An investigation into the use of a commonly available fabric dye as a routine stain for tissue samples to be used as a first line, low cost, diagnostic adjunct for the diagnosis of anaphylactic death at autopsy, in a resource-challenged environment

A retrospective study of deaths attributable to anaphylaxis at the Salt River Forensic Pathology Laboratory was undertaken, with a view to determine if eosinophilia was present in tissue samples of the spleen, in accordance with previously published research. Suitable cases of non-anaphylactic death were used as controls. Use was made of two commonly available fabric dyes as alternative stains to the traditional Haematoxylin -Eosin ["H&E"]

Proficient replication of the yeast genome by a viral DNA polymerase

DNA replication in eukaryotic cells requires minimally three B-family DNA polymerases: Pol α, Pol δ, and Pol ϵ. Pol δ replicates and matures Okazaki fragments on the lagging strand of the replication fork. Saccharomyces cerevisiae Pol δ is a three-subunit enzyme (Pol3-Pol31-Pol32). A small C-terminal domain of the catalytic subunit Pol3 carries both iron-sulfur cluster and zinc-binding motifs, which mediate interactions with Pol31, and processive replication with the replication clamp proliferating cell nuclear antigen (PCNA), respectively. We show that the entire N-terminal domain of Pol3, containing polymerase and proofreading activities, could be effectively replaced by those from bacteriophage RB69, and could carry out chromosomal DNA replication in yeast with remarkable high fidelity, provided that adaptive mutations in the replication clamp PCNA were introduced. This result is consistent with the model that all essential interactions for DNA replication in yeast are mediated through the small C-terminal domain of Pol3. The chimeric polymerase carries out processive replication with PCNA in vitro; however, in yeast, it requires an increased involvement of the mutagenic translesion DNA polymerase ζ during DNA replication

Towards Quantum Teleportation from a Spontaneous Parametric Down-Conversion Source to a Quantum Dot Spin by Hong-Ou-Mandel Interference.

In this thesis, we will discuss results on quantum optical measurements in InAs self-assembled quantum dots (QDs). A single electron confined to a QD provides an attractive ground state qubit for many quantum information architectures. The excitation of an exciton in the QD structure via optical radiation will produce an electron-hole pair and, when combined with the ground state electron, form a quasi three-particle state called a trion. The trion transitions allow us to optically manipulate and readout the spin state. Narrow linewidth continuous wave lasers are used to probe the properties of the trion transitions in the frequency domain. We recover atomic like behavior for the QD resonances. The time dynamics of the trion system are studied by time gating a CW laser with an electro-optic modulator (EOM) and time tagging the scattered single photons from the transition using single photon detectors. These techniques are utilized in an entanglement protocol where a photon emitted during decay from the trion state to one of the electron spin states creates an entangled state between the electron spin and the photon. Under the influence of a transverse magnetic field the optical transitions from the spin states to the trion states are orthogonally polarized. The entangled state we are demonstrating is between the polarization state of the photon and the polarization state of the electron spin. We extract a corresponding fidelity of 0.6. The entangled nature of the spin state and the photon allows for the possibility to transport entanglement by means of this photonic channel. We study the coherence properties of the emitted photon using Hong Ou Mandel interference, which shows the indistinguishability of the photons (a required property for quantum information transport). Finally, we develop an experimental protocol to teleport quantum states from cavity enhanced photon pairs created in spontaneous parametric down conversion (SPDC) to a QD spin state by the interference between entangled photons from the QD and the SPDC photons.PhDPhysicsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113343/1/aburgers_1.pd

Lagging strand maturation factor Dna2 is a component of the replication checkpoint initiation machinery

