Dissertations / Theses on the topic 'Deoxyribonucleic acid (DNA)'

DNA analysis is now a sufficiently sensitive technique to enable identification of an individual from an extremely small amount of biological material. Exhibits are routinely submitted to forensic laboratories for recovery and analysis of ‘touch DNA’, in order to link an offender to the crime scene. One such exhibit type is spent cartridge cases, where DNA transferred from the handler to the exterior surface of the casing may be the only evidence available for identification of the handler. Alternatively the firearm itself may be recovered, which could also have potential for uncovering the identity of the shooter by means of ‘touch DNA’ profiling. However, the analysis of minute amounts of DNA introduces additional interpretational challenges. The ability to identify the source of a low level DNA sample and the relevance of a recovered DNA profile to the crime scene are not comprehensively understood. The variations in DNA deposition, recovery, transfer and persistence were examined, through a series of controlled laboratory experiments. Volunteers were asked to take part in DNA deposition studies that involved handling items for set periods of time, to determine the variability in the quality of DNA deposited. They were also asked to take part in handshaking studies, where the persistence of DNA, as well as the primary and secondary transfer of DNA, was studied. Additional variables were considered in relation to DNA recovered from spent cartridge cases, including the effect of firing and gunshot residue on DNA quality. DNA was extracted using QIAamp® DNA Mini Kit (Qiagen) and Chelex® (Bio-Rad) protocols and amplified with the AmpFlSTR® SGM Plus® Kit and the AmpFlSTR® Identifiler® Kit (both Applied Biosystems). DNA profiles were analysed on the ABI PRISM™ 310 Genetic Analyser and the ABI PRISM™ 3500 Genetic Analyser (both Applied Biosystems). It was possible to recover a usable DNA profile from a handled item and the quality of DNA deposited after repeated contacts was comparable. The quality of DNA recovered from ‘touch DNA’ samples from different individuals varied, and specific methods for recovery based on surface type were found to increase the likelihood of generating a successful DNA profile. Where an item was handled by more than one individual, the major contributor to the profile was not always that of the final handler. Furthermore, secondary transfer of DNA was observed to some degree in every test sample. This research also highlighted the challenges of interpreting mixed profiles, especially with low levels of DNA present. Identification of the handler of a spent cartridge case was not possible using DNA profiling techniques, due to the increased DNA degradation as a result of conditions experienced during the firing process. However, where a higher yield of DNA was present prior to firing, there was the possibility of recovering an interpretable DNA profile from this type of evidence. The findings of this research should be considered when submitting items for DNA analysis, when considering best practice for recovery of ‘touch DNA’ samples and when attempting to interpret ‘touch DNA’ evidence profiles.

The fact that loosely bonded DNA bases could tolerate large structural fluctuations, form a dissipative environment for a charge traveling through the DNA. Nonlinear stochastic nature of structural fluctuations facilitates rich charge dynamics in DNA. We study the complex charge dynamics by solving a nonlinear, stochastic, coupled system of differential equations. Charge transfer between donor and acceptor in DNA occurs via different mechanisms depending on the distance between donor and acceptor. It changes from tunneling regime to a polaron assisted hopping regime depending on the donor-acceptor separation. Also we found that charge transport strongly depends on the feasibility of polaron formation. Hence it has complex dependence on temperature and charge-vibrations coupling strength. Mismatched base pairs, such as different conformations of the G・A mispair, cause only minor structural changes in the host DNA molecule, thereby making mispair recognition an arduous task. Electron transport in DNA that depends strongly on the hopping transfer integrals between the nearest base pairs, which in turn are affected by the presence of a mispair, might be an attractive approach in this regard. I report here on our investigations, via the I –V characteristics, of the effect of a mispair on the electrical properties of homogeneous and generic DNA molecules. The I –V characteristics of DNA were studied numerically within the double-stranded tight-binding model. The parameters of the tight-binding model, such as the transfer integrals and on-site energies, are determined from first-principles calculations. The changes in electrical current through the DNA chain due to the presence of a mispair depend on the conformation of the G・A mispair and are appreciable for DNA consisting of up to 90 base pairs. For homogeneous DNA sequences the current through DNA is suppressed and the strongest suppression is realized for the G(anti)・A(syn) conformation of the G・A mispair. For inhomogeneous (generic) DNA molecules, the mispair result can be either suppression or an enhancement of the current, depending on the type of mispairs and actual DNA sequence.

The diagnostic value of extracellular occurring DNA (eoDNA) is limited by our lack of understanding its biological function. eoDNA exists in a number of forms, namely vesicle bound DNA, histone/DNA complexes or nucleosomes and virtosomes. These forms of DNA can also be categorized under the terms circulating DNA, cell free DNA, free DNA and extracellular DNA. The DNA can be released by means of form–specific mechanisms and seem to be governed by cell cycle phases and apoptosis. Active release is supported by evidence of energy dependant release mechanisms and various immunological– and messenger functions. Sequencing has shown that eoDNA sequences present in the nucleome reflects traits and distribution of genome sequences and are regulated by ways of release and/or clearance. eoDNA enables the horizontal transfer of gene sequences from one cell to another, over various distances. The ability of eoDNA to partake in horizontal gene transfer makes it an important facet in the field of epigenetic variation. Clinical implementation of eoDNA diagnostics requires that all of the subgroups of eoDNA be properly investigated. It is suggested that eoDNA is the result of the metabolic fraction of DNA that is released by the cell. Various observations indicate that eoDNA may also be incorporated into the genome of a cell, from where it may affect cell function. Therefore horizontal gene transfer in higher organisms is a real possibility. In this study, variations and increases in eoDNA levels over time correlate with stressors that are subjected to 143B human osteosarcoma cells. It seems viable to assume that a stressor is met by a change in the molecular machinery of a cell, required to neutralise the onset of metabolic instability. This may be done by amplification of necessary cistrons, producing metabolic DNA, that may then be observed after its release as eoDNA. The presence of hydrolysing enzymes gives an updated real time picture of the state of eoDNA. The eogenics hypothesis emanating from this study, suggests that amplification and horizontal transfer of cistrons affect tissue and organ function over long periods of time, in order for an organism to evolve one or more a specialized genomes.<br>Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.

Herbal medicines are growing in popularity in the Western world and are becoming more stringently regulated under new EU legislation. Within the arena of herbal medicines, St. John’s Wort (SJW), Hypericum perforatum, is a top ten best seller with clinical evidence to support its use as an anti-depressant. A fundamental requirement of the new legislation is to prove the identity of the plant material in question. This is currently achieved via morphological and chemical methods, neither of which are ideal. A wide range of DNA based methods have been applied to this arena, standardisation is required to realise the potential of DNA based techniques. The DNA barcoding initiative aims to produce sequence data for all plant species, capable of species identification. The proposal is to use these data to design fast and effective DNA based methods of identification. For assay design, the putative barcode region nrITS was selected as a platform. Three assays were designed; • A PCR assay designed to hyper variable sequences within a barcode region. This assay is capable of distinguishing SJW from other closely related species. • A quantitative qPCR assay designed to measure total DNA and specific SJW DNA within a mixed sample. • A multiplex PCR incorporating fluorescently labelled primers, allowing amplicon detection by capillary electrophoresis. This assay identifies four separate Hypericum species, including SJW, with a mixed sample in one reaction. The suitability of the nrITS and three other barcode regions is assessed based on sequence data generated for 32 vouchered samples of different Hypericum species, and a Lithuanian sample set of 22 and 16 H. perforatum and H. maculatum samples respectively. The matK is currently unusable, the rbcL highly conserved, trnH-psbA problematically variable and the nrITS proved to be ideal for assay design.

Magister Scientiae - MSc (Biotechnology)<br>The objective of this study is to deduce paternal ancestry using ancestry informative single nucleotide polymorphisms (SNPs) by means of High Resolution Melting (HRM). This was completed by producing a multiplex system that was designed in a hierarchical manner according to the YSNP tree. This project mainly focused on African ancestry and was used to infer paternal ancestral lineages on the Johannesburg Coloured population. South Africa has a diverse population that has ancestral history from across the globe. The South African Coloured population is the most admixed population as it is derived from at least five different population groups: these being Khoisan, Bantu, Europeans, Indians and Southeast Asians. There have been studies done on the Western Cape/ Cape Town Coloured populations before but this study focused on the Johannesburg Coloured population. The first step was to design the multiplex system. This was done by using inhouse SNPs. A total of seven multiplexes were designed and optimised, each consisting of two, three or four different SNPs respectively. A total of 143 saliva and buccal samples were collected from male Johannesburg Coloureds. DNA was extracted from the saliva samples using an optimised organic method. DNA was extracted from the buccal samples using an optimised salting out method. DNA was successfully extracted from 77 of the male samples. A total of 69 samples were screened using Multiplex 1; of the 69 samples 56 samples were successfully screened to infer the paternal lineage of the samples. The results show that the most frequent haplogroup of the Johannesburg male samples was haplogroup CF (39%). The second most frequent haplogroup was haplogroup DE (38%). Under further analysis of haplogroup DE it was seen that 37% of those samples were derived for the haplogroup E1b1b.

Feline babesiosis, first described in domestic cats in South Africa in 1937, is regarded to be of great importance in the coastal regions although isolated cases also occur on the eastern highlands of Mpumalanga Province. Babesia felis (described from domestic cats) and B. leo (described from lions) are the two best characterised Babesia species in felids. These two parasites are morphologically similar when examined under a light microscope, but are serologically and genetically distinct. In this study the prevalence of these two Babesia species in various wild and domestic felid species was determined. A total of 358 samples were tested using the reverse line blot hybridization (RLB) assay. This assay makes it possible to simultaneously detect and differentiate between blood parasites using DNA probes. The RLB consists of three basic steps, the first being amplification of the variable region (V4) in the 18S rRNA gene using genus-specific primers where one is labelled with biotin. This is followed by a blotting step, where the amplicons are hybridized to oligonucleotides bound to a nitrocellulose membrane. The third and last step is the detection of the hybridized amplicons by using chemiluminescence reagents. This assay is a screening tool utilizing the variable (V4) region in the 18S rRNA gene to detect and differentiate between blood parasites. A new B. felis-specific DNA probe was developed to use in the RLB assay. Results demonstrated that these two parasites not only occur in the felid species from which they have been described, but also in other felid species. Babesia microti was also detected in various felid species, while B. rossi was detected in 1 of the lion samples. Two hundred and twelve samples tested positive for Babesia spp., of which only 54.24% of the samples reacted with the genus-specific probe. This indicates the presence of a novel Babesia or Theileria species or variant of a species.<br>Dissertation (MSc)--University of Pretoria, 2010.<br>Veterinary Tropical Diseases<br>unrestricted

