Browsing by Author "Petrache, Horia"

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    A Computational Framework for Investigating mRNA Localization Patterns in Pancreatic Beta-Cells During Type 1 Diabetes Progression
    (2024-12) Chang, Hok Wai; Petrache, Horia; Liu, Jing; Wassall, Stephen; Vemuri, Gautam; Syed, Farooq
    Spatial transcriptomics improves transcriptomic studies by incorporating RNA localization information, which provides a more profound insight into cellular functions, interactions between cells, and their reactions to external stimuli. Single-molecule fluorescent in situ hybridization (smFISH) is a commonly utilized technique in spatial transcriptomics that allows for the accurate visualization of mRNA distribution in cells. This method aids in the quantitative evaluation of mRNA localization patterns by utilizing various physical properties, thereby illuminating processes such as transcription, nuclear export, and localized translation. Nevertheless, existing computational approaches for analyzing smFISH images often have constraints, concentrating primarily on cellular expression or specific biological contexts while overlooking broader physical analysis. In my PhD research, I created STProfiler, a comprehensive tool aimed at an unbiased physical examination of mRNA distribution. STProfiler includes an image analysis workflow that processes raw biological images to effectively detect mRNA and nuclei. It also employs machine learning techniques to biologically interpret mRNA spatial characteristics and categorize cells based on these features. My dissertation illustrates the use of STProfiler in multiple studies investigating the transcriptomic profiles of β-cells during the progression of type 1 diabetes (T1D), uncovering spatial transcriptomic diversity in β-cells. These investigations involve analyzing mRNA clusters and stress granules in pancreatic β-cells, measuring the physical characteristics of mRNAs linked to cellular stress and inflammation in mice developing T1D, evaluating the rise in HLA-DMB mRNA spliced variant in T1D, and exploring miRNA as a potential biomarker for T1D. Furthermore, STProfiler has also proven beneficial in tissue-wide spatial transcriptomics by creating masks for nuclei and cells from biological images and assigning mRNA transcripts to develop subcellular expression profiles. This capability allows for more thorough bioinformatic evaluations. In summary, STProfiler serves as a robust tool for both cell- and tissue-level spatial transcriptomics, offering an unbiased platform for researchers to investigate complex transcriptomic variations within cells.
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    Advancing super-resolution microscopy for quantitative in-vivo imaging of chromatin nanodomains
    (2024-12) Seitz, Clayton; Liu, Jing; Vemuri, Gautam; Cheng, Ruihua; Wassall, Stephen; Petrache, Horia
    Single molecule localization microscopy (SMLM) techniques, such as direct stochastic optical reconstruction microscopy (dSTORM), can be used to produce a pointillist representation of fluorescently-labeled biological structures at diffraction-unlimited precision. This class of techniques permits localization of fluorescent molecules in the cell with nanometer precision and thus is commonly referred to as nanoscopy. Conventional nanoscopy utilizes the deactivation of standard fluorescent tags, followed by spontaneous or photoinduced reactivation, to resolve fluorophores at distances below the diffraction limit. While powerful, this approach has limited throughput and requires localization in sparse scenes. This dissertation introduces fluorescence nanoscopy and covers its innovation and application as discussed in the following papers: Quantum enhanced localization microscopy with a single photon avalanche diode array [ 1 ] leverages recent advancements in single photon avalanche diode array technology to count fluorescent emitters using a widefield microscope. Uncertainty-aware localization microscopy by variational diffusion [ 2 ] describes a novel algorithm that applies a diffusion model in order to model a posterior distribution on high resolution localization microscopy images, given low resolution inputs. role of the BRD4 phosphoswitch in the maintenance of chromatin nanodomains via super resolution microscopy and molecular dynamics simulation. We demonstrate that BRD4 phosphorylation regulates chromatin packing and mobility in mammalian nuclei.
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    Biophysical studies of cholesterol in unsaturated phospholipid model membranes
    (2013) Williams, Justin A.; Wassall, Stephen R.; Decca, Ricardo; Petrache, Horia; Zhu, Fangqiang; Todd, Brian A.
