Thema dieser Arbeit ist die evolutive Optimierung der T7-RNA-Polymerase. Zur Stabilisierung technischer oder therapeutischer RNA-Moleküle gegenüber RNAsen wäre es wünschenswert eine RNA-Polymerase zu generieren, welche RNA vollständig aus 2’- modifizierten Nucleotiden synthetisieren kann. Zu diesem Zweck wurde ein kombiniertes Selektions- und Screeningverfahren zur Durchmusterung von Variantenbibliotheken der T7- RNA-Polymerase hinsichtlich des Einbaus von 2’-Methoxy-modifizierten Nucleotiden in RNA entwickelt. Es wurden ein gut handhabbarer, cis-regulierter Expressionsvektor sowie ein Selektionsplasmid erzeugt, die zusammen in E. coli ein in-vivo-Selektionssystem bilden, mit dessen Hilfe man Zellen, welche T7-RNA-Polymerase-Aktivität zeigen anhand ihrer grünen Fluoreszenz identifizieren konnte. Durch error-prone PCR wurden Mutantenbibliotheken generiert, und diese in das Selektionssystem eingesetzt. So konnte die Anzahl der potentiell zu testenden Varianten erheblich gesenkt werden. Zur Bestimmung der T7-RNA-Polymerase- Aktivität mit 2’-Methoxy-modifizierten Nucleotiden wurde ein Fluoreszenz-basierendes Assay etabliert. Dieses Assay, das nicht mit radioaktiv-markierten Nucleotiden arbeitete und keinen gelelektrophoretischen Separationsschritt benötigte, konnte in allen Schritten zur parallelen Bearbeitung von 96 Proben in einem Mikrotiterplatten-Format angepasst werden, so dass es prinzipiell hochdurchsatzfähig war und sich zum Screening umfangreicher Variantenbibliotheken eignete. Die Assay-Reaktion kann dabei auch unkompliziert auf ein Screening von RNA- oder DNA-Polymerase-Bibliotheken hinsichtlich anderer Eigenschaften der Polymerase-Aktivität übertragen werden.

RNA interference (RNAi) in the nematode Caenorhabditis elegans is a type of homology-dependent post-transcriptional gene silencing induced by dsRNA. This dissertation describes the genetic analysis of the RNA interference pathway and inheritance properties associated with this phenomenon. We demonstrate that the RNAi effect can be observed in the progeny of the injected animal for at least two generations. Transmission of the interference effect occurs through a dominant extragenic agent. The wild-type activities of the RNAi pathway genes rde-l and rde-4 are required for the formation of this interfering agent but are not needed for interference thereafter. In contrast, the rde-2 and mut-7 genes are required downstream for interference. These findings provide evidence for germline transmission of an extragenic sequence-specific silencing factor and implicate rde-l and rde-4in the formation of the inherited agent. Other forms of homology-dependent silencing in C. elegansinclude co-suppression and transcriptional silencing of transgenes in the germline. We demonstrate that silencing of a germline transgene can be initiated by injected dsRNA, via the RNAi pathway, and then maintained on a different level. This observation indicates that post-transcriptional and transcriptional silencing of homologous genes could be connected. This dissertation also describes the connection between RNAi and developmental pathways of gene regulation in C. elegans. We show that inactivation of genes related to RNAi pathway genes, a homolog of Drosophila Dicer (dcr-l), and two homologs of rde-1 (alg-l and alg-2) cause heterochronic phenotypes similar to lin-4 and let-7 mutations. Further we show that dcr-l, alg-l, and alg-2 are necessary for the maturation and activity of the lin-4 and let-7small temporal RNAs that regulate stage-specific development. Our findings suggest that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation. Finally, this study illustrates the detection of small interfering RNAs (siRNAs), intermediates in the RNAi process, and describes requirements for their accumulation. We show that, in the course of RNAi induced by feeding dsRNA, C. elegans accumulate only siRNAs complementary to the target gene. This accumulation depends on the presence of the target sequence and requires activities of several RNAi-pathway genes. We show that selective retention or amplification of RNAi-active molecules can create a reservoir of memory antisense siRNAs that prevent future expression of the genes with complementary sequence. This suggests a parallel at the molecular level with the clonal selection of antibody forming cells and in the vertebrate immune system.

