Regulation of DNA Replication Origins in Fission Yeast: A Dissertation

Cells need to complete DNA replication in a timely and error-free manner. To ensure that replication is completed efficiently and in a finite amount of time, cells regulate origin firing. To prevent any errors from being transmitted to the next generation, cells have the checkpoint mechanism. The S-phase DNA damage slows replication to allow the cell to repair the damage. The mechanism of replication slowing by the checkpoint was not clear in fission yeast, Schizosaccharomyces pombe, at the start of my thesis. The downstream targets of the DNA damage checkpoint in fission yeast were also unclear. I worked on identifying the downstream targets for the checkpoint by studying if Cdc25, a phosphatase, is a target of the checkpoint. Work from our lab has shown that origin firing is stochastic in fission yeast. Origins are also known to be inefficient. Inefficient origins firing stochastically would lead to large stretches of chromosome where no origins may fire randomly leading to long replication times, an issue called the random gap problem. However, cells do not take a long time to complete replication and the process of replication itself is efficient. I focused on understanding the mechanism by which cells complete replication and avoid the random gap problem by attempting to measure the firing efficiency of late origins. Genome-wide origin studies in fission yeast have identified several hundred origins. However, the resolution of these studies can be improved upon. I began a genome-wide origin mapping study using deep sequencing to identify origins at a greater resolution compared to the previous studies. We have extended our origin search to two other Schizosaccharomyces species- S. octosporus and S. japonicus.There have been no origin mapping studies on these fission yeasts and identifying origins in these species will advance the field of replication. My thesis research shows that Cdc25 is not a target of the S-phase DNA damage checkpoint. I showed that DNA damage checkpoint does not target Cdc2-Y15 to slow replication. Based on my preliminary observation, origin firing might be inhibited by the DNA damage checkpoint as a way to slow replication. My efforts to measure the firing efficiency of a late replicating sequence were hindered by potentially unidentified inefficient origins firing at a low rate and replicating the region being studied. Studying the origin efficiency was maybe further complicated by neighboring origins being able to passively replicate the region. To identify origins in recently sequenced Schizosaccharomyces species, we initiated the genome-wide origin mapping. The mapping was also done on S. pombe to identify inefficient origins not mapped by other mapping studies. My work shows that deep sequencing can be used to map origins in other species and provides a powerful tool for origin studies

Predicting Critical Heat Flux With Multiphase CFD: 4 Years in the Making

Advancement in the experimental techniques have brought new insights into the microscale boiling phenomena, and provide the base for a new physical interpretation of flow boiling heat transfer. A new modeling framework in Computational Fluid Dynamics has been assembled at MIT, and aims at introducing all necessary mechanisms, and explicitly tracks: (1) the size and dynamics of the bubbles on the surface; (2) the amount of microlayer and dry area under each bubble; (3) the amount of surface area influenced by sliding bubbles; (4) the quenching of the boiling surface following a bubble departure and (5) the statistical bubble interaction on the surface. The preliminary assessment of the new framework is used to further extend the portability of the model through an improved formulation of the force balance models for bubble departure and lift-off. Starting from this improved representation at the wall, the work concentrates on the bubble dynamics and dry spot quantification on the heated surface, which governs the Critical Heat Flux (CHF) limit. A new proposition is brought forward, where Critical Heat Flux is a natural limiting condition for the heat flux partitioning on the boiling surface. The first principle based CHF is qualitatively demonstrated, and has the potential to deliver a radically new simulation technique to support the design of advanced heat transfer systems.United States. Department of Energy. Consortium for Advanced Simulation of Light Water Reactor

The ventrolateral medulla and medullary raphe in sudden unexpected death in epilepsy

