Enteroviruses affect a broad segment of the population throughout the world and have been suspected to play a major role in waterborne disease for quite some time. The presence of these viruses in drinking water supplies constitutes a major health risk to the population because of their low infectious dose. The monitoring and study of these viruses in the environment have been limited by the current standard detection methodologies. Nucleic acid probe hybridization is a new and effective approach for the study and detection of these viruses in the environment. An important step in the detection of viruses in concentrated water samples by nucleic acid probes is the isolation of the viral genome from the water sample for hybridization. Previously, a series of time consuming organic extract ions was used to isolate viral RNA. This study reports the development of an alternative method for the isolation and preservation of viral RNA in environmental samples. Briefly, the sample is heated in the presence of an RNase inhibitor, and then applied to a hybridization membrane. This procedure has greatly reduced the time and difficulty of the assay while maintaining sensitivity and increasing consistency. This study reports the development and modification of a nonradioactive labeling system for the detection of viruses in water. Nonradioactive labels such as biotin offer several advantages over radioactive labels including unlimited shelf life, reduced cost and time of assay, and elimination of the radiation hazard. However, radioactive labels are generally the more sensitive method of detection. By combining direct and indirect labeling strategies, the sensitivity of this nonradioactive assay has been increased ten-fold. This assay can detect as little as 100 plaque forming units of poliovirus, only one order of magnitude less sensitive than radiolabeled probes. This assay is also ten-fold less sensitive than radiolabeled probes for the detection of enteroviruses in water samples. Nonradioactive probes offer a safe, inexpensive alternative to radiolabeled probes and tissue culture for the detection of viruses in the environment when ultrasensitivity is not required.

Glycine-N-acyltransferase (GLYAT, EC 2.3.1.13) has been characterised in a number of species including: humans, chimpanzees, rhesus monkeys and bovines. The characterisation of GLYAT from various species contributes to a better understanding of the diversity of the enzyme which in turn might help improve the current understanding of detoxification in mammals. The GLYAT enzyme of both the chacma baboon and vervet monkey has not been characterised. In this project, tissue samples were obtained from a chacma baboon (Papio ursinus) and a vervet monkey (Chlorocebus pygerythrus) to determine the nucleic acid sequence that encodes GLYAT in these two species to broaden our current understanding on the diversity of GLYAT in primates. A liver of a chacma baboon was used to extract total RNA. Complementary DNA (cDNA) was synthesised using an oligo (dT) primer. An open reading frame (ORF) encoding GLYAT of the chacma baboon was amplified with a PCR (polymerase chain reaction) using primers designed from a human GLYAT transcript. The PCR product containing an ORF encoding GLYAT of the chacma baboon was cloned, sequenced and expressed. The recombinant GLYAT of the chacma baboon expressed well in bacteria, but was insoluble and did not have enzyme activity. A crude cytoplasmic extract was prepared from the liver of a chacma baboon. The objective was to compare enzyme activity between the native and recombinant GLYAT. The prepared liver extract from the chacma baboon was assayed for enzyme activity and compared to the activity in a liver extract from bovine, previously prepared by Ms M Snyders. Both the chacma baboon and bovine liver extracts had GLYAT enzyme activity. To obtain sequence information on vervet monkey GLYAT, leukocytes were isolated from blood obtained from a living vervet monkey. A human GLYAT gene sequence was used as a reference DNA sequence in the design of PCR primers that were used to amplify the exons of GLYAT of the vervet monkey. All six GLYAT exons were individually amplified and PCR products were sequenced. The sequences were combined to reconstruct an ORF encoding GLYAT of the vervet monkey. The ORFs coding the GLYAT of both chacma baboon and vervet monkey were found to be 888 bp long (excluding stop codon) and encoded a protein of 296 amino acids. A fragment of 1256 bp of the chacma baboon GLYAT transcript was sequenced. The two GLYAT ORF sequences were translated to amino acid sequences and aligned to that of GLYAT of primates obtained from the Ensembl sequence database. The GLYAT amino acid sequences of the chacma baboon, vervet monkey and rhesus monkey formed a related group, distinct from other primates. The chacma baboon and vervet monkey sequences were 99 % identical to the rhesus monkey sequence and 92.6 % identical to the human sequence. There were 4 new variations introduced by GLYAT amino acid sequences from the chacma baboon and the vervet monkey. The vervet monkey introduced an isoleucine in place of a valine at position 32 and an arginine in place of a histidine or glutamine at position 224. The chacma baboon introduced a tyrosine in place of isoleucine at position 201 and an arginine in place of histidine or glutamine at position 240. The knowledge generated in this project will broaden the understanding of GLYAT diversity relating to GLYAT in primates. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011

