Bayesian Statistical Models of Cell-Cycle Progression at Single-Cell and Population Levels

<p>Cell division is a biological process fundamental to all life. One aspect of the process that is still under investigation is whether or not cells in a lineage are correlated in their cell-cycle progression. Data on cell-cycle progression is typically acquired either in lineages of single cells or in synchronized cell populations, and each source of data offers complementary information on cell division. To formally assess dependence in cell-cycle progression, I develop a hierarchical statistical model of single-cell measurements and extend a previously proposed model of population cell division in the budding yeast, <italic>Saccharomyces cerevisiae</italic>. Both models capture correlation and cell-to-cell heterogeneity in cell-cycle progression, and parameter inference is carried out in a fully Bayesian manner. The single-cell model is fit to three published time-lapse microscopy datasets and the population-based model is fit to simulated data for which the true model is known. Based on posterior inferences and formal model comparisons, the single-cell analysis demonstrates that budding yeast mother and daughter cells do not appear to correlate in their cell-cycle progression in two of the three experimental settings. In contrast, mother cells grown in a less preferred sugar source, glycerol/ethanol, did correlate in their rate of cell division in two successive cell cycles. Population model fitting to simulated data suggested that, under typical synchrony experimental conditions, population-based measurements of the cell-cycle were not informative for correlation in cell-cycle progression or heterogeneity in daughter-specific G1 phase progression.</p>Thesi

Molecular and Genetic Analysis of a Conserved Transcription Factor with a Role in Promoting the Completion of Cytokinesis in Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe activates regulatory networks that promote the faithful execution of cytokinesis in response to drugs that perturb the cytokinesis machinery. In order to identify novel components of these networks, a screen for mutants hyper-sensitive to the actin depolymerizing drug LatrunculinA (LatA) was previously performed. This screen identified a transcription factor, Pap1p, which is orthologous to the mammalian stress activated transcription factor, AP-1. Through molecular and genetic analysis, I showed that the deletion mutant of pap1 is sensitive to LatA and that it cannot maintain the integrity of the actomyosin ring upon LatA treatment leading to cytokinesis failure. I also demonstrated that Pap1p is imported to the nucleus upon LatA challenge. Finally, I showed that nuclear translocation of Pap1p is important for its role in the cytokinesis monitoring system. Translocation was also important to the colony forming abilities of S. pombe cells and for their viability. In summary, I concluded that nuclear import of Pap1p is crucial for the proper function and regulation of the cytokinesis monitoring system

Clozapine-induced paroxysmal discharges

PhD ThesisThe atypical antipsychotic clozapine is a widely prescribed and effective treatment for the positive and negative symptoms of schizophrenia, but reports of side effects are common. In one study EEG abnormalities were observed in 53% of patients treated with clozapine, and the absence or presence of EEG abnormalities correlated with the plasma clozapine concentration. Here, epileptiform activity was present in conventional EEG recordings from a 32 year old male patient with psychiatric illness taking clozapine for 3 weeks. Brief (ca.100ms), transient epileptiform spikes occurred at a frequency of approximately 2 per h and originated primarily in parietal cortex. One month after withdrawal of clozapine, epileptiform spikes were no longer present. An in vitro model was developed using the equivalent region of association cortex, namely 2⁰ somatosensory cortex, in normal rat brain slices to probe such activity with increased spatial and temporal resolution, and to investigate mechanisms underlying its generation. Wide band in vitro recordings revealed that clozapine (10-20µM) induced regular, frequent very fast oscillations (VFO, > 70Hz) in this region. These VFO comprised short transient high frequency discharges and were maximal in patches along layer V. The atypical antipsychotic olanzapine, but not the classical antipsychotic haloperidol, also induced prominent VFO in this region. Sharp electrode intracellular recordings revealed that there was almost no correlation between the somatic activity of layer V regular spiking (RS) pyramidal cells and field VFO, but layer V intrinsically bursting (IB) cells did correlate to some extent with the local field. Interestingly, IB cell spikelets were also weakly correlated with field VFO suggesting a role for axonal hyperexcitability in this cell type in the mechanism. Clozapine-induced VFO persisted following blockade of AMPA, NMDA, and GABAA chemical synaptic receptors, and the gap junction blockers carbenoxolone and quinine also failed to significantly attenuate the power of this activity. Although octanol abolished clozapine-induced VFO, it was not clear that this effect resulted from blockade of gap junctions as this drug also blocks spikes. In addition to VFO events, clozapine (10-20µM) also induced occasional, spontaneous transient paroxysmal discharges, similar to the EEG phenomena, in 33% (11/33 slices) of slices in vitro. Sharp electrode intracellular recordings revealed that clozapine- induced full paroxysmal discharges were associated with spikes, EPSPs and IPSPs in layer V RS and IB cells, suggesting that these events were mediated via chemical synaptic transmission in both of these cell types. Multi-electrode array recordings of local field potentials and units suggested that clozapine-induced paroxysmal events started superficially in association cortex, moved deeper and then propagated horizontally along these deep layers. The onset of clozapine-induced VFO was accompanied by a significant elevation in parvalbumin immunoreactivity, particularly in layer II-IV, where there was a greater than twofold increase in the signal, and this may be relevant to the therapeutic action of the drug