Sudden unexpected death in epilepsy (SUDEP) is a leading cause of premature death in patients with epilepsy. One hypothesis proposes that sudden death is mediated by post-ictal central respiratory depression, which could relate to underlying pathology in key respiratory nuclei and/or their neuromodulators. Our aim was to investigate neuronal populations in the ventrolateral medulla (which includes the putative human pre-Bötzinger complex) and the medullary raphe. Forty brainstems were studied comprising four groups: 14 SUDEP, six epilepsy controls, seven Dravet syndrome cases and 13 non-epilepsy controls. Serial sections through the medulla (from obex 1 to 10 mm) were stained for Nissl, somatostatin, neurokinin 1 receptor (for pre-Bötzinger complex neurons) and galanin, tryptophan hydroxylase and serotonin transporter (neuromodulatory systems). Using stereology total neuronal number and densities, with respect to obex level, were measured. Whole slide scanning image analysis was used to quantify immunolabelling indices as well as co-localization between markers. Significant findings included reduction in somatostatin neurons and neurokinin 1 receptor labelling in the ventrolateral medulla in sudden death in epilepsy compared to controls (P < 0.05). Galanin and tryptophan hydroxylase labelling was also reduced in sudden death cases and more significantly in the ventrolateral medulla region than the raphe (P < 0.005 and P < 0.05). With serotonin transporter, reduction in labelling in cases of sudden death in epilepsy was noted only in the raphe (P ≤ 0.01); however, co-localization with tryptophan hydroxylase was significantly reduced in the ventrolateral medulla. Epilepsy controls and cases with Dravet syndrome showed less significant alterations with differences from non-epilepsy controls noted only for somatostatin in the ventrolateral medulla (P < 0.05). Variations in labelling with respect to obex level were noted of potential relevance to the rostro-caudal organization of respiratory nuclear groups, including tryptophan hydroxylase, where the greatest statistical difference noted between all epilepsy cases and controls was at obex 9-10 mm (P = 0.034), the putative level of the pre-Bötzinger complex. Furthermore, there was evidence for variation with duration of epilepsy for somatostatin and neurokinin 1 receptor. Our findings suggest alteration to neuronal populations in the medulla in SUDEP with evidence for greater reduction in neuromodulatory neuropeptidergic and mono-aminergic systems, including for galanin, and serotonin. Other nuclei need to be investigated to evaluate if this is part of more widespread brainstem pathology. Our findings could be a result of previous seizures and may represent a pathological risk factor for SUDEP through impaired respiratory homeostasis during a seizure

Mineralogy, geochemistry, and dispersal of opaque oxides on the continental shelf of the Cascadia margin

Opaque oxide minerals (ilmenite, chromite, and magnetite) in sands from the Oregon continental shelf have been studied to establish the provenance, dispersal, and grade of potential shelf placer deposits. The study area extends southward from offshore of the Columbia River in northern Oregon to the Klamath River in northern California

Development and assessment of a physics-based model for subcooled flow boiling with application to CFD

Thesis: Ph. D. in Mechanical Engineering and Computation, Massachusetts Institute of Technology, Department of Mechanical Engineering, 2020Cataloged from student-submitted PDF of thesis.Includes bibliographical references (pages 113-119).Boiling is an extremely efficient mode of heat transfer and is the preferred heat removal mechanism in power systems in general and, more recently, in electronics cooling. Physics-based models that describe boiling heat transfer, when coupled with Computational Fluid Dynamics (CFD), can be an invaluable tool to increase the performance of such systems. Existing modeling approaches do not incorporate all relevant heat transfer mechanisms at the wall, limiting their predictive capability and general applicability. These shortcomings restrict the application of CFD in the design process. For the nuclear industry, this means having to rely on expensive experimental campaigns to develop and license new reactor designs. A second-generation mechanistic heat flux partitioning framework developed in our group provides an enhanced physical description of flow boiling.It introduces several mechanisms not accounted for in previous formulations, such as 1) bubbles sliding on the heater surface, 2) interaction of nucleation sites and 3) microlayer evaporation. The framework requires describing the complete bubble ebullition cycle, including bubble nucleation, growth, and departure through closure models, which are currently lacking. This thesis extends the framework into a closed-formulation by developing closure models that adequately represent the underlying physics. New models for predicting the bubble departure diameter and frequency are developed based on insights gathered from experiments and direct numerical simulations. An assessment against existing approaches to model boiling heat transfer demonstrates the model's ability to predict over 80% of the boiling curves within a 20% error, while also capturing the correct trends with flow conditions.The model implementation in a commercial CFD software is demonstrated using data from the Bartolomei experiment. The extendability of the model to novel heater surfaces is further demonstrated for a sapphire heater substrate, where fewer cavities for nucleation shift the boiling curves to considerably higher wall superheats. This mechanistic representation of boiling heat transfer has the potential to support predictive design with optimal boiling heat transfer for improved system efficiency, with the specific objective to accelerate the development of novel nuclear fuel concepts.by Ravikishore Kommajosyula.Ph. D. in Mechanical Engineering and ComputationPh.D.inMechanicalEngineeringandComputation Massachusetts Institute of Technology, Department of Mechanical Engineerin

Light on Electric Light!