Glioblastoma multiforme is the most aggressive form of malignant brain tumor with poor prognosis. The efficacy of brain cancer treatment by chemotherapeutics is limited by the blood-brain barrier (BBB) which allows less than 2% of the small molecules and blocks almost all the macromolecules to transport into the brain. Delivery of the large molecules such as proteins and nucleic acids across the BBB is a great challenge for brain-targeted drug delivery. To overcome this obstacle, cell-penetrating peptides (CPPs) were used as vectors for delivery of nucleic acids across the BBB targeting glioblastomas. The CPPs have shown such promising carriers to deliver various cargoes ranging from small molecules to large molecules into the cells. This thesis is focused on the development of glioblastoma-targeting vectors based on modifications of the CPPs and the targeting peptides. The peptide-based vectors were developed to improve the transport of the nucleic acids across the BBB and specifically target glioblastomas. In this thesis, a series of peptide-based vectors targeting glioblastomas were synthesized and modified with targeting peptides by either covalent conjugation or non-covalent complex formation. The delivery of plasmid DNA (pDNA) in the complex with the peptide-based vectors was studied in the in vitro model of the BBB. The role of receptors expressed on the BBB was investigated. Scavenger receptors class A and B were found to be expressed on the BBB, and they were involved in the delivery of the pDNA across the BBB model. Moreover, various targeting peptides were modified with hexaglutamate to form non-covalent complexes with the CPPs for small interfering RNA (siRNA) delivery to glioblastoma cells. The non-covalent complex of the CPP and the targeting peptide showed greater gene-silencing efficiency than the consecutively covalent conjugation of the CPP and the targeting peptide for siRNA delivery to glioblastoma cells. Lastly, a number of novel, amphipathic peptides were developed based on the model amphipathic peptide. The prediction of the biological effect of the designed peptides using quantitative structure-activity relationship model showed a correlation with the experimental data. Finally, the CPP-based nucleic acid delivery vectors with homing peptide strategy have a potential for the BBB shuttle and the future use as a glioblastoma-targeted drug carrier in the in vivo studies and the clinical applications. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Bacteria classified in the family Methylomonadaceae must derive their carbon from one-carbon compounds. They are characterized by the possession of internal membranes of two types. Type I membranes are layered and fill the middle of the cells while type II membranes form concentric layers around the periphery of the cells. Also, there are two metabolic pathways by which the methylobacteria assimilate one-carbon compounds. Further evidence of this dichotomy was sought by DNA-DNA saturation hybridization of DNAs from both types of methylobacteria. Very low DNA-DNA homology was seen between types I and II or within the types. It was not possible, therefore, to correlate the degree of genetic relatedness with either the nature of the internal membranes or the pathway of carbon assimilation.

Although the nucleon acid oscillatory spectra have been studied for a long period of time, many of oscillating modes have not been properly explained regarding their geometric sensitivity and weak binding. One of the possibilities how to acquire experimental data for interpretation of theese modes is to confront oscillating spectra of isotopes. The simplest is proton - deutron exchange. When molecules dissolve in heavy water they rapidly exchange the heteroatoms, but this is simultaneous for more stages, which leads to dynamic equilibrium state among various isotope forms. Statistic analysis of temporal progression of Raman spectra give us the possibility to analyze the spectra and to obtain unique experimental data. This technique is very promising for more complex nucleon acid segments to gain more structural information. This diploma thesis represents pilot study of introduced methodics. The objective is to implement adaptation of Raman spectrometr for given types of expiments and undertake first series of measurements. The results should clarify application bounds of proposed method refering to sensitivity, time constant of incident isotope exchange, severity of read-out in comparasion of achieved accuracy of the measurement.