Mistimed malaria parasites re‐synchronise with host feeding‐fasting rhythms by shortening the duration of intra‐erythrocytic development

AIMS: Malaria parasites exhibit daily rhythms in the intra‐erythrocytic development cycle (IDC) that underpins asexual replication in the blood. The IDC schedule is aligned with the timing of host feeding‐fasting rhythms. When the IDC schedule is perturbed to become mismatched to host rhythms, it readily reschedules but it is not known how. METHODS: We intensively follow four groups of infections that have different temporal alignments between host rhythms and the IDC schedule for 10 days, before and after the peak in asexual densities. We compare how the duration, synchrony and timing of the IDC differs between parasites in control infections and those forced to reschedule by 12 hours and ask whether the density of parasites affects the rescheduling process. RESULTS AND CONCLUSIONS: Our experiments reveal parasites shorten the IDC duration by 2–3 hours to become realigned to host feeding‐fasting rhythms with 5–6 days, in a density‐independent manner. Furthermore, parasites are able to reschedule without significant fitness costs for them or their hosts. Understanding the extent of, and limits on, plasticity in the IDC schedule may reveal targets for novel interventions, such as drugs to disrupt IDC regulation and preventing IDC dormancy conferring tolerance to existing drugs

Regulation of Thalamic and Cortical Network Synchrony by Scn8a.

Voltage-gated sodium channel (VGSC) mutations cause severe epilepsies marked by intermittent, pathological hypersynchronous brain states. Here we present two mechanisms that help to explain how mutations in one VGSC gene, Scn8a, contribute to two distinct seizure phenotypes: (1) hypoexcitation of cortical circuits leading to convulsive seizure resistance, and (2) hyperexcitation of thalamocortical circuits leading to non-convulsive absence epilepsy. We found that loss of Scn8a leads to altered RT cell intrinsic excitability and a failure in recurrent RT synaptic inhibition. We propose that these deficits cooperate to enhance thalamocortical network synchrony and generate pathological oscillations. To our knowledge, this finding is the first clear demonstration of a pathological state tied to disruption of the RT-RT synapse. Our observation that loss of a single gene in the thalamus of an adult wild-type animal is sufficient to cause spike-wave discharges is striking and represents an example of absence epilepsy of thalamic origin

Innovative Techniques of Neuromodulation and Neuromodeling Based on Focal Non-Invasive Transcranial Magnetic Stimulation for Neurological Disorders

This dissertation aims to develop alternative technology that improves the current range of application of transcranial magnetic stimulation (TMS), on a scale that would permit defining specific non-invasive treatments for Parkinson’s disease and other neurological disorders. This is accomplished through three specific objectives. 1) The design of a neurostimulation system that increases the focality in TMS to regions of narrow target areas and variable depths in the brain cortex. 2) The assessment of the feasibility of novel high-frequency neuromodulation techniques that would allow increasing the focality in deeper areas beyond the cortical surface. 3) The development of a computational model of the motor pathway that allows studying the underlying mechanisms that originate PD symptoms, and the effects of TMS for the development of new treatments. The results successfully demonstrated the feasibility of using the novel high-frequency neuromodulation technique as an effective manner to reduce the necessary current in TMS coils. This reduction, which reached an order of magnitude of 100 times compared to commercial TMS technology, made it possible to reduce the coil sizes, making them more focal to targets (in the order of a few millimeters square). Finally, our innovative oscillatory model of the motor pathway allowed us to conclude that an internal regulatory mechanism that we believe neurons activate in advanced PD stages seems to be the pathological response of some neural subpopulations to dopamine depletion, trying to compensate for the downstream effects in the system. We also found that such a mechanism seems to the burstiness in PD

