No description available.

A peptide nucleic acid (PNA) encoded 1296 member peptide library was synthesised and incubated with a variety of cell types. Library members entering cells were extracted, hybridised onto DNA microarrays and the peptide identity was determined via deconvolution. Global consensus analysis highlighted the tetrapepide, Glu-Llp- Glu-Glu (Llp is 6-hexamine-N-aminoacetic acid), a surprise in view of the basic residues typically observed in cell penetrating peptides. When evaluated, Glu-Llp- Glu-Glu revealed cellular uptake comparable to a known basic peptide (tetraLlp). In depth delineation via clustering analysis allowed assessment of differential cellular uptake, with the identified peptides showing clear cellular specificity. This was verified by peptide synthesis and cellular uptake analysis by flow-cytometry, and in all cases an endocytic uptake mechanism was confirmed. This approach establishes a strategy for the identification of short peptides as tools for selective delivery into specific cell types. The incubation of a 10,000 member PNA-encoded peptide library with D54 and HEK293T transfected with CCR6 cells followed by microarray analysis allowed detailed information on the interaction between peptide-ligands and cell surface receptors to be extracted. This allowed the identification of new ligands for integrins and G-protein coupled receptors and offers a novel approach to ligand discovery allowing the comparative analysis of different cell types for the identification of differences in surface-receptor ligands and/or receptor expression between various cell types. In addition, this work included the development of a novel method for the indirect amplification of a PNA library by amplification of a complementary DNA library hybridised to the PNA. The generation of 10,000 defined pieces of DNA would have a myriad of applications, not least in the area of defined or directed sequencing and synthetic biology, but also in applications associated with encoding and tagging. By this approach DNA microarrays were used to allow the linear amplification of immobilised DNA sequences on an array followed by PCR amplification. Arrays of increasing sophistication (1; 10; 3875; 10,000 defined oligonucleotides) were used to validate the process, with sequences verified by selective hybridisation to a complementary DNA microarray with DNA sequencing demonstrating error rates of ca ≈ 0.2%. This technique offers an economical and efficient way of producing hundreds to thousands of specific DNA primers, while the DNA-arrays can be used as “factories” allowing specific DNA oligonucleotide pools to be generated with or without masking. This study also demonstrated a significant variance observed between the sequence frequencies found via Solexa sequencing compared to microarray analysis.

Aberrant expression of short non-coding micro RNAs (miRNA) in many human diseases, along with remarkable stability in physiological media, has made them attractive clinical biomarkers. In particular, miRNA-122 is substantially elevated in plasma of patients with established drug-induced liver injury and can also be used to identify early liver injury when current markers, such as alanine aminotransferase (ALT), still show normal levels. The development of a rapid test for miRNA-122 e.g. in drug poisoning would allow earlier and more sensitive clinical diagnosis of liver injury. Nucleic acids are traditionally analysed by polymerase chain reaction (PCR), which has a high degree of sensitivity but suffers from high cost and is prone to sample contamination. The aim of this project is to develop a PCR free method to directly detect miRNA- 122 in biological samples using SMART technology. The SMART technology takes advantage of dynamic chemistry for sequence specific recognition of nucleic acids using aldehyde-modified nucleobases (SMART nucleobases), and target-complementary peptide nucleic acid (PNA) probe containing an “abasic” position (so called modified PNA probe). In this study, this unique detection method was used in a fluorescent detection with the use of light up probes, which are probes with an environmental dye as nucleobase; a FRET system was also designed to allow the discrimination between perfect match target and mismatched one. The SMART technology was also transferred onto magnetic beads to develop an ELISA like assay allowing sensitive and rapid detection of single stranded DNA mimic of the miRNA-122. With its potential PCR free approach, this easily adapted platform promises to transform and expand routine clinical diagnostic testing and screening for circulating miRNAs.

