD mutations,the pyrE mutation also showed that the bacteria were deficient in producing virulence factors when compared to the wild type. The broader impact of this research would be the possibility of finding drugs that could treat patients infected with P. aeruginosa and possibly extend the lives of chronically infected patients with cystic fibrosis.

African trypanosomiasis remains a major health problem to both humans and animals due to lack of effective treatment or vaccine to control the disease. Animal trypanosomiasis is considered one of the most important diseases affecting livestock production and agricultural development in sub-Saharan Africa. Although the use of trypanocides remain the most important method for controlling the disease in animals, the mechanisms of action of these compounds are not completely known. The overall aim of this thesis is to decipher the molecular mechanisms involved in Trypanosoma congolense-induced cytokine production and how this is modulated by the trypanocide, Diminazene aceturate (Berenil). First, I investigated the molecular and biochemical mechanisms of action of Berenil to determine whether in addition to trypanolytic effect, it exerts a modulatory effect on the host immune system. Although it is known that T. congolense infection in mice is associated with increased production of pro-inflammatory cytokines by macrophages, the intracellular signaling pathways leading to the production of these cytokines remain unknown. Therefore, I investigated the innate receptors and intracellular signaling pathways that are involved in T. congolense-induced pro-inflammatory cytokine production in macrophages. Next I further determined whether the inhibitory effect of Berenil on proinflammatory cytokine production in macrophages is specific to T. congolense. I found that Berenil treatment significantly reduced the immune activation and proinflammatory cytokine production in infected mice suggesting that in addition to its direct trypanolytic effect, Berenil also modulates the host immune response to the parasite. Next, I show that T. congolense induced pro-inflammatory cytokine production in macrophages is dependent on phosphorylation of mitogen-activated protein kinase (MAPK) and signal transducer and activation of transcription (STAT) proteins in a TLR2-dependent manner. I further show that Berenil treatment downregulates T. congolense as well as LPS induced cytokine production by affecting the phosphorylation of MAPK and STAT proteins. Collectively, the results from this thesis provide novel insights into T. congolense-induced activation of the innate immune system and modulation of host immune response by Berenil. These findings are significant and could help in developing newer and better therapeutic strategies against the disease, in particular, and inflammatory responses in general.
October 2016

Ce travail, réalisé en collaboration avec Rhône-Poulenc santé, a consisté d'une part, à étudier la régulation de l'expression du gène cloné de la pénicilline amidase (PA) dans une souche mutante, d'autre part, à développer une technique de mutagenèse chimique adaptée à l'étude d'enzyme, à l'appliquer au gène PA et à caractériser les mutants obtenus. La souche transformée présente les caractéristiques suivantes par rapport à la souche non transformée: une exigence plus importante en source de carbone, un effet non pas négatif mais positif du glucose sur la production de PA, une expression en partie constitutive, une stimulation par l'acide phenylacetique n'est observée qu'en présence de glucose. La majorité des mutants PA obtenus est affectée dans le processus de maturation protéolytique interne. Un grand nombre des mutations responsables de ce phénotype est localise dans la région c-terminale de la grande sous-unité qui doit donc jouer un rôle important dans ce processus

Aspartokinase is the first enzyme of the aspartate family amino acids biosynthetic pathway. Cephamycin C is a &
#946
-lactam antibiotic produced as a secondary metabolite via the enzymatic reactions in the lysine branch of this pathway in Streptomyces clavuligerus. The aspartokinase activity of S. clavuligerus is under concerted feedback inhibition by two of the end product amino acids, lysine plus threonine. It is also known that carbon flow through the lysine branch of the aspartate pathway is rate limiting step in the formation of cephamycin C. Therefore, genetic alterations in the regulatory regions of the aspartokinase enzyme are expected to lead to an increased cephamycin C production. The aim of this study was to obtain S. clavuligerus mutants that possess aspartokinase enzyme insensitive to feedback inhibition by lysine and threonine, identification of the mutation(s) accounting for the resistance being the ultimate goal. For this aim, chemical mutagenesis was employed to increase random mutation rate and a population of lysine anti-metabolite resistant S. clavuligerus mutants that can grow in the presence of S-(2-aminoethyl)-L-cysteine was obtained. The mutants were screened for their aspartokinase insensitivity via enzyme assays and one mutant exhibiting the highest level of deregulation was assessed for its cephamycin C production. The results revealed a 2-fold increase in specific production of the antibiotic. As a member of &
#946
-lactam class antibiotics, cephamycin C has an importance in medicine. Therefore, the mutant strain obtained might be a candidate for industrial production of the compound.

Thesis (Ph. D.)--Ohio State University, 2005.
Title from first page of PDF file. Document formatted into pages; contains xv, 220 p.; also includes graphics (some col.). Includes bibliographical references (p. 189-201). Available online via OhioLINK's ETD Center

The AbsA two-component system is a negative regulator of antibiotic biosynthesis in Streptomyces coelicolor. To gain further insight into the role of the AbsA system different in-frame deletion mutations were made in the absA1A2 operon in S. coelicolor MT1110, MT1110DeltacdaR and S. lividans 1326. Phenotypic analysis of DeltaabsA1A2 and DeltaabsA2 in the MT1110 derivatives showed that CDA was produced early on Oxoid nutrient agar relative to the parent strain. The absA mutants in S. lividans 1326 (both DeltaabsA1 and DeltaabsA1A2) did not show early CDA production. On solid R2YE both MT1110DeltaabsA1A2 and MT1110DeltaabsA2 produced undecylprodigiosin and actinorhodin earlier. In liquid R2YE medium MT1110DeltaabsA1A2 produced undecylprodigiosin and actinorhodin earlier compared to MT1110 wild-type. RNA was extracted from surface-grown cultures of MT1110 and its DeltaabsA1A2 derivative on R2YE agar at different stages of growth. Cy3-labelled cDNA and Cy5-labelled genomic DNA (gDNA) were hybridised on in-house printed 50-mer oligo arrays representing all S. coelicolor open reading frames. Differentially expressed genes were identified by the 'Rank Product' method. Transcriptomic analysis revealed that many genes encoding enzymes of central metabolism were up-regulated in MT1110DeltaabsA1A2 compared to MT1110 wild-type. Three genes, SCO4089, SCO3829 and SCO1270 encoding valine dehydrogenase, dihydrolipoamide acyltransferase component E2 and the alpha-subunit of pyruvate dehydrogenase complex respectively, were found to be more highly expressed in MT1110DeltaabsA1A2. These genes encode enzymes whose reactions produce possibly precursors for CDA, Act and Red biosynthesis. The gene encoding pyruvate kinase 2 (SCO5423) showed increased expression in the mutant at and after 24 h of growth. Genes encoding subunits of ATP synthases showed increased expression in the mutant that indicates that an increase oxidative metabolism is occurring in MT1110DeltaabsA1A2. A gene, SCO6195, encoding a stationary phase-active enzyme, acyl-coenzyme A synthetase was induced in MT1110DeltaabsA1A2 at 23 and 24 h of cultivation. Two genes in the cda cluster were found to be repressed in mutant: SCO3224 and SCO3229, encoding an ABC transporter ATP-binding protein and 4-hydroxymandelate synthase, respectively. The gene encoding AfsS (SCO4425) was found to be up-regulated in the DeltaabsA1A2 mutant at the late phase of the growth. Four genes encoding enzymes putatively involved in the pentose phosphate pathway were found to be up-regulated in MT1110DeltaabsA1A2 at certain time points. Genes encoding ribosomal protein showed a more relaxed growth phase dependent pattern of expression in the mutant while one r- protein encoding gene, rpsK (SCO4728), showed higher expression in the mutant except at stationary phase. It is deduced from the global gene expression analysis that the possible role of the AbsA system in S. coelicolor is to regulate precursor supply for antibiotic production rather than to directly regulate the genes of the respective antibiotic biosynthetic clusters.