    Cellular membranes contain a staggering diversity of lipids. The lipids are heterogeneously distr ibuted to create regions, or domains, whose physical properties differ from the bulk membrane and play an essential role in modulating the function of resident proteins. Many basic questions pertaining to the formation of these lateral assemblies remain. T his research employs model membranes of well - defined composition to focus on the potential role of polyunsaturated fatty acids (PUFAs) and their interaction with cholesterol (chol) in restructuring the membrane environment. Omega - 3 (n - 3) PUFAs are the main bioactive components of fish oil, whose consumption alleviates a variety of health problems by a molecular mechanism that is unclear. We hypothesize that the incorporation of PUFAs into membrane lipids and the effect they have on molecular organization may be, in part, responsible. Chol is a major constituent in the plasma membrane of mammals. It determines the arrangement and collective properties of neighboring lipids, driving the formation of domains via differential affinity for different lipids . T he m olecular organization of 1 -[ 2 H 31 ]palmitoyl -2- eicosapentaenoylphosphatidylcholine (PEPC - d 31 ) and 1 -[ 2 H 31 ]palmitoyl -2- docosahexaenoylphosphatidylcholine (PDPC -d 31 ) in membran es with sphingomyelin (SM) and chol (1:1:1 mol) was compared by solid - state 2 H NMR spectroscopy. Eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are the two major n - 3 PUFAs found in fish oil, while PEPC -d 31 and PDPC -d 31 are phospholipids containing the respective PUFAs at the sn - 2 position and a perdeuterated palmitic acid a t the sn - 1 position . Analysis of s pectra recorded as a function of temperature indicate s that in both cases, formation of PUFA - rich (less ordered) and SM - rich (more ordered) domains occurred. A surprisingly substantial proportion of PUFA was found to infil trate the more ordered domain. There was almost twice as much DHA (65%) as EPA (30%) . The implication is that n - 3 PUFA s can incorporate into lipid rafts, which are domains enriched in SM and chol in the plasma membrane, and potentially disrupt the activity of signaling proteins that reside therein. DHA, furthermore, may be the more potent component of fish oil. PUFA - chol interactions were also examined through affinity measurements. A novel method utilizing electron paramagnetic resonance (EPR) was develope d, to monitor the partitioning of a spin - labeled analog of chol , 3β - doxyl - 5α - cholestane (chlstn), between large unilamellar vesicles (LUVs) and met hyl - β - cyclodextrin (mβCD). The EPR spectra for chlstn in the two environments are distinguishable due to the substantial differences in tumbling rates , allowing the population distribution ratio to be determined by spectral simulation. Advantages of this approach include speed of implementation and a vo idance of potential artifact s associated with physical separation of LUV and mβCD . Additionally, in a check of the method, t he relative partition coefficients between lipids measured for the spin label analog agree with values obtained for chol by isothermal titration calorimetry (ITC). Results from LUV with different composition confirmed a hierarchy of decreased sterol affinity for phospholipids with increasing acyl chain unsaturation , PDPC possessing half the affinity of the corresponding monounsaturated phospholipid. Taken together, the results of these studies on model membranes demonstrate the potential for PUFA - driven alteration of the architecture of biomembranes, a mechanism through which human health may be impacted.