It has been well documented that mRNA is associated with the cytoskeleton, and that this relationship is involved in translation and mRNA sorting. The molecular components involved in the attachment of mRNA to the cytoskeleton are only poorly understood. The objective of this thesis was to directly visualize the interaction of mRNA with the cytoskeleton, with sufficient resolution to identify the filament systems and structures involved. This work required the development of novel in situ hybridization methods for use with electron microscopy. This allowed resolution to visualize single mRNA molecules and individual filaments. The development of a silver enhancement methodology for both the light and electron microscopic detection of biotinated oligo-dT probes permitted a synoptic view of the intracellular distribution of poly(A) mRNA. At the light microscope, the distribution of poly(A) mRNA did not resemble the individual distribution patterns of microfilaments, intermediate filaments or microtubules. Ultrastructural examination revealed that poly(A) mRNA was not uniformly distributed along cytoskeletal filaments, but clustered at their intersections. The composition of these mRNA containing structures was investigated by both morphologic and in situ hybridization analysis using antibodies to cytoskeletal proteins. In thin sections, polysomes were observed attached to both microfilaments and intermediate filaments. To permit the simultaneous detection of oligo-dT hybridization and specific cytoskeletal proteins, a double labelling method using colloidal gold conjugated antibodies was developed. The majority of poly(A) mRNA was associated with the actin cytoskeleton, with 72% of the hybridization localized within 5nm of a labelled microfilament. Within the actin cytoskeleton, poly(A) mRNA was localized to intersections of orthogonal networks. Greater than 50% of poly(A) colocalized with the actin crosslinking proteins, filamin and α-actinin, but not vinculin. A significant amount of poly(A) mRNA was found to be associated with intermediate filaments. The double label gold analysis demonstrated that 33% of the hybridization signal localized within 5nm of labelled vimentin filaments. Prior disorganization of the actin cytoskeleton using cytochalasin did not disrupt the association of mRNA with vimentin. These observations are consistent with our morphologic results of polysome-intermediate filament associations, and indicate that microfilaments are not the only filament system to which mRNA is bound. Furthermore, a small amount of hybridization signal (12%) consistently was observed along microtubules, providing an additional cytoskeletal network to distribute mRNA. To further characterize the spatial organization of mRNA within the cytoskeleton, ultrastructural methods were developed to directly visualize individual mRNA molecules. First, oligonucleotide probes chemically modified with a single hapten and directly conjugated primary reagents were used to permit detection of an individual hybridized probe molecule by a single gold particle. Second, biotin and digoxigenin labelled oligonucleotide probes were used to simultaneously visualize the intermolecular and intramolecular relationships of two nucleic acid sequences. Third, reverse transcriptase was used to extend hybridized primers in situ which permitted visualization of the poly(A) sequence concomittant with the conformation of an mRNA molecule. These methods have permitted analysis of how single mRNA molecules may be positioned with respect to each other within the cytoskeleton. The ultrastructural visualization of mRNA within its structural environment has demonstrated heterogeneous interactions with the cytoskeleton. Future work will be needed to further characterize the mechanism of mRNA attachment. The proteins which bridge nucleic acid sequences to specific intersections can be identified. It will be interesting to learn how the identified mRNA-cytoskeletal interactions might be involved in the regulation of both mRNA translation and intracellular location. Lastly, and perhaps the most challenging goal, is to investigate whether the identified mRNA-cytoskeletal interactions are used by the cell to influence its own shape, polarity and architecture.