La découverte de petites molécules capables de moduler les systèmes biologiques présente un intérêt majeur dans l'étude des mécanismes cellulaires et la mise au point de nouvelles méthodes thérapeutiques. Bien que les techniques de criblage haut débit soient régulièrement utilisées, il y a un vrai besoin de réduire le coût et le temps associés à la découverte de ligands, afin de valider la fonction de nombreuses cibles potentielles dans notre protéome ou ceux d' organismes pathogènes. A cette fm, l'émergence de technologies basées sur l'encodage de chimiothèques par des acides nucléique offre une alternative répondant à ces critères. Nous avons développé un système permettant une synthèse rapide de bibliothèques de structures variées,conjuguées à des codes PNA (acide peptidique nucléique) uniques, ainsi qu'une technologie de criblage basée sur la sélection par affinité, qui permet une étude rapide de l'interaction avec une protéine-cible et par conséquent l'identification de nouveaux ligands. Plusieurs chimiothèques ont déjà été synthétisées et criblées, et compte tenu de la stabilité chimique remarquable du PNA, nous avons également développé une nouvelle gamme de réactions compatibles avec la synthèse PNA-encodée, la voie étant maintenant ouverte pour la génération de chimiothèques plus complexes et pour l'étude de cibles biologiques très variées. / The discovery of srnall molecules capable of modulating biological systems is of major interest for the understanding of cellular mechanisms as weil as for the drug discovery process. In spite of established high throughput techniques routinely used, there is a clear need to reduce the time and cost •associated to ligand discovery, in order to validate the function of numerous potential targets in our proteome or the one of pathogens. In this perspective, the emergence of technologies based on nucleic acid encoding of chemical libraries presents an alternative that fulfills these criteria. We have developed a system enabling the rapid synthesis of libraries containing various structures, conjugated to unique PNA (peptide nucleic acid) tags, a weil as a screening technique based on affinity selection that allows for the rapid study of the interaction witb a target protein and the consequent identification of new ligands. Several libraries have already been synthesized and screened, and based on the remarkable chemical stability of PNA, we have also developed a new palette of reactions compatible with PNA-encoded synthesis, the path now being open for the generation of more complex libraries, and the study of various biological targets

The cell membrane is an impermeable barrier for most macromolecules. Recently discovered cell-penetrating peptides (CPPs) have gained lot of attention because they can cross the membrane, and even more, carry cargoes with them. How CPPs enter cells is still not clear, while the delivery of different cargoes has been convincingly shown. This thesis concentrates on evaluating CPPs as vectors for different biologically relevant cargoes. Proposed internalisation mechanisms are reviewed as well as cargo coupling strategies. Biological activities of antisense oligonucleotides delivered by CPPs have been of particular interest and are explained in greater details. A new CPP, pIsl, was derived from Islet-1 transcription factor, and compared to archetypical CPPs like penetratin and transportan. All three peptides resided in the headgroup region of lipid bilayers in model membranes. However, penetratin and pIsl did only interact with negatively charged membranes, while transportan did not distinguish negatively charged and neutral membranes. This suggests different translocation pathways for different CPPs. Biotinylated pIsl and penetratin were complexed with avidin, and uptake of avidin into the human melanoma cell line Bowes was observed in both cases. This means that the protein is not unfolded during the translocation process, which is important in delivery of other, biologically active proteins. Transportan and its analogue TP10 were used for peptide nucleic acid (PNA) antisense oligonucleotide delivery. First, eight human galanin receptor type 1 targeting PNA oligomers were designed, conjugated to transportan and assayed for antisense efficiency. In contrary to avidin-biotinylated peptide conjugate, a covalent bond between PNA oligomers and the transport peptide was necessary for cellular uptake of oligomers. A common problem in antisense technology is inactivity of antisense oligonucleotides due to the secondary structure of the target. Efficiencies of tested galanin receptor type 1 targeting PNA oligomers varied over two orders of magnitude. The most efficient oligomers were targeting coding sequence regions 24-38 and 27-38, and had EC50 values 70 and 80 nM, respectively. TP10-antisense PNA oligomer conjugates were targeted also to L-type voltage dependent Ca2+ channel subunits CaV1.2 and CaV1.3. Specific down-regulation of respective proteins was demonstrated by immunohistochemistry. Physiological response to the down-regulation of either of Ca2+ channels was studied by alteration of flexor reflex sensitisation. Rats treated with either of the antisense PNA, but not with scrambled PNA lost the action potential windup phenomenon. In conjunction with a variety of drugs, modulating the conductivity and excitability of neuronal membranes, a central role of L-type CaV channels in sensitisation was confirmed. Nevertheless, also N-methyl-D-aspartate and glycine receptors were found to be required. Finally, delivery of plasmids by TP10 was evaluated. In contrary to many similar CPPs, TP10 was incapable to translocate plasmids to cells. However, addition of TP10 or a TP10-PNA conjugate to polyethyleneimine-condensed plasmids increased the expression of reporter genes. In summary, different types of cargoes have been delivered by CPPs and different cargo coupling strategies have been used. CPP-mediated antisense oligonucleotide delivery has been used to identify accessible sites in human galanin receptor type 1 mRNA and to determine the role of L-type voltage dependent Ca2+ channels in axon potential windup.

