Chronic administration of glucocorticoids (GC) leads to characteristic features of type 2 diabetes in mammals. The main action of dexamethasone in target cells occurs through modulation of gene expression, although the exact mechanisms are still unknown. We therefore investigated the gene expression profile of pancreatic islets from rats treated with dexamethasone using a cDNA array screening analysis. The expression of selected genes and proteins involved in mitochondria] apoptosis was further analyzed by PCR and immunoblotting. Insulin, triglyceride and free fatty acid plasma levels, as well as glucose-induced insulin secretion, were significantly higher in dexamethasone-treated rats compared with controls. Out of 1176 genes, 60 were up-regulated and 28 were down-regulated by dexamethasone treatment. Some of the modulated genes are involved in apoptosis, stress response, and proliferation pathways. RT-PCR confirmed the cDNA array results for 6 selected genes. Bax alpha protein expression was increased, while Bcl-2 was decreased. In vivo dexamethasone treatment decreased the mitochondrial production of NAD(P)H, and increased ROS production. Concluding, our data indicate that dexamethasone modulates the expression of genes and proteins involved in several pathways of pancreatic-islet cells...
Kinesin-1 is a dimeric motor that transports cargo along microtubules, taking 8.2-nm steps in a hand-over-hand fashion. The ATP hydrolysis cycles of its two heads are maintained out of phase by a series of gating mechanisms, which lead to processive runs averaging ∼1 μm. A key structural element for inter-head coordination is the neck linker (NL), which connects the heads to the stalk. To examine the role of the NL in regulating stepping, we investigated NL mutants of various lengths using single-molecule optical trapping and bulk fluorescence approaches in the context of a general framework for gating. Our results show that, although inter-head tension enhances motor velocity, it is crucial neither for inter-head coordination nor for rapid rear-head release. Furthermore, cysteine-light mutants do not produce wild-type motility under load. We conclude that kinesin-1 is primarily front-head gated, and that NL length is tuned to enhance unidirectional processivity and velocity.
Cellular physiology is implemented by formidably complex biochemical systems with highly nonlinear dynamics, presenting a challenge for both experiment and theory. Time-scale separation has been one of the few theoretical methods for distilling general principles from such complexity. It has provided essential insights in areas such as enzyme kinetics, allosteric enzymes, G-protein coupled receptors, ion channels, gene regulation and post-translational modification. In each case, internal molecular complexity has been eliminated, leading to rational algebraic expressions among the remaining components. This has yielded familiar formulas such as those of Michaelis-Menten in enzyme kinetics, Monod-Wyman-Changeux in allostery and Ackers-Johnson-Shea in gene regulation. Here we show that these calculations are all instances of a single graph-theoretic framework. Despite the biochemical nonlinearity to which it is applied, this framework is entirely linear, yet requires no approximation. We show that elimination of internal complexity is feasible when the relevant graph is strongly connected. The framework provides a new methodology with the potential to subdue combinatorial explosion at the molecular level.
We propose an approach for approximating electrostatic charge distributions with a small number of point charges to optimally represent the original charge distribution. By construction, the proposed optimal point charge approximation (OPCA) retains many of the useful properties of point multipole expansion, including the same far-field asymptotic behavior of the approximate potential. A general framework for numerically computing OPCA, for any given number of approximating charges, is described. We then derive a 2-charge practical point charge approximation, PPCA, which approximates the 2-charge OPCA via closed form analytical expressions, and test the PPCA on a set of charge distributions relevant to biomolecular modeling. We measure the accuracy of the new approximations as the RMS error in the electrostatic potential relative to that produced by the original charge distribution, at a distance the extent of the charge distribution–the mid-field. The error for the 2-charge PPCA is found to be on average 23% smaller than that of optimally placed point dipole approximation, and comparable to that of the point quadrupole approximation. The standard deviation in RMS error for the 2-charge PPCA is 53% lower than that of the optimal point dipole approximation...