Cocaine is a widely abused drug without an FDA-approved medication. It has been recognized as an ideal anti-cocaine medication to accelerate cocaine metabolism producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e. butyrylcholinesterase (BChE)-catalyzed hydrolysis. However, the native BChE has a low catalytic activity against cocaine. We recently designed and discovered a set of BChE mutants with a high catalytic activity specifically for cocaine. An ideal, therapeutically valuable mutant of human BChE should have not only a significantly improved catalytic activity against cocaine, but also certain selectivity for cocaine over neurotransmitter acetylcholine (ACh) such that one would not expect systemic administration of the BChE mutant to interrupt cholinergic transmission. Through integrated computational-experimental studies, several BChE mutants were identified to have not only a considerably improved catalytic efficiency against cocaine, but also the desirable selectivity for cocaine over ACh. Representative BChE mutants have been confirmed to be potent in actual protection of mice from acute toxicity (convulsion and lethality) of a lethal dose of cocaine (180 mg/kg, LD100). Pretreatment with the BChE mutant (i.e. 1 min prior to cocaine administration) dose-dependently protected mice against cocaine-induced convulsions and lethality. The in vivo data reveal the primary factor, i.e. the relative catalytic efficiency, determining the efficacy in practical protection of mice from the acute cocaine toxicity and future direction for further improving the efficacy of the enzyme in the cocaine overdose treatment. For further characterization in animal models, we successfully developed high-efficiency stable cell lines efficiently expressing the BChE mutants by using a lentivirus-based repeated-transduction method. The large-scale protein production enabled us to further characterize the in vivo profiles of the BChE mutant concerning the biological half-life and potency in accelerating cocaine clearance. In particular, it has been demonstrated that the BChE mutant can rapidly metabolize cocaine and completely eliminate cocaine-induced hyperactivity in rodents, implying that the BChE mutant may be developed as a promising therapeutic agent for cocaine abuse treatment.

Myxococcus xanthus est une bactérie à gram-négatif qui sécrète de nombreuses activités enzymatiques durant la phase exponentielle de croissance. Nous avons tenté dans une première étape de cloner un gène de structure de l'une des trois activités sécrétées par cette bactérie: protéase, endoprotéase acide et glucanase en utilisant le promoteur mobile Ptac permettant de créer des phénotypes conditionnels inductibles par l'IPTG. Les résultats que nous avons obtenus ont montré que le clonage d'un fragment d'ADN de M xanthus situé en aval du Ptac dans E. Coli est faisable. Mais l'obtention conditionnelle par cette méthode n'a pas été possible faute de méthode de criblage suffisamment fiable. L'analyse des mutants d'insertion obtenus montre que la majorité sont affectés dans la production de certaines des activités seulement et sont vraisemblablement des mutants de régulation. Une du mutant paraît toucher le mécanisme de sécrétion et le cycle de développement multicellulaire et pourrait faire partie d'une famille comprenant au moins 6 gènes suggérant une relation entre mécanisme de sécrétion et le développement

Mestrado em Biotecnologia - Biotecnologia Molecular
Parkinson´s disease (PD), the second most common progressive neurodegenerative disorder, is a multifactorial disease caused by both genetic and environmental factors. Among the genes associated with PD, DJ-1 is a multifunctional protein involved in oxidative stress response and neuroprotection. DJ-1 mutations, such as L166P, M26I and E163K lead to loss of protein function causing early onset autosomal recessive PD. Moreover, the residue C106 is considered crucial in DJ-1 function as a sensor of oxidative stress. In this study, one missense mutations (E163K) and two engineered mutations in the residue C106 (C106A and C106DD) were produced and characterized in order to evaluate the neuroprotective effect of each mutation and also characterize their dynamic interactome. Structural analysis confirmed the production of all the mutants in the dimeric form, with a molecular weight of approximately 43kDa. Moreover, protein´s thermal stability was assessed by thermal shift and the results showed that the mutant E163K was the less stable and the C106A the most stable. Secondary structure analysis was performed by circular dichroism and revealed similar secondary structures between DJ-1 WT and mutants. In addition, a LC-MS/MS was performed to determine proteins´ contaminants and the majority of the protein contaminants were coming from the expression system and culture medium used in proteins´ production. Moreover, neuroprotection assays revealed that DJ-1 WT did not protect SH-SY5Y cells under oxidative stress conditions. The dynamic interactome of DJ-1 WT and mutants C106DD, E163K and C106A was characterized under oxidative stress conditions. A wide number of binding partners were identified and for some of them quantification in the different conditions was also determined. These interactors have a broad range of functions but the majority are associated with cellular response to oxidative stress. The study of DJ-1 mutations is very important, since it gives elucidations into DJ-1 WT functions and related disease mechanisms. In this way, the putative DJ-1 WT interactors identified still lack validation, but from these characterized dynamic interactomes further elucidations can be obtained into Parkinson’s Disease pathology and potential new targets for PD prevention and therapy, like ATP-dependent RNA helicase DDX3X herein identified as new dynamic interactor of DJ-1.
A Doença de Parkinson, a segunda doença neurodegenerativa progressiva mais comum, é uma doença multifatorial causada conjuntamente por fatores genéticos e fatores ambientais. De entre os inúmeros genes associados à Doença de Parkinson, a DJ-1 é uma proteína multifuncional envolvida na resposta ao stress oxidativo e neuroproteção. Mutações na DJ-1, tais como L166P, M26I e E136K levam à perda de função da proteína causando a forma de Parkinson autossomal recessiva com desenvolvimento precoce. De salientar que o resíduo C106 é considerado crucial na função de sensor de stress oxidativo que a DJ-1 desempenha. Neste estudo, foram produzidas e caracterizadas duas mutações sintéticas no resíduo C106 (C106A e C106DD) e uma mutação natural (E163K) de modo a avaliar o efeito neuroprotetor de cada mutação bem como caracterizar o seu interactoma dinâmico. Análises estruturais confirmaram a produção de todos os mutantes na forma dimérica, apresentando um peso molecular de aproximadamente 43kDa. A estabilidade térmica das proteínas foi ainda avaliada por thermal shift e os resultados revelaram que o mutante E163K foi o menos estável enquanto que o mutante C106A foi o mais estável. Análise da estrutura secundária foi realizada por dicroísmo circular revelando elevada semelhança entre as estruturas secundárias da DJ-1 nativa e mutantes. Por fim, foi realizada uma análise de LC-MS/MS de modo a determinar os contaminantes das proteínas produzidas e verificou-se que a maioria dos contaminantes era proveniente do sistema de expressão e meio de cultura utilizados na produção das proteínas. Seguidamente, ensaios de neuroproteção revelaram que a DJ-1 nativa não exercia um efeito neuroprotetor nas células SH-SY5Y em condições de stress oxidativo. O interactoma dinâmico da DJ-1 nativa e mutantes C106DD, E163K e C106A foi caracterizado sob condições de stress oxidativo. Um elevado número de interactores foram identificados e para alguns deles foi possível obter uma quantificação nas diferentes condições. Os referidos interactores apresentam uma enorme variedade de funções, contudo a grande maioria está associada à resposta celular ao stress oxidativo. O estudo das mutações na DJ-1 é considerado muito relevante visto que fornece importantes elucidações relativamente às funções e mecanismos da DJ-1 nativa associados à doença. Neste sentido, os supostos interactores da DJ-1 nativa identificados ainda carecem de validação, mas da caracterização dos interactomas dinâmicos, elucidações podem ser obtidas sobre a patologia da Doença de Parkinson e identificação de novos potenciais alvos para prevenção e terapia desta doença, tal como a RNA helicase DDX3X dependente de ATP aqui identificada como novo interactor dinâmico da DJ-1.