This thesis examines the structure and regulated expression of the gene encoding the neuroendocrine peptides neurotensin and neuromedin N (NT/N gene). Previous studies have shown that expression of NT/N mRNA and NT peptide in PC12 cells are strictly dependent on simultaneous exposure to combinations of nerve growth factor (NGF), glucocorticoids, activators of adenylate cyclase, and lithium ion. My objective was to characterize the cis-regulatory DNA sequences involved in regulated expression of this gene. The initial focus of this study was an analysis of the structure, tissue-specific expression, and exon evolution of the rat NT/N gene. Nucleotide sequence comparisons between the rat gene and the canine and bovine cDNA sequences indicated that the predicted structure of a 170 amino acid precursor protein is highly conserved. Furthermore, the close similarity between the two cDNAs suggested that identical precursor proteins are expressed in neural and endocrine tissues. RNA analysis revealed that the gene is transcribed to yield two distinct mRNAs, 1.0 kb and 1.5 kb in size. The two mRNA species differ only in the size of their 3' untranslated regions. Interestingly, the smaller mRNA is predominant in the gastrointestinal tract, while both mRNAs are equally abundant in all neural tissues examined, except the cerebellum, where no expression was observed. Transient transfection assays were used to delineate the rat NT/N gene cis-regulatory DNA sequences. Progressive deletion of the NT/N 5' flanking region revealed that sequences between -216 and +56 of the NT/N gene are sufficient to confer the full spectrum of responses of the endogenous gene to either of two reporter genes. A detailed mutational analysis of the NT/N control region indicated that it is composed of an array of inducible cis-regulatory elements, including an AP-1 site, two cAMP-responsive elements (CREs), and a glucocorticoid-responsive element (GRE). Specific mutations to the AP-1 site and either CRE suggested that these elements are functionally interdependent. I propose that this array of cis-regulatory sequences in the NT/N transcriptional control region serves to integrate multiple environmental stimuli into a unified transcriptional response. To further examine the role of the AP-1 site and CREs in the NT/N promoter, reporter genes containing either a single or multiple AP-1 or CRE sites were expressed in PC12 cells and protein kinase A-deficient PC12 cells treated with forskolin, NGF, and lithium, either individually, or in combination. The results indicated that lithium and NGF markedly activate promoters containing multiple AP-1 sites, but not a single site, and that these effects were additive. Both agents potentiated forskolin-induced activation of promoters containing a single or multiple CREs, but had no effect, individually. Also, in contrast to the activation of multiple AP-1 sites by lithium and NGF, activation of the NT/N promoter and promoters containing CREs is absolutely dependent on protein kinase A activity. These results suggested that promoters containing multiple AP-1 sites, or a single AP-1 site in the context of nearby active CREs, are selectively activated by lithium and NGF in PC12 cells. Based on the results of this thesis I have proposed a model to account for the complex transcriptional regulation of the NT/N gene in PC12 cells. I have also addressed the relevance of these findings to the mechanisms of phenotypic plasticity of embryonic neural crest cells, NGF-induced neuronal differentiation, and the pharmacological actions of lithium.

