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Molecular characterization of IBDV-induced apoptosis in vitro using cDNA microarraysWong, Tsz-yeung. January 2005 (has links)
Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.
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Improved Techniques for High-Throughput Molecular DiagnosticsCurcio, Mario January 2002 (has links)
The amount of information derived from sequencing the humangenome is leading to an exponential increase in the rate atwhich genes and genetic disorders are mapped and characterized.As a consequence, the demand for genetic testing is alsodramatically increasing. Screening and assaying methods, otherthan direct sequencing, are gradually becoming available,although with different robustness, sensitivity and throughput.In the work summarized in this thesis, attention was devotedprimarily to the improvement and the development of newtechniques for some of these methods. Considering the role of capillary electrophoresis inmolecular diagnostics and an associated important phenomenon -electroosmotic flow (EOF) - a robust, reproducible procedurefor surface modification of the inner wall of capillaries wasreported (Paper I) and this method was beneficially employed insubsequent projects. The effort to screen and characterize point mutations in theCACNA1A gene, responsible for the Familial Hemiplegic Migraine(FHM) disease, led to the optimization and validation of a verysensitive technique on slab gel called Double GradientDenaturing Gradient Gel Electrophoresis (DG-DGGE) (Paper II). A more reliable and robust method forSingle-Strand Conformation Polymorphism analysis (SSCP) bycapillary electrophoresis, making use of neutral pH buffers,was also developed (Paper III), while the next project resulted in thedevelopment of a high-throughput method for assaying knownpolymorphisms by multiplex solid-phase minisequencing inmulti-capillary format using a detection system based on liquidcore waveguiding (Paper IV). As these and other methods, as well as most applications inmolecular diagnostics and molecular biology, depend on thepolymerase chain reaction (PCR), an effort was made to enhancethe throughput of this technology and to minimize reactionvolumes and costs. For this, the concept of a dynamic reactorwas employed, instead of static systems where the reactionmixture is exposed to temperature cycles in a confined space. Acontinuous flow of small-volume reaction mixtures, separated byan immiscible hydrophobic carrier fluid such as aperfluorocarbon, is transported in a hydrophobic tube throughthree zones, which are kept at constant temperatures optimizedfor denaturation, annealing and elongation (Paper V). If combined with a technique for automatedsample loading and collection (Chapter 7), this method should allow veryhigh-throughput miniaturized DNA amplification. A samplehandling concept using hydrophilic anchors is proposed, whichshould also be useful for other miniaturized reactions andchemical processing. Finally, some possible alternative methods are discussed aswell as future trends. <b>Keywords:</b>Amplification, anchor, array, assay,capillary, DGGE, disease, disorder, DNA, droplet, dynamicreactor, electrophoresis, fluorescence, fluorocarbon, gel,genetic alteration, genomics, genotyping, high-throughput,hydrophobic, liquid lid, miniaturized reaction, minisequencing,molecular diagnostics, mutation, PCR, polymerase chainreaction, screening, segmented flow, single nucleotidepolymorphism, SNP, SSCP, test.
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The effects of pH on the torsional flexibility of DNA bound to a nucleosome core particleWinzeler, Elizabeth A. 20 July 1990 (has links)
The effects of pH on the torsional flexibility of DNA bound to a
nucleosome core particle were investigated by studying the time-resolved
fluorescence anisotropy decays of ethidium bromide intercalated into the DNA
of the core particle. As the torsional flexibility of DNA is affected by the
presence of an intercalating dye, the decays were studied at different ethidium
bromide to core particle binding ratios. The anisotropy decays were collected
using the method of time-resolved single-photon counting and were fit to a
model developed by J. M. Schurr (Schurr, 1984) using a non-linear least
squares fitting algorithm developed by the author for this purpose. It was
shown that below a binding ratio of 0.1 there was no demonstrable change in
the anisotropy as a function of binding ratio. Our results show, that the
apparent torsional flexibility of DNA of to a nucleosome core particle is
dependent on the number of base pairs of the DNA between points of
attachment to the histone core. If this number is as high as 30 base pairs, then
the torsional flexibility of DNA on a nucleosome core particle is as high or
higher than DNA free in solution. Also, for reasonable values of N, the friction
felt by the DNA on a core particle is much higher than that felt by free DNA.
This indicates that the DNA on a core particle is highly constrained in its
motions. The hydrogen ion concentration was shown to have a substantial
effect on the fluorescent anisotropy decays, particularly in the early regions of
the decay. These analyses indicated that the observed change could be
attributed to either a loosening of the contacts between the DNA and the
histone core, or a relaxing of the torsional flexibility of the DNA. / Graduation date: 1991
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Characterization of the DNA Damage Resistance Gene RTT107Roberts, Tania 28 July 2008 (has links)
In Saccharomyces cerevisiae, RTT107 (ESC4, YHR154W) encodes a BRCT-domain protein that is important for recovery from DNA damage during S phase. I have found that Rtt107 forms a complex with the Slx1/Slx4 structure-specific nuclease. Deletion of SLX4 confers many of the same phenotypes observed in rtt107∆, including DNA damage sensitivity, prolonged DNA damage checkpoint activation, and increased spontaneous DNA damage, suggesting that Slx4 and Rtt107 function in concert. These defects are not shared by Slx1 indicating that the function of Slx4 and Slx1 in the DNA damage response is not entirely overlapping. Furthermore, I found that Slx4 regulates the phosphorylation of Rtt107 by the checkpoint kinase Mec1.