Organometallic chemotherapeutic agents, many of which target DNA, have been shown to be effective in the treatment of cancer. With that said though, these compounds have a number of side affects such as nephrotoxicity. Two novel compounds, Ferrocene [ferrocenoyltrichloroacetone] and Rhodium-ferrocene [(1.5 cyclooctadiene)(1-ferrocenyl- 4,4,4-trichloro-1,3-butanedionate], synthesised by the research group of J Swarts (University of the Free State) were evaluated to determine their mechanism of action and their potential use as novel therapeutic agents. It is hypothesized, by merit of their chemical structures, that these compounds’ anti-cancer activity is due to their interaction with DNA. Both drugs were evaluated from a cellular to a molecular level, in vitro, to validate this hypothesis. Linearised DNA was exposed to both drugs and digested with a variety of restriction enzymes. It was found that the compounds bind to the PstI restriction site; thereby inhibiting the enzyme’s restriction activity. From this point it was necessary to show that the compounds are able to interact with DNA in a cellular system. By exposing a transformed breast epithelial cell line (MCF-12A) and a cancerous breast epithelial cell line (MCF-7) to the compounds, for various times, followed by flow cytometric analyses, it was found that both affect progression through the cell cycle. Cells accumulated at various phases of the cell cycle, as a result of checkpoint gene activation. Further flow cytometric analyses showed that both drugs induce necrosis in MCF-7 cells. The “normal” cell line however did not show this response as it is believed that cell cycle arrest and repair mechanisms were initiated, which would delay cell death. Gene expression analyses were performed by reverse transcriptase real-time PCR in which panels of cell cycle related genes as well as DNA damage associated genes were probed in two separate array formats. These studies revealed that a number of DNA damage and repair genes are activated; specifically those associated with excision repair and free-radical induced DNA damage. Members of the RAD family as well as the genes GADD45A, XPC and OGG1 were found to be upregulated as a result of Ferrocene treatment. This could be expected as it was shown that ferrocene binds to DNA, and it logically then follows that this would lead to excision repair being attempted by the cell. Similar gene expression patterns were found following Rhodium-ferrocene treatment with the up-regulation of genes such as OGG1, ATM and GADD45G, albeit to a lesser extent. It is hypothesised that the larger molecule may not interact as effectively with DNA, due to steric hinderance. Arrest mechanisms, for both drugs, were more pronounced in the “normal” cell line and it is believed that this is due to the fact that many of these genes have been inactivated in the cancerous cell line. We have shown, on multiple levels, that both compounds’ therapeutic action is as a result of their interaction with the cell’s DNA. This interaction leads to cell death in both the transformed and the cancerous cell line. In order to clarify these mechanisms it is suggested that proteomic and metabolomic studies should be performed.<br>Dissertation (MSc)--University of Pretoria, 2012.<br>Genetics<br>unrestricted

Aufgrund einer komplexen Wechselwirkung mit Proteinen ist das Genom in einer Zelle ständig mechanischer Spannung und Torsion ausgesetzt. Daher ist es wichtig die Mechanik und die Dynamik von verdrillter DNA unter Spannung zu verstehen. Diese Situation wurde experimentell mittels einer sog. magnetischen Pinzette nachgestellt, indem sowohl Kraft als auch Drehmoment auf ein einzelnes DNA Molekül ausgeübt und gleichzeitig die mechanische Antwort des Polymers aufgezeichnet wurde. Als erstes Beispiel wurde der Übergang von linearer zu sog. plectonemischer DNA untersucht, d.h. die Absorption eines Teils der induzierten Verdrillung in einer superhelikalen Struktur. Eine abrupte Längenänderung am Anfang dieses Übergangs wurde bereits im Vorfeld publiziert. In der vorliegenden Arbeit wird gezeigt, dass diese abrupte DNA Verkürzung insbesondere von der Länge der DNA und der Ionenkonzentration der Lösung abhängt. Dieses Verhalten kann mittels eines Modells verstanden werden, in dem die Energie pro Verwringung der ersten Schlinge innerhalb der Superhelix größer ist als die aller nachfolgenden. Des Weiteren wurden DNA-DNA Wechselwirkungen in der Umgebung monovalenter Ionen durch die Analyse des Superspiralisierungsverhaltens einzelner DNA Moleküle bei konstanter Kraft charakterisiert. Solche Wechselwirkungen sind für die Kompaktierung des Genoms und die Regulation der Transkription wichtig. Oft wird DNA als gleichmäßig geladener Zylinder modelliert und ihre elektrostatischen Wechselwirkungen im Rahmen der Poisson-Boltzmann-Gleichung mit einem Ladungsanpassungsfaktor berechnet. Trotz erheblicher Anstrengung ist eine präzise Bestimmung dieses Parameters bisher nicht gelungen. Ein theoretisches Modell dieses Prozesses zeigte nun eine erstaunlich kleine effektive DNA Ladung von ~40% der nominalen Ladungsdichte. Abgesehen von Gleichgewichtsprozessen wurde auch die Dynamik eines Faltungsvorgangs von DNA untersucht. Spontane Branch Migration einer homologen Holliday-Struktur wurde genutzt, um die intramolekulare Reibung der DNA zu erforschen. Mittels einer magnetischen Pinzette wurde eine torsionslimitierte Holliday-Struktur gestreckt während die Längenfluktuationen der Zweige mit schneller Videomikroskopie bei ~3 kHz aufgezeichnet wurden. Einzelne diffusive Schritte der Basenpaare sollten auf einer sub-Millisekunden Zeitskala auftreten und viel kleiner als die Gesamtfluktuationen der DNA sein. Eine Analyse der spektralen Leistungsdichte der Längenfluktuationen ermöglicht eine eindeutige Beschreibung der Dynamik der Branch Migration. Die Holliday-Struktur wurde außerdem als nanomechanischer Linearversteller eingesetzt, um einen einzelnen fluoreszierenden Quantenpunkt durch ein exponentiell abfallendes evaneszentes Feld zu bewegen. Durch die Aufzeichnung der Emission des Quantenpunkts sowohl in dem evaneszenten Feld als auch unter gleichmäßiger Beleuchtung kann die Intensitätsverteilung des Anregungsfelds ohne weitere Dekonvolution bestimmt werden. Diese neue Technik ist von besonderem wissenschaftlichen Interesse, weil die Beschreibung dreidimensionaler inhomogener Beleuchtungsfelder eine große Herausforderung in der modernen Mikroskopie darstellt. Die Ergebnisse dieser Arbeit werden dem besseren Verständnis einer Vielzahl biologischer Prozesse, die in Verbindung mit DNA Superspiralisierung stehen, dienen und weitere technische Anwendungen des DNA-basierten Linearverstellers hervorbringen<br>The genome inside the cell is continuously subjected to tension and torsion primarily due to a complex interplay with a large variety of proteins. To gain insight into these processes it is crucial to understand the mechanics and dynamics of twisted DNA under tension. Here, this situation is mimicked experimentally by applying force and torque to a single DNA molecule with so called magnetic tweezers and measuring its mechanical response. As a first example a transition from a linear to a plectonemic DNA configuration is studied, i.e. the absorption of part of the applied twist in a superhelical structure. Recent experiments revealed the occurrence of an abrupt extension change at the onset of this transition. Here, it is found that this abrupt DNA shortening strongly depends on the length of the DNA molecule and the ionic strength of the solution. This behavior can be well understood in the framework of a model in which the energy per writhe for the initial plectonemic loop is larger than for subsequent turns of the superhelix. Furthermore DNA-DNA interactions in the presence of monovalent ions were comprehensively characterized by analyzing the supercoiling behavior of single DNA molecules held under constant tension. These interactions are important for genome compaction and transcription regulation. So far DNA is often modeled as a homogeneously charged cylinder and its electrostatic interactions are calculated within the framework of the Poisson-Boltzmann equation including a charge adaptation factor. Despite considerable efforts, until now a rigorous quantitative assessment of this parameter has been lacking. A theoretical model of this process revealed a surprisingly small effective DNA charge of ~40% of the nominal charge density. Besides describing equilibrium processes, also the dynamics during refolding of nucleic acids is investigated. Spontaneous branch migration of a homologous Holliday junction serves as an ideal system where the friction within the biomolecule can be studied. This is realized by stretching a torsionally constrained Holliday junction using magnetic tweezers and recording the length fluctuations of the arms with high-speed videomicroscopy at ~3 kHz. Single base pair diffusive steps are expected to occur on a sub-millisecond time scale and to be much smaller than the overall DNA length fluctuations. Power-spectral-density analysis of the length fluctuations is able to clearly resolve the overall dynamics of the branch migration process. Apart from studying intramolecular friction, the four-arm DNA junction was also used as a nanomechanical translation stage to move a single fluorescent quantum dot through an exponentially decaying evanescent field. Recording the emission of the quantum dot within the evanescent field as well as under homogeneous illumination allows to directly obtain the intensity distribution of the excitation field without additional deconvolution. This new technique is of particular scientific interest because the characterization of three-dimensional inhomogeneous illumination fields is a challenge in modern microscopy. The results presented in this work will help to better understand a large variety of biological processes related to DNA supercoiling and inspire further technical applications of the nanomechanical DNA gear

Of the many existing eukaryotic gene finding software programs, none are able to guarantee accurate identification of genomic protein coding regions and other biological signals central to pathway from DNA to the protein. Eukaryotic gene finding is difficult mainly due to noncontiguous and non-continuous nature of genes. Existing approaches are heavily dependent on the compositional statistics of the sequences they learn from and are not equally suitable for all types of sequences. This thesis firstly develops efficient digital signal processing-based methods for the identification of genomic protein coding regions, and then combines the optimum signal processing-based non-data-driven technique with an existing data-driven statistical method in a novel system demonstrating improved identification of acceptor splice sites. Most existing well-known DNA symbolic-to-numeric representations map the DNA information into three or four numerical sequences, potentially increasing the computational requirement of the sequence analyzer. Proposed mapping schemes, to be used for signal processing-based gene and exon prediction, incorporate DNA structural properties in the representation, in addition to reducing complexity in subsequent processing. A detailed comparison of all DNA representations, in terms of computational complexity and relative accuracy for the gene and exon prediction problem, reveals the newly proposed ?paired numeric? to be the best DNA representation. Existing signal processing-based techniques rely mostly on the period-3 behaviour of exons to obtain one dimensional gene and exon prediction features, and are not well equipped to capture the complementary properties of exonic / intronic regions and deal with the background noise in detection of exons at their nucleotide levels. These issues have been addressed in this thesis, by proposing six one-dimensional and three multi-dimensional signal processing-based gene and exon prediction features. All one-dimensional and multi-dimensional features have been evaluated using standard datasets such as Burset/Guigo1996, HMR195, and the GENSCAN test set. This is the first time that different gene and exon prediction features have been compared using substantial databases and using nucleotide-level metrics. Furthermore, the first investigation of the suitability of different window sizes for period-3 exon detection is performed. Finally, the optimum signal processing-based gene and exon prediction scheme from our evaluations is combined with a data-driven statistical technique for the recognition of acceptor splice sites. The proposed DSP-statistical hybrid is shown to achieve 43% reduction in false positives over WWAM, as used in GENSCAN.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-10-02T20:17:04Z No. of bitstreams: 1 ThiagoSantos.pdf: 1103502 bytes, checksum: 16ad7405a0ab31d83423293c43110ee8 (MD5)<br>Made available in DSpace on 2017-10-02T20:17:04Z (GMT). No. of bitstreams: 1 ThiagoSantos.pdf: 1103502 bytes, checksum: 16ad7405a0ab31d83423293c43110ee8 (MD5) Previous issue date: 2017-09-11<br>Coordenação de Aperfeiçoamento de Pessoal de Nível Superior<br>Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão<br>Instituto Nacional de Ciência e Tecnologia em Bioanalítica<br>A photoelectroanalytical sensor was developed, based on deoxyribonucleic acid (DNA) and anatase titanium dioxide (TiO2) nanoparticles sensitized with bis(ethylenedithio)tetrathiofulvalene (BEDT-TTF) for determination of the adrenaline, also denominated as epinephrine. The photosensor composite developed was denominated as BEDT-TTF/DNA/TiO2/ITO and shows a high photocurrent for the adrenaline under light emitting diode (LED) irradiation in comparison to each component of the composite material. Under optimized conditions, the BEDTTTF/DNA/TiO2/ITO sensor shows a linear response range from 10 nmol L-1 up to 100 μmol L-1 with a sensitivity of 8,1 nA L μmol-1 and limit of detection of 1 nmol L-1 for the adrenaline. The photoelectrochemical sensor showed high photocurrent to adrenaline in comparison to photocurrent response to ascorbic acid and uric acid. The BEDT-TTF/DNA/TiO2/ITO photoelectrochemical sensor was successfully applied to urine samples, with recovery values between 96 and 106%.<br>Um sensor fotoeletroanalítico foi desenvolvido, baseado em ácido desoxirribonucleico (DNA) e nanopartículas de dióxido de titânio anatase (TiO2) sensibilizadas com bis(etilenoditio)tetratiofulvaleno (BEDT-TTF) para a determinação de adrenalina, também denominada como epinefrina. O fotossensor compósito desenvolvido foi denominado como BEDT-TTF/DNA/TiO2/ITO e exibiu uma elevada fotocorrente para a adrenalina sob a irradiação do diodo emissor de luz (LED) em comparação com cada componente do material compósito. Sob condições otimizadas, o sensor BEDT-TTF/DNA/TiO2/ITO exibiu um intervalo de resposta linear de 10 nmol L-1 para 100 μmol L-1 com uma sensibilidade de 8,1 nA L μmol-1 e limite de detecção de 1 nmol L-1 para a adrenalina. O sensor fotoeletroquímico mostrou elevada fotocorrente para a adrenalina em comparação com a resposta de fotocorrente para ácido ascórbico e ácido úrico. O fotossensor BEDTTTF/DNA/TiO2/ITO foi aplicado com sucesso em amostras de urina, com valores de recuperação entre 96 e 106%.