6-(p-Hydroxyphenylhydrazino) uracil (H2-HPUra) is a selective and potent inhibitor of the replication-specific DNA polymerase III (pol III) of Gram+ bacteria such as Bacillus subtilis. Although a pyrimidine, H2-HPUra derives its inhibitory activity from its specific capacity to mimic the purine nucleotide, dGTP. The project described in this thesis dissertation involves the use of H2-HPUra-like inhibitors to probe the structure and function of the pol III active site. It consists of two separate problems which are summarized below. Production of a potent bona fide dGTP form of inhibitor. A method was devised to successfully convert the H2-HPUra inhibitor prototype to a bona fide purine, using N2-benzyl guanine as the basis. Structure-activity relationships of benzyl guanines carrying a variety of substituents on the aryl ring identified N2-(3,4-dichlorobenzyl) guanine (DCBG) as a compound equivalent to H2-HPUra with respect to potency and inhibitor mechanism. DCBdGTP, the 2'-deoxyribonucleoside 5'-triphosphate form of DCBG, was synthesized and characterized with respect to its action on wild-type and mutant forms of pol III. DCBdGTP acted on pol III by the characteristic inhibitor mechanism and formally occupied the dNTP binding site with a fit which permitted its polymerization. The latter experiment identified the site for the binding of the inhibitor's aryl moiety as a distinct site located at a distance of approximately 6-7 Å from the base-paired 2-NH group of a bound dGTP. Attempt to covalently label amino acid residue 1175, a putative participant in inhibitor binding. Azp-12, a point mutation of serine 1175, yields a form of pol III whose inhibitior sensitivity varies specifically as a function of the composition of the para substituent of the inhibitor's aryl ring. On the basis of the latter behavior, residue 1175 was hypothesized to be a residue directly involved in the binding of the inhibitor's aryl moiety. To test this hypothesis, residue 1175 was specifically mutated to either cysteine or lysine, each of which presents a side chain amenable to covalent bond formation with appropriately reactive inhibitor forms. Of the two mutant pol III forms, only the cysteine form (pol III-cys) was catalytically active. The kinetic properties and inhibitor sensitivity profile of pol III-cys identified it as a target suitable for potentially irreversible inhibitor forms containing the following groups in the meta position of the aryl ring: -CH2Br, -CH2C1, and -CH2SH. None of the several inhibitors tested selectively or irreversibly inactivated pol III-cys. Possible bases for the failure of this group of inhibitors and for the redesign of more useful covalently reactive inhibitor forms are considered.

In diverse eukaryotic organisms, double-stranded RNA (dsRNA) induces robust silencing of cellular RNA cognate to either strand of the input dsRNA; a phenomenon now known as RNA interference (RNAi). Within the RNAi pathway, small, 21 nucleotide (nt) duplexed RNA, dubbed small interfering RNAs (siRNAs), derived from the longer input dsRNA, guide the RNA induced silencing complex (RISC) to destroy its target RNA. Due to its ability to silence virtually any gene, whether endogenous or exogenous, in a variety of model organisms and systems, RNAi has become a valuable laboratory tool, and is even being heralded as a potential therapy for an array of human diseases. In order to understand this complex and unique pathway, we have undertaken the biochemical characterization of RNAi in the model insect, Drosophila melanogaster. To begin, we investigated the role of ATP in the RNAi pathway. Our data reveal several ATP-dependent steps and suggest that the RNAi reaction comprises as least five sequential stages: ATP-dependent processing of double-stranded RNA into siRNAs, ATP-independent incorporation of siRNAs into an inactive ~360 kDa protein/RNA complex, ATP-dependent unwinding of the siRNA duplex to generate an active complex, ATP-dependent activation of RISC following siRNA unwinding, and ATP-independent recognition and cleavage of the RNA target. In addition, ATP is used to maintain 5´ phosphates on siRNAs, and only siRNAs with these characteristic 5´ phosphates gain entry into the RNAi pathway. Next, we determined that RISC programmed exogenously with an siRNA, like that programmed endogenously with microRNAs (miRNAs), is an enzyme. However, while RISC behaves like a classical Michaelis-Menten enzyme in the presence of ATP, without ATP, multiple rounds of catalysis are limited by release of RISC-produced cleavage products. Kinetic analysis of RISC suggests that different regions of the siRNA play distinct roles in the cycle of target recognition, cleavage and product release. Bases near the siRNA 5´ end disproportionately contribute to target RNA-binding energy, whereas base pairs formed by the central and 3´ region of the siRNA provide helical geometry required for catalysis. Lastly, the position of the scissile phosphate is determined during RISC assembly, before the siRNA encounters its RNA target. In the course of performing the kinetic assessment of RISC, we observed that when siRNAs are designed with regard to 'functional asymmetry' (by unpairing the 5´ terminal nucleotide of the siRNA's guide strand, i.e. the strand anti-sense to the target RNA), not all of the RISC formed was active for target cleavage. We observed, somewhat paradoxically, that increased siRNA unwinding and subsequent accumulation of single-stranded RNA into RISC led to reduced levels of active RISC formation. This inactive RISC did not act as a competitor for the active fraction. In order to characterize this non-cleaving complex, we performed a series of protein-siRNA photo-crosslinking assays. From these assays we found that thermodynamic stability and termini structure plays a role in determining which proteins an siRNA will associate with, and how association occurs. Furthermore, we have found, by means of the photo-crosslinking assays, that siRNAs commingle with components of the miRNA pathway, particularly Ago1, suggesting overlapping functions or crosstalk for factors thought to be involved in separate, distinct pathways.

