Investigation of the heat shock response in yeast: quantitative modeling and single-cell microfluidic studies

Heat shock response (HSR) is an ancient and highly conserved signaling pathway in cells that regulates the expression of heat shock proteins (HSPs) in the presence of thermal and other environmental stresses. HSPs function to prevent the formation of non-specific protein aggregates and to assist proteins in acquiring their native structures. Although HSR has been extensively studied, key aspects of this pathway remain a mystery. In particular, how HSR is activated and regulated by the master transcription factor HSF1 is not well understood. The broad goal of this thesis is to develop a quantitative framework aimed at elucidating the HSF1-mediated activation of HSR in yeast cells. Understanding this process has important implications for development, physiology and disease. Indeed, HSF1 is conserved from yeast to human, has been shown to play an important role in stress resistance, health and disease, and is a therapeutic target for neurodegenerative diseases. Broadly, there are two putative (not mutually exclusive) models for activation in response to heat shock: (1) HSF1 dissociation from chaperone proteins and (2) hyper-phosphorylation and the subsequent activation of HSF1. However the relative contribution of each of these events in the activation process is not characterized. Thus far, there is no direct evidence linking either of these two events to activation, and the relative contribution of each mechanism to the activation process has not been quantitatively characterized. To address these issues, we develop a quantitative model of HSR in yeast cells. We use the model to make a series of quantitative predictions and, in a collaborative effort, experimentally test these predictions in a yeast model of HSR. Critically, we provide the first direct evidence for chaperone dissociation of HSF1 in response to heat shock. Moreover, we find that HSF1 phosphorylation is dispensable for activation of HSR, but is able to modulate its activity. Taken together, our work leads to a model for two “orthogonal” mechanisms regulating HSR in yeast, in which chaperone dissociation acts as an ON/OFF switch, whereas phosphorylation functions to tune the gain of the response. Finally, to complement and further test this quantitative model, we develop a novel microfluidic system to explore in more depth the behavior of individual cells in the presence of heat shock inputs. This includes (1) a microfluidic device with microscale on-chip heaters enabling programmable thermal perturbations and (2) a custom image analysis platform to follow single cells through heat shock time courses. In preliminary single-cell studies, we find a relationship between HSF1 phosphorylation state and cell-to-cell variability in HSR activation level (as measured by a transcriptional reporter). These preliminary results suggest that HSF1 phosphorylation may be generating and tuning noise in the HSR in order to promote phenotypic plasticity and increased survivability of a cell population in the face of stress

Facile preparation of a nanostructured functionalized catalytically active organosalt

We report a novel nanostructured organosalt, based on sulfonic acid functionalized pyrazinium {[H-pyrazine–SO3H]Cl2} that was synthesized and characterized by several techniques including Fourier transform infrared (FT- IR) spectroscopy, X-ray diffraction (XRD), thermal gravimetric analysis (TGA), differential thermal gravimetric (DTG) analysis, transmission electron microscopy (TEM), mass spectrometry (MS), proton NMR (1H NMR), carbon-13 NMR (13C NMR) and also electron diffraction (ED) patterns. Results proved that the unprecedented sulfonated pyrizinium organosalt is indeed nanostructured and highly crystalline as supported by TEM, ED and XRD studies, having an average nanoparticle size of 50 nm according to TEM micrographs. The novel nano- organocatalyst was proved to be an efficient catalyst in the synthesis of 1,2,4,5-tetrasubstituted imidazoles by a one-pot multi-component condensation of benzil, a broad range of aldehydes, primary amines and ammonium acetate at 90 °C under solvent-free conditions

On-chip polyelectrolyte coating onto magnetic droplets-towards continuous flow assembly of drug delivery capsules

Polyelectrolyte (PE) microcapsules for drug delivery are typically fabricated via layer-by-layer (LbL) deposition of PE layers of alternating charge on sacrificial template microparticles, which usually requires multiple incubation and washing steps that render the process repetitive and time-consuming. Here, ferrofluid droplets were explored for this purpose as an elegant alternative of templates that can be easily manipulated via an external magnetic field, and require only a simple microfluidic chip design and setup. Glass microfluidic devices featuring T-junctions or flow focusing junctions for the generation of oil-based ferrofluid droplets in an aqueous continuous phase were investigated. Droplet size was controlled by the microfluidic channel dimensions as well as the flow rates of the ferrofluid and aqueous phases. The generated droplets were stabilised by a surface active polymer, polyvinylpyrrolidone (PVP), and then guided into a chamber featuring alternating, co-laminar PE solutions and wash streams, and deflected across them by means of an external permanent magnet. The extent of droplet deflection was tailored by the flow rates, the concentration of magnetic nanoparticles in the droplets, and the magnetic field strength. PVP-coated ferrofluid droplets were deflected through solutions of polyelectrolyte and washing streams using several iterations of multilaminar flow designs. This culminated in an innovative "Snakes-and-Ladders" inspired microfluidic chip design that overcame various issues of the previous iterations for the deposition of layers of anionic poly(sodium-4-styrene sulfonate) (PSS) and cationic poly(fluorescein isothiocyanate allylamine hydrochloride) (PAH-FITC) onto the droplets. The presented method demonstrates a simple and rapid process for PE layer deposition in <30 seconds, and opens the way towards rapid layer-by-layer assembly of PE microcapsules for drug delivery applications.The authors thank the Royal Embassy of Saudi Arabia Cultural Bureau in London and Albaha University in Saudi Arabia for funding. J.G.-P., E.B. and I.O. acknowledge financial support from the Spanish Ministry of Economy and Competitiveness (project CTQ2015-66078-R (MINECO/FEDER) and FPI postgraduate research grant (BES-2013-064415). The authors thank Dr Stephen Clark for fabrication of the microfluidic devices