Les métaux sont indispensables à la vie cellulaire car ils sont constitutifs des protéines. Les ions Ni, font partie intégrante des hydrogénases, enzymes primordiales pour le métabolisme énergétique. Paradoxalement, en excès, les métaux deviennent toxiques pour la cellule. Les bactéries luttent contre cette toxicité en produisant des systèmes de résistance ou d’adaptation. Les cellules procaryotes peuvent équilibrer les teneurs en métaux en contrôlant leur entrée ou leur efflux grâce à la biogenèse de transporteurs spécifiques. L’objectif de ces travaux de thèse a consisté à comprendre les mécanismes principaux permettant à la bactérie modèle Escherichia coli de s’adapter à de fortes variations en ions métalliques, en prenant comme modèle un stress provoqué par un excès d’ions Ni. Afin d’appréhender l’ensemble de la réponse cellulaire, l’effet de ce stress a été évalué sur l’expression de l’ensemble des gènes d’E. coli par des approches de transcriptomique couplées à une validation fonctionnelle. L’excès d’ions Ni induit le système d’efflux RcnRAB. En plus de la pompe d’efflux RcnA, ce système comporte une protéine périplasmique, RcnB, qui module le trafic des ions Ni ou Co via RcnA. Ces travaux ont montré que RcnB n’interagit pas avec les ions Ni ou Co mais de façon inattendue avec les ions Cu, définissant une nouvelle classe de cupro-protéines. Nous montrons que si RcnB n’intervient pas dans le contrôle de l’homéostasie du Cu, l’interaction avec ces ions est essentielle à sa fonction dans la modulation de l’efflux des ions Ni et Co. Ces résultats suggèrent des connexions entre les différents systèmes de maintien des homéostasies métalliques. Les résultats d’analyse transcriptomique montrent une forte modulation de l’expression des gènes impliqués dans les homéostasies du Cu et du Fe en présence d’un excès d’ions Ni, corrélée à une augmentation cellulaire de leur teneur mesurée par spectrométrie plasma. Ces métaux sont responsables de la production d’espèces réactives oxygénées entraînant de sérieux dégâts cellulaires, une des cibles privilégiée étant l’ADN. Nous montrons que les ions Ni ne provoquent pas de cassures de l’ADN et n’ont pas d’effet mutagène, par contre ils provoquent une modification importante de l’état de repliement de l’ADN. Nous proposons que ce relâchement de l’ADN soit dû à l’induction indirecte d’un stress oxydant. Ces travaux ont aboutis à l’identification du premier système de transport spécifique des ions Ni à travers la membrane externe chez E. coli. En résumé, un excès d’ions Ni affecte les systèmes spécifiques d’entrée et d’efflux des ions métalliques troublant les teneurs intracellulaires des autres métaux comme le Cu et le Fe. Ces métaux sont en partie responsables de la production de ROS létaux pour les cellules bactériennes. L’excès de Ni va induire une profonde reprogrammation génétique entraînant des changements physiologiques multifactoriels importants pour la survie bactérienne dans ces conditions de stress<br>Metals are necessary components of all living cells because they are constitutive of many essential proteins. Nickel, for example, is required for hydrogenase activity, which is essential for the energetic metabolism. However, metals become toxic when present in excess. Prokaryotes can overcome this toxicity by using several systems of resistance or adaptation. Import systems must be repressed whereas export pathways activated. This work consists in bringing out the principal strategies established by Escherichia coli for accommodating a stress caused by an excess of Ni ions. In order to understand the cellular response, the effect of nickel stress has been evaluated in E. coli by a transcriptomic approach coupled to functional validation. Excess Ni induces the biosynthesis of the efflux system RcnRAB. In addition to the RcnA efflux pump, this system contains a periplasmic protein called RcnB. This protein modulates Ni and Co traffic. RcnB displayed no Ni or Co binding capacity but was shown to bing Cu ions. RcnB was characterized as a new family of cupro-protein. We showed that RcnB is not involved in the control of Cu homeostasis but that Cu binding is essential for its Ni and Co efflux function. Our results suggest connections between different systems of metals homeostasis. Indeed, RNA-Seq data analysis revealed that exposure to Ni induces strong variations of the expression of genes involved in Cu and Fe homeostasis. Our results correlated with an increase of intracellular Cu and Fe pools as assayed by plasma spectrometry. Both metals are involved in reactive oxygen species (ROS) production and generate serious cell damages, targeting DNA for example. We showed that Ni ions do not trigger DNA breakage and are not mutagenic. On the other hand, Ni stress has a strong effect on DNA folding. We propose that excess Ni causes DNA relaxation by the indirect induction of oxidative stress. Furthermore, we identified the first transport system specific for Ni ions localized in the outer membrane. This system, composed of YddA and YddB, allows the transfer of Ni ions accross the two membranes. The genes encoding these proteins are expressed in conditions evocative of a biofilm lifestyle. Moreover, this work showed that Ni stress promotes biofilm growth instead of a planktonic one. Indeed, in the presence of an excess of Ni ions, genes encoding flagella are down regulated whereas genes encoding adherence structures are up regulated. To conclude, an excess of Ni ions affects specific metals import and efflux systems unbalancing intracellular Fe and Cu contents. These metals in turn generate ROS that are toxic for the bacterial cells. Ni stress induces large transcriptomic modifications causing major physiological changes important for the survival of the bacteria

The mechanism for the hydrolysis of 2′-deoxyribonucleosides is examined using computational chemistry techniques. Initially, a model capable of accurately predicting the mechanism and activation barrier for the uncatalyzed hydrolysis of 2′-deoxyuridine is designed. It is found that the smallest model includes both explicit and implicit solvation during the optimization step. Next, this hybrid solvation model is applied to four natural nucleosides, namely 2′-deoxyadenosine, 2′-deoxycytidine, 2′-deoxyguanosine and thymidine. The hybrid model correctly predicts the trend in activation Gibbs energies for the pyrimidines and purines, separately. Finally, the concepts developed during the generation of the uncatalyzed hydrolysis model are applied to the mechanism of action of a glycosylase enzyme, namely human uracil DNA glycosylase. A hybrid ONIOM approach is utilized to study the experimentally proposed two-step mechanism. Results regarding the protonation state of His148 are inconclusive, and future directions are proposed.<br>xiii, [131] leaves : ill. (some col.) ; 29 cm

Software to display and analyse DNA sequences is a crucial tool for bioinformatics research. The data of a DNA sequence has a relatively simple format but the length and sheer volume of data can create difficulties in navigation while maintaining overall context. This is one reason that current bioinformatics applications can be difficult to use. This research examines techniques for navigating through large single DNA sequences and their annotations. Navigation in DNA sequences is considered here in terms of the navigational activities: exploration, wayfinding and identifying objects. A process incorporating user-centred design was used to create prototypes involving panning and zooming of DNA sequences. This approach included a questionnaire to define the target users and their goals, an examination of existing bioinformatics applications to identify navigation designs, a heuristic evaluation of those designs, and a usability study of prototypes. Three designs for panning and five designs for zooming were selected for development. During usability testing, users were asked to perform common navigational activities using each of the designs. The “Connected View” design was found to be the most usable for panning while the “Zoom Slider” design was best for zooming and most useful zooming tool for tasks involving browsing. For some tasks the ability to zoom was unnecessary. The research provides important insights into the expectations that researchers have of bioinformatics applications and suitable methods for designing for that audience. The outcomes of this type of research can be used to help improve bioinformatics applications so that they will be truly usable by researchers.