Antibiotics are routinely used in modern livestock production systems to treat and prevent disease as well as to enhance livestock production and/or outputs. This widespread use of antibiotics has led to a growing worldwide interest in antibioticfree animal production. The addition of feed supplements such as nucleotides to improve intestinal health as well as the early development of the immune system needs to be investigated as an environmentally-friendly nutritional management activity. In this study four (4) groups of newborn Holstein calves (n=24) were evaluated after being treated as follows: Group 1 (Negative control), Group 2 (Investigational Veterinary Product (IVP) 1 – Oral supplement containing nucleotides, vitamins, essential fatty acids, essential amino acids, pre-biotics and trace-minerals), Group 3 (IVP 2 – Oral supplement containing nucleotides only at concentration and dosage equal to IVP1) and Group 4 (IVP 3 - Parenteral glucocorticoids). Results of the study indicated that: Pre-colostral Serum IgG titres – After titration of the serum the anti-BVDV results showed no difference between the study groups. All calves recorded a SP ratio of zero value (0.00 ± 0.2) indicating that they had not been nursed by their mothers prior to the start of the study. This was an entrance requirement for the calves to be enrolled into the study. Apparent Efficiency of Absorption % (AEA%) – Literature shows that an AEA% result of between 20%-30% is good and 35% is excellent. The AEA% recorded for this study fell within this range although there was no statistically significant difference shown between the groups. In spite of a strictly controlled study protocol, 7 of the 24 calves (29%) still suffered from FPT in this study. The result falls within the range of 19% - 37% reported on United States of America (USA) farms by Doepel and Bartier in 2014. Serum cortisol - When compared to the control group, there were no statistical significant difference evident for group 2 and group 3. However, the difference between the Glucocorticoid – Group 4 and the control and other groups was statistically significant (P = 0.0001; table 12) at the various time intervals. It was also evident that the inclusion of parenteral glucocorticoids (group 4) may have assisted in prolonging the natural “gut closure”. Gut closure – a positive 2 point linear regression forecast line indicated an increasing trend in IgG absorption post-24 hours for group number 4 whilst groups 1, 2 and 3 each had a negative 2 point linear regression forecast line (figure 23). Weight / ADG (D42) - Group 2 showed a statistically significant increase in mean D42 weight (P = 0.0042) of 59.167 kg ± 3.545 kg when compared to the other study groups and compared to the control group (P = 0.0227). A comparison of the relative increase in mass of groups indicated that Group 2 efficiently achieved this result with a statistically significant ADG of 0.536 kg (P = 0.014) compared to the other study groups and compared to the control group (P = 0.022). In addition, weight / FCR – FCR was calculated for all groups as follows: G1 – 4.000 kg, G2 - 2.593 kg, G3 – 2.703 kg and G4 - 3.012 kg feed required for the production of 1 kg meat. The results indicated that the group 2 feed input was the most economical followed by groups 3, 4 and control respectively. In conclusion, it is evident that a nutritional supplement containing nucleotides, vitamins, trace- minerals, essential fatty acids, essential amino acids and pre-biotics may contribute significantly to important economic indicators such as weight gain, ADG and FCR on a commercial dairy farm. / Environmental Sciences / Ph. D. (Environmental Science)