Initiation of the DNA replication checkpoint in yeast is mainly mediated by Mec1 protein kinase, the ortholog of human ATR, while its homolog Tel1, the ortholog of human ATM, has a minor replication checkpoint function. Checkpoint initiation requires stimulation of Mec1 kinase activity by specific activators. Saccharomyces cerevisiae Dna2, a nuclease-helicase that is essential for Okazaki fragment maturation, is employed specifically during S phase to stimulate Mec1 kinase and initiate the replication checkpoint. Mutations (W128A and Y130A) in the unstructured N terminus of Dna2 abrogate its checkpoint function in vitro and in vivo. Dna2 shows partial redundancy for the replication checkpoint with checkpoint initiators 9-1-1 (S. cerevisiae Ddc1–Mec3–Rad17 and human Rad9–Rad1–Hus1) and Dpb11, the ortholog of human TopBP1. A triple mutant that eliminates the checkpoint functions of all three initiators abrogates the Mec1-dependent checkpoint

Pif1 Helicase Lengthens Some Okazaki Fragment Flaps Necessitating Dna2 Nuclease/Helicase Action in the Two-nuclease Processing Pathway

We have developed a system to reconstitute all of the proposed steps of Okazaki fragment processing using purified yeast proteins and model substrates. DNA polymerase δ was shown to extend an upstream fragment to displace a downstream fragment into a flap. In most cases, the flap was removed by flap endonuclease 1 (FEN1), in a reaction required to remove initiator RNA in vivo. The nick left after flap removal could be sealed by DNA ligase I to complete fragment joining. An alternative pathway involving FEN1 and the nuclease/helicase Dna2 has been proposed for flaps that become long enough to bind replication protein A (RPA). RPA binding can inhibit FEN1, but Dna2 can shorten RPA-bound flaps so that RPA dissociates. Recent reconstitution results indicated that Pif1 helicase, a known component of fragment processing, accelerated flap displacement, allowing the inhibitory action of RPA. In results presented here, Pif1 promoted DNA polymerase δ to displace strands that achieve a length to bind RPA, but also to be Dna2 substrates. Significantly, RPA binding to long flaps inhibited the formation of the final ligation products in the reconstituted system without Dna2. However, Dna2 reversed that inhibition to restore efficient ligation. These results suggest that the two-nuclease pathway is employed in cells to process long flap intermediates promoted by Pif1

Parallel analysis of ribonucleotide-dependent deletions produced by yeast Top1 in vitro and in vivo

Ribonucleotides are the most abundant non-canonical component of yeast genomic DNA and their persistence is associated with a distinctive mutation signature characterized by deletion of a single repeat unit from a short tandem repeat. These deletion events are dependent on DNA topoisomerase I (Top1) and are initiated by Top1 incision at the relevant ribonucleotide 3′-phosphodiester. A requirement for the re-ligation activity of Top1 led us to propose a sequential cleavage model for Top1-dependent mutagenesis at ribonucleotides. Here, we test key features of this model via parallel in vitro and in vivo analyses. We find that the distance between two Top1 cleavage sites determines the deletion size and that this distance is inversely related to the deletion frequency. Following the creation of a gap by two Top1 cleavage events, the tandem repeat provides complementarity that promotes realignment to a nick and subsequent Top1-mediated ligation. Complementarity downstream of the gap promotes deletion formation more effectively than does complementarity upstream of the gap, consistent with constraints to realignment of the strand to which Top1 is covalently bound. Our data fortify sequential Top1 cleavage as the mechanism for ribonucleotide-dependent deletions and provide new insight into the component steps of this process

Efficient non-linear data assimilation in geophysical fluid dynamics

New ways of combining observations with numerical models are discussed in which the size of the state space can be very large, and the model can be highly nonlinear. Also the observations of the system can be related to the model variables in highly nonlinear ways, making this data-assimilation (or inverse) problem highly nonlinear. First we discuss the connection between data assimilation and inverse problems, including regularization. We explore the choice of proposal density in a Particle Filter and show how the ’curse of dimensionality’ might be beaten. In the standard Particle Filter ensembles of model runs are propagated forward in time until observations are encountered, rendering it a pure Monte-Carlo method. In large-dimensional systems this is very inefficient and very large numbers of model runs are needed to solve the data-assimilation problem realistically. In our approach we steer all model runs towards the observations resulting in a much more efficient method. By further ’ensuring almost equal weight’ we avoid performing model runs that are useless in the end. Results are shown for the 40 and 1000 dimensional Lorenz 1995 model