This dissertation discusses the development of inexpensive, easy-to-use, and field-deployable diagnostic techniques and devices for the early detection of various pathogens, commonly found in clinical samples and contaminated food and water. Infectious diseases account for about 90% of world health problems, killing approximately 14 million people annually, the majority of which reside in developing countries. In 2012, the World Health Organization (WHO) published data on the top 10 causes of death across the globe. Although communicable disease is a prevalent cause of fatality, both low-income and high-income countries, pathogen species and transmission are very different. Nearly 60% of deaths in developing countries are caused by food, water, air or blood-borne pathogens. The most prevalent illnesses are diarrheal disease, malaria, and HIV/AIDS. By contrast, the leading causes of death in developed countries (heart disease, cancer, and stroke) are not communicable and are often preventable. However, there is an increasing need for the development of rapid and accurate methods for pathogen identification in clinical samples, due to the growing prevalence of antibiotic-resistant strains. Incorrect, or unneeded antibiotic therapies result in the evolution of extremely aggressive nosocomial (hospital-acquired) infections, such as methicillin- (MRSA) and vancomycin-resistant Staphylococcus aureus (VRSA). The implementation of rapid, easy to use and cost-effective diagnostics will reduce the frequency of pathogen-related deaths in underdeveloped countries, and improve targeted antibiotic treatment in hospital settings, thus decreasing the potential development of more treatment-resistant "super bugs". This research includes novel techniques utilizing two major sensing modalities: serological (i.e. immunological), and nucleic acid amplification testing (NAATs). We first developed a highly sensitive (limit-of-detection = 100 CFU mL-1) particle immunoassay that takes advantage of elastic and inelastic light scatter phenomena, for optical detection of target antigens. This assay is performed upon a unique nanofibrous substrate that promotes multiplexing on a user-friendly platform. We then developed a novel technique, termed emulsion loop-mediated isothermal amplification (eLAMP), in which the target amplicon is detected in real-time, again utilizing light scattering detection and quantification. Both techniques require no sample pre-treatments, and can be combined with smartphone imaging for detection of targets in under 15 minutes. These methods have the potential to improve the speed and sensitivity of early pathogenic identification, thus leading to a reduction in preventative deaths and a decrease in global economic costs associated with infectious disease in clinical and other settings.

<p>Electrostatic interaction is a strong force that attracts positively and negatively charged molecules to each other. Such an interaction is formed between positively charged polycationic polymers and negatively charged nucleic acids. In this dissertation, the electrostatic attraction between polycationic polymers and nucleic acids is exploited for applications in oral gene delivery and nucleic acid scavenging. An enhanced nanoparticle for oral gene delivery of a human Factor IX (hFIX) plasmid is developed using the polycationic polysaccharide, chitosan (Ch), in combination with protamine sulfate (PS) to treat hemophilia B. For nucleic acid scavenging purposes, the development of an effective nucleic acid scavenging nanofiber platform is described for dampening hyper-inflammation and reducing the formation of biofilms.</p><p>Non-viral gene therapy may be an attractive alternative to chronic protein replacement therapy. Orally administered non-viral gene vectors have been investigated for more than one decade with little progress made beyond the initial studies. Oral administration has many benefits over intravenous injection including patient compliance and overall cost; however, effective oral gene delivery systems remain elusive. To date, only chitosan carriers have demonstrated successful oral gene delivery due to chitosan’s stability via the oral route. In this study, we increase the transfection efficiency of the chitosan gene carrier by adding protamine sulfate to the nanoparticle formulation. The addition of protamine sulfate to the chitosan nanoparticles results in up to 42x higher in vitro transfection efficiency than chitosan nanoparticles without protamine sulfate. Therapeutic levels of hFIX protein are detected after oral delivery of Ch/PS/phFIX nanoparticles in 5/12 mice in vivo, ranging from 3 -132 ng/mL, as compared to levels below 4 ng/mL in 1/12 mice given Ch/phFIX nanoparticles. These results indicate the protamine sulfate enhances the transfection efficiency of chitosan and should be considered as an effective ternary component for applications in oral gene delivery.