The phenotypes conferred by deletion of RTT107 and the spectrum of its synthetic genetic interactions indicates that Rtt107 may function at stalled replication forks. I have shown that Rtt107 is recruited to chromatin in the presence of DNA damaging agents that cause DNA replication forks to stall. Recruitment of Rtt107 to chromatin requires Rtt109, an acetyltransferase, and the cullin Rtt101, but is not dependent on Slx4 or the checkpoint kinases. Rtt109 acetylates histone H3 on lysine 56 (H3-K56), yet recruitment of Rtt107 to chromatin does not require acetylation of H3-K56, indicating that Rtt109 may have additional targets. Chromatin immunoprecipitation indicates that the sites of Rtt107 binding correspond to regions at or near stalled replication forks throughout the genome. I propose that Rtt107 acts in the recovery from DNA damage by localizing to stalled replication forks and acting as a scaffold for assembly of DNA damage response proteins, ultimately promoting replication fork restart.
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Characterization of APLF in the Nonhomologous End-joining PathwayMacrae, Chloe Jean 25 July 2008 (has links)
Nonhomologous end-joining (NHEJ) is a major DNA double-strand break (DSB) repair pathway. NHEJ is initiated through DSB recognition by the DNA end-binding heterodimer, Ku, while end-joining is accomplished by the XRCC4-DNA ligase IV (X4L4) complex. This thesis reports that APLF (Aprataxin and Polynucleotide kinase-Like Factor), an endo/exonuclease with a forkhead-associated (FHA) domain and two unique zinc fingers (ZF), interacts with both Ku and X4L4. The APLF-X4L4 interaction is FHA- and phospho-dependent, and is mediated by CK2 phosphorylation of XRCC4 in vitro. APLF binds Ku independently of the FHA and ZF domains, and complexes with Ku at DNA ends. APLF undergoes ionizing radiation induced ATM-dependent hyperphosphorylation and ATM phosphorylates APLF in vitro. Downregulation of APLF is associated with defective NHEJ and impaired DSB repair kinetics. These results suggest that APLF is an ATM target that is involved in NHEJ and facilitates DSB repair, likely via interactions with Ku and X4L4.
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DNA Palindrome Revision in Mammalian CellsBelsito, Tara Anne 14 July 2009 (has links)
A DNA palindrome is a sequence of DNA followed by an exact inverted copy of itself. Palindromes are associated with gross chromosomal instability in mammalian cells. This may be related to their ability to extrude a double-stranded cruciform structure. In mammalian cells, palindromes have been shown to undergo centre-directed rearrangements resulting in a central region of asymmetry. This process occurs via a mechanism termed â centre break palindrome revisionâ . In this thesis, I have investigated palindrome revision in mammalian cells using two existing assays. In the first, performed by transfection of an extrachromosomal palindromic dimer, I have shown that joining of palindrome-mediated double-strand breaks does not depend solely on NHEJ and instead relies heavily on an alternate end-joining pathway. Using the second assay, the Line78 mouse model which contains a 15.4kb transgenic palindrome, I have shown that small modifications near the centre of the palindrome prevent these centre-directed rearrangements possibly by inhibiting cruciform formation.
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Sequence conversion during adenovirus DNA replicationBennett, Kelly L. 30 April 1991 (has links)
The work in this thesis has provided conclusive genetic evidence
that "panhandle" intermediates form during adenovirus replication.
Adenovirus chromosomes lacking 51 by from their left -hand termini
are infectious and capable of regenerating the missing origin
sequence. Yet if an entire inverted terminal repeat is removed, the
adenovirus chromosome is no longer viable. This first suggested, but
did not prove, that "panhandles" formed during adenovirus
replication. Homologous recombination or postreplicative overlap
recombination could generate the same outcome. Analysis of the
segregation of markers in the inverted repeats of adenovirus
minichromosomes shows that homologous recombination does not
mediate end repair. A special case was also found where
postreplicative overlap recombination failed to transfer sequences
between the inverted repeats, but similar molecules could exchange
sequence information during "panhandle" formation. The exchange of
information between inverted repeats is referred to as sequence
conversion. A number of length and/or orientation constraints on
sequence conversion during adenovirus DNA replication were
identified. A length- and orientation-dependent constraint was found
for gap filling close to "panhandle" loops. Polymerization towards the
loop could occur even when the gap was only 6 by away. In contrast,
polymerization away from the "panhandle" loop at a gap at 6 bp, did not
take place. This steric constraint could reflect an asymmetry in the
action of adenovirus DNA polymerase. A similar length and/or
orientation dependent constraint was found for the removal of bulges (3
by and 4 by mismatches). Incision in the bulge of the 5' inverted repeat
caused a block to the completion of sequence conversion at that site.