The nucleus is an organelle of central importance to the mammalian cell. However, our understanding of the organizations and dynamics of many nuclear structures and processes remains inadequate, largely due to the difficulty in probing them in situ, with single-molecule sensitivity as well as ultra-high resolutions in space and time. In this dissertation, we develop approaches to interrogate, through imaging and modeling, the spatio-temporal organizations and dynamics of two key nuclear processes: gene expression and DNA replication. We first describe a novel fluorescence imaging technique, named reflected light-sheet (RLS) microscopy, that is capable of detecting single molecules with superior signal-to-background ratio inside the mammalian nucleus. By selectively illuminating only a thin section of the nucleus using a light-sheet reflected off a miniature mirror, RLS microscopy combines the capabilities of 3D optical sectioning, fast imaging speed, and applicability to single, normal-sized adherent cells. As demonstration, we apply RLS microscopy to directly monitor the DNA binding dynamics and spatio-temporal colocalization of single mammalian transcription factor molecules in live cells. By measuring their diffusion constants, DNA-bound fraction...
Estimating the effects of missense mutations is a problem with many important applications in a variety of fields, including medical genetics, evolutionary theory, population genetics, and protein structure and design. Many popular methods exist to solve this problem, the most widely used of which are PolyPhen-2 and SIFT. These methods, along with most other popular methods, rely on multiple sequence alignments of orthologous protein sequences. Based on the amino acids observed in each column of the alignment, they produce a profile describing how tolerated each amino acid is at each position. They then compare the wild-type and variant amino acids to this profile to produce a prediction.
In practice, these methods are fast, robust, and relatively reliable. However, from a theoretical perspective, they have at least three significant shortcomings:
1. They use effects on selection as a proxy for effects on phenotype and protein structure and function.
2. They treat each position as independent, ruling out most forms of interactions between sites.
3. They do not explicitly model the process of evolution, instead assuming that sequences we observe more or less represent an equilibrium state.
With the recent explosion of sequencing technology...
The precise delivery and organization of intracellular factors in space and time relies on a set of molecules that move along and regulate the dynamics of cytoskeletal filaments. The two families of microtubule-based motors-- dyneins and kinesins-- power vital biological processes such as intracellular transport, chromosome segregation and more broadly cell-cell communication and cell polarization. Despite their role in such diverse activities, their molecular mechanisms remain poorly understood. Combining biochemistry, cryo-electron microscopy, molecular dynamics simulations and single molecule biophysics, we provide novel insights into the mechanistic basis of how dynein and kinesin-8 interact with microtubules (MTs) to regulate their function.
Cytoplasmic dynein is a homodimer that moves for long distances along MTs without dissociating, a property known as processivity. Its movement requires coupling cycles of ATP binding and hydrolysis to changes in affinity for its track. Intriguingly, the main site of ATP hydrolysis in the motor is separated from the microtubule binding domain (MTBD) by 25 nm. How do these sites communicate with each other? What are the changes responsible for modulating the affinity between the motor and its track during dynein’s mechanochemical cycle? Furthermore...
Inner-ear mechanotransduction relies on tip links, fine protein filaments made of cadherin-23 and protocadherin-15 that convey tension to mechanosensitive channels at the tips of hair-cell stereocilia. The tip-link cadherins are thought to form a heterotetrameric complex, with two cadherin-23 molecules forming the upper part of the filament and two protocadherin-15 molecules forming the lower end. The interaction between cadherin-23 and protocadherin-15 is mediated by their N-terminal tips. Missense mutations that modify the interaction interface impair binding and lead to deafness. We have developed molecular tools to perform single-molecule force spectroscopy on the tip-link bond. Self-assembling DNA nanoswitches are functionalized with the interacting tips of cadherin-23 and protocadherin-15 using the enzyme sortase under conditions that preserve protein function. These tip-link-functionalized nanoswitches are designed to provide a signature force-extension profile, which allows us to identify single-molecule rupture events that result from applying force. Using this system, we have been able to measure the cadherin-23-protocadherin-15 single-molecule force-dependent off rate, as well as the concentration-dependent on rate for a single pair of these proteins. The rates suggest that a single bond is inadequate to withstand physiological forces for physiological times...
The study of chromosome, and genome, organization is a both an ongoing challenge, and one with a long history. Following the advent of high-throughput sequencing and genomic technologies, much research has focused on the one-dimensional, or sequence-level, organization of genomes, with many successes. Nonetheless, genomes are physically organized as chromosomes in the three-dimensional confines of the cell nucleus, with implications for processes including gene regulation, DNA replication, and cell division.