</p><p>Dying cells release nucleic acids (NA) and NA-complexes that activate the inflammatory pathways of immune cells. Sustained activation of these pathways contributes to chronic inflammation related to autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, and inflammatory bowel disease. Studies have shown that certain soluble, cationic polymers can scavenge extracellular nucleic acids and inhibit RNA-and DNA-mediated activation of Toll-like receptors (TLRs) and inflammation. In this study, the cationic polymers are incorporated onto insoluble nanofibers, enabling local scavenging of negatively charged pro-inflammatory species such as damage-associated molecular pattern (DAMP) molecules in the extracellular space, reducing cytotoxicity related to unwanted internalization of soluble cationic polymers. In vitro data show that electrospun nanofibers grafted with cationic polymers, termed nucleic acid scavenging nanofibers (NASFs), can scavenge nucleic acid-based agonists of TLR 3 and TLR 9 directly from serum and prevent the production of NF-ĸB, an immune system activating transcription factor while also demonstrating low cytotoxicity. NASFs formed from poly (styrene-alt-maleic anhydride) conjugated with 1.8 kDa branched polyethylenimine (bPEI) resulted in randomly aligned fibers with diameters of 486±9 nm. NASFs effectively eliminate the immune stimulating response of NA based agonists CpG (TLR 9) and poly (I:C) (TLR 3) while not affecting the activation caused by the non-nucleic acid TLR agonist pam3CSK4. Results in a more biologically relevant context of doxorubicin-induced cell death in RAW cells demonstrates that NASFs block ~25-40% of NF-ĸβ response in Ramos-Blue cells treated with RAW extracellular debris, ie DAMPs, following doxorubicin treatment. Together, these data demonstrate that the formation of cationic NASFs by a simple, replicable, modular technique is effective and that such NASFs are capable of modulating localized inflammatory responses. </p><p>An understandable way to clinically apply the NASF is as a wound bandage. Chronic wounds are a serious clinical problem that is attributed to an extended period of inflammation as well as the presence of biofilms. An NASF bandage can potentially have two benefits in the treatment of chronic wounds by reducing the inflammation and preventing biofilm formation. NASF can prevent biofilm formation by reducing the NA present in the wound bed, therefore removing large components of what the bacteria use to develop their biofilm matrix, the extracellular polymeric substance, without which the biofilm cannot develop. The NASF described above is used to show the effect of the nucleic acid scavenging technology on in vitro and in vivo biofilm formation of P. aeruginosa, S. aureus, and S. epidermidis biofilms. The in vitro studies demonstrated that the NASFs were able to significantly reduce the biofilm formation in all three bacterial strains. In vivo studies of the NASF on mouse wounds infected with biofilm show that the NASF retain their functionality and are able to scavenge DNA, RNA, and protein from the wound bed. The NASF remove DNA that are maintaining the inflammatory state of the open wound and contributing to the extracellular polymeric substance (EPS), such as mtDNA, and also removing proteins that are required for bacteria/biofilm formation and maintenance such as chaperonin, ribosomal proteins, succinyl CoA-ligase, and polymerases. However, the NASF are not successful at decreasing the wound healing time because their repeated application and removal disrupts the wound bed and removes proteins required for wound healing such as fibronectin, vibronectin, keratin, and plasminogen. Further optimization of NASF treatment duration and potential combination treatments should be tested to reduce the unwanted side effects of increased wound healing time.</p> / Dissertation

Structural and mechanical properties of nucleic acids play a key role in a wide range of biological processes, as well as in the field of nucleic acid nanotechnology. The thesis presents results of several studies focused on modelling these properties. Extensive unrestrained atomic-resolution molecular dynamics (MD) simulations are used to investigate structural dynamics of nucleic acids, and to parametrize their mechanical models. The deformation energy is assumed to be a general quadratic function of suitably chosen internal coordinates. Two types of models are employed which differ in the level of coarse- graining. The first one is based on the description of conformation at the level of individual bases and the second, coarser one is used to study global bending and twisting flexibility. The models are applied to explain mechanical properties of A-tracts in the context of DNA looping and nucleosome positioning, to characterize twist-stretch cou- pled deformations in DNA and RNA, and to predict changes in the properties of damaged DNA that are likely to be relevant for damage recognition and repair. Besides that, we propose a general model of DNA allostery, applied to study the effect of minor groove binding of small ligands and the allosteric coupling between proteins mediated by the DNA. A careful...