Previous research in our laboratory established that pyrB, pyrC or pyrD knock-out mutants in Pseudomonas aeruginosa required pyrimidines for growth. Each mutant was also discovered to be defective in the production of virulence factors. Moreover, the addition of exogenous uracil did not restore the mutant to wild type virulence levels. In an earlier study using non-pathogenic P. putida, mutants blocked in one of the first three enzymes of the pyrimidine pathway produced no pyoverdine pigment while mutants blocked in the fourth, fifth or sixth steps produced copious quantities of pigment, just like wild type P. putida. The present study explored the correlation between pyrimidine auxotrophy and pigment production in P. aeruginosa. Since the pigment pyoverdine is a siderophore it may also be considered a virulence factor. Other virulence factors tested included casein protease, elastase, hemolysin, swimming, swarming and twitching motilities, and iron binding capacity. In all cases, these virulence factors were significantly decreased in the pyrB, pyrC or pyrD mutants and even in the presence of uracil did not attain wild type levels. In order to complete this comprehensive study, pyrimidine mutants blocked in the fifth (pyrE) and sixth (pyrF) steps of the biosynthetic pathway were examined in P. aeruginosa. A third mutant, crc, was also studied because of its location within 80 base pairs of the pyrE gene on the P. aeruginosa chromosome and because of its importance for carbon source utilization. Production of the virulence factors listed above showed a significant decrease in the three mutant strains used in this study when compared with the wild type. This finding may be exploited for novel chemotherapy strategies for ameliorating P. aeruginosa infections in cystic fibrosis patients.

La chaine respiratoire de bacillus subtilis contient au moins quatre oxydases terminales. Deux de ces oxydases terminales, la quinol oxydase aa#3-600 et la cytochrome c oxydase caa#3-605 ont ete purifiees. Les genes de l'operon ctacdef qui codent les sous-unites de l'oxydase caa#3-605 ont ete isoles et sequences. Dans notre laboratoire, nous avons isole et sequence les genes de l'operon qoxabcd codant les sous-unites de l'oxydase aa#3-600. L'etude de l'expression des deux operons montre que l'expression de l'operon qoxabcd est constitutive, tandis que celle de l'operon ctacdef est dereprimee quand la source de carbone est non fermentescible. Pendant la croissance vegetative et en l'absence de l'oxydase aa#3-600, b. Subtilis utilise une oxydase alternative de type o. La reduction des parametres de croissance en milieu minimum glucose a 37c, ainsi que la reduction de la valeur du rapport atp/adp suggerent que la voie des electrons jusqu'a cette oxydase alternative pourrait etre responsable d'une diminution de l'efficacite de la chaine respiratoire chez les mutants qox ; pour compenser la diminution de la production d'atp par phosphorylation oxydative, les mutants qox augmentent la phosphorylation au niveau du substrat, dirigeant le carbone vers la formation d'acetate. Nous avons clone et sequence les genes de l'operon atp qui code les sous-unites de l'enzyme membranaire, l'atp synthase. Cette enzyme utilise le gradient de protons etabli lors de flux d'electrons dans la chaine respiratoire pour la synthese d'atp. Deux mutants de deletion de cet operon, montrent une diminution du taux et du rendement de croissance en milieu minimum glucose a 37c par rapport a la souche sauvage. Cette reduction peut etre correlee avec une diminution du rapport atp/adp et avec un doublement de la production d'acetate chez ces mutants. Donc, les mutants atp dirigent aussi le carbone vers la formation d'acetate et augmentent le flux glycolytique. L'augmentation du flux catabolique conduit a l'augmentation de la production de nadh, et les mutants atp augmentent le taux specifique de respiration ; l'expression de genes de respiration est augmentee. L'expression de ces genes augmente aussi dans un contexte qox, suggerant qu'un meme signal de regulation de l'expression de ces genes (peut-etre le rapport atp/adp et/ou le rapport nadh/nad#+) est active dans les deux cas

Deux mutants de levure saccharomyces cerevisiae, vl134 et vl135, auxotrophes pour l'ergosterol et bloques dans l'activite farnesyl diphosphate (erg20) ont ete isoles. Les mutations affectant la farnesyl diphosphate synthetase des souches vl134 et vl135 ont ete notees respectivement erg20-1 et erg20-2. La particularite de ces souches mutantes est leur capacite a excreter des terpenes aromatiques (farnesol et geraniol dans le cas de vl134 et farnesol dans le cas de vl135). L'analyse genetique de ces souches a montre que d'autres mutations sur des genes de la voie de biosynthese de l'ergosterol etaient presentes et indispensables a leur viabilite. Le role est tres vraisemblablement de diminuer la synthese du geraniol, toxique pour les cellules de levure. Nous avons isole le gene fonctionnel erg20 de levure par complementation de la mutation erg20-2. Le fragment isole a une taille de 5,7 kb qui a pu etre reduit a 3 kb sans perte de l'activite. Nous avons montre qu'une souche haploide portant une copie chromosomique du gene erg20 et une copie extrachromosomique, presente une augmentation de 28% du taux de sterol, ce qui suggere fortement que la farnesyl diphosphate synthetase pourrait etre une enzyme limitante dans la voie de biosynthese des sterols chez la levure. Par contre, une souche haploide porteuse du gene erg20 sur un plasmide replicatif (de 20 a 50 copies du plasmide par cellule), presente une teneur en sterols identique a celle de la souche sauvage, ce qui peut s'expliquer par l'existence de mecanismes de regulation qui auraient pour role de prevenir une eventuelle accumulation de sterols