Cette thèse porte sur le développement d’un capteur plasmonique intégrant une fonction d’actuation des objets visés. L’objectif est de passer outre la limite de diffusion rencontrée à basse concentration en piégeant les particules sur la surface de détection. La stratégie adoptée est de structurer le film d’or servant à la détection de manière à pouvoir l’utiliser pour mettre en mouvement le fluide et les molécules par le biais de champs électriques. Le transfert de masse est réalisé par diélectrophorèse et électroosmose, deux effets électrocinétiques mis en oeuvre par des électrodes servant à la fois d’actuateur et de capteur plasmonique. Un état de l’art exhaustif et des simulations multiphysiques ont permis de concevoir un prototype de capteur intégré constitué d’électrodes interdigitées en or permettant la détection plasmonique. Le dispositif proposé a été obtenu par microfabrication en salle blanche puis caractérisé avant l’étude de ses performances. Une première phase de tests sur un système modèle, des billes de polystyrène dans de l’eau, a permis d’apporter la preuve de concept du fonctionnement du capteur, qui est effectivement capable de piéger rapidement les objets visés à sa surface afin de les détecter. Les mécanismes de transfert de masse ont été expliqués et la preuve de l’amélioration de la limite de détection par un facteur supérieur à 100 a été apportée. Dans un second temps, les performances du capteur appliqué à des objets biologiques ont été évaluées. Celui-ci piège efficacement des levures et des protéines, mais aucune amélioration n’a été observée dans le cas de la détection spécifique de l’hybridation entre deux brins d’acide désoxyribonucléique (ADN). Les causes de ce résultat ont été discutées et comprises et deux solutions différentes ont été explorées : l’adaptation de la fréquence d’opération et l’optimisation de la géométrie des électrodes. Ainsi, cette étude a permis de souligner la problématique de la mise en oeuvre d’effets électrocinétiques dans des milieux biologiques et de réfléchir aux pistes pertinentes pour sa résolution<br>This thesis focuses on the development of an integrated plasmonic sensor capable to perform mass transport on targeted objects. The goal is to overcome the diffusion limit by trapping particules directly on the sensing surface. The adopted strategy was to structure the gold layer used for plasmonic detection in order to use the sofabricated structures to set the fluid and the molecules in motion by applying electric fields in the fluid. The mass transfer is realized through dielectrophoresis and electroosmosis, those two electrokinetic effects being operated by electrodes acting as sensor and actuator at the same time. An exhaustive state of the art as well as multiphysical simulations allowed us for designing a prototype for an integrated sensor consisting in gold interdigitated electrodes enabling plasmoninc sensing. The proposed device was obtained through microfabrication in clean room facilities and was characterized before the study of its performances. A first sequence of tests on a model system – polystyrene microbeads in water – brought the proof of concept we needed to validate the correct operation of the sensor, which is indeed capable of quickly trapping targeted objects on its surface and detecting them. The mass transfer mechanisms were explained and we showed the enhancement of the limit of detection by a factor greater than 100. In a second phase, performances of the sensor applied to biological objects were evaluated. It can effectively trap yeasts and proteins but no enhancement has been observed while detecting DNA hybridization events. Causes for this result were discussed and understood and two different solutions were explored: the adaptation of the operating frequency and the optimization of the electrodes geometry. Thus, this study highlighted the problematic of operating electrokinetic effects in biological media and suggested relevant leads towards its resolution

The majority of structural studies on DNA have been carried out using fibre diffraction, while studies of its dynamics and thermal behaviour have been mainly performed in solution. When the DNA double helix is heated, it exhibits local separation of the two strands that grow in size with temperature and lead to their complete separation. This work has investigated various aspects of this phenomenon. The experiments reported in this thesis were carried out on films of oriented fibres of DNA prepared with the Wet Spinning Apparatus. Thus, sample preparation and characterisation are essential parts of the research. The structures of two forms of DNA, A and B, have been explored as a function of relative humidity at fixed ionic conditions. A method to eliminate traces of ever-present B-form contamination in A-form samples was established. The high orientation of the DNA molecules within the samples allowed us to investigate dynamical fluctuations and the melting transition of DNA using neutron scattering, which can provide the spatial information crucial to understand a phase transition, probing the static correlation length along the molecule as a function of temperature. The transition has been investigated for A and B-forms in order to understand its dependence on molecular configuration.Furthermore, after the first melting, denatured DNA films show typical glass behaviour. Their thermal relaxation has been explored using calorimetry.Neutron and X-ray inelastic scattering (INS and IXS) were used in the past to measure longitudinal phonons in fibre DNA, and the results shown disagreement. Recent INS measurements supported with phonon simulations have been crucial to understand the different dispersion curves reported to date. Experiments using INS and IXS have been carried out to continue with this investigation. Attempts to observe the transverse fluctuations associated to the thermal denaturing of DNA, never experimentally investigated before, have been made.

Fu Annie Yuen Yee.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.<br>Includes bibliographical references (leaves 97-108).<br>Abstracts in English and Chinese.<br>ABSTRACT (ENGLISH) --- p.iii<br>ABSTRACT (CHINESE) --- p.iv<br>ACKNOWLEDGMENTS --- p.v<br>TABLE OF CONTENTS --- p.vi<br>LIST OF FIGURES --- p.ix<br>LIST OF TABLES --- p.xi<br>Chapter CHAPTER ONE: --- BACKGROUND INFORMATION --- p.1<br>Chapter 1.1 --- Introduction --- p.1<br>Chapter 1.2 --- Deoxyribonucleic Acid --- p.1<br>Chapter 1.2.1 --- Nomenclature and Symbols --- p.1<br>Chapter 1.2.2 --- Torsion Angles --- p.4<br>Chapter 1.2.3 --- Conformation --- p.5<br>Chapter 1.3 --- DNA Studies --- p.8<br>Chapter 1.3.1 --- Base --- p.8<br>Chapter 1.3.2 --- Base-Pair --- p.10<br>Chapter 1.3.3 --- Summary --- p.11<br>Chapter 1.4 --- Cisplatin Studies --- p.11<br>Chapter 1.4.1 --- Reaction --- p.12<br>Chapter 1.4.2 --- Cisplatin-DNA Products --- p.14<br>Chapter 1.4.3 --- Summary --- p.15<br>Chapter 1.5 --- Scope of This Thesis --- p.15<br>Chapter CHAPTER TWO: --- COMPUTATION AND METHODOLOY --- p.17<br>Chapter 2.1 --- Introduction --- p.17<br>Chapter 2.2 --- Hartree-Fock Approximation --- p.17<br>Chapter 2.3 --- Geometry Optimization --- p.18<br>Chapter 2.4 --- Molecular Orbital (MO) Calculation --- p.20<br>Chapter 2.5 --- Verification of Methodology --- p.20<br>Chapter 2.5.1 --- Backbone Torsion Angles --- p.20<br>Chapter 2.5.2 --- N7-N7 Distance --- p.26<br>Chapter 2.5.3 --- Location of HOMO --- p.30<br>Chapter 2.6 --- Summary --- p.31<br>Chapter CHAPTER THREE: --- UNDERSTANDING OF THE CISPLATIN-DNA CROSSLINKS --- p.33<br>Chapter 3.1 --- Introduction --- p.33<br>Chapter 3.2 --- MO Analysis --- p.33<br>Chapter 3.3 --- Potential Binding Sites of DNA --- p.34<br>Chapter 3.3.1 --- "1,2-d(GpG) Intrastrand Crosslink" --- p.40<br>Chapter 3.3.2 --- "l,2-d(ApG) Intrastrand Crosslink" --- p.40<br>Chapter 3.3.3 --- "l,3-d(GpXpG) Intrastrand Crosslink" --- p.41<br>Chapter 3.3.4 --- d(GpC)d(GpC) Interstrand Crosslink --- p.41<br>Chapter 3.3.5 --- d(GpXpC)d(GpXpC) Interstrand Crosslink --- p.41<br>Chapter 3.3.6 --- Summary --- p.42<br>Chapter 3.4 --- Empirical Selection Rule --- p.44<br>Chapter 3.4.1 --- Convention --- p.44<br>Chapter 3.4.2 --- Selection of Potential HOMO Location (or Active Site) --- p.45<br>Chapter 3.4.3 --- Selection of Potential HOMO-Nearby Active Site --- p.47<br>Chapter 3.4.4 --- Applications --- p.48<br>Chapter 3.5 --- Cisplatin --- p.51<br>Chapter 3.6 --- Cisplatin-DNA Crosslinks --- p.52<br>Chapter 3.6.1 --- "l,2-d(GpG) and l,2-d(ApG) Intrastrand Crosslinks" --- p.52<br>Chapter 3.6.2 --- "l,2-d(ApG) versus l,2-d(GpA) Intrastrand Crosslinks" --- p.53<br>Chapter 3.6.3 --- "l,3-d(GpXpG) Intrastrand and d(GpXpC)d(GpXpC) Interstrand Crosslinks" --- p.54<br>Chapter 3.6.4 --- Platination at Terminal Positions --- p.55<br>Chapter 3.7 --- Structural Parameters --- p.59<br>Chapter 3.7.1 --- Optimized Geometries --- p.59<br>Chapter 3.7.2 --- DNA Sequences from PDB --- p.67<br>Chapter 3.7.3 --- Backbone Torsion Angles --- p.70<br>Chapter 3.8 --- Summary --- p.70<br>Chapter CHAPTER FOUR: --- CONCLUDING REMARKS --- p.72<br>APPENDIX I BACKBONE TORSION ANGLES AND SUGAR RING CONFORMATION OF THE OPTIMIZED GEOMETRIES --- p.74<br>APPENDIX II BACKBONE TORSION ANGLES OF THE EXPERIMENTAL SEQUENCES FROM NUCLEIC ACID DATABASE (NDB) --- p.82<br>REFERENCES --- p.97

The central theme in this thesis is Empirical Bayes. It starts off with application of Bayes and Empirical Bayes methods to deoxyribonucleic acid fingerprinting. Different Bayes factors are obtained and an alternative Bayes factor using the method of Savage is studied both for normal and non- normal priors. It then moves on to deeper methodological aspects of Empirical Bayes theory. A 1983 conjecture by Carl Morris on the parametric empirical Bayes prediction intervals for the normal regression model is studied and an improvement suggested. Carlin and Louis’ (1996) parametric empirical Bayes prediction interval for the same model is also dealt with analytically while their approach had been primarily numerical. It is seen that both of these intervals have the same coverage probability up to a certain order of approximation and they have the same expected length up to the same order of approximation. Both the intervals are equal tailed up to the same order of approximation. Then the corrected proof of an important published result by Datta, Ghosh and Mukerjee (2000) is provided using first principles of probability matching. This result is relevant to our work on parametric empirical Bayes prediction intervals.

碩士<br>國立清華大學<br>動力機械工程學系<br>105<br>Brain cancer is a lethal disease which is quite difficult to cure by traditional therapy such as surgery since human brain consists of very complicated nervous system. It is very likely to make undesirable mistakes during an operation. Proton therapy is a very effective method to destroy brain cancer cells because it has a special characteristic: the Bragg Peak. This special feature allows the dose deposit in the premier part of the incident path to remain at a very low value and then suddenly soar to the climax. With this magnificent phenomenon, it is possible to kill the tumor cells without damaging other normal cells, thus avoiding unwanted side effects after the cure. In this paper, we use Monte-Carlo method and quantum chemistry to simulate the behavior of protons traveling through substrates such as water and calculate the dose distribution and other properties. To build a DNA model and calculate its bonding energy through quantum chemistry methods, Gaussian is a tool required. As to obtain the dose distribution and proton range by using Monte-Carlo method, Geant4 would be an adequate computer program for the task. According to our research, the dose deposit in the front part of the model actually seems to be too low to break the bonding of DNA. Only the dose near the Bragg Peak is capable of breaking the backbone of the DNA structure. Therefore, proton therapy is proved to be a safe way to remedy brain cancer by now.