O objetivo deste trabalho foi avaliar o efeito dos diferentes níveis de nucleotídeos sobre o desempenho de frangos de corte alimentados com probióticos. Para o trabalho, foi utilizado um delineamento inteiramente casualizado com cinco repetições dentro de cada tratamento. Foram utilizados 1050 frangos machos da linhagem Ross 308 totalizando trinta e cinco aves por boxe. As aves foram criadas até 42 dias de idade que receberam as rações experimentais a base de milho e farelo de soja contendo seis diferentes níveis de nucleotídeos (0; 100; 200; 300; 400 e 500 gramas por tonelada de ração). Os diferentes níveis de nucleotídeos foram utilizados na fase inicial (1 a 21 dias de idade) e crescimento (22 a 35 dias de idade). Durante a fase final (36 a 42 dias de idade) foi fornecido rações sem nucleotídeo para todos os tratamentos. Os resultados experimentais demonstraram que houve melhora linear no desempenho dos frangos de corte no período de 1 a 21 dias de idade, indicando que, quanto maior o nível de nucleotídeos na dieta de frangos de corte, maior foi o peso corporal das aves. A conversão alimentar também é melhorou linearmente no período de 1 a 21 dias de idade à medida que aumentou o nível de nucleotídeos na ração. O peso no período de 35 dias de idade, também teve um comportamento linear, semelhante ao período de 1 a 21 dias, indicando que, quanto maior o nível de nucleotídeos na dieta de frangos de corte, maior o desempenho das aves. Quanto aos níveis plasmáticos de ácido úrico, pôde-se observar efeito quadrático no período de 1 a 21 dias de idade, indicando o valor de 231,59 gramas de nucleotídeos por tonelada de ração, como o melhor, em níveis mínimos de ácido úrico, por outro lado, no período de 35 dias de idade, estima-se o nível de 208,99 g de nucleotídeos por tonelada de ração; como o melhor em níveis mínimos de ácido úrico no sangue. No período final (35 a 42 dias de idade) e período total (1 a 42 dias de idade) não foi possível o observar efeito dos contrastes testados em nenhum dos parâmetros avaliados. / The objective of this experiment was to evaluate the influence of different nucleotides levels in the rations of broilers containing probiotics on response of the birds and its influence on the performance. Birds were allocated in a randomized experimental design with five replications of each treatment. It was used 1,050 chicks of a day of age, males, distributed in 30 experimental boxes with 35 birds each. The chickens were reared from 1 to 42 days of age and the diets contained corn and soybean meal with one of six different nucleotídes levels (0; 100; 200; 300; 400 and 500 grams for ton of ration). The different nucleotides levels were used in the initial phase (1 to 21 days of age) and growth (22 to 35 days of age). During the final phase (36 to 42 days of age) it was supplied rations without nucleotides for all of the treatments. The experimental results demonstrated that there was improvement on broilers performance in the period from 1 to 21 days of age, demonstrating proportionality between nucleotides level in the diet of broilers and body weight of the birds. Feed conversion at 21 days of age was directly proportional to nucleotides level in the diet. Body weight at 35 days of age, also had a linear behavior, similar to the period from 1 to 21 days, indicating that, as higher the nucleotides level in the diet of broilers, higher the acting of the birds. Acid plasmatic uric levels, demonstrated quadratic effect at 21 days of age, indicating 231,59 grams of nucleotides for ton of ration, and at 35 days of age, it was considered the level of 208,99 g of nucleotides for ton of ration. In the final period (35 to 42 days of age) and total period (1 to 42 days of age) it was not demonstrated effect of the contrasts tested in none of the appraised parameters.