La presente etude porte sur l'amelioration de la production de diacetyle par lactococcus lactis subsp. Lactis biovar diacetylactis. Dans la premiere partie, les effets du controle de la concentration en oxygene dissous ainsi que de l'addition de citrate dans le milieu ont ete etudies. L'oxygene augmente considerablement la production de diacetyle, dont la concentration maximale varie de 0,015 mm sous azote a 0,45 mm sous une pression de 2 atmospheres d'oxygene. L'augmentation de la production de diacetyle est probablement due a la decarboxylation chimique oxydative de l'acetolactate et a la diminution de l'activite de la lactate deshydrogenase. La production de diacetyle augmente egalement en presence de citrate. Egale a 0,08 mm en absence de citrate, la concentration en diacetyle atteint 0,85 mm en presence de 26 mm de citrate. Cependant, une chute de rendement de bioconversion du citrate en molecules d'aromes est observee. La deuxieme partie de ce travail a consiste a selectionner des mutants de lactococcus lactis subsp. Lactis diacetylactis cnrz 483 capables de produire du diacetyl et de l'acetoine a partir du glucose. En aerobiose, les mutants selectionnes produisent environ 0,25 mm de diacetyle alors que la souche parentale n'en produit pas.

Sélection de mutants plus tolérants vis-à-vis du butanol et produisant plus de solvants, et de mutants ne produisant pas d'acétone en l'absence d'acide acétique dans le milieu. Étude des effets des acides acétique, butyrique et pyruvique sur la production de solvants et sur les activités de différentes enzymes (acétate kinase, butyrate kinase, coenzyme à transférase, acétoacétate décarboxylase,. . . ). Étude des effets du PH

Dans la fermentation acétonobutylique, la conversion du sucre en solvants est de l'ordre de 33 % ; le rapport habituel butanol/acétone/éthanol est 6/3/1/ ; la concentration maximale de glucose pouvant être dégradé est de 60 g/1 pour une teneur finale en solvants de 20 g/1. La principale limitation de cette fermentation réside dans une inhibition par les produits, en particulier le butanol, qui perturbe le métabolisme bactérien. Ce travail s'est focalisé sur l'obtention de mutants résistants au butanol, et sur le mécanisme d'action des acides acétique et butyrique dans la formation de solvants. En utilisant différents cribles de mutation, tels que l'alcool allylique, le butyraldéhyde, le pentanol et le butanol, on a pu isoler différentes sortes de mutants. Certains ont perdu leur phase solvantogène, d'autres se sont montrés plus résistants au butanol que la souche sauvage. L'addition des acides acétique et butyrique a montré que le premier augmente la production d'acétone, sans toucher à celle du butanol, tandis que le deuxième augmente la production d'acétone et de butanol, d'une manière équivalente. Par la suite, nous avons proposé un mécanisme d'action de ces deux acides dans la formation de solvants. Dans la dernière partie, nous avons montré que ces deux acides modifiaient l'activité spécifique des enzymes acétate et butyrate kinase, au cours de la première phase de fermentation

Isolement d'un mutant de clostridium thermocellum non producteur d'acide lactique en utilisant le fluoropyruvate comme agent de selection, pour empecher l'inhibition causee par cet acide et augmenter le rendement en ethanol. Clostridium thermocellum est capable de convertir en ethanol la lignocellulose pretraitee par hydrothermolyse ainsi que la cellulose pure

Spoilage induced by Pseudomonas strains is commonly found in a wide range of food products as a result of the ubiquitous presence of these strains and their ability to induce alteration through different mechanisms. Particular attention has been recently paid on those P. fluorescens strains able to induce a blue discolouration on several food matrices (e.g. dairy or meat products). Actually, poor data are available about this curious event that draw the attention of European consumer from 2010. In the present manuscript a step-by-step investigation of the spoilage potential of Pseudomonas fluorescens species complex strains is reported, focusing in particular on the ability to produce an unpleasant blue pigment in food. Firstly, some general information is given to the reader to understand the P. fluorescens group as food spoiler. Then, the application of a polyphasic approach is described with the aim to investigate 136 Pseudomonas fluorescens group strains. Additionally, the achievement and the analyses of draft genomes and transcriptomes for 4 P. fluorescens strains are described to investigate the biosynthetic pathways involved in the blue pigment production. The attempt to chemically characterise the blue molecule using MALDI-TOF mass spectrometry is also reported. Finally, the execution of a transposon-mediated mutagenesis is described to confirm previously obtained genomic data and to highlight further genes involved in the blue-pigment production. The phenotypic and genotypic characterisation, based on the combination of classical microbiological tests and a MLST scheme, allowed the reconstruction of phylogenetic relationships among the isolates and the identification of a monophyletic group (named “the blue branch”) grouping all the blue-pigmenting and few uncoloured strains. The real involvement of these strains in the blue mozzarella event was confirmed by their ability to induce a blue discolouration on mozzarella cheese during a challenge test. The genomic investigation confirmed the strict phylogenetic relationship between the strains belonging to the “blue branch”. Additionally, comparative genomic tools revealed the presence of a genetic cluster unique to the blue pigmenting strains containing a second copy of five trp genes, clearly involved in the blue pigment production. The biochemical characterisation of the pigment, hampered by strong issues of solubility, led to the conclusion that the molecule is an indigo-derivative. Transposon-induced mutants confirmed the involvement of the previously identified unique cluster and the association of several genes affecting directly or indirectly the blue molecule production. Furthermore, the phenotypic characterisation of the mutants revealed a key role of iron in the production of the pigment, such as absence of any advantage of the wild-type strain in co-culture with a non-pigmenting mutant. To conclude, the present work represents an exhaustive investigation of the spoilage potential of the blue-pigmenting P. fluorescens strains, giving to food industry reliable approaches to identify, track and prevent spoilage related to the growth of these interesting bacteria.
Le alterazioni causate da ceppi di Pseudomonas sono solitamente riscontrate in una grande varietà di alimenti a causa del loro essere ubiquitari e dalla loro capacità di indurre modificazioni organolettiche negli alimenti mediante diversi meccanismi. Particolare attenzione è stata posta su alcuni ceppi di P. fluorescens in grado di indurre una colorazione blu in diverse matrici alimentari (quali prodotti lattiero-caseari o carne). In realtà, poche informazioni sono ad oggi disponibili riguardo al curioso caso che ha attirato l’attenzione pubblica a partire dal 2010. In questo lavoro è riportata un’analisi a più livelli del potenziale alternate dei ceppi appartenenti allo Pseudomonas fluorescens species complex, ponendo particolare attenzione alla capacità di produrre un indesiderato pigmento blu negli alimenti. In primo luogo, ai lettori sono date delle informazioni generali per una migliore comprensione di P. fluorescens come alterante alimentare. In seguito, è descritta la messa a punto e applicazione di un approccio polifasico con l’obbiettivo di indagare 136 ceppi appartenenti al gruppo P. fluorescens. Inoltre, sono descritti l’ottenimento e le analisi dei genomi draft e dei trascrittomi di 4 ceppi di P. fluorescens con la finalità di comprendere il pathway biosintetico coinvolto nella produzione del pigmento blu. In aggiunta, è riportato il tentativo di caratterizzare chimicamente il pigmento mediante la metodica della spettrometria di massa MALDI-TOF. Infine, è riportata l’esecuzione della mutagenesi random con la finalità di confermare i risultati genomici precedentemente ottenuti e di individuare ulteriori geni coinvolti nella produzione del pigmento blu. La caratterizzazione fenotipica e genotipica, basata sulla combinazione di metodiche di microbiologia classica e di uno schema MLST, ha permesso la ricostruzione delle relazioni filogenetiche tra gli isolati e l’identificazione di un gruppo monofiletico (chiamato “ramo blu”) che raggruppa tutti i ceppi pigmentanti e pochi ceppi non-pigmentanti. Il reale coinvolgimento dei ceppi blu nei casi di mozzarella blu è stato confermato dalla possibilità degli stessi di indurre un’anomala colorazione blu su mozzarella durante un challenge test. Le analisi genomiche hanno confermato la stretta vicinanza filogenetica tra i ceppi del “ramo blu”. Inoltre, analisi di genomica comparativa hanno rivelato la presenza di un cluster genico unicamente presente nei ceppi blu, contenente una seconda copia di cinque dei sette geni per la biosintesi del triptofano, chiaramente coinvolto nella produzione del pigmento blu. La caratterizzazione biochimica del pigmento, resa difficoltosa da problemi di solubilità, ha portato alla conclusione che la molecola blu sia un derivato dell’indigo. I mutanti ottenuti mediante l’applicazione di trasposoni hanno confermato il coinvolgimento del cluster genico precedentemente identificato nella produzione del pigmento e l’associazione di ulteriori geni che influenzano direttamente o indirettamente la produzione della molecola blu. Inoltre, la caratterizzazione dei mutanti ha rivelato il ruolo importante del ferro nella produzione del pigmento e l’assenza di un effettivo vantaggio del ceppo wild-type posto in co-cultura con un mutante non pigmentante. In conclusione, questo studio rappresenta un’indagine esaustiva del potenziale alterante dei ceppi blu, dando inoltre all’industria alimentare sistemi efficaci per identificare, tracciare e prevenire l’alterazione indotta da questi interessanti ceppi.