This study analyses how DNA evidence can be distorted by the behaviour of criminal investigators and role-players within the Criminal Justice System (CJS). This has a negative impact on justice resulting in further criminality. The study has resulted in revelatory weaknesses owing to constitutional violations which cause sound evidence to become futile as it will not be admissible in court. Justice is aborted. The researcher has further explained the properties of the pertinent terms, such as: mental illness, psycho-social functioning, DNA, forensic investigator, forensic psychology, and courts. Concepts are building blocks, hermeneutical distortion leads to the frustrating of what justice intends and this, in turn, leads to poor criminal investigation performance. It is submitted that not only ineptness, but also deception possibly evolves from genotypic to phenotypic type which causes unwelcome behaviour within the criminal justice system to surface. The frequency of monitoring psychological behaviour amongst criminal investigations is low, and it, therefore, also contributes to delict and the miscarriage of justice occurs.<br>Police Practice<br>M.A. (Criminal Justice)

Yan, Changqing.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2009.<br>Includes bibliographical references (leaves 92-97).<br>Abstracts in English and Chinese.<br>ABSTRACT (ENGLISH) --- p.iii<br>ABSTRACT (CHINESE) --- p.iv<br>ACKNOWLEDGMENTS --- p.v<br>LIST OF ABBREVIATIONS --- p.vi<br>TABLE OF CONTENTS --- p.vii<br>LIST OF FIGURES --- p.ix<br>LIST OF TABLES --- p.x<br>Chapter CHAPTER ONE: --- BACKGROUND INFORMATION --- p.1<br>Chapter 1.1 --- Introduction --- p.1<br>Chapter 1.2 --- Deoxyribonucleic Acid --- p.2<br>Chapter 1.3 --- DNA Studies --- p.9<br>Chapter 1.4 --- Cisplatin Studies --- p.11<br>Chapter 1.5 --- Scope of the Thesis --- p.13<br>Chapter CHAPTER TWO: --- METHODOLOY AND COMPUTATION --- p.16<br>Chapter 2.1 --- Introduction --- p.16<br>Chapter 2.2 --- Molecular Dynamics Simulation --- p.16<br>Chapter 2.3 --- Quantum Mechanics Calculation --- p.23<br>Chapter 2.4 --- Verification of Methodology --- p.25<br>Chapter 2.4.1 --- Backbone Torsion Angles --- p.25<br>Chapter 2.4.2 --- N7-N7 Distance --- p.30<br>Chapter 2.4.3 --- Location of HOMO --- p.33<br>Chapter 2.5 --- Summary --- p.35<br>Chapter CHAPTER THREE: --- UNDERSTANDING OF THE CISPLATIN-DNA CROSSLINKS --- p.36<br>Chapter 3.1 --- Introduction --- p.36<br>Chapter 3.2 --- MO Analysis --- p.37<br>Chapter 3.3 --- Potential Binding Products with the Ligand --- p.37<br>Chapter 3.3.1 --- "1,2-d(GpG) Intrastrand Crosslink" --- p.43<br>Chapter 3.3.2 --- "l,2-d(ApG) Intrastrand Crosslink" --- p.43<br>Chapter 3.3.3 --- "l,3-d(GpXpG) Intrastrand Crosslink" --- p.44<br>Chapter 3.3.4 --- d(GpC)d(GpC) Interstrand Crosslink --- p.44<br>Chapter 3.3.5 --- d(GpXpC)d(GpXpC) Interstrand Crosslink --- p.44<br>Chapter 3.3.6 --- Summary --- p.45<br>Chapter 3.4 --- Potential Binding Products Analysis --- p.47<br>Chapter 3.4.1 --- Site Identification Convention --- p.47<br>Chapter 3.4.2 --- Potential Binding Products Analysis --- p.48<br>Chapter 3.4.3 --- Applications --- p.53<br>Chapter 3.5 --- Cisplatin-DNA Crosslink Products Analysis --- p.56<br>Chapter 3.5.1 --- "1,2-d(GpG) and l,2-d(ApG) Intrastrand Crosslinks" --- p.61<br>Chapter 3.5.2 --- "l,3-d(GpXpG) Intrastrand and d(GpXpC)d(GpXpC) Interstrand Crosslinks" --- p.62<br>Chapter 3.5.3 --- d(GpC)d(GpC) Interstrand Crosslinks --- p.63<br>Chapter 3.5.4 --- Platination at Terminal Positions --- p.65<br>Chapter 3.6 --- Summary --- p.65<br>Chapter CAHPTER FOUR: --- CONCLUDING REMARKS --- p.67<br>APPENDIX I: BACKBONE TORSION ANGLES AND SUGAR RING CONFORMATIONS OF THE OPTIMIZED GEOMETRIES --- p.69<br>APPENDIX II: BACKBONE TORSION ANGLES OF THE EXPERIMENTAL SEQUENCES FROM NUCLEIC ACID DATABASE (NDB) --- p.77<br>REFERENCES --- p.92

The application of Molecular Biochemistry for transfection studies in eukaryotic systems is well documented. Of the numerous methods employed for the introduction of foreign DNA into eukaryotic cells, the use of low density lipoproteins (LDL) as carriers of DNA into cells has not been reported. LDL was isolated, characterized with respect to its protein and lipid components, and then variously modified in an attempt to enhance its affinity for DNA. It was found that both unmodified and modified LDL could interact with DNA, at physiological pH. The carbodiimide modified LDL (ECDI - LDL) showed the greatest affinity for DNA. LDL and ECDI - LDL were used to study LDL receptor binding in skin fibroblasts. This was followed by a study of receptor binding activities of both unmodified LDL and ECDI - LDL complexed to DNA (pBR322). Although the extent of binding of ECDI - LDL and ECDI - LDL - DNA complexes to plasma membranes was greater, the internalization and degradation of both modified and unmodified LDL complexes were equivalent. This additional binding was attributed to non - receptor - specific affinity of the carbodiimide modified complexes for the plasma membrane. The transfection of foreign DNA into eukaryotic cells in culture was monitored by assaying for the expression of the cloning vector, pSV2cat, complexed to LDL or ECDI - LDL and introduced into the cells by LDL receptor - mediated endocytosis. Of the cell lines in which the expression of the pSV2cat recombinant DNA was monitored, the human lung fibroblasts showed the greatest activity of the expressed chloramphenicol acetyl transferase enzyme. Although transfection efficiency was lower than that of the calcium phosphate - DNA coprecipitation procedure, the LDL receptor - mediated transfection of eukaryotic cells was carried out under physiological conditions and may be applicable in vivo.<br>Thesis (Ph.D)-University of Durban-Westville, 1987.

Genetic polymorphisms can be seen as the occurrence of more than one form of a DNA- or protein sequence at a single locus in a group of organisms, where these different forms occur more frequently than can be attributed to mutation alone. The combination of genetic polymorphisms present in the genome of a particular individual is referred to as its genotype. A wide range of genotyping techniques have been developed to detect and visualize genetic polymorphisms. One such technique examines highly polymorphic repetitive DNA regions called microsatellites, also called “short tandem repeats” (STRs) and sometimes “simple sequence repeats” (SSRs) or “simple-sequence length polymorphisms” (SSLPs). A microsatellite region consists of a DNA sequence of identical units of usually 2-6 base pairs strung together to produce highly variable numbers of tandem repeats among individuals of a population. Microsatellite genotyping is a popular choice for many types of studies including individual identification, paternity testing, germplasm evaluation, genome mapping and diversity studies and can be used in many commercial, academic, social, and agricultural applications. There are, however, many obstacles in effectively managing and analysing microsatellite genotype data. Currently, researchers are struggling to effectively manage and analyse rapidly growing volumes of genotyping data. Management problems range from simply the lack of a secure, easily accessible central data repository to more complex issues like the merging and standardization of data from multiple sources into combined datasets. Due to these issues, genetic fingerprinting applications such as identity matching and relatedness studies can be challenging when data from different experiments or laboratories have to be combined into a central database. The main aim of this M.Sc study in Bioinformatics was to develop a bioinformatics resource for the management and analysis of genetic fingerprinting data from microsatellite marker genotyping studies, and to apply the software to the analysis of microsatellite marker data from ramets of Pinus patula clones with the purpose of analysing clonal identity in pine breeding programmes. The software resource developed here is called GenoSonic. It is a web application that provides users with a secure, easily accessible space where genotyping project data can be managed and analysed as a team. Users can upload and download large amounts of marker genotype data. Once uploaded to the system, DNA fingerprint data needs to be standardised before it can be used in further analyses. To do this, a two-step approach was implemented in GenoSonic. The first step is to assign standardized allele sizes to all of the input allele sizes of the microsatellite fingerprints automatically using a novel automated binning algorithm called CSMerge-1, which was designed specifically to bin data from multiple experiments. The second step is to manually verify the results from the automated binning function and add the verified data to a standardized dataset. Once the genetic fingerprints have been standardized, allele- and genotype frequencies can be viewed for any given marker. GenoSonic also provides functionalities for identity matching. One or more DNA fingerprints from unknown samples can be matched against a standardized dataset to establish identities or infer relatedness. Finally, GenoSonic implements a genetic distance tree construction function, which can be used to visualize relatedness among samples in a selected dataset. The bioinformatics resource developed in this study was applied to a microsatellite DNA fingerprinting project aimed at the re-establishment or confirmation of clonal identity of Pinus patula ramets from pine clonal seed orchards developed by a South African forestry company at one of their new agricultural estates in South Africa. The results from GenoSonic‟s automated binning function (CSMerge-1) and the results from the identity matching and tree construction exercise were compared to results obtained by human experts who have analysed the data manually. It was demonstrated that the results from GenoSonic equalled or surpassed the manual results in terms of accuracy and consistency, and far surpasses the manual effort in terms of the speed at which analyses could be completed. GenoSonic was developed with specific focus on reusability, and the ability to be modified or extended to solve future genotyping-related problems. This study not only provides a solution to current genotype data management and analysis needs of researchers, but is aimed at serving as a basic framework, or component library for future software development projects that may be required to address specific needs of researchers dealing with high-throughput genotyping data.<br>Dissertation (MSc)--University of Pretoria, 2011.<br>Biochemistry<br>unrestricted

The correction of human genetic disorders by transfer of genetic material to cells is under intensive investigation in a number of 1aboratories. One possible way of trying to achieve the transfer of nucleic acid is by attaching DNA to a protein which has specific receptors on cells and which undergoes receptor-mediated endocytosis. In order to make use of the ligand protein-receptor approach for DNA transfer, iron-loaded human serum transferrin has been modified with the water soluble carbodiimides N-ethy1-N I -(3-dilllethy1aminopropyl) carbodiimide (CDI) and its quaterary analogue (ECDI) to give modified N-acy1urea transferrins. N-Acy1urea CDI (Fe 3+) transferrin and N-acy1urea CDI (Fe ) transferrin have been found to interact with and bind DNA in a reversible manner which i! dependent on ionic strength. [1251] N-Acy1urea CDI+(Fe3+) transferrin binds to transferrin receptors on Hea cells in culture and undergoes internalization through receptor-mediated endocytosis. Binding of the modified transferrin in the presence of calf thymus DNA to transferrin receptors also takes place. However, although internalization in the presence of DNA doe! appear to take place, the results of the internalization are not fully understood. Transfection studies with N-acy1urea CDI (Fe ) transferrin and plasmid pBR322 DNA as well as plasmid ptkNEO DNA complexes in the HeLa cell system are reported. The results of a number of transfection experiments suggests that N-acy1urea transferrins are capable of transfecting DNA (ptkNEO DNA), carrying genes for resistance to the antibiotic Geneticin (G41S) in the HeLa cell system. However, further development of the transfection system is necessary in order that consistantly reproducible results may be achievd.<br>Thesis (M.Sc.)-University of Durban-Westville, 1986.