Active control of microdroplets and particles in microfluidics

152 p.The manipulation of small amounts of fluids in the order of nano and pico liters has emerged as a new scientific field of microfluidics. This field has potentially a huge im-pact on chemical synthesis, biological analysis, optics and information technology. At the beginning, all microftuidic devices were designed to manipulate continuous flow, while re-cently there has been a shift towards working with discreet droplets. Droplet manipulation allows the use of non mechanical methods for actuation and in some cases elimination of micro and nanochannels in the chips. The field is new and therefore still needs improve-ments in the actuation schemes. The purpose of this research is using magnetic force for active control and manipulation of droplets.MASTER OF ENGINEERING (MAE

Programmable two-dimensional actuation of ferrofluid droplet using planar microcoils

This paper reports the concept and the device for two-dimensional magnetic actuation of a ferrofluid droplet. Four planar microcoils were etched on one side of a printed circuit board (PCB). The magnetic field was digitally controlled by adjusting the magnitude and the polarity of the driving current in the coils. A computer programme generates the control signals, which are conditioned by an external amplifier circuit and transferred to the coils. The ferrofluid droplet is attracted to the field maximum. With the controlled magnetic field, the location of the field maximum can be changed electronically allowing the droplet to move in a closed loop on the planar platform. The concept presented in this paper can have a variety of applications in digital microfluidics such as sample transport or mixing.Accepted versio

Modeling and optimization of planar microcoils

Magnetic actuation has emerged as a useful tool for manipulating particles, droplets and biological samples in microfluidics. A planar coil is one of the suitable candidates for magnetic actuation and has the potential to be integrated in digital microfluidic devices. A simple model of microcoils is needed to optimize their use in actuation applications. This paper first develops an analytical model for calculating the magnetic field of a planar microcoil. The model was validated by experimental data from microcoils fabricated on printed circuit boards (PCB). The model was used for calculating the field strength and the force acting on a magnetic object. Finally, the effect of different coil parameters such as the magnitude of the electric current, the gap between the wires and the number of wire segments is discussed. Both analytical and experimental results show that a smaller gap size between wire segments, more wire segments and a higher electric current can increase both the magnitude and the gradient of the magnetic field, and consequently cause a higher actuating force. The planar coil analyzed in the paper is suitable for applications in magnetic droplet-based microfluidics.Accepted versio

Kinematics and deformation of ferrofluid droplets under magnetic actuation

This paper reports the experimental results on kinematics and deformation of ferrofluid droplets driven by planar coils. Ferrofluid droplets act as liquid magnets, which can be controlled and manipulated by an external magnetic field. In our experiments, the magnetic field was generated by two pairs of planar coils, which were fabricated on a double-sided printed circuit board. The first pair of coils constrains the ferrofluid droplet to a one-dimensional motion. The second pair generates the magnetic gradient needed for the droplet motion. The direction of the motion can be controlled by changing the sign of the gradient or of the driving current. Kinematic characteristics of the droplet such as the velocity–position diagram and the aspect ratio of the droplet are investigated. The analysis and discussion are based on the different parameters such as the droplet size, the viscosity of the surrounding medium, and the driving current. This simple actuation concept would allow the implementation of lab-on-a-chip platforms based on ferrofluid droplets

Hsf1 Phosphorylation Generates Cell-to-Cell Variation in Hsp90 Levels and Promotes Phenotypic Plasticity

SUMMARY Clonal populations of cells exhibit cell-to-cell variation in the transcription of individual genes. In addition to this noise in gene expression, heterogeneity in the proteome and the proteostasis network expands the phenotypic diversity of a population. Heat shock factor 1 (Hsf1) regulates chaperone gene expression, thereby coupling transcriptional noise to proteostasis. Here we show that cell-to-cell variation in Hsf1 activity is an important determinant of phenotypic plasticity. Budding yeast cells with high Hsf1 activity were enriched for the ability to acquire resistance to an antifungal drug, and this enrichment depended on Hsp90, a known phenotypic capacitor and canonical Hsf1 target. We show that Hsf1 phosphorylation promotes cell-to-cell variation, and this variation, rather than absolute Hsf1 activity, promotes antifungal resistance. We propose that Hsf1 phosphorylation enables differential tuning of the proteostasis network in individual cells, allowing populations to access a range of phenotypic states