碩士
國立成功大學
生物化學研究所
97
Integrins are a family of heterodimeric receptors and modulate many cellular processes, including growth, death, adhesion, and migration. Rhodostomin (Rho) is a potent platelet aggregation inhibitor and belongs to the family of disintegrins. Rho consists of 68 amino acids with a 48PRGDM53P motif and six disulfide bonds. In previous study we expressed Rho in Pichia pastrois (P. pastoris) and used it as the scaffold to design integrin αvβ3-specific drugs. In this study I optimized the fermentation culture condition to increase the protein production of Rho mutants expressed in P. pastoris. The protein production of Rho mutants using shake-flask culture is ~100 mg/L. I increased the production of a Rho mutant, HSA-ARLDDL, up to 9-fold with a yield of ~900 mg/L by optimizing pH, temperature, medium composition, and oxygen level of the fermentation process. The final yields of proteins produced from shake-flask and fermentation cultures after purification were 30 mg/L and 170 mg/L, respectively. The recovery of proteins produced from shake-flask and fermentation cultures after purification were 30% and 17%, respectively. It is likely due to higher mis-folding proteins produced from the fermentation process. In our previous study we found that Rho mutants with a 39KKKRTICR47I linker sequence affected their binding affinity and selectivity to integrins α5β1, αvβ3, and αIIbβ3. Therefore, I have successfully expressed three Rho mutants with the 39KKKRT linker sequence (R46A, I47R, and R46A/I47R mutants) and one mutant with the 39SRAGK linker sequence (R46A mutant). The analysis of platelet aggregation and cell adhesion assays showed that the R46A mutant with the 39SRAGK linker sequence exhibited 5.4-, 7.6-, 2.2-, and 5.6-fold decreases in their inhibitory activity to platelet aggregation and integrins αIIbβ3,αvβ3, and α5β1, respectively. In addition, the R46A mutant with the 39KKKRT linker sequence exhibited 4.1-, 24.8-, 1.8-, and 1.8-fold decreases in their inhibitory activity to platelet aggregation and integrins αIIbβ3, αvβ3, and α5β1. In contrast, the I47R mutant with the 39KKKRT linker sequence exhibited 3- and 7-fold increases in their inhibitory activity to integrins αvβ3 and α5β1, respectively. Interestingly, we found that Rho mutant containing the 39KKKRTICARARGDN53P sequence inhibited integrins αIIbβ3, αvβ3, and α5β1 with the IC50 values of 956.0, 5.7, and 6.6 nM, respectively. The finding indicated that this mutant can abolish its activity to integrin αIIbβ3. The mutational study on Rho will provide new insight into design potent integrin-specific disintegrins, and the optimization of fermentation condition of HSA-ARLDDL will serve the basis to produce integrin-specific disintegrins expressed in P. pastoris.