Thymidine oligomers were chemically synthesised and linked to available amino functions of transferrin in alternative orientations: (a) A CMP residue attached to the 3' end of (pT)₁₀ with terminal deoxynucleotidyl transferase was oxidised with NaI0 and linked to transferrin via a Schiff base formation. (b) The 5' terminal phosphate group of (pT)₅ was activated with imidazole and reacted with transferrin to form a phosphoramide bond. The (pT)₅ thus attached to the protein was elongated to (pT)₃₀₀ using terminal deoxnucleotidyl transferase and TTP. The latter conjugate was capable of hybridising poly(A) tailed linear PBR322 DNA. The binding of this hybridisation complex to the transferrin receptor on various cell types was investigated.<br>Thesis (M.Sc.)-University of Durban-Westville, 1986.

by Lau Tze Chin, Gene.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 1991.<br>Includes bibliographical references (Leaves 170-195).<br>Abstract --- p.1<br>Acknowledgement --- p.3<br>List of tables --- p.4<br>List of figures --- p.6<br>List of abbreviations --- p.7<br>Chapter Chapter One - --- Introduction --- p.9<br>Chapter 1.1. --- Historical Aspects --- p.9<br>Chapter 1.2. --- Classification of hepatitis B virus --- p.12<br>Chapter 1.2.1. --- Hepadnaviruses --- p.12<br>Chapter 1.2.2. --- Comparative properties of hepadnaviruses --- p.13<br>Chapter 1.2.2.1. --- Physical properties --- p.13<br>Chapter 1.2.2.2. --- Genetic relatedness --- p.15<br>Chapter 1.2.2.3. --- Pathogenesis --- p.16<br>Chapter 1.3. --- Structural and morphological properties of HBV --- p.17<br>Chapter 1.4. --- Molecular biology of HBV --- p.20<br>Chapter 1.4.1. --- Molecular structure of HBV --- p.20<br>Chapter 1.4.1.1. --- Biochemistry of the virion envelope --- p.20<br>Chapter 1.4.1.2. --- The nucleocapsid --- p.21<br>Chapter 1.4.1.3. --- Structural features of HBV genome --- p.23<br>Chapter 1.4.2. --- Genetic organization of HBV --- p.24<br>Chapter 1.4.3. --- Infection cycle of HBV --- p.29<br>Chapter 1.4.3.1. --- Viral attachment and internalization --- p.29<br>Chapter 1.4.3.2. --- Replication of HBV --- p.30<br>Chapter 1.4.3.3. --- Gene expression and regulation --- p.31<br>Chapter 1.4.3.4. --- Host-virus DNA interaction --- p.33<br>Chapter 1.5. --- Epidemiology and transmission of HBV --- p.34<br>Chapter 1.5.1. --- World wide prevalence --- p.35<br>Chapter 1.5.1.1. --- HBsAg prevalence --- p.35<br>Chapter 1.5.1.2. --- Cumulative rate of HBV infection --- p.35<br>Chapter 1.5.1.3. --- Age specific pattern of HBV infection --- p.36<br>Chapter 1.5.2. --- Epidemiological pattern of HBV in Hong Kong --- p.37<br>Chapter 1.5.3. --- Mode of transmission --- p.38<br>Chapter 1.6. --- Clinical outcomes of HBV infection --- p.38<br>Chapter 1.6.1. --- Acute infection --- p.41<br>Chapter 1.6.2. --- Chronic infection --- p.42<br>Chapter 1.6.3. --- Primary hepatocellular carcinoma --- p.43<br>Chapter 1.7. --- Laboratory diagnosis of hepatitis B --- p.44<br>Chapter 1.7.1. --- The HBV markers --- p.47<br>Chapter 1.7.1.1. --- HBsAg and anti-HBs --- p.47<br>Chapter 1.7.1.2. --- HBcAg and Anti-HBc --- p.47<br>Chapter 1.7.1.3. --- HBeAg and anti-HBe --- p.49<br>Chapter 1.7.1.4. --- HBV-associated DM polymerase --- p.49<br>Chapter 1.7.1.5. --- HBV-DNA --- p.49<br>Chapter 1.7.2. --- Methodology in the detection of hepatitis B markers --- p.50<br>Chapter 1.7.2.1. --- Direct detection of HBV and HBV antigens --- p.50<br>Chapter 1.7.2.2. --- Serological detection of HBV markers --- p.51<br>Chapter 1.7.2.3. --- HBV-associated DNA polymerase assay --- p.51<br>Chapter 1.7.2.4. --- Molecular technique for the detection and quantitation of HBV-DNA --- p.52<br>Chapter 1.8. --- Antiviral therapy in hepatitis B --- p.52<br>Chapter 1.8.1. --- Therapeutic agents for treatment of HBV infection --- p.53<br>Chapter 1.8.1.1. --- Steroids --- p.53<br>Chapter 1.8.2.2. --- Nucleoside analogs --- p.54<br>Chapter 1.8.1.3. --- Interferon --- p.55<br>Chapter 1.8.2. --- Clinical trials of interferons --- p.55<br>Chapter 1.9. --- Extrahepatic tissue tropism of HBV --- p.62<br>Chapter 1.10. --- Objective and design of study --- p.65<br>Chapter 1.10.1. --- Objectives of study --- p.65<br>Chapter 1.10.2. --- Study design --- p.66<br>Chapter 1.10.2.1. --- Cross-sectional study --- p.67<br>Chapter 1.10.2.2. --- Longitudinal study --- p.67<br>Chapter 2.1. --- Materials --- p.71<br>Chapter 2.1.1. --- Patients recruitment and clinical materials --- p.71<br>Chapter 2.1.1.1. --- Cross-sectional study --- p.71<br>Chapter 2.1.1.2. --- Longitudinal study --- p.71<br>Chapter 2.1.2. --- Bacteria] stock --- p.71<br>Chapter 2.1.3. --- "Chemicals, equipments and consumables" --- p.72<br>Chapter 2.1.4. --- Buffers and solutions --- p.72<br>Chapter 2.1.4.1. --- Phosphate buffer saline (PBS) --- p.72<br>Chapter 2.1.4.2. --- Leucocyte lysis buffer (X 5)(LLB) --- p.72<br>Chapter 2.1.4.3. --- Buffer equilibrated phenol (BEP) --- p.76<br>Chapter 2.1.4.4. --- Phenol-Chloroform mixture --- p.76<br>Chapter 2.1.4.5. --- 3.0M sodium acetate (pH 5.2) --- p.76<br>Chapter 2.1.4.6. --- Tris-EDTA buffer (pH 8.0) (TE) --- p.76<br>Chapter 2.1.4.7. --- Stock salmom sperm DNA solution --- p.77<br>Chapter 2.1.4.8. --- Tracking dye --- p.77<br>Chapter 2.1.4.9. --- Tris-borate electrophoresis buffer (TBE) --- p.77<br>Chapter 2.1.4.10. --- Luria-Bertani Broth (LB) --- p.77<br>Chapter 2.1.4.11. --- Solution ] --- p.78<br>Chapter 2.1.4.12. --- Solution ]] --- p.78<br>Chapter 2.1.4.13. --- Potassium acetate buffer (pH 5.4) --- p.78<br>Chapter 2.1.4.14. --- Column elution buffer (CEB) --- p.78<br>Chapter 2.1.4.15. --- NPMEB solution --- p.79<br>Chapter 2.1.4.16. --- Neutralizing solution --- p.79<br>Chapter 2.1.4.17. --- Standard saline citrate (SSC) --- p.79<br>Chapter 2.1.4.18. --- Denhardt solution --- p.79<br>Chapter 2.1.4.19. --- Prehybridization solution (PS) --- p.80<br>Chapter 2.1.4.20. --- NETFAP Solution --- p.80<br>Chapter 2.1.4.21. --- Heparin solution --- p.81<br>Chapter 2.1.4.22. --- Hybridization mix for oligo-nucleotide probe --- p.81<br>Chapter 2.1.4.23. --- NEPS solution (pH 7.0) --- p.81<br>Chapter 2.1.4.24. --- Restriction endonuclease and buffer --- p.82<br>Chapter 2.2. --- Methods --- p.82<br>Chapter 2.2.1. --- Sample preparations --- p.82<br>Chapter 2.2.1.1. --- Isolation of plasma and peripheral blood leucocytes (PBL) --- p.82<br>Chapter 2.2.1.2. --- Extraction of DNA from Peripheral blood leucocytes --- p.83<br>Chapter 2.2.1.3. --- Quantitation of Peripheral blood leucocyte DNA --- p.83<br>Chapter 2.2.2. --- Preparation of radio-labelled HBV-DNA probe --- p.84<br>Chapter 2.2.2.1. --- Plating and selection of bacterial stock --- p.84<br>Chapter 2.2.2.2. --- Growth of E. coli HB101 and amplification of pAM6 --- p.84<br>Chapter 2.2.2.3. --- Harvesting of E. coli and extraction of plasmid pAM6 --- p.84<br>Chapter 2.2.2.4. --- Purification of plasmid pAM6 --- p.86<br>Chapter 2.2.2.5. --- Large scale isolation and purification of HBV genome from plasmid pAM6 --- p.86<br>Chapter 2.2.2.6. --- Radio-labelling of HBV-DNA --- p.88<br>Chapter 2.2.2.6.1. --- Nick-translation of total HBV-DNA genome --- p.88<br>Chapter 2.2.2.6.2. --- Multi-primer labelling of total HBV- DNA genome --- p.88<br>Chapter 2.2.2.6.3. --- End-labeling of 21-base HBV oligo- nucleotide --- p.88<br>Chapter 2.2.2.6.4. --- Determination of labelling efficiency --- p.89<br>Chapter 2.2.2.7. --- Purification of labelled HBV-DNA probe --- p.90<br>Chapter 2.2.2.7.1. --- Total genomic HBV-DNA probe (pAM6 probe) --- p.90<br>Chapter 2.2.2.7.2. --- Oligo-nucleotide HBV-DNA probe (oligo probe) --- p.90<br>Chapter 2.2.3. --- Hybridization study of clinical samples --- p.91<br>Chapter 2.2.3.1. --- Solution hybridization of sera samples --- p.91<br>Chapter 2.2.3.2. --- Spot hybridization of sera samples --- p.91<br>Chapter 2.2.3.2.1. --- "Pre-hybridization treatment of sera samples (adapted from Lin et al.,1987)" --- p.91<br>Chapter 2.2.3.2.2. --- Pre-hybridization and hybridization of the membrane --- p.92<br>Chapter 2.2.3.2.3. --- Washing of membrane --- p.92<br>Chapter 2.2.3.2.4. --- Final treatment and autoradiography: --- p.92<br>Chapter 2.2.3.3. --- Quantitation of HBV-DNA in the sera samples: --- p.93<br>Chapter 2.2.4. --- Assay for serological Hepatitis B marker --- p.93<br>Chapter Chapter Three - --- Results --- p.93<br>Chapter 3.1. --- Preparation of HBV-DNA probes --- p.95<br>Chapter 3.2. --- Radiolabelling of HBV-DNA --- p.95<br>Chapter 3.3. --- Hybridization methodology --- p.98<br>Chapter 3.4. --- Comparison of the performance of HBV-DNA probes --- p.100<br>Chapter 3.4.1. --- Quantitation of serum HBV-DNA --- p.100<br>Chapter 3.4.2. --- Comparative hybridization performance of different HBV-DNA probes --- p.105<br>Chapter 3.5. --- Clinical application of HBV-DNA probe:Detection of HBV-DNAin serum and peripheral blood leucocytes (PBL) --- p.109<br>Chapter 3.5.1. --- Cross-sectional study --- p.112<br>Chapter 3.5.1.1. --- Frequency of HBV-DNA detection in relation to different clinical manifestations --- p.112<br>Chapter 3.5.1.2. --- Frequency of HBV-DNA detection in relation to the serological status --- p.114<br>Chapter 3.5.1.3. --- Distribution of serum and PBL HBV-DNA level in chronic hepatitis B patients in relation to the different HBV-related manifestations --- p.119<br>Chapter 3.5.2. --- Longitudinal study of patients with chronic hepatitis B under interferon therapy with prednisolone pretreatment --- p.123<br>Chapter 3.5.2.1. --- Features of patients under study --- p.123<br>Chapter 3.5.2.2. --- Correlation between the occurrence of HBV- DNA and HBeAg in serum --- p.123<br>Chapter 3.5.2.3. --- Outcome of clinical trial: --- p.126<br>Chapter 3.5.2.3.1. --- Number of patients responding to therapy: --- p.126<br>Chapter 3.5.2.3.2. --- Variation in serum HBV markers during the course of study --- p.128<br>Chapter 3.5.2.3.3. --- Change of HBV-DNA statusin peripheral blood leucocytes --- p.134<br>Chapter Chapter Four - --- Dicussion --- p.140<br>Chapter 4.1. --- Preparation of HBV-DNA hybridization probes --- p.140<br>Chapter 4.1.1. --- Source of HBV-DNA --- p.140<br>Chapter 4.1.2. --- Raidolabelling of HBV-DNA --- p.141<br>Chapter 4.2. --- Hybridization methodology --- p.141<br>Chapter 4.2.1. --- Optimization of hybridization conditions --- p.141<br>Chapter 4.2.2. --- Comparison of the performance among different HBV- DNA probes --- p.144<br>Chapter 4.3. --- Detection of HBV-DNA in clinical serum samples --- p.148<br>Chapter 4.3.1. --- Crossectional study of patients with various categories of HBV related diseases --- p.148<br>Chapter 4.3.1.1. --- HBV-DNA detection in serum --- p.148<br>Chapter 4.3.1.2. --- Detection of HBV-DNA in peripheral blood mononuclear cells --- p.153<br>Chapter 4.3.2. --- Longitudinal studies of patients undergoing antiviral therapy --- p.159<br>Chapter 4.3.2.1. --- Serum HBV-DNA and HBeAg --- p.159<br>Chapter 4.3.2.2. --- HBV-DNA in peripheral blood leucocytes --- p.163<br>Conclusion --- p.166<br>Future perspectives --- p.168<br>References --- p.170