Extended-spectrum β-lactamases (ESBLs) constitute a major problem given their broad substrate specificity and ability to hydrolyse many of the extended-spectrum third-generation cephalosporins currently in use in hospital settings. Guiana extended-spectrum-type (GES-1 – GES-9) ESBL enzymes have mainly been found in Pseudomonas aeruginosa (P. aeruginosa) and only at a limited number of geographical sites, mainly France, Greece and South Africa. Detection of GES-type ESBL-producing P. aeruginosa isolates in the clinical microbiology laboratory using conventional methods is problematic with molecular methods yielding better results. The aim of this study was to utilise various molecular techniques to determine the prevalence of GES-type ESBLs, characterise their genetic determinants and determine their clonal relatedness. The study further aimed to apply a sequence-selective, competitive PNA-based multiplex PCR in real-time for the identification and differentiation of GES-type enzymes. The prevalence of GES-type ESBLs was determined successfully through DNA sequencing. An increase in GES-2 prevalence since 2000 was noted which emphasised the importance of constant surveillance to monitor antibiotic determinants, their spread and overall prevalence. The knowledge on prevalence could be used in turn to monitor the efficacy of infection control measures and antibiotic regimens. Repeated sequencing confirmed the presence of blaGES-5 in P. aeruginosa isolates. As far as could be established, this study reported the first occurrence of GES-5 in South Africa and was the second description of GES-5 in P. aeruginosa. Application of a sequence-specific, competitive PNA-based multiplex PCR in real-time utilising SYBR Green was not suitable for the identification and differentiation of the blaGES genes. Although the method achieved different melting temperatures for the bla<GES genes tested, these temperatures were not suitable for accurate differentiation. Melting temperatures obtained for the same blaGES gene varied and those for different genes overlapped. An approach exploiting the high temperature shift caused by the PNA-probe rather than its competitive nature might be more successful. Random amplified polymorphic DNA typing has been described as a fast and simple method with high discriminatory power for the typing of P. aeruginosa and was thus used to determine the clonal relatedness of the bla<GES positive P. aeruginosa isolates. The occurrence of identical or similar P. aeruginosa isolates producing ESBLs in a single hospital setting emphasised the importance of constant surveillance. The study further identified identical P. aeruginosa clones that occurred in different hospitals indicating spread from a common external reservoir into these hospitals. The occurrence of highly drug-resistant P. aeruginosa in the environment has serious implications in a country with an ever increasing immune-compromised population. These finding were of concern since they demonstrated that acquired GES ESBLs can rapidly emerge and become a major cause of broad-spectrum β-lactam resistance among nosocomial pathogens. The information obtained in this study should be used to create awareness of the potential ESBL problem threatening current antimicrobial regimens in South Africa. / Dissertation (MSc (Medical Microbiology))--University of Pretoria, 2008. / Medical Microbiology / unrestricted