When the bulge was in the 3' inverted repeat, a length requirement for
successful removal was demonstrated. When 6 by or 39 by separated
the bulge from the "panhandle" loop, removal of the bulge was not
detected. When the distance was 79 bp, 184 bp, or 217 bp, bulges were
successfully removed. The molecular basis for this obstruction
remains to be determined. Moreover, incision in bulges located in the
3' inverted repeat triggers directional coconversion. Finally, small
loops placed close to the site of polymerization did not cause the same
length and orientation dependent constraints as did the "panhandle"
loop. / Graduation date: 1991
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DNA Adsorption, Desorption, and Fluorescence Quenching by Graphene Oxide and Related Analytical ApplicationHuang, Po-Jung Jimmy January 2011 (has links)
Graphene is a single layer of graphite with many unique mechanical, electrical, and optical properties. In addition, graphene is also known to adsorb wide range of biomolecules including single-stranded DNA. On the other hand, the adsorption of double-stranded DNA was much weaker. To properly disperse in water, graphene oxide (GO) is often used due to its oxygen-containing groups on the surface. Recently, it was discovered that it could efficiently quench the fluorescence of fluorophores that were adsorbed. With these properties, it is possible to prepare DNA-based optical sensors using GO. Majority of the DNA/GO-based fluorescent sensors reported so far were relied on the complete desorption of DNA probes. Even though all these reports demonstrated the sensitivity and selectivity of the system, the fundamentals of binding between DNA and GO were hardly addressed.
Understanding and controlling binding between biomolecules and inorganic materials is very important in biosensor development. In this thesis, adsorption and desorption of DNA on the GO surface under different buffer conditions including ionic strength, pH, and temperature were systematically evaluated. For instance, adsorption is favored in a lower pH and a higher ionic strength buffer. It was found that once a DNA was adsorbed on the surface, little desorption occurred even in low salt buffers. Even with high pH or temperature, only small percentage of adsorbed DNA can be desorbed. To completely desorb the DNA, complementary DNA is required. The energies and activation energies associated with DNA adsorption/desorption were measured and molecular pictures of these processes were obtained. With the fundamental understanding of the DNA/GO interaction, we demonstrated that it is possible to achieve sensor regeneration without covalent immobilization. In addition, we also achieved the separation of double-stranded DNAs from single-stranded ones without using gel electrophoresis.
We also studied the fluorescence property of DNA near the GO surface using covalently attached DNA probes. It was found that the fluorophore quantum yield and lifetime changed as a function of DNA length. This study is important for rational design of covalently linked DNA sensors. This study confirmed that fluorescence quenching by GO occurs in a distance-dependent manner. Energy transfer occurred between the fluorophore and GO to result in reduced quantum yield, shorter lifetime, and lower fluorescence intensity. Although fluorescent sensors based on covalently attached DNA probes on GO have not yet been reported, the study presented here clearly supported its feasibility.
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Characterization of APLF in the Nonhomologous End-joining PathwayMacrae, Chloe Jean 25 July 2008 (has links)
Nonhomologous end-joining (NHEJ) is a major DNA double-strand break (DSB) repair pathway. NHEJ is initiated through DSB recognition by the DNA end-binding heterodimer, Ku, while end-joining is accomplished by the XRCC4-DNA ligase IV (X4L4) complex. This thesis reports that APLF (Aprataxin and Polynucleotide kinase-Like Factor), an endo/exonuclease with a forkhead-associated (FHA) domain and two unique zinc fingers (ZF), interacts with both Ku and X4L4. The APLF-X4L4 interaction is FHA- and phospho-dependent, and is mediated by CK2 phosphorylation of XRCC4 in vitro. APLF binds Ku independently of the FHA and ZF domains, and complexes with Ku at DNA ends. APLF undergoes ionizing radiation induced ATM-dependent hyperphosphorylation and ATM phosphorylates APLF in vitro. Downregulation of APLF is associated with defective NHEJ and impaired DSB repair kinetics. These results suggest that APLF is an ATM target that is involved in NHEJ and facilitates DSB repair, likely via interactions with Ku and X4L4.
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DNA Palindrome Revision in Mammalian CellsBelsito, Tara Anne 14 July 2009 (has links)
A DNA palindrome is a sequence of DNA followed by an exact inverted copy of itself. Palindromes are associated with gross chromosomal instability in mammalian cells. This may be related to their ability to extrude a double-stranded cruciform structure. In mammalian cells, palindromes have been shown to undergo centre-directed rearrangements resulting in a central region of asymmetry. This process occurs via a mechanism termed â centre break palindrome revisionâ . In this thesis, I have investigated palindrome revision in mammalian cells using two existing assays. In the first, performed by transfection of an extrachromosomal palindromic dimer, I have shown that joining of palindrome-mediated double-strand breaks does not depend solely on NHEJ and instead relies heavily on an alternate end-joining pathway. Using the second assay, the Line78 mouse model which contains a 15.4kb transgenic palindrome, I have shown that small modifications near the centre of the palindrome prevent these centre-directed rearrangements possibly by inhibiting cruciform formation.
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