DNA is identified as a powerful tool in the solving of rape cases, but it is often destroyed either by members of the public or the police officials who attend to the scene. The aim of the study was to evaluate the management of DNA evidence in rape cases in the Bishop Lavis Policing Area. To address the research topic under investigation, research questions, a legal framework and policies were used. The outcome of the study indicated poor performance in securing the crime scene and ensuring that physical evidence is preserved and not tampered with. On this basis, it was recommended that developmental workshops and intensive training on the management of DNA evidence be conducted to all members of the South African Police Service attend to rape crime scenes. This should be done to equip them with knowledge and an understanding of the management of DNA evidence.<br>Police Practice<br>M. Tech. (Forensic Investigation)

Aufgrund einer komplexen Wechselwirkung mit Proteinen ist das Genom in einer Zelle ständig mechanischer Spannung und Torsion ausgesetzt. Daher ist es wichtig die Mechanik und die Dynamik von verdrillter DNA unter Spannung zu verstehen. Diese Situation wurde experimentell mittels einer sog. magnetischen Pinzette nachgestellt, indem sowohl Kraft als auch Drehmoment auf ein einzelnes DNA Molekül ausgeübt und gleichzeitig die mechanische Antwort des Polymers aufgezeichnet wurde. Als erstes Beispiel wurde der Übergang von linearer zu sog. plectonemischer DNA untersucht, d.h. die Absorption eines Teils der induzierten Verdrillung in einer superhelikalen Struktur. Eine abrupte Längenänderung am Anfang dieses Übergangs wurde bereits im Vorfeld publiziert. In der vorliegenden Arbeit wird gezeigt, dass diese abrupte DNA Verkürzung insbesondere von der Länge der DNA und der Ionenkonzentration der Lösung abhängt. Dieses Verhalten kann mittels eines Modells verstanden werden, in dem die Energie pro Verwringung der ersten Schlinge innerhalb der Superhelix größer ist als die aller nachfolgenden. Des Weiteren wurden DNA-DNA Wechselwirkungen in der Umgebung monovalenter Ionen durch die Analyse des Superspiralisierungsverhaltens einzelner DNA Moleküle bei konstanter Kraft charakterisiert. Solche Wechselwirkungen sind für die Kompaktierung des Genoms und die Regulation der Transkription wichtig. Oft wird DNA als gleichmäßig geladener Zylinder modelliert und ihre elektrostatischen Wechselwirkungen im Rahmen der Poisson-Boltzmann-Gleichung mit einem Ladungsanpassungsfaktor berechnet. Trotz erheblicher Anstrengung ist eine präzise Bestimmung dieses Parameters bisher nicht gelungen. Ein theoretisches Modell dieses Prozesses zeigte nun eine erstaunlich kleine effektive DNA Ladung von ~40% der nominalen Ladungsdichte. Abgesehen von Gleichgewichtsprozessen wurde auch die Dynamik eines Faltungsvorgangs von DNA untersucht. Spontane Branch Migration einer homologen Holliday-Struktur wurde genutzt, um die intramolekulare Reibung der DNA zu erforschen. Mittels einer magnetischen Pinzette wurde eine torsionslimitierte Holliday-Struktur gestreckt während die Längenfluktuationen der Zweige mit schneller Videomikroskopie bei ~3 kHz aufgezeichnet wurden. Einzelne diffusive Schritte der Basenpaare sollten auf einer sub-Millisekunden Zeitskala auftreten und viel kleiner als die Gesamtfluktuationen der DNA sein. Eine Analyse der spektralen Leistungsdichte der Längenfluktuationen ermöglicht eine eindeutige Beschreibung der Dynamik der Branch Migration. Die Holliday-Struktur wurde außerdem als nanomechanischer Linearversteller eingesetzt, um einen einzelnen fluoreszierenden Quantenpunkt durch ein exponentiell abfallendes evaneszentes Feld zu bewegen. Durch die Aufzeichnung der Emission des Quantenpunkts sowohl in dem evaneszenten Feld als auch unter gleichmäßiger Beleuchtung kann die Intensitätsverteilung des Anregungsfelds ohne weitere Dekonvolution bestimmt werden. Diese neue Technik ist von besonderem wissenschaftlichen Interesse, weil die Beschreibung dreidimensionaler inhomogener Beleuchtungsfelder eine große Herausforderung in der modernen Mikroskopie darstellt. Die Ergebnisse dieser Arbeit werden dem besseren Verständnis einer Vielzahl biologischer Prozesse, die in Verbindung mit DNA Superspiralisierung stehen, dienen und weitere technische Anwendungen des DNA-basierten Linearverstellers hervorbringen.<br>The genome inside the cell is continuously subjected to tension and torsion primarily due to a complex interplay with a large variety of proteins. To gain insight into these processes it is crucial to understand the mechanics and dynamics of twisted DNA under tension. Here, this situation is mimicked experimentally by applying force and torque to a single DNA molecule with so called magnetic tweezers and measuring its mechanical response. As a first example a transition from a linear to a plectonemic DNA configuration is studied, i.e. the absorption of part of the applied twist in a superhelical structure. Recent experiments revealed the occurrence of an abrupt extension change at the onset of this transition. Here, it is found that this abrupt DNA shortening strongly depends on the length of the DNA molecule and the ionic strength of the solution. This behavior can be well understood in the framework of a model in which the energy per writhe for the initial plectonemic loop is larger than for subsequent turns of the superhelix. Furthermore DNA-DNA interactions in the presence of monovalent ions were comprehensively characterized by analyzing the supercoiling behavior of single DNA molecules held under constant tension. These interactions are important for genome compaction and transcription regulation. So far DNA is often modeled as a homogeneously charged cylinder and its electrostatic interactions are calculated within the framework of the Poisson-Boltzmann equation including a charge adaptation factor. Despite considerable efforts, until now a rigorous quantitative assessment of this parameter has been lacking. A theoretical model of this process revealed a surprisingly small effective DNA charge of ~40% of the nominal charge density. Besides describing equilibrium processes, also the dynamics during refolding of nucleic acids is investigated. Spontaneous branch migration of a homologous Holliday junction serves as an ideal system where the friction within the biomolecule can be studied. This is realized by stretching a torsionally constrained Holliday junction using magnetic tweezers and recording the length fluctuations of the arms with high-speed videomicroscopy at ~3 kHz. Single base pair diffusive steps are expected to occur on a sub-millisecond time scale and to be much smaller than the overall DNA length fluctuations. Power-spectral-density analysis of the length fluctuations is able to clearly resolve the overall dynamics of the branch migration process. Apart from studying intramolecular friction, the four-arm DNA junction was also used as a nanomechanical translation stage to move a single fluorescent quantum dot through an exponentially decaying evanescent field. Recording the emission of the quantum dot within the evanescent field as well as under homogeneous illumination allows to directly obtain the intensity distribution of the excitation field without additional deconvolution. This new technique is of particular scientific interest because the characterization of three-dimensional inhomogeneous illumination fields is a challenge in modern microscopy. The results presented in this work will help to better understand a large variety of biological processes related to DNA supercoiling and inspire further technical applications of the nanomechanical DNA gear.