Recently, chromosome conformation capture (3C) based methods have enabled new high-resolution and genome-wide views (Hi-C) of chromosome organization in three-dimensions. 3C methods convert direct spatial contacts between pairs of genomic loci into molecular products that can be assayed using high-throughput sequencing. The new views of chromosomal organization enabled by 3C techniques have been the principal motivation for my graduate research. In particular, 3C technologies now pose multiple important computational and theoretical challenges, including how to: (1) process and filter large quantities of experimental data; (2) develop computational models of chromosomes that agree with and help the understanding of experimental data; and (3) integrate views from 3C technologies with other genomic datasets...
Cette thèse porte sur l’étude de la relation entre la structure et la fonction chez les cotransporteurs Na+/glucose (SGLTs). Les SGLTs sont des protéines membranaires qui se servent du gradient électrochimique transmembranaire du Na+ afin d’accumuler leurs substrats dans la cellule.
Une mise en contexte présentera d’abord un bref résumé des connaissances actuelles dans le domaine, suivi par un survol des différentes techniques expérimentales utilisées dans le cadre de mes travaux.
Ces travaux peuvent être divisés en trois projets. Un premier projet a porté sur les bases structurelles de la perméation de l’eau au travers des SGLTs. En utilisant à la fois des techniques de modélisation moléculaire, mais aussi la volumétrie en voltage imposé, nous avons identifié les bases structurelles de cette perméation. Ainsi, nous avons pu identifier in silico la présence d’une voie de perméation passive à l’eau traversant le cotransporteur, pour ensuite corroborer ces résultats à l’aide de mesures faites sur le cotransporteur Na/glucose humain (hSGLT1) exprimé dans les ovocytes.
Un second projet a permis d’élucider certaines caractéristiques structurelles de hSGLT1 de par l’utilisation de la dipicrylamine (DPA)...
The physics of self-organization and complexity is manifested on a variety of biological scales, from large ecosystems to the molecular level. Protein molecules exhibit characteristics of complex systems in terms of their structure, dynamics, and function. Proteins have the extraordinary ability to fold to a specific functional three-dimensional shape, starting from a random coil, in a biologically relevant time. How they accomplish this is one of the secrets of life. In this work, theoretical research into understanding this remarkable behavior is discussed. Thermodynamic and statistical mechanical tools are used in order to investigate the protein folding dynamics and stability. Theoretical analyses of the results from computer simulation of the dynamics of a four-helix bundle show that the excluded volume entropic effects are very important in protein dynamics and crucial for protein stability. The dramatic effects of changing the size of sidechains imply that a strategic placement of amino acid residues with a particular size may be an important consideration in protein engineering. Another investigation deals with modeling protein structural transitions as a phase transition. Using finite size scaling theory, the nature of unfolding transition of a four-helix bundle protein was investigated and critical exponents for the transition were calculated for various hydrophobic strengths in the core. It is found that the order of the transition changes from first to higher order as the strength of the hydrophobic interaction in the core region is significantly increased. Finally...
Expertise in physics has been traditionally studied in cognitive science, where physics expertise is understood through the difference between novice and expert problem solving skills. The cognitive perspective of physics experts only create a partial model of physics expertise and does not take into account the development of physics experts in the natural context of research. This dissertation takes a social and cultural perspective of learning through apprenticeship to model the development of physics expertise of physics graduate students in a research group. I use a qualitative methodological approach of an ethnographic case study to observe and video record the common practices of graduate students in their biophysics weekly research group meetings. I recorded notes on observations and conduct interviews with all participants of the biophysics research group for a period of eight months. I apply the theoretical framework of Communities of Practice to distinguish the cultural norms of the group that cultivate physics expert practices. Results indicate that physics expertise is specific to a topic or subfield and it is established through effectively publishing research in the larger biophysics research community. The participant biophysics research group follows a learning trajectory for its students to contribute to research and learn to communicate their research in the larger biophysics community. In this learning trajectory students develop expert member competencies to learn to communicate their research and to learn the standards and trends of research in the larger research community. Findings from this dissertation expand the model of physics expertise beyond the cognitive realm and add the social and cultural nature of physics expertise development. This research also addresses ways to increase physics graduate student success towards their PhD. and decrease the 48% attrition rate of physics graduate students. Cultivating effective research experiences that give graduate students agency and autonomy beyond their research groups gives students the motivation to finish graduate school and establish their physics expertise.^
Les canaux potassiques dépendants du voltage sont formés de quatre sous-unités, chacune possédant six segments transmembranaires (S1-S6) et une boucle (p-loop) qui se trouve entre le cinquième et le sixième segment au niveau du pore. Il est connu que le segment senseur du voltage (S1-S4) subit un mouvement lorsque le potentiel membranaire change. Pour ouvrir le canal, il est nécessaire de transférer l'énergie du senseur du voltage (généré par le mouvement des charges positives de S4) au pore. Le mécanisme exact de ce couplage électromécanique est encore sous étude. Un des points de liaison entre le senseur de voltage et le pore est le lien physique fait par le segment S4-S5 (S45L). Le but de cette étude est de déterminer l'influence de la flexibilité du segment S45L sur le processus de couplage. Dans le S45L, trois glycines sont distribuées dans des positions différentes. Elles sont responsables de la flexibilité des hélices-alpha. Ces glycines (mais pas leurs positions exactes) sont conservées pour tous les canaux potassiques dépendants de potentiel. En utilisant la technique de mutagènes dirigé, la glycine a été remplacée dans chacune de ces différentes positions par une alanine et dans une deuxième étape...