Extended-spectrum beta-lactamases (ESBLs) in Pseudomonas aeruginosa tend to be geographically scattered, such as GES-2, which partially compromises the efficacy of imipenem. The G170N mutation, ascribed to a CC to AA base pair substitution on positions 493-494 of the blaGES-2 coding region, distinguishes this ESBL from blaGES-1 and the blaIBC-type genes, making it an ideal target for developing a novel sequence-specific, peptide nucleic acid (PNA)-based, multiplex-PCR detection method. Utilizing two primer pairs in conjunction with a PNA probe, this novel method delivered accurate identification of blaGES-2 compared to standard PCR and gene sequencing techniques, when tested against one hundred (n = 100) P. aeruginosa clinical isolates as well as previously published, well-described control strains. This method has the potential to be used in large-scale, cost-effective screening programmes for specific or geographically restricted ESBLs. To date, in addition to being only described in South Africa, GES-2 is notoriously difficult to identify in P. aeruginosa, using standard methodology. A real-time PCR method using the LightCycler™ was compared to a two-step nested-PCR assay for the detection of blaGES and blaIBC genes from one hundred P. aeruginosa clinical isolates collected over a four-year period from two teaching hospitals in Pretoria, South Africa. Real-time PCR amplification was monitored through hybridisation of fluorescently labelled probes followed by melting curve analysis to detect the relevant G170N mutation occurring in the omega loop region of blaGES-2. Nested-PCR products were subjected to automated DNA sequencing and compared to melting point (Tm) analyses results obtained from the LightCycler assay. Real time and nested-PCR assays detected a blaIBC gene product from 83 and 88 clinical isolates respectively, with the LightCycler thus exhibiting a sensitivity of 94.3% compared to the nested-PCR assay. Comparison of Tm and gene sequencing data however revealed 100% specificity for sequence specific detection of blaGES-2 with the LightCycler. One clinical isolate was found to harbour a blaGES-1 gene, making this the first report of this specific ESBL from South Africa. Selective antibiotic pressure has recently been implicated as a possible driving force behind point mutations observed in blaGES–type genes. This part of the study subjected two well-characterized clinical isolates with class 1 integron-borne blaGES-type genes to five days incubation in the presence of sub-inhibitory concentrations of 15 different antibiotics, including beta-lactams, aminoglycosides and quinolones. Restriction enzyme analysis and DNA sequencing of blaGES-1, blaGES-2 and their immediate upstream genetic environments failed to demonstrate any changes compared to non-exposed controls. Short-term exposure to a sub-inhibitory level of a single antimicrobial agent is thus unlikely to select significant mutations in these beta-lactamase genes or their regulatory mechanisms. / Thesis (PhD (Medical Microbiology))--University of Pretoria, 2004. / Medical Microbiology / unrestricted

No description available.

Investigating novel treatment approaches to combat Clostridioides difficile Rusha Pal ABSTRACT Clostridioides difficile is the leading cause of antibiotic-induced diarrhea and colitis in hospitals and communities worldwide. The enteric pathogen, classified to be an "urgent threat" by the United States Center for Disease Control and Prevention (CDC), capitalizes on disrupted intestinal microbiome to establish infection with disease symptoms ranging from mild diarrhea to potentially fatal conditions. Disruption of the intestinal microbiome, caused mostly by antibiotic use, enables C. difficile to colonize and proliferate within the host. Paradoxically, antibiotics are used to treat C. difficile infection. These antibiotics decimate the gut microbial community further, thus priming the gastrointestinal tract to become more prone to recurrence of infection. To tackle this clinical setback, we utilized a combination of traditional and non-traditional drug discovery approaches and identified chemical entities and targeted treatment options effective against this toxin-producing intestinal pathogen. Herein, we exploited the strategy of high-throughput screening to identify leads that harbor anticlostridial activity. Our primary phenotypic screen of FDA-approved drugs and natural product libraries led to the identification of novel molecules that were further characterized for their anticlostridial efficacy both in vitro and in vivo. The most potent scaffolds identified were those of mitomycin C, mithramycin A, aureomycin, NP-003875, NAT13-338148, NAT18-355531, and NAT18-355768. Of these, mithramycin A, aureomycin, and NP-003875 were also found to harbor anti-virulence properties as they inhibited toxin production by the pathogen. Furthermore, natural product NP-003875 could confer protection to 100% of the infected mice from clinical manifestations of the disease in a primary infection model of C. difficile. Our final approach has been to develop targeted therapeutics called peptide nucleic acids (PNAs). PNAs are antisense agents capable of inhibiting gene expression in bacteria. In this study, antisense inhibition of the RNA polymerase  subunit gene (rpoA) of C. difficile was found to be bactericidal for the pathogen and could also inhibit the expression of its virulence factors. Additionally, antisense inhibition of the C. difficile rpoA gene was found to be non-deleterious for the tested commensal microflora strains. Given their intriguing anticlostridial properties, it can be concluded that our research opened exciting possibilities that can be further evaluated to uncover new treatments for CDI. / Doctor of Philosophy / Investigating novel treatment approaches to combat Clostridioides difficile Rusha Pal LAYMAN LANGUAGE ABSTRACT Clostridioides difficile is a prominent human pathogen that can colonize the gut and cause fatal infections. C. difficile is the most common cause of microbial healthcare-associated infection and results in substantial morbidity and mortality. The "most urgent worldwide public health threat" label has been assigned to C. difficile by the United States Centers for Disease Control and Prevention (CDC). There is a pressing need to develop new classes of antibiotics with improved efficacy to treat C. difficile infections (CDI). To address the need for novel strategies to combat the growing problem of CDI, we screened FDA-approved drugs and natural products library in search of novel drugs that possess potent and specific anticlostridial activity. Several promising hits were identified and evaluated successfully both in vitro and in vivo. The most potent and novel hits that displayed exceptional activity were mitomycin C, mithramycin A, aureomycin, NP-003875, NAT13-338148, NAT18-355531, and NAT18-355768. Furthermore, a murine model of C. difficile infection revealed that compound NP-003875 conferred 100% protection to the infected mice from clinical manifestations of the disease. Interestingly, these compounds were non-toxic to the gut microflora and human cells. Our final approach has been to develop non-traditional therapeutics to target specific genes in C. difficile. These novel therapeutics are called peptide nucleic acids (PNA). Herein, we designed a PNA targeting RNA polymerase  subunit gene (rpoA) of C. difficile. The designed PNA could successfully inhibit the growth of the pathogen and expression of its virulence factors. In conclusion, our research opened exciting possibilities that can be further evaluated to uncover new treatments for CDI.