The recent advent of bioinformatics has given rise to the central and recurrent problem of optimally aligning biological sequences. Many techniques have been proposed in an attempt to solve this complex problem with varying degrees of success. This thesis investigates the application of a computational intelligence technique known as particle swarm optimization (PSO) to the multiple sequence alignment (MSA) problem. Firstly, the performance of the standard PSO (S-PSO) and its characteristics are fully analyzed. Secondly, a scalability study is conducted that aims at expanding the S-PSO’s application to complex MSAs, as well as studying the behaviour of three other kinds of PSOs on the same problems. Experimental results show that the PSO is efficient in solving the MSA problem and compares positively with well-known CLUSTAL X and T-COFFEE.<br>Dissertation (MSc)--University of Pretoria, 2009.<br>Computer Science<br>Unrestricted

Genes comprised of DNA (Deoxyribonucleic Acid) molecules contain the blueprint of any living organism. Expression levels of genes are the measures of genes activity under certain biochemical conditions. DNA microarray technology is widely used to measure gene expression levels and study gene interactions. This technology provides access to thousands of genes at once by recording expression levels simultaneously. The data generated by microarray technology is important because it has been shown that from gene expression changes we are able to extract different types of cancer information. Support Vector Machine (SVM) algorithms have recently been extensively applied to the DNA microarray applications. However, cancer classification using gene expression data with SVM is a non trivial task due to the very nature of this kind of data as they have very high dimensionality, usually in the order of thousands to tens of thousands of genes. The situation is more complicated with comparatively few number of sample sizes- usually below hundred. The high dimensionality of the features and low population size usually cause over-fitting of the classifier. Problem of feature selection is, hence, an important issue in this research context. Feature selection process comprises selecting relevant features and eliminating irrelevant and redundant features from the data, and training the SVM classifier on reduced dimensionality. The objective of the current research was to introduce an effective solution for the feature selection problem. The preliminary set of experiments investigated the impact of feature selection on the SVM performance. The experimental outcome showed that feature selection had a positive impact on classification accuracy of SVM. Subsequently this research addressed the solution of feature selection problem through the clustering based approach. The key idea of the proposed FSFC (Feature Selection through Feature Clustering) method was to transpose the gene expression data matrix and to consider the resultant rows as the points in a reduced dimensional space. An optimal number of clusters through the application of the well known k-means clustering method were made from these data points. Those points were selected in the final subset of features whose distances were greater than the average distances of the points from the cluster centers of the respective clusters. The performance of this novel strategy was studied extensively with other standard approaches on various benchmark gene expression datasets. Experiments on real world datasets showed that this method was computationally less expensive than the other standard approaches and selected moderately less features for which the SVM classification accuracy was relatively high. The advantage of this method was that no parameter tuning was required for the purpose of feature selection but dealt effectively with the high dimensional nature of the gene expression data. The next steps of the current research introduced three simple but intuitive hybrid approaches of feature selection based on FSFC. These hybrid approaches achieved further reduction in data dimensionality while maintained, or in some cases improved, the SVM classification accuracy. These three hybrid feature selection methods, namely, Combined Distance Metrics, FSFC followed by SVM-RFE (Support Vector Machine - Recursive Feature Elimination) and Recursive FSFC, clearly showed the suitability of the proposed FSFC method in hybrid feature selection paradigm. The determination of the impact of feature selection on the SVM, proposed FSFC - parameterless feature selection environment and the hybrid approaches of feature selection based on FSFC will make statistical learning theorem SVM more effective in the domain of microarray analysis for cancer biology.<p></p>

In this Diploma Thesis, an interaction of genotoxic environmental pollutant 2-nitrofluorene with a double-stranded calf thymus DNA has been studied using a hanging mercury drop electrode (HMDE) as an electrochemical sensor. Two types of DNA damage were investigated and electrochemically detected (using cyclic voltammetry and differential pulse voltammetry): (i) The DNA damage caused by the direct interaction with 2-nitrofluorene and (ii) the DNA damage caused by short-lived radicals generated by the electrochemical reduction of the nitro group in 2-nitrofluorene. For the study of direct interaction, HMDE was modified by DNA and the interaction of DNA with 2-nitrofluorene was studied, after their incubation, right at the HMDE surface (adsorptive transfer stripping technique) or the DNA was preincubated with 2-nitrofluorene and, subsequently, the interaction was studied voltammetrically (DNA titration technique). Using both detection techniques, the formation of DNA - 2-nitrofluorene complex was observed and the mutual interaction was interpreted as an intercalation between the DNA base pairs, although such interaction was not clearly confirmed by UV-visible absorption spectroscopy. An electrostatic binding of 2-nitrofluorene on DNA sugar-phosphate backbone was partially formed at low concentrations of...

Ho Cheuk-nang.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.<br>Includes bibliographical references (leaves 89-94).<br>Abstracts in English and Chinese.<br>Acknowledge --- p.ii<br>Table of Contents --- p.iii<br>Lists of Tables --- p.vi<br>Lists of Figures --- p.vii<br>Abstract (English Version) --- p.x<br>Abstract (Chinese Version) --- p.xii<br>Chapter 1. --- Literature Survey of Phosphorus Chemical Shift Studies of DNA --- p.1<br>Chapter 1.1 --- Introduction --- p.1<br>Chapter 1.2 --- General Review of DNA Structures --- p.2<br>Chapter 1.2.1 --- "Nomenclature, Symbols and Numbering Scheme" --- p.2<br>Chapter 1.2.2 --- Conformations of DNA --- p.7<br>Chapter 1.2.3 --- Random Coil State --- p.8<br>Chapter 1.3 --- Phosphorus Chemical Shift Studies of DNA --- p.9<br>Chapter 1.4 --- Purpose of This Work --- p.10<br>Chapter 2. --- Methodology for Studying the Sequence Effect on Random Coil Phosphorus Chemical Shift --- p.12<br>Chapter 2.1 --- Introduction --- p.12<br>Chapter 2.2 --- Determination of Sequence Effect --- p.12<br>Chapter 2.3 --- Design of Sequence --- p.13<br>Chapter 2.4 --- Sample Preparation --- p.14<br>Chapter 2.5 --- Measurement of Phosphorus Chemical Shift --- p.16<br>Chapter 2.5.1 --- Proton Resonance Assignments --- p.16<br>Chapter 2.5.2 --- Phosphorus Resonance Assignments --- p.20<br>Chapter 2.6 --- Determination of Sugar Conformation --- p.23<br>Chapter 2.7 --- Determination of Backbone Conformation --- p.25<br>Chapter 3. --- Results and Discussion of Random Coil Phosphorus Chemical Shift of DNA --- p.28<br>Chapter 3.1 --- Introduction --- p.28<br>Chapter 3.2 --- Resonance Assignments --- p.28<br>Chapter 3.3.1 --- Proton Resonance Assignments --- p.31<br>Chapter 3.3.2 --- Phosphorus Resonance Assignments --- p.31<br>Chapter 3.3 --- Verification of Random Coil State --- p.31<br>Chapter 3.2.1 --- Variable Temperature Proton Chemical Shift --- p.31<br>Chapter 3.2.2 --- Sugar Conformation --- p.32<br>Chapter 3.2.3 --- Backbone Conformation --- p.33<br>Chapter 3.4 --- Random Coil Phosphorus Chemical Shift of DNA --- p.33<br>Chapter 3.4.1 --- Temperature Effect --- p.33<br>Chapter 3.4.2 --- Neighbor Effect --- p.36<br>Chapter 3.5 --- Random Coil Phosphorus Chemical Shift Prediction --- p.42<br>Chapter 3.5.1 --- Dimer Model Prediction Protocol --- p.42<br>Chapter 3.5.2 --- Trimer Model Prediction Protocol --- p.45<br>Chapter 4. --- Conclusions and Future Work --- p.48<br>Appendix I H6/H8-H1' regions of 2D NOESY spectra of SS1-SS16 --- p.49<br>"Appendix II 'H and 31P resonance assignments and 3Jhi1,h2,3Jh1，h2´ح and 3Jh3，p coupling constants of SS1-SS16" --- p.57<br>Appendix III H3' regions 1H-31P HSQC spectra of SS1-SS16 --- p.65<br>Appendix IV H1，-H2，/H2´حregions of DQF-COSY spectra of SS1- SS16 --- p.73<br>Appendix V H3' regions of 1H-31P selective heteronuclear COSY spectra of SS1-SS16 --- p.81<br>References --- p.89

Zhong, Yangliu.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2011.<br>Includes bibliographical references (leaves 50-57).<br>Abstracts in English and Chinese.<br>Title Page --- p.i<br>Thesis Committee --- p.ii<br>Abstract (English Version) --- p.iv<br>Abstract (Chinese Version) --- p.V<br>Acknowledgement --- p.vi<br>Table of Contents --- p.viii<br>List of Tables --- p.X<br>List of Figures --- p.xii<br>List of Abbreviations and Symbols --- p.xiii<br>Chapter 1 --- Introduction --- p.1<br>Chapter 1.1 --- DNA methylation --- p.1<br>Chapter 1.2 --- Repair of m3T --- p.2<br>Chapter 1.3 --- Objectives of this work --- p.3<br>Chapter 1.4 --- DNA structure --- p.3<br>Chapter 1.4.1 --- Nomenclature scheme for DNA --- p.3<br>Chapter 1.4.2 --- Base pair scheme --- p.4<br>Chapter 1.4.3 --- Sugar conformation --- p.5<br>Chapter 1.4.4 --- Backbone conformation --- p.7<br>Chapter 2 --- Materials and Methods --- p.9<br>Chapter 2.1 --- Sample design --- p.9<br>Chapter 2.2 --- Sample preparation --- p.10<br>Chapter 2.3 --- NMR analysis --- p.10<br>Chapter 2.3.1 --- Resonance assignment --- p.12<br>Chapter 2.3.2 --- Determination of sugar conformation --- p.13<br>Chapter 2.3.3 --- Determination of backbone conformation --- p.14<br>Chapter 2.4 --- UV melting study --- p.15<br>Chapter 3 --- Effect of m3T on Double-Helical Structures and Stabilities --- p.17<br>Chapter 3.1 --- Resonance assignments --- p.17<br>Chapter 3.2 --- Effect of m3T on double-helical DNA structures --- p.19<br>Chapter 3.2.1 --- Base pairing mode --- p.19<br>Chapter 3.2.2 --- Sugar conformation --- p.21<br>Chapter 3.2.3 --- Backbone conformation --- p.22<br>Chapter 3.3 --- Effect of m3T on double-helical DNA stabilities --- p.25<br>Chapter 3.4 --- Discussion --- p.26<br>Chapter 3.4.1 --- Single-strand requirement in FTO repair --- p.26<br>Chapter 3.4.2 --- Relationship between m3T pairing structure and stability --- p.27<br>Chapter 4 --- Effect of m3T Mispair on Double-Helical DNA Structures and Stabilities --- p.28<br>Chapter 4.1 --- Resonance assignments --- p.28<br>Chapter 4.2 --- Effect of m3T mispair on double-helical DNA structures --- p.32<br>Chapter 4.2.1 --- Pairing mode of T m3T --- p.34<br>Chapter 4.2.2 --- Pairing mode of G m3T --- p.35<br>Chapter 4.2.3 --- Pairing mode of C.m3T --- p.35<br>Chapter 4.3 --- Effect of m3T mispair on double-helical DNA stabilities --- p.36<br>Chapter 4.4 --- Discussion --- p.36<br>Chapter 4.4.1 --- Predominant mutation --- p.37<br>Chapter 4.4.2 --- Relationship between m3T pairing structure and stabilities --- p.37<br>Chapter 5 --- Conclusion and Future Work --- p.39<br>Chapter Appendix I --- Proton chemical shift values (ppm) of AmT --- p.40<br>Chapter Appendix II --- Proton chemical shift values (ppm) of RefAT --- p.41<br>Chapter Appendix III --- Proton chemical shift values of NmT samples --- p.42<br>Chapter Appendix IV --- "Σ1' and %S of TmT, GmT and CmT" --- p.45<br>Chapter Appendix V --- "1H-31P HSQC spectra of (a) TmT, (b) GmT and (c) CmT" --- p.46<br>Chapter Appendix VI --- "1H-31P COSY spectra of (a) TmT, (b) GmT and (c) CmT" --- p.47<br>Chapter Appendix VII --- "31P chemical shifts, 3JH3'P and %Bi of TmT, GmT and CmT" --- p.48<br>Chapter Appendix VIII --- "UV melting curves of RefAT, AmT, TmT, GmT and CmT" --- p.49<br>References --- p.50