An ellipsoidally shaped body, or more commonly, an ellipsoid of revolution, is generally assumed to serve as a convenient model for evaluating the rotational diffusion properties of macromolecules. If Perrin's equations for the rotational diffusion coefficients of general ellipsoids can be shown to generate all possible rotational diffusion coefficients, then there would exist at least one equivalent ellipsoidal shape for every arbitrarily shaped rigid body. We investigated the problem by first generating a space, r-space, representing all possible ellipsoidal shapes. We then generated another space, D-space, representing all possible combinations of rotational diffusion coefficients. We then mapped r-space into D-space by using Perrin's equations. Ellipsoidal shapes map into diffusion space in a well-defined manner. The mapping is either 1:1, 2:1, or 3:1; several distinctly different regions of r-space map onto the same regions of D-space. Thus, for some combinations of rotational diffusion coefficients, more than one ellipsoid can be used as a model. Not all of D-space is covered by the mapping of r-space. Therefore, there are combinations of rotational diffusion coefficients that cannot be generated from ellipsoidally shaped bodies. Several examples of rigid body shapes with nonellipsoidal diffusion properties are described.
The measurement of Förster resonance energy transfer (FRET) at the single-molecule level provides a powerful method for monitoring the structural dynamics of biomolecular systems in real time. These single-molecule FRET (smFRET) assays enable the characterization of the transient intermediates that form during enzymatic processes, providing information about the mechanism and regulation of the enzymes involved. In this dissertation, I develop and use smFRET assays to study two processes driven by motor proteins—reverse transcription and chromatin remodeling—and reveal novel features of their mechanism and regulation.
Reverse transcription of the human immunodeficiency virus genome initiates from a cellular tRNA primer that is bound to a specific sequence on the viral RNA (vRNA). During initiation, reverse transcriptase (RT) exhibits a slow mode of synthesis characterized by pauses at specific locations, and RT transitions to a faster mode of synthesis after the extension of the tRNA primer by six nucleotides. By using smFRET to examine how RT interacts with the tRNA-vRNA substrate, we found that RT binds to its substrate in either an active or inactive orientation and samples the two orientations during a single binding event. The equilibrium between these two orientations is a major factor influencing the activity and pausing of the enzyme...
The human body is composed of hundreds of specialized cell types, each fulfilling distinct functions that are together essential for normal tissue homeostasis. This thesis is aimed at identifying genes that contribute to to cell type-specific functions, with major projects focused on (1) a specialized epithelial transport pathway called transcytosis and (2) the challenge of measuring cell type-specific gene expression. In both projects, we applied high-throughput methods to narrow down from the ~25,000 protein coding genes to distinguish the subset that contribute to specialized cellular functions. Common themes include the development of enabling technology and the value of integrating diverse genomic datasets. The results described here implicate new genes in cell type-specific processes and provide a starting place for subsequent investigation into the individual genes and pathways.
In the first project, we performed an RNA interference (RNAi) screen to identify genes necessary for receptor-mediated transcytosis, a specialized endosomal pathway in epithelial cells. We developed high-throughput assays to measure the transcytosis of immunoglobulin G (IgG) across cultured epithelial cells in conjunction with gene knockdown. Then we selected a set of 582 candidate genes to screen using a combination of literature review and integrated high-throughput evidence...