Espécies autopoliploides são extremamente importantes na agricultura. No entanto, a estrutura complexa de seus genomas não é bem compreendida. Apesar de todos os avanços no mapeamento genético de autotetraploides, a grande maioria dos modelos utilizados para a construção de mapas em espécies autopoliploides com elevado nível de ploidia, tais como a canade- açúcar, são aproximações daqueles usados para organismos diplóides. Assim, este trabalho teve como objetivo o desenvolvimento de um novo modelo para construção de mapas genéticos em espécies autopoliploides com qualquer nível de ploidia e incluindo marcadores com todas as dosagens possíveis. Para tanto foi utilizada a tecnologia dos modelos de Markov ocultos. O modelo aqui apresentado pode ser aplicado a dados de marcadores dominantes e codominantes, com comportamento bialélico ou multialélico. O método baseia-se no cálculo das probabilidades condicionais que compõem a matriz de transição seguido da redução de sua dimensão usando uma abordagem computacional. O uso do método foi ilustrado em uma população de mapeamento de cana-de-açúcar proveniente do cruzamento entre duas variedades pré-comerciais (IACSP 95-3018 × IACSP 93-3046) e genotipadas com três tipos de marcadores: SNPs, microssatélites e AFLPs. Os resultados indicam que o novo método é muito eficiente na obtenção de mapas genéticos, mesmo em situações com níveis de ploidia altos e marcadores com altas doses, particularmente quando estes marcadores têm comportamento codominante. Também foi possível estimar a verossimilhança, as frações de recombinação e as fases de ligação usando a abordagem multiponto, a qual leva em consideração todos os marcadores do grupo de ligação analisado simultaneamente. O novo modelo aqui proposto representa um importante passo para realizar futuramente a localização de regiões genômicas associadas à variação das características quantitativas, no entendimento da arquitetura genética de tais características e na montagem de genomas de espécies autopoliploides. / Autopolyploid species are extremely important in agriculture. However, the complex structure of their genomes is not well understood. Despite all advances in genetic mapping of autotetraploids, the vast majority of the models used for autopolyploid species with high ploidy level, such as sugarcane, are approximations of those used in diploid organisms. Thus, the aim of this work was to develop a new model to build genetic linkage maps in autopolyploid species with any ploidy level, including markers with all possible dosages. For doing so, hiddenMarkov model technology was used. The new model presented herein can be applied to dominant and codominantmarkers data, with biallelic or multiallelic behavior. The method is based on the calculation of conditional probabilities that comprise the transition matrix followed by a reduction of its dimension using a computer-based approach. An application of the method was illustrated with a sugarcane mapping population derived from a cross between two pre-commercial varieties (IACSP 95-3018 × IACSP 93-3046), scored with three types of markers: SNPs, microssatellite and AFLPs. The results indicate that the new method is very efficient in obtaining genetic maps, even for high ploidy levels and for markers with high dosages, particularly when these markers have codominant behavior. It was also possible to estimate the likelihood, the recombination fractions and the linkage phases between allmarkers using themultipoint approach, which takes into account all markers of the linkage group simultaneously. The new model proposed in this work represents a major step towards the location of genomic regions associated with variation in quantitative traits and its genetic architecture, and assembling autopoliploid genome sequences.