Tin-hung Ho. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 137-151. / ACKNOWLEDGEMENT --- p.1 / CONTENT --- p.3 / ABBREVIATIONS --- p.7 / ABSTRACT --- p.8 / Chapter CHAPTER 1 --- INTRODUCTION --- p.10 / Chapter CHAPTER 2 --- LITERATURE REVIEW / Chapter 2.1 --- The cervix and cervical cancer --- p.12 / Chapter 2.2 --- Human papillomaviruses --- p.23 / Chapter 2.3 --- Culture of cancer cells --- p.36 / Chapter 2.4 --- Methods for the detection of HPV infection --- p.40 / Chapter CHAPTER 3 --- MATERIALS AND METHODS / Chapter 3.1 --- Characterization of cervical carcinoma cell lines / Chapter 3.1.1 --- Materials and solutions --- p.47 / Chapter 3.1.2 --- Establishment of cervical carcinoma cell lines --- p.50 / Chapter 3.1.3 --- Morphological studies of cervical carcinoma cells --- p.52 / Chapter 3.1.4 --- Examination of cervical carcinoma cells cultured on collagen gel --- p.54 / Chapter 3.1.5 --- Growth kinetics study --- p.55 / Chapter 3.1.6 --- Plating efficiency test --- p.56 / Chapter 3.1.7 --- Spheroid formation assay --- p.56 / Chapter 3.1.8 --- Chromosome number study --- p.57 / Chapter 3.2 --- Immunocytochemical studies / Chapter 3.2.1 --- Materials and solutions --- p.58 / Chapter 3.2.2 --- Immunocytochemical test for keratin --- p.59 / Chapter 3.2.3 --- Test for HPV capsid antigens --- p.60 / Chapter 3.3 --- Molecular studies of HPV in cervical carcinoma cells / Chapter 3.3.1 --- Materials and solutions --- p.62 / Chapter 3.3.2 --- Preparation of HPV DNA probes --- p.66 / Chapter 3.3.3 --- DNA extraction from the cervical carcinoma cells --- p.74 / Chapter 3.3.4 --- Detection of HPV DNA sequences by the use of hybridization techniques --- p.76 / Chapter 3.3.5 --- Copy number and physical state studies of HPV --- p.81 / Chapter 3.3.6 --- Study of the transcriptional activity of HPV DNA in cultured cervical carcinoma cells --- p.83 / Chapter CHAPTER 4 --- RESULTS / Chapter 4.1 --- Characterization of cervical carcinoma cell lines / Chapter 4.1.1 --- Morphological studies --- p.89 / Chapter 4.1.2 --- Examination of cervical carcinoma cells cultured on collagen gel --- p.90 / Chapter 4.1.3 --- Growth kinetics study --- p.93 / Chapter 4.1.4 --- Plating efficiency test --- p.94 / Chapter 4.1.5 --- Spheroid formation assay --- p.95 / Chapter 4.1.6 --- Chromosome number study --- p.98 / Chapter 4.2 --- Immunocytochemical studies / Chapter 4.2.1 --- Immunocytochemical test for keratin --- p.99 / Chapter 4.2.2 --- Test for HPV capsid antigen --- p.99 / Chapter 4.3 --- Molecular studies of HPV in cervical carcinoma cell lines / Chapter 4.3.1 --- Preparation of HPV DNA probes --- p.101 / Chapter 4.3.2 --- Detection of HPV DNA by the use of hybridization techniques --- p.102 / Chapter 4.3.3 --- Copy number and physical state studies --- p.105 / Chapter 4.3.4 --- Analysis of the transcriptional activity --- p.108 / Chapter CHAPTER 5 --- DISCUSSIONS / Chapter 5.1 --- Characterization of cervical carcinoma cell lines / Chapter 5.1.1 --- Morphological features of two cervical carcinoma cell lines --- p.110 / Chapter 5.1.2 --- Other characteristics of the cell lines --- p.112 / Chapter 5.2 --- Immunocytochemical studies / Chapter 5.2.1 --- Test for keratin antigens --- p.117 / Chapter 5.2.2 --- Test for HPV capsid antigens --- p.117 / Chapter 5.3 --- Molecular studies of HPV in cervical carcinoma cell lines / Chapter 5.3.1 --- Establishment of methods --- p.121 / Chapter 5.3.2 --- Detection of HPV DNA sequences by nucleic acid hybridizations --- p.123 / Chapter 5.3.3 --- Copy number and physical state studies --- p.128 / Chapter 5.3.4 --- Transcriptional analysis of HPV DNA in cervical carcinoma cell lines --- p.132 / CONCLUSION --- p.134 / REFERENCES --- p.137 / ILLUSTRATIONS --- p.152