Dissertação de mestrado em Biotecnologia Farmacêutica, apresentada à Faculdade de Farmácia da Universidade de Coimbra
Nos últimos anos têm sido desenvolvidas várias ferramentas para permitir o controlo de neurónios específicos, possibilitando o estudo da sua função. Estas novas ferramentas superam a falta de selectividade e o fraco controlo temporal proveniente do uso de estimulação eléctrica no controlo de actividade neuronal. A optogenética refere-se á integração de óptica e genética para obter um ganho ou perda de função em eventos bem definidos dentro de células específicas em tecido vivo. A capacidade de “ligar” e “desligar” neurónios utilizando luz é de facto uma tecnologia inovadora que oferece uma solução para limitações passadas. A optogenética, considerada por vários especialistas como ‘’método do ano’’ e ‘’inovação da década’’, em 2010, é utilizada para hiperpolarizar ou despolarizar neurónios alvo, de uma forma menos invasiva, utilizando luz e usufruindo de uma alta resolução espacial e escala temporal na ordem dos milissegundos. Esta técnica tem permitido o mapeamento e estudo de redes neuronais com uma grande eficácia. A ‘’Channelrhodopsin-2’’ (ChR2) é um canal catiónico sensível à luz, derivado da microalga Chlamydomonas reinhardtii. Na última década, a ChR2 tornou-se o arquétipo central e a principal ferramenta da optogenética. Actualmente, a caixa de ferramentas optogenética está em contínua actualização, com contribuições de estratégias de engenharia protéica, tais como mutagénese dirigida e a construção de quimeras com troca de domínios de diferentes espécies de channelrhodopsin. No entanto, alguns aspectos da forma ‘’wild-type’’ da ChR2 ainda requerem atenção e melhoramento. Estes incluem o seu espectro de acção, cinética, níveis de expressão, inactivação, condutância e exactidão de pico de absorção. Em termos de propriedades espectrais, poucas variantes desta proteína têm sido geradas e completamente caracterizadas com sucesso. No entanto, o aprimorar do espectro de activação da ChR2 e do formato do respectivo pico de absorção são algumas das propriedades mais desejadas. A ChR2 é excitada preferencialmente com comprimentos de onda de luz azul (470nm), o que limita o seu uso em material biológico de alta taxa de difusão, tal como o cérebro. Luz de excitação com maiores comprimentos de onda diminui a difusão de luz produzida por tecidos biológicos, e não é absorvida pela hemoglobina, assim, formas da ChR2 ‘’red-shifted’’, a absorver luz vermelha ou mesmo perto de infravermelha, são ferramentas desejáveis para a excitação de tecidos profundos. Alem disto, variantes ‘’blue-shifted’’ são também ferramentas atrativas para desenvolver, XXI dado que a combinação de várias ChR2 que apresentem sensibilidades a diversos comprimentos de onda permitiriam a estimulação de diferentes populações neuronais sem interferência entre si. Neste projecto, realizámos um desenho ab-initio para produzir quatro novas variantes de ChR2, usando uma abordagem de mutagénese dirigida no ambiente do cromófero da ChR2 alterando de forma radical os resíduos alvo. As mutações foram selecionadas com a aplicação de Time Dependent – Density Functional Theory (TDDFT) para prever o espectro de absorção dos mutantes selecionados da ChR2. O ‘’colour tuning’’ da ChR2 foi alcançado em quatro novas variantes criadas. Em particular, fomos capazes de gerar três variantes ‘’red-shifted’’ e uma ‘’blue-shifted’’. Após caracterização espectral, as variantes F217D e F269D apresentaram um ‘’red-shift’’ significativo de 90nm, a variante L221D apresentou um ‘’red-shift’’ de 180nm, a variante F269H apresentou um ‘’blue-shift’’ de 20nm. Apesar dos nossos resultados, é necessária uma caracterização protéica adicional, tal como a avaliação do tráfego membranar em neurónios e as características electrofisiológicas destes novos mutantes para determinar as proriedades cinéticas do canal. Neste trabalho, também conseguimos definir e descrever com sucesso a expressão e purificação da ChR2 ‘’wild-type’’ e de todas as quatro novas variantes no sistema eucariótico de expressão heteróloga - Pichia pastoris. Por fim, o nosso estudo valida as previsões de Time- Dependent Density Functional Theory e revela que abordagens de simulação biofísica podem ser utilizadas com vista à criação de variantes de ChR2 inteligentemente desenhadas. O desenho de novas variantes ChR2, seguindo a lógica racional aplicada, é uma abordagem poderosa e fiável para obter proteínas optimizadas para estratégias biotecnológicas. Os resultados originais obtidos com este trabalho demonstram potential para aplicações futuras, já que novas e melhoradas variantes de ChR2 continuarão a desempenhar um papel central no desenvolvimento e implementação da optogenética
Over the last few years, several tools have been developed to allow the control over specific types of neuron to enable the study of their function. These novel tools aim to overcome the lack of selectivity and the poor temporal control that derives from trying to control neuronal activity with electrical stimulation. Optogenetics refers to the integration of optics and genetics to obtain gain or loss of function in well-defined events and within specific cells in living tissue. The capacity to turn neurons “on and off” using light is indeed a groundbreaking technology that has become a solution for past limitations. Considered by many, “method of the year” and “breakthrough of the decade”, in 2010, optogenetics is used to hyperpolarize or depolarize specific targeted neurons using light in a less invasive manner, with high spatial resolution and a temporal resolution on the scale of milliseconds. This technique has allowed the mapping and study of neuronal networks with demonstrated efficacy. Channelrhodopsin-2 (ChR2) is a light-gated cation channel, derived from the microalga Chlamydomonas reinhardtii. In the last decade, ChR2 has become the central archetype and the main tool of optogenetics. Presently, the optogenetic toolbox is under continuous update, with contributions from protein engineering strategies, such as site-directed mutagenesis and construction of chimeras with domain swaps between channelrhodopsins of different species. However, some aspects of the wild-type form of ChR2 still require attention and enhancement. These include its action spectra, kinetics, expression levels, inactivation, conductance and absorption peak sharpness. In terms of spectral properties, few variants of this protein have been successfully generated and fully characterized. Nevertheless, tuning of ChR2 activation spectra and absorption peak sharpness are one of the most sought after properties. ChR2 is optimally excitable at a wavelength of blue light (470nm), which limits its use in high light-scattering biologic material, such as the brain. However, long-wavelength excitation light decreases the scattering of light produced by biological tissues and is not absorbed by haemoglobin. Thus, a red-shifted form of ChR2, absorbing red or even near infrared light would be a desirable tool for the excitation of relatively deep tissues. Furthermore, blue-shifted variants would also be attractive tools to develop, since the combination of ChR2 proteins with well separate wavelength sensitivities, combined with multicoloured optics, would permit the stimulation of different neuronal populations with no XXIII interference between them. In this project, we performed ab-initio design to produce four new ChR2 variants, using a radical site-directed mutagenesis approach on target residues in the environment of the ChR2 chromophore. The mutations were selected with the application of Time Dependent – Density Functional Theory (TDDFT) to predict the absorption spectra of ChR2 selected mutants. We achieved successful colour tuning of ChR2 with our four newly created variants. In particular, we were able to generate three red-shifted and one blue–shifted variant. After spectral characterization, the F217D and F269D variants presented a significant 90nm red shift, the L221D variant had a 180nm red shift and the F269H variant presented a 20nm blue shift. Despite our results, additional protein characterization is needed, such as the assessment of membrane trafficking in neurons and an electrophysiological characterization to determine channel kinetic proprieties for each of the variants. In this work, we were also able to define and describe the successful expression and purification of wild type ChR2 and of all the new four variants using the eukaryotic Pichia pastoris heterologous expression system. Finally, our study validates Time-Dependent Density Functional Theory predictions and reveals that biophysical simulation approaches may be used towards the creation of intelligently designed ChR2 variants. The design of new ChR2 variants, following our applied rationale, is a powerful and reliable approach to obtain enhanced proteins for biotechnological strategies. The original output obtained here shows potential for future optogenetic application, as new and improved ChR2 variants will continue to play a central role in the development and implementation of optogenetics