Approximately 33.4 million people are living with HIV/AIDS. Of those, 97% live in low and middle income countries, with 22.4 million in sub-Saharan Africa. Only 42% of the people who require anti-retrovirals (ARVs) in low to middle income countries are receiving anti-retroviral therapy (ART). There is a need to develop novel and cost effective methods for producing antiretroviral drugs. Stavudine and azidothymidine (AZT) were identified as potential targets because they could both be produced through a common intermediate – 5 methyluridine (5-MU). It has been established that the biocatalytic production of 5-methyluridine is possible through a reaction known as transglycosylation, in a process which has not previously been demonstrated as commercially viable. A selection of biocatalysts were expressed either in recombinant E. coli strains or in the wild type organisms, purified and then screened for their ability to produce 5-MU. A combination of Bacillus halodurans purine nucleoside phosphorylase 1 (BHPNP1) and E. coli uridine phosphorylase (EcUP) gave the highest 5-MU yield (80%). This result represents the first combination of free enzymes from different organisms, giving high yields of 5-MU under high substrate conditions. Both enzymes were purified and successfully characterised. The established pH optimum was pH 7.0 for both enzymes. Temperature optima and stability data for BHPNP1 (70 C and t1/2 at 60 C of 20.8 h) indicated that the biocatalytic step was operating within the capabilities of this enzyme and would operate well at elevated temperatures (up to 60 C). Conversely, the temperature optimum and stability data for EcUP (optimum of 40 C and t1/2 at 60 C of 9.9 h) indicated that the enzyme remained active at 40 C for the duration of a 25 h biotransformation, but at 60 C would only be operating at 20% of its optimum activity and would lose activity rapidly. BHPNP1 and EcUP were used in a bench scale (650 ml) transglycosylation for the production of 5-MU. A 5-MU yield of 79.1% was obtained at this scale with a reactor productivity of 1.37 g.l-1.h-1. Iterative saturation mutagenesis was used to rapidly evolve EcUP for improved thermostability. A moderately high throughput colorimetric method was developed for screening the mutants based on the release of p-nitrophenol upon phosphorolysis of a pyrimidine nucleoside analogue. By screening under 20 000 clones the mutant UPL8 was isolated. The mutant enzyme showed an optimum temperature of 60 C and improved stability at 60 C (t1/2 = 17.3 h). The increase in stability of UPL8 is due to only 2 mutations (Lys235Arg, Gln236Ala). These mutations may have caused an increase in stability due to interactions with other structural units in the protein, stabilization of the entrance to the binding pocket, or by decreasing the flexibility of the α-helix at the N-terminus. Transglycosylation experiments showed that the mutant enzyme UPL8 is a superior catalyst for the production of 5-MU. A 300% increase in reactor productivity was noted when free enzyme preparations of UPL8 was combined with BHPNP1 at 1.5% m.m-1 substrate loading. The high yield of 5-MU (75-80% mol.mol-1) was maintained at 9% m.m-1 substrate loading. A commercially viable productivity of 31 g.l-1.h-1 was thus realised. Further optimisation of the process could produce still higher productivities. Future work in directed evolution of nucleoside phosphorylases is envisaged for improved stability and enhanced substrate range for application to other commercially relevant transglycosylation reactions.

A PEGlação é uma bem sucedida estratégia nano-biotecnológica que envolve a ligação covalente do polietilenoglicol (PEG) a uma droga para melhorar sua farmacocinética, farmacodinâmica e perfil imunológico, e portanto, aumentar seu efeito terapêutico. Atualmente, a PEGlação é usada para modificar proteínas, peptídeos, oligonucleotídeos e fragmentos de anticorpos. A Uricase (EC 1.7.3.3, UC) é uma enzima pertencente à classe das oxidorredutases, responsável pela oxidação do ácido úrico, produzindo alantoína. Essa enzima é encontrada em muitos organismos vivos como: bactérias, leveduras, fungos, vegetais e animais. Entretanto, durante a evolução das espécies o gene da UC tornou-se inativo, por isso, em humanos a UC é inativa. Nesse sentido, a UC adquiriu destaque como um potencial fármaco uricolítico, devido à necessidade do desenvolvimento de novos agentes terapêuticos no tratamento de hiperuricemia e gota. Neste estudo, a uricase recombinante purificada de Candida sp (UC-r) e a de rim bovino (UC-b) foram modificadas por PEGlação com mPEG-p-nitrofenil carbonato (mPEG-pNP) e 2-O-mPEG-4,6-dicloro-s-triazina (mPEG-CN), produzindo conjugados com considerável atividade enzimática residual UC-r-mPEG-pNP (87%), UC-r-mPEG-CN (75%) e UC-b-mPEG-pNP (75%), UC-b-mPEG-CN (50%).Além disso, os conjugados obtidos com a UC-r e UC-b apresentaram valores de KM menores do que as enzimas nativas, indicando que a PEGlação conferiu uma interessante propriedade aos conjugados, que permitiu um aumento da afinidade da UC-r e UC-b pelo ácido úrico. O efeito do pH e da temperatura sobre a UC-r e UC-b modificadas indicou que os conjugados obtidos foram mais ativos em pH próximo ao fisiológico e mais estáveis do que a respectiva enzima nativa. As formas PEGladas da UC-r e UC-b foram mais resistentes à ação de diferentes proteases e mantiveram-se estáveis em soro humano, indicando que a PEGlação favoreceu a resistência a degradação proteolítica. Análises espectroscópicas de dicroísmo circular (CD) e infravermelho (FTIR) não apresentaram nenhuma diferença relevante entre a estrutura protéica da UC-r nativa e PEGlada. Estudos in vivo com coelho e camundongos Balb/c mostraram que a UC-r nativa induziu uma intensa resposta imune sendo altamente imunogênica. Por outro lado, a UC-r PEGlada quando injetada cronicamente em camundongos não induziu qualquer resposta detectável de anticorpos. Esses resultados indicam uma suficiente redução da imunogenicidade dessa enzima, devido à conjugação do mPEG-pNP ou mPEG-CN, tornando-a adequada para um possível uso terapêutico. Portanto, nesse trabalho, os resultados obtidos com a UC-r de Candida sp, mostram que dois conjugados apresentaram interessantes propriedades físico-química, biológicas e imunológicas, que permitiram um significativo avanço na transformação de uma enzima de origem fúngica em uma droga, com uma possível aplicação terapêutica no tratamento de hiperuricemia e gota. / PEGylation is a successful nanobiotechnology strategy that involves the covalent attachment of polyethylene glycol (PEG) to a drug to improve its pharmacokinetic, pharmacodynamic, and immunological profiles, and thus, enhance its therapeutic effect. Currently, PEGylation is used to modify proteins, peptides, oligonucleotides, antibody fragments, and small organic molecules. Uricase (EC 1.7.3.3, UC) is an enzyme belonging to the class of oxidorreductases responsible for the oxidation of uric acid, producing allantoin. This enzyme is found in many living organisms such as bacteria, yeasts, fungi, plants and animals. However, during the evolution of the species gene became inactive UC, therefore, in humans UC is inactive. Accordingly, UC has acquired prominence as a potential drug uricolytic due to the need of developing new therapeutic agents for the treatment of hyperuricemia and gout. In this study, purified recombinant uricase from Candida sp (UC-r) and ox kidney (UC-b) were modified by PEGylation with mPEG-p-nitrophenyl-carbonate (mPEG-pNP) and 2-O-mPEG-4,6-dichloro-s-triazine (mPEG-CN), producing conjugates with considerable residual enzyme activity UC-r-mPEG-pNP (87%), UC-r-mPEG-CN (75%) and UC-b-mPEG-pNP (75%),UC-b-mPEG-CN (50%). In addition, conjugates obtained with the UC-r and UC-b had lower KM values than native enzymes, indicating that the PEGylation gave an interesting property the conjugate that increased the affinity of UC-r and UC-b by uric acid. The effect of pH and temperature on the modified UC-r and UC-b indicated that the conjugates were more active at pH close to the physiological and more stable than its native enzyme. PEGylated forms of UC-r and UC-b were more resistant to the action of different proteases and remained stable in human serum, indicating that the PEGylation favored resistance to proteolytic degradation. Spectroscopic analysis of circular dichroism (CD) and infrared (FTIR) did not show any relevant difference in protein structure between native and PEGylated UC-r. In vivo studies with rabbit and Balb/c mice showed that UC-r native elicited an intense immune response being highly immunogenic. On the other hand, the PEGlated UC-r when chronically injected into mice did not induce any detectable response to antibodies. These results indicate a sufficient reduction of immunogenicity of this enzyme, due to conjugation of mPEG-pNP or mPEG-CN, making it suitable for possible therapeutic use. Therefore, the results obtained with the UC-r of Candida sp, showed that two conjugates have interesting physical-chemical, biological and immunological, which allowed a significant advance in the transformation of an enzyme of fungal origin in a drug with a possible application therapeutic in the treatment of hyperuricemia and gout.