Développement et évaluation de nouvelles stratégies pour le traitement des hépatites B chroniques, dans le modèle du canard de Pékin infecté par le DHBVL’infection chronique par le HBV est la cause majeure de cirrhose hépatique et de carcinome hépatocellulaire, conduisant à plus d’un million de décès chaque année. Le faible taux de réussite des thérapies actuelles des hépatites B montre la nécessité du recours à des méthodes thérapeutiques alternatives. Ainsi, nous avons étudié une stratégie pertinente reposant sur l’utilisation de molécules antisens (PNAs) couplées à des peptides perméabilisants (CPPs). Nous avons démontré que les PNAs ciblant le signal d’encapsidation du DHBV couplés au CPP pénétraient dans les cellules et conduisait à une inhibition de la réplication virale. De plus, nous avons mis en évidence une activité antivirale du CPP (Arg)8 seul. Nous avons ensuite évaluer le mécanisme d’action antivirale du CPP in vitro et avons démontré qu’il inhibait les stades tardifs de la morphogénèse virale, conduisant à une inhibition forte de la sécrétion des particules virales. Par ailleurs, nous nous sommes intéressés à l’évaluation de stratégies immunothérapeutiques, reposant sur la vaccination génétique. Nous avons démontré les bénéfices de la co-administration de cytokines (IFNγ), avec un vaccin à ADN dirigé contre la grande protéine d’enveloppe du DHBV (preS/S), sur l’amplitude de la réponse humorale et sur le pouvoir neutralisant des anticorps induits. Enfin nous avons évalué les bénéfices d’une approche d’immunisation hétérologue « prime-boost » associant l’immunisation à ADN et un vecteur viral (AdénoCELO) recombinant, codant la protéine preS/S du DHBV et l’IFNγ. Nous avons montré que l’immunisation hétérologue induisait une réponse humorale plus forte que celle induite par l’immunisation homologue. / Development and evaluation of new strategies for treating chronic hepatitis B in the model of Peking duck infected with DHBVChronic infection with Hepatitis B virus (HBV) is the major cause of liver cirrhosis and hepatocellular carcinoma, leading to more than one million deaths each year. The low success rate of current therapies against HBV infection shows the need of alternative therapeutics. Thus, we studied a new strategy based on the use of antisense molecules (PNAs) coupled with cell penetrating peptides (CPPs). We have shown that PNAs targeting the DHBV encapsidation signal coupled to CPPs penetrated into the cells and led to an inhibition of viral replication. In addition, we have demonstrated an antiviral activity of the CCP (Arg)8 itself. We then evaluate the mechanism of antiviral action of this CPP in vitro and have shown that it inhibited the late stages of viral morphogenesis, leading to a strong inhibition of the release of viral particles. Furthermore, we were interested in evaluating immunotherapeutic strategies, based on DNA vaccination. We have demonstrated the benefits of co-administration of cytokines (IFNy), with a DNA vaccine directed against the DHBV large envelope protein (preS/S), enhancing the magnitude of humoral response and enhancing neutralizing anti-DHBV antibody response. Finally we evaluated the benefits of a heterologous immunization approach or prime-boost immunization involving DNA vaccination and a recombinant viral vector (AdenoCELO) encoding the DHBV preS/S and IFNy proteins. We have shown that heterologous immunization induced a humoral response stronger than that induced by homologous immunization. By contrast, the heterologous prime-boost strategy was less effective than homologous DNA immunization for therapy of chronic DHBV-carrier ducks.