37-43The journey of electric light from the incandescent lamp to LEDs has been fascinating

Letter to a Daughter

29-32A father’s letter to a daughter to instill creativity in her child through examples from the world of science

NEUROACTIVE AGENTS-MEDIATED CHANGES IN NEURONAL NETWORK ACTIVITY CONTROLS SUSCEPTIBILITY TO SUDDEN UNEXPECTED DEATH IN EPILEPSY (SUDEP)

The incidence of sudden death is higher in epileptic people compared to the general population and sudden unexpected death in epilepsy (SUDEP) is second only to stroke in the years of potential life loss among the major neurological disorders. In the majority of observed human SUDEP cases, respiratory dysfunction post-seizure is shown to be the primary initiating event leading to cardiac asystole and death. During seizures, several neuroactive agents are shown to be released, including serotonin and adenosine. Previous research has shown the effects of these neuroactive agents on seizure and respiratory function independently. A role of adenosine in triggering death post-seizures in a chemically-induced seizure model has been shown, but the mechanism of death is not clear. Studies from our lab have shown the role of fluoxetine (selective serotonin-reuptake inhibitor) in preventing seizure-induced respiratory arrest (S-IRA) in DBA/1 mouse model of SUDEP, but the neuronal networks mediating S-IRA and the brain structures involved in the fluoxetine-mediated blockade of S-IRA are not known. Data from human SUDEP imaging has underlined the role of periaqueductal gray (PAG), which is also implicated in audiogenic seizure (AGSz) network and respiratory modulation in other models. The goal of my dissertation is to understand the mechanisms by which adenosine could cause SUDEP susceptibility, the neuronal networks in the DBA/1 mice that lead to S-IRA and how fluoxetine modulates the neuronal activity at these neuronal network structures to prevent S-IRA. A better understanding of these mechanisms may lead to development of potentially important targeted therapies to prevent SUDEP in future. In the first aim, I have examined the role of adenosine in mediating SUDEP. Genetically epilepsy prone rats (GEPR-9s) exhibit AGSz but the incidence of death post-seizure is very low. I tested whether decreasing adenosine breakdown could increase the incidence of death in GEPR-9s. My study shows that adenosine metabolic blockers, which prevent the metabolism of released adenosine during seizures significantly increased the duration of respiratory dysfunction, post-ictal depression, decreased the peripheral oxygen saturation and subsequently, increased the incidence of death post-seizure in GEPR-9s. These findings on the role of adenosine and role of specific adenosine receptors in SUDEP are required to be validated in another SUDEP model. This formed the core of my second specific aim and since DBA/2 mice are susceptible to AGSz, and after seizures a large percent of these DBA/2 mice show S-IRA, while the rest don’t show S-IRA. Therefore, I tested if adenosine antagonism could prevent S-IRA post AGSz in DBA/2 mice, and found that caffeine a non-selective adenosine antagonist significantly decreased the incidence of S-IRA post AGSz. Administration of adenosine metabolic blockers increased the incidence of S-IRA in DBA/2 mice similar to GEPR-9s. Parallel studies from our lab have shown that administration of selective A2a antagonist but not A1 antagonist also decreased S-IRA incidence in DBA/2 mice. These data from GEPR-9s and DBA/2 mice suggests for a potentially important role of selective adenosine receptors in mediating the susceptibility to SUDEP by acting on respiratory function. In the third specific aim, I have examined the role of subcortical neuronal network structures including the PAG in mediating S-IRA and the quantitative differences in respiratory function elicited by electrical stimulation at PAG between DBA/1 and C57 mice. While the role of neuroactive agents in SUDEP has received attention, the neuronal networks mediating SUDEP in pre-clinical models are not known, specifically in DBA/1 mice an established SUDEP model susceptible to AGSz. The role of subcortical neuronal network structures including PAG in AGSz has been well-studied in other AGSz models. To decipher the neuronal networks that lead to S-IRA in DBA/1 mice, I exposed both DBA/1 mice that show AGSz and S-IRA and C57 mice that are non-susceptible to AGSz to acoustic stimulus and performed an ex vivo manganese-enhanced magnetic resonance imaging (MEMRI). Data analyses revealed the role of several brain structures in auditory, sensorimotor-limbic, respiratory networks and serotonergic raphe nuclei in DBA/1 mice. Of interest the PAG, a region implicated in other models of AGSz, respiratory modulation and human SUDEP has shown a significant increase in MEMRI signal intensity compared to C57 mice. These findings formed the rationale for the fourth specific aim to examine the quantitative differences in PAG-mediated respiratory modulation in response to electrical stimulation between C57 and DBA/1 mice. The threshold of current needed at PAG for a significant increase in respiration in DBA/1 mice is four times greater than C57 mice. Electrical stimulation at amygdala (AMG) showed marginal differences between DBA/1 and C57 mice suggesting the least possible pathological role of AMG in DBA/1 mice to mediate S-IRA. These data support a reduced respiratory function of PAG in DBA/1 mice compared to C57 mice. Taken together, these findings suggest that a reduced respiratory function of PAG in DBA/1 mice could lead to S-IRA and support a potentially critical compensatory role of PAG in DBA/1 mice. In the fifth specific aim, I examined the effect of fluoxetine on the subcortical neuronal network structures in DBA/1 mice that may lead to blockade of S-IRA. Fluoxetine has been shown to prevent S-IRA in DBA/1 mice effectively, but where in the brain does this drug act to prevent the susceptibility to SUDEP in DBA/1 mice is not known. To address this question, I used ex vivo MEMRI in DBA/1 mice that received fluoxetine at a dose which selectively blocks S-IRA but not AGSz. Fluoxetine treated DBA/1 mice that didn’t show S-IRA have shown a potential compensatory increase in activity at several sub-cortical structures including PAG compared to DBA/1 mice that showed S-IRA. In summary, these data suggest the PAG as a critical compensatory structure among the other sub-cortical neuronal network structures identified for SUDEP in this mice model. Differential modulation of these subcortical neuronal network structures by adenosine or serotonin released during seizures could determine the susceptibility to SUDEP

