Characterizing the regulation of mitochondrial nucleoids

Mitochondria contain a 16.6 kb circular genome encoding 13 proteins as well as mitochondrial tRNAs and rRNAs. Copies of the genome are organized into nucleoids containing both DNA and proteins, including the machinery required for mtDNA replication and transcription. Although mtDNA integrity is essential for cellular and organismal viability, regulation of proliferation of the mitochondrial genome is poorly understood. To elucidate the mechanisms behind this, we chose to study the interplay between mtDNA copy number and the proteins involved in mitochondrial fusion, another required function in cells. Strikingly, we found that mouse embryonic fibroblasts lacking fusion also had a mtDNA copy number deficit. To understand this phenomenon further, we analyzed the binding of mitochondrial transcription factor A, whose role in transcription, replication, and packaging of the genome is well-established and crucial for cellular maintenance. Using ChIP-seq, we were able to detect largely uniform, non-specific binding across the genome, with no occupancy in the known specific binding sites in the regulatory region. We did detect a single binding site directly upstream of a known origin of replication, suggesting that TFAM may play a direct role in replication. Finally, although TFAM has been previously shown to localize to the nuclear genome, we found no evidence for such binding sites in our system.

To further understand the regulation of mtDNA by other proteins, we analyzed publicly available ChIP-seq datasets from ENCODE, modENCODE, and mouseENCODE for evidence of nuclear transcription factor binding to the mitochondrial genome. We identified eight human transcription factors and three mouse transcription factors that demonstrated binding events with the classical strand asymmetrical morphology of classical binding sites. ChIP-seq is a powerful tool for understanding the interactions between proteins and the mitochondrial genome, and future studies promise to further the understanding of how mtDNA is regulated within the nucleoid.

Part I. Influence of membrane morphology on ion-exchange and charge propagation in composite polyelectrolyte electrode coatings. Part II. Dynamic surface tension measurements of polarization relaxation at mercury pool electrodes by the method of Wilhelmy.

Part I. Novel composite polyelectrolyte materials were developed that exhibit desirable charge propagation and ion-retention properties. The morphology of electrode coatings cast from these materials was shown to be more important for its electrochemical behavior than its chemical composition.

Part II. The Wilhelmy plate technique for measuring dynamic surface tension was extended to electrified liquid-liquid interphases. The dynamical response of the aqueous NaF-mercury electrified interphase was examined by concomitant measurement of surface tension, current, and applied electrostatic potential. Observations of the surface tension response to linear sweep voltammetry and to step function perturbations in the applied electrostatic potential (e.g., chronotensiometry) provided strong evidence that relaxation processes proceed for time-periods that are at least an order of magnitude longer than the time periods necessary to establish diffusion equilibrium. The dynamical response of the surface tension is analyzed within the context of non-equilibrium thermodynamics and a kinetic model that requires three simultaneous first order processes.

A model for energy and morphology of crystalline grain boundaries with arbitrary geometric character

It has been well-established that interfaces in crystalline materials are key players in the mechanics of a variety of mesoscopic processes such as solidification, recrystallization, grain boundary migration, and severe plastic deformation. In particular, interfaces with complex morphologies have been observed to play a crucial role in many micromechanical phenomena such as grain boundary migration, stability, and twinning. Interfaces are a unique type of material defect in that they demonstrate a breadth of behavior and characteristics eluding simplified descriptions. Indeed, modeling the complex and diverse behavior of interfaces is still an active area of research, and to the author's knowledge there are as yet no predictive models for the energy and morphology of interfaces with arbitrary character. The aim of this thesis is to develop a novel model for interface energy and morphology that i) provides accurate results (especially regarding "energy cusp" locations) for interfaces with arbitrary character, ii) depends on a small set of material parameters, and iii) is fast enough to incorporate into large scale simulations.

In the first half of the work, a model for planar, immiscible grain boundary is formulated. By building on the assumption that anisotropic grain boundary energetics are dominated by geometry and crystallography, a construction on lattice density functions (referred to as "covariance") is introduced that provides a geometric measure of the order of an interface. Covariance forms the basis for a fully general model of the energy of a planar interface, and it is demonstrated by comparison with a wide selection of molecular dynamics energy data for FCC and BCC tilt and twist boundaries that the model accurately reproduces the energy landscape using only three material parameters. It is observed that the planar constraint on the model is, in some cases, over-restrictive; this motivates an extension of the model.

In the second half of the work, the theory of faceting in interfaces is developed and applied to the planar interface model for grain boundaries. Building on previous work in mathematics and materials science, an algorithm is formulated that returns the minimal possible energy attainable by relaxation and the corresponding relaxed morphology for a given planar energy model. It is shown that the relaxation significantly improves the energy results of the planar covariance model for FCC and BCC tilt and twist boundaries. The ability of the model to accurately predict faceting patterns is demonstrated by comparison to molecular dynamics energy data and experimental morphological observation for asymmetric tilt grain boundaries. It is also demonstrated that by varying the temperature in the planar covariance model, it is possible to reproduce a priori the experimentally observed effects of temperature on facet formation.

Finally, the range and scope of the covariance and relaxation models, having been demonstrated by means of extensive MD and experimental comparison, future applications and implementations of the model are explored.