Segmentation, tracking and cell cycle analysis of live-cell imaging data with Cell-ACDC

Background: High-throughput live-cell imaging is a powerful tool to study dynamic cellular processes in single cells but creates a bottleneck at the stage of data analysis, due to the large amount of data generated and limitations of analytical pipelines. Recent progress on deep learning dramatically improved cell segmentation and tracking. Nevertheless, manual data validation and correction is typically still required and tools spanning the complete range of image analysis are still needed. Results: We present Cell-ACDC, an open-source user-friendly GUI-based framework written in Python, for segmentation, tracking and cell cycle annotations. We included state-of-the-art deep learning models for single-cell segmentation of mammalian and yeast cells alongside cell tracking methods and an intuitive, semi-automated workflow for cell cycle annotation of single cells. Using Cell-ACDC, we found that mTOR activity in hematopoietic stem cells is largely independent of cell volume. By contrast, smaller cells exhibit higher p38 activity, consistent with a role of p38 in regulation of cell size. Additionally, we show that, in S. cerevisiae, histone Htbl concentrations decrease with replicative age. Conclusions: Cell-ACDC provides a framework for the application of state-of-the-art deep learning models to the analysis of live cell imaging data without programming knowledge. Furthermore, it allows for visualization and correction of segmentation and tracking errors as well as annotation of cell cycle stages. We embedded several smart algorithms that make the correction and annotation process fast and intuitive. Finally, the open-source and modularized nature of Cell-ACDC will enable simple and fast integration of new deep learning-based and traditional methods for cell segmentation, tracking, and downstream image analysis.Peer reviewe

Regulation of Zygotic Transcription and Cell Cycle Checkpoints in Early Embryogenesis

For many organisms, the first goal of embryogenesis is to accumulate a large cell population to accommodate gastrulation. To achieve this quickly, embryos employ specialized cell cycles called cleavages that consist of continuous rounds of DNA replication and division. Cell proliferation occurs rapidly because cleavage cycles lack the gap phases and cell cycle checkpoints found in canonical cell cycles. Further, the genetic materials required to sustain cleavage cycles are preloaded during oogenesis, aiding efficient cell cycle progression. After a constant, organism-specific number of cleavages, many metazoan embryos undergo the mid-blastula transition (MBT), which initiates extensive cell cycle remodeling. Cell cycles lengthen, gap phases appear and checkpoint function is acquired. At the same time, the nearly quiescent zygotic genome is activated and transcriptional activity dramatically increases. This dissertation describes how these simultaneous MBT events are regulated. Chapter 2 addresses how zygotic transcription and cell cycle remodeling are coordinated. By artificially slowing cleavage cycles in zebrafish embryos, I demonstrate that increases in transcriptional activity are independent of cell cycle elongation and embryo age. I conclude that zygotic transcription is regulated by the nuclear-to-cytoplasmic (N:C) ratio, which increases after each round of replication in cleavage-stage embryos. Chapter 2 also shows the mechanisms governing DNA damage checkpoint acquisition at the MBT. DNA damage checkpoint acquisition does not require zygotic transcription. Instead, using immunostaining to examine checkpoint signaling, I show that cleavage-stage embryos cannot activate the checkpoint protein Chk1 kinase after damage induction. I conclude that the lack of Chk1 activity prior to the MBT limits DNA damage checkpoint function during cleavage cycles. Chapter 3 investigates how the spindle assembly checkpoint (SAC) is acquired at the MBT. I show that SAC acquisition is independent of the N:C ratio and other MBT events like cell cycle elongation and zygotic transcription. I conclude that SAC acquisition is age-dependent, and relies on a timer mechanism to regulate maternally-supplied SAC components. The studies reported in this dissertation demonstrate the various mechanisms embryos use to orchestrate simultaneous MBT events