Dissertação de mestrado em Bioengenharia
Curcumin has been reported for its beneficial therapeutic properties including as anti-cancer agent. However, it has poor bioavailability and it is quickly metabolized in the human body, implying a repetitive oral administration if a therapeutic effect is envisaged. Besides, its extraction from plants is very expensive. For these reasons, the use of microorganisms to produce it on large scale and with greater yields constitutes an interesting alternative. With this aim, Escherichia coli K-12 MG1655 (DE3) was previously engineered with three enzymatic steps (4-coumarate-CoA ligase, diketide-CoA synthase and curcumin synthase 1) that catalyze the production of curcumin from ferulic acid. In the present study, the optimal strain, operational conditions and media composition for the production of curcumin by E. coli harboring the artificial biosynthetic pathway were established. Previously, a standard two-step fermentation strategy (LB+M9 minimal medium) was used. Although feasible at the laboratory scale, the biomass separation is much more difficult, laborious and expensive in large-scale fermentations. Therefore, herein a single medium formulation more suitable for the production of curcumin at an industrial set-up was implemented. MOPS minimal medium, TB and LB were evaluated. Using the optimized conditions, the curcumin concentration obtained in this study was the highest reported to be produced by a heterologous organism, 686.7±59.7 µM in TB (43 h) and 822.6±28.1 µM in LB+M9 (63 h). These results were obtained using E. coli BL21 (DE3) that was identified as the best producer since it produced 3.7 times more curcumin than E. coli K-12 MG1655 (DE3). Moreover, curcumin toxicity against E. coli cells was evaluated. The tests performed showed that curcumin concentrations above 400 µM influence negatively the E. coli cells growth. Furthermore, one of the purposes of the current work was to construct and validate several E. coli mutants (e.g. ΔfumA,fumB,fumC) previously identified by an in silico approach as the most promising towards an increased production of curcumin from ferulic acid. The deletion of fumB gene from E. coli K-12 MG1655 (DE3) genome was accomplished and it resulted in a faster curcumin production in the initial 21 h, but after 63 h, the curcumin production by this mutant was 2.6 times lower as compared to the ‘original’ strain (i.e. the strain harboring the curcuminoids biosynthetic pathway but with no gene knockout). The same deletion in E. coli BL21 (DE3) genome resulted in a more significant decrease in curcumin production. In the future, the triple knock-out (ΔfumA,fumB,fumC) should be constructed to evaluate if curcumin production can indeed be improved as predicted in silico.
A curcumina tem sido reportada pelas suas propriedades benéficas incluindo como agente anticancerígeno. Apesar da curcumina apresentar um alto potencial terapêutico, tem uma baixa biodisponibilidade e é rapidamente metabolizada no organismo humano, o que implica uma repetitiva administração oral para atingir o efeito terapêutico pretendido. Além disso, a sua extração a partir das plantas é muito dispendiosa. Por estas razões, o uso de microrganismos para a produzir em larga escala e com melhores rendimentos constitui uma alternativa atraente. Neste sentido, Escherichia coli K-12 MG1655 (DE3) foi previamente geneticamente modificada adicionando três reações enzimáticas (4-cumarato-CoA ligase, dicetídeo-CoA sintase e curcumina sintase 1) que catalisam a produção de curcumina a partir do ácido ferúlico. No presente trabalho foi estabelecida a estirpe, as condições operacionais e a composição de meio ótimas para a produção de curcumina por E. coli contendo a via biossintética artificial. Anteriormente, utilizou-se uma estratégia comum de dois passos (LB+meio mínimo M9). Apesar de ser praticável numa escala laboratorial, a separação de biomassa é muito mais difícil, trabalhosa e dispendiosa numa fermentação em grande escala. Assim, foi implementada uma única formulação de meio mais adequada para a produção de curcumina a nível industrial. O meio mínimo MOPS, TB e LB foram avaliados. Usando as condições otimizadas, a concentração de curcumina produzida neste estudo foi mais elevada do que as previamente descritas na literatura, 686,7±59,7 µM em TB (43 h) e 822,6±28,1 µM em LB+M9 (63 h). Estes resultados foram obtidos usando a estirpe E. coli BL21 (DE3) que foi a identificada como melhor produtora após produzir 3,7 vezes mais curcumina do que a E. coli K-12 MG1655 (DE3). Além disso, a toxicidade da curcumina para células de E. coli foi avaliada. Os resultados mostraram que concentrações de curcumina acima de 400 µM influenciam negativamente o crescimento de E. coli. Adicionalmente, um dos objetivos do presente trabalho foi construir e validar vários mutantes de E. coli (p. ex., ΔfumA,fumB,fumC) que foram previamente identificados in silico como os mais promissores no sentido de aumentar a produção de curcumina a partir do ácido ferúlico. A deleção do gene fumB do genoma de E. coli K-12 MG1655 (DE3) foi efetuada e resultou numa produção mais rápida de curcumina nas 21 h iniciais, mas após 63 h, a produção de curcumina usando este mutante foi 2,6 vezes mais baixa do que a da estirpe que lhe deu origem. A mesma deleção no genoma de E. coli BL21 (DE3) resultou num decréscimo ainda mais significativo na produção de curcumina. No futuro, é necessário ainda construir o mutante triplo (ΔfumA,fumB,fumC) para avaliar se a produção de curcumina é efetivamente aumentada como previsto in silico.

碩士
國立臺灣大學
植物科學研究所
96
Ralstonia solanacearum causes a deadly wilting disease on a wide range of crops. To achieve a successful infection, R. solanacearum is well equipped with many pathogenesis machineries, including swimming and twitching motility (MO), biofilm formation (BF), production of exopolysaccharides (EPS) and avoidance or suppression of host defenses. The general objective of this study is to characterize mutants of candidate genes involved in R. solanacearum virulence and fitness. Firstly, a group of transposon (Tn5)-insertional R. solanacearum mutants defective in EPS, MO or BF were characterized. Allelic mutants of these mutants displayed consistent phenotypic changes, confirming that the transposon insertions are responsible for the altered phenotypes. Studies on two novel genes related to BF and MO revealed that RSc0727 is related to BF and twitching motility, and that RSp0199 is related to swimming motility. Further cross-examination of a range of mutants suggested that (1) EPS production is required for BF, but its role in swimming and twitching motility is inconclusive; (2) BF and swimming motility seem to be independent of each other, but twitching motility is important to BF. Secondly, previous systematic screens of Tn5-insertional R. solanacearum mutants led to the identification of two mutants displaying normal pathogenesis on tomato but decreased pathogenesis on Arabidopsis. Allelic mutants exhibited similar in vitro characteristics and differential pathogenesis on tomato and Arabidopsis, verifying the transposon insertions are responsible for the altered phenotypes. Multiplication of these mutants in Nicotiana benthamiana and tomato was not significantly different from that of the wild-type strains. However, the significantly decreased mutants’ colonization in Arabidopsis was found to be correlated with differential pathogenesis on tomato and Arabidopsis. Complementation tests further evidenced RSc1206 is essential for cell membrane/envelope integrity. Results gathered from this study suggested that RSc1206 is required for adaptation of this bacterium in different host environments, and the decreased pathogenesis might be due to the alterations in nutrient utilization, reduced stress tolerance, biofilm formation and swimming motility. On the other hand, the mutant 18A7 displayed mostly normal phenotypes under normal and stress conditions, except for its reduced swimming motility which might be related to the decreased pathogenesis on Arabidopsis. However, whether other factors and how they may contribute to the decreased pathogenesis of 18A7 on Arabidopsis still remains to be determined in the future.