This paper presents yet another personal reflection on one the most important
concepts in both science and the humanities: time. This elusive notion has been
not only bothering philosophers since Plato and Aristotle. It goes throughout
human history embracing all analytical and creative (anthropocentric)
disciplines. Time has been a central theme in physical and life sciences,
philosophy, psychology, music, art and many more. This theme is known with a
vast body of knowledge across different theories and categories. What has been
explored concerns its nature (rational, irrational, arational),
appearances/qualia, degrees, dimensions and scales of conceptualization
(internal, external, fractal, discrete, continuous, mechanical, quantum, local,
global, etc.). Of particular interest have been parameters of time such as
duration ranges, resolutions, modes (present, now, past, future), varieties of
tenses (e.g. present perfect, present progressive, etc.) and some intuitive,
but also fancy phenomenological characteristics such as arrow, stream, texture,
width, depth, density, even scent. Perhaps the most distinct characteristic of
this fundamental concept is the absolute time constituting the flow of
consciousness according to Husserl, the reflection of pure (human) nature
without having the distinction between exo and endo. This essay is a personal
reflection upon the meaning of time in modern physics and phenomenological
philosophy.; Comment: 35 pages...
Improving the scientific literacy of non-scientists is an important goal,
both because of the ever-increasing impact of science and technology on our
lives, and because understanding science enriches our experience of the natural
world. One route to improving scientific literacy is via general education
undergraduate courses -- i.e. courses intended for students not majoring in the
sciences or engineering -- which in many cases provide these students' last
formal exposure to science. I describe here a course on biophysics for
non-science-major undergraduates recently developed at the University of Oregon
(Eugene, OR, USA). Biophysics, I claim, is a particularly useful vehicle for
addressing scientific literacy. It involves important and general scientific
concepts, demonstrates connections between basic science and tangible, familiar
phenomena related to health and disease, and illustrates that scientific
insights develop by applying tools and perspectives from disparate fields in
creative ways. In addition, biophysics highlights the far-reaching impact of
physics research. I describe the general design of this course, which spans
both macroscopic and microscopic topics, and the specific content of a few of
its modules. I also describe evidence-based pedagogical approaches adopted in
teaching the course...
This study is focused on designing and characterizing protein-based building blocks in order to construct self-assembled nano-structured biomaterials. In detail, this research aims to: (1) investigate a new class of proteins that possess nanospring behaviors at a single-molecule level, and utilize these proteins along with currently characterized elastomeric proteins as building blocks for nano-structured biomaterials; (2) develop a new method to accurately measure intermolecular interactions of self-assembling two or more arbitrary (poly)peptides, and select some of them which have appropriate tensile strength for crosslinking the proteins to construct elastomeric biomaterials; (3) construct well-defined protein building blocks which are composed of elastomeric proteins terminated with self-oligomerizing crosslinkers, and characterize self-assembled structures created by the building blocks to determine whether the elasticity of proteins at single-molecule level can be maintained.
Primary experimental methods of this research are (1) atomic force microscope (AFM) based single-molecule force spectroscopy (SMFS) that allows us to manipulate single molecules and to obtain their mechanical properties such as elasticity, unfolding and refolding properties...
Understanding the interconversion between the thermodynamically distinguishable states present in a protein folding pathway provides not only the kinetics and energetics of protein folding but also insights into the functional roles of these states in biological systems. The protein component of bacterial RNase P holoenzyme from Bacillus subtilis (P protein) was used as a model system to elucidate the general folding/unfolding of an intrinsically unstructured protein (IUP) both in the absence and presence of ligands.
P protein was previously characterized as an intrinsically unstructured protein, and it is predominantly unfolded in the absence of ligands. Addition of small anions can induce the protein to fold. Therefore, the folding and binding are tightly coupled. Trimethylamine-N oxide (TMAO), an osmolyte that stabilizes the unliganded folded form of the protein, enabled us to study the folding process of P protein in the absence of ligand. Transient stopped-flow kinetic time courses at various final TMAO concentrations showed multiphase kinetics. Equilibrium "cotitration" experiments were performed using both TMAO and urea to obtain a TMAO-urea titration surface of P protein. Both kinetic and equilibrium studies show evidence of an intermediate state in the P protein folding process. The intermediate state is significantly populated and the folding rate constants involved in the reaction are slow relative to similar size proteins.
NMR spectroscopy was used to characterize the structural properties of the folding intermediate of P protein. The results indicate that the N-terminal (residues 2-19) and C-terminal regions (residues 91-116...