Cdc2 tyrosine phosphorylation is not required for the S-phase DNA damage checkpoint in fission yeast

The S-phase DNA damage checkpoint slows replication when damage occurs during S phase. Cdc25, which activates Cdc2 by dephosphorylating tyrosine-15, has been shown to be a downstream target of the checkpoint in metazoans, but its role is not clear in fission yeast. The dephosphorylation of Cdc2 has been assumed not to play a role in S-phase regulation because cells replicate in the absence of Cdc25, demonstrating that tyrosine-15 phosphorylated dc2 is sufficient for S phase. However, it has been reported recently that Cdc25 is involved in the slowing of S phase in response to damage in fission yeast, suggesting a modulatory role for Cdc2 dephosphorylation in S phase. We have investigated the role of Cdc25 and the tyrosine phosphorylation of Cdc2 in the S-phase damage checkpoint, and our results show that Cdc2 phosphorylation is not a target of the checkpoint. The checkpoint was not compromised in a Cdc25 overexpressing strain, a strain carrying nonphosphorylatable form of Cdc2, or in a strain lacking Cdc25. Our results are consistent with a strictly Cdc2-Y15 phosphorylation-independent mechanism of the fission yeast S-phase DNA damage checkpoint

A reassessed model for mechanistic prediction of bubble departure and lift off diameters

Heat transfer models in multiphase flow with wall boiling rely on closure relations for bubble departure and lift-off diameters. The approach proposed in this paper reassesses the physical representation of each term of the force balance model, eliminating inconsistent assumptions and redundant calibration, leading to a more general methodology to predict lift-off and departure diameters. The validation against avail- able datasets shows improved applicability when compared to existing models. The mechanistic model proposed in this work is expected to be implemented in CFD codes, to improve predictive performance of heat partitioning models