碩士
國立中興大學
生命科學系所
104
Green algae have a photosynthetic system that can produce H2 by hydrogenase (HydA, encoded by hydA) using sun light under certain condition. In the previous studies, an HydA-overexpressed mutant and some O2 tolerant HydA-mutated-overexpressed Chlorella sp. DT (DT) mutants were obtained to enhance the H2 production in this laboratory. However, the increased fold of HydA expression was not compatible to that of H2 production. In order to understand whether Ferredoxin-NADP+-oxidoreductase (FNR) competes electrons with HydA of DT mutants, NADPH reduction and H2 photoproduction of isolated chloroplasts of mutants were measured. The mutated-hydA sequence and H2 production of O2 tolerant 3-year-old HydA-mutated-overexpressed DT mutants were confirmed. DT mutants still carried mutated-hydA gene and had O2 tolerance so that they could produce more H2 than DT-WT. The chloroplasts of DT mutants were isolated. With addition of NADP+ as FNR substrate, the NADPH reduction rates of isolated chloroplasts of one HydA-mutated-overexpressed DT mutants were 0.5 fold of DT-WT. Without addition of NADP+, the H2 photoproduction of isolated chloroplasts of DT mutants was about 0.3-0.4 fold higher than that with addition of NADP+ condition. The percentages of NADPH reduction and H2 production of one HydA-mutated-overexpressed DT mutants were 32% and 68%. The percentages of NADPH reduction and H2 production of DT-WT were 44% and 56%. The results suggested that the FNR-mediated NADPH reduction limited HydA-mediated H2 photoproduction in HydA-mutated-overexpressed DT mutants. Moreover, in order to measure the activity of HydA for further investigation in the future, the wild type HydA protein was genetically attached a StrepII-tag. The gene of strepII-tag was constructed into plasmid pHyg3-HydA-StrepII, ligated to the 3'' end of hydA gene, and electrotransformed into DT. The observation of hydA-strepII fragment in DT transformants by PCR indicated that the plasmid was successfully transformed into DT. The detectable HydA-StrepII protein by western blot suggested that HydA-StrepII could be expressed in DT transformants. The HydA-StrepII DT mutant cells were broken by sonication and the total proteins were extracted by centrifugation. Furthermore, the HydA-StrepII was purified by Strep-Tactin affinity column. However, no detectable signal of HydA-StrepII was visualized. The results implied that there may be no enough HydA-StrepII extracted from HydA-StrepII DT mutant at present stage.

The soil-borne, rhizosphere-competent, filamentous fungus Trichoderma virens is a well-known biocontrol agent able to control pathogenic fungi through the production of antibiotics, the induction of systemic resistance in host plants, or by directly parasitizing the competing fungus. Competition for iron is another means by which Trichoderma can hinder competing microorganisms, and siderophores are a means by which microorganisms obtain iron. In silico analysis of the T. virens genome suggested that two genes putatively encoding extracellular siderophore-producing nonribosomal peptide synthetases (NRPSs) were present. In this study, a disruption was created in one of the genes, TvNPS6, to create a mutant unable to produce the NRPS TvNps6 (DeltaTvnps6). Previously, a mutant (DeltaTvsidD) had been generated with a disruption in the second gene (TvSIDD) encoding an NRPS thought to be involved in siderophore biosynthesis. A double mutant (DeltaDeltaTvsidDTvnps6) was generated by transformation of a DeltaTvsidD strain with a vector targeting disruption of TvNPS6. This resulted in transformants disrupted within both the putative siderophore-producing NRPSs. Thus, three mutants were available for analysis of the role of these genes in the ecology of T. virens. Transformants were confirmed by PCR and Southern blotting analysis. Phenotypic characterization of the mutants included both HPLC analysis of siderophore production, growth on agar and in liquid media, conidiation, germination in the presence of hydrogen peroxide, biocontrol against Pythium ultimum, in vitro confrontation against Rhizoctonia solani and growth with iron chelators to determine the contribution of reductive iron assimilation (RIA) compared to that of siderophores. The HPLC analysis demonstrated that T. virens Gv 29-8 (wild-type) produced a single siderophore peak when grown in an iron-depleted medium. This peak was not present in the DeltaTvnps6 and DeltaDeltaTvsidDTvnps6 mutants but was apparent with the DeltaTvsidD mutants. From the HPLC analysis, T. virens evidently produces a coprogen-type siderophore. Few differences were observed in the other phenotypic tests, though hydrogen peroxide showed some small inhibitory effects towards the DeltaTvnps6 mutants. The addition of chelators, which inhibit RIA, exerted some negative effects on all strains growing under iron-limited media, particularly the DeltaTvnps6 and DeltaDeltaTvsidDTvnps6 strains. This study demonstrated that although T. virens has two genes putatively encoding siderophore producing NRPSs, only the TvNPS6 gene was required for extracellular siderophore production. The greater sensitivity of the mutants towards the iron chelators suggests that unlike other other fungi studied, Trichoderma virens utilizes RIA, rather than siderophore production, as the primary means by which the fungus obtains iron in an iron-limited environment.

碩士
國立中興大學
生物科技學研究所
106
Abstract Infectious Bursal Disease Virus (IBDV) mainly infects young chickens aged 3-6 weeks and pathologically causes necrosis of the B lymphocytes and suppression of the immune system. The infection is often accompanied by high morbidity and high mortality and so that often cause the poultry farmers suffering from huge economic loss. Recently, we concluded that the exposed residue His253 of a VP2-formed SVP is crucial for binding affinity of SVP to Ni-NTA. In order to effectively purify IBDV virus particles by IMAC, in this study, predict the exposure area of each amino acid of IBDV rD78 according to the protein structure software. Substitution of a specific amino acid at three residues in VP2 including Ser251, Ser317 and Gln320 with histidine may increase the ability of recombinant IBDV to bind to nickel ions, making them useful for IMAC purification. The recombinant IBDV mutants were subjected to DF- 1 cells and Vero cells for virus production and purification. It was found that Vero cells were 23-68 times more potent than the DF-1 cell mutant recombinant virus. However, since the medium of DF-1 cells contains fetal bovine serum (FBS), which may interfere with the ability of virus particles to bind and hence affect the results. Vero cells culture in serum-free medium to improver purification efficiency. The virus solution were further purified by IMAC. During purification, virus particles were eluted at the step of pH4.0 elution meanwhile rD78-WT and rD78- S371H were eluted earlier at the step of pH 7.8 equilibrium, indicating that rD78-S251H and rD78-Q320H virus particle are more capable of binding nickel ions than WT and S251H. However, more complete virus particle of rD78-WT were observed by transmission electron microscopy. In addition, the sequence of p300-D78A plasmid and PBD9 Segment A fragment of IBDV native strain was completed. The P3009 recombinant virus strain is produced in the same manner, which facilitates the subsequent research to provide a vaccine for the prevention and treatment of IBDV infection, and can also be applied to the production and purification of other viruses or virus-like particles.