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    Characterization and Measurement of Hybrid Gas Journal Bearings
    (2012-08-28) Lawrence, Tom Marquis; Kemple, Marvin D.; Decca, Ricardo; Joglekar, Yogesh; Petrache, Horia; Akay, Hassan; Krousgrill, Charles Morton
    This thesis concentrates on the study of hybrid gas journal bearings (bearings with externally pressurized mass addition). It differs from most work in that it goes back to “basics” to explore the hydrodynamic phenomena in the bearing gap. The thesis compares geometrically identical bearings with 2 configurations of external pressurization, porous liners where mass-addition compensation is varied by varying the liner’s permeability, and bushings with 2 rows of 6 feedholes where the mass-addition compensation is varied by the feedhole diameter. Experimentally, prototype bearings with mass-addition compensation that spans 2 orders of magnitude with differing clearances are built and their aerostatic properties and mass addition characteristics are thoroughly tested. The fundamental equations for compressible, laminar, Poiseuille flow are used to suggest how the mass flow “compensation” should be mathematically modeled. This is back-checked against the experimental mass flow measurements and is used to determine a mass-addition compensation parameter (called Kmeas) for each prototype bushing. In so doing, the methodology of modeling and measuring the mass addition in a hybrid gas bearing is re-examined and an innovative, practical, and simple method is found that makes it possible to make an “apples-to-apples” comparison between different configurations of external pressurization. This mass addition model is used in conjunction with the Reynolds equation to perform theory-based numerical analysis of virtual hybrid gas journal bearings (CFD experiments). The first CFD experiments performed use virtual bearings modeled to be identical to the experimental prototypes and replicate the experimental work. The results are compared and the CFD model is validated. The ontological significance of appropriate dimensionless similitude parameters is re-examined and a, previously lacking, complete set of similitude factors is found for hybrid bearings. A new practical method is developed to study in unprecedented detail the aerostatic component of the hybrid bearings. It is used to definitively compare the feedhole bearings to the porous liner bearings. The hydrostatic bearing efficiency (HBE) is defined and it is determined that the maximum achievable hydrostatic bearing efficiency (MAHBE) is determined solely by the bearing’s mass addition configuration. The MAHBE of the porous liner bearings is determined to be over 5 times that of the feedhole bearings. The method also presents a means to tune the Kmeas to the clearance to achieve the MAHBE as well as giving a complete mapping of the hitherto misunderstood complex shapes of aerostatic load versus radial deflection curves. This method also rediscovers the obscure phenomenon of static instability which is called in this thesis the “near surface effect” and appears to be the first work to present a practical method to predict the range of static instability and quantify its resultant stiffness fall-off. It determines that porous liner type bearings are not subject to the phenomenon which appears for feedhole type bearings when the clearance exceeds a critical value relative to its mass-addition compensation. The standing pressure waves of hydrostatic and hybrid bearings with the 2 configurations of external pressurization as well as a geometrically identical hydrodynamic bearing are studied in detail under the methodology of the “CFD microscope”. This method is used to characterize and identify the development, growth, and movement of the pressure wave extrema with increased hydrodynamic action (either increasing speed or increasing eccentricity). This method is also used to determine the “cause” of the “near surface effect”. A gedanken experiment is performed based on these results which indicates that a bearing with a “stronger aerostatic strength” component should be more stable than one with a low aerostatic strength component. Numerical instability “speed limits” are found that are also related to the hydrostatic strength of the bearing. The local conditions in the standing waves are characterized in terms of their local Mach number, Knudsen number, Reynolds number, and Taylor Number. It is concluded that low eccentricity bearing whirl can be attributed to the off load-line orientation of the bearing load force caused by the overlay of the hydrodynamic bearing standing wave onto the hydrostatic bearing wave of the hybrid bearing, whereas it is hypothesized that aperiodic and random self-excited vibration which occurs at high eccentricity, as reported in the literature, is probably due to shock waves, turbulence, near surface effect, and slip at local areas of the standing wave.
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    Developing an approach to improve beta-phase properties in ferroelectric pvd-hfp thin films
    (2020-05) Dale, Ashley S.; Cheng, Ruihua; Petrache, Horia; Wassall, Stephen
    Improved fabrication of poly(vinylindenefluoride)-hexafluoropropylene (PVDF-HFP) thin films is of particular interest due to the high electric coercivity found in the beta-phase structure of the thin film. We show that it is possible to obtain high-quality, beta-phase dominant PVDF-HFP thin films using a direct approach to Langmuir-Blodgett deposition without the use of annealing or additives. To improve sample quality, an automated Langmuir-Blodgett thin film deposition system was developed; a custom dipping trough was fabricated, a sample dipping mechanism was designed and constructed, and the system was automated using custom LabVIEW software. Samples were fabricated in the form of ferroelectric capacitors on substrates of glass and silicon, and implement a unique step design with a bottom electrode of copper with an aluminum wetting layer and a top electrode of gold with an aluminum wetting layer. Samples were then characterized using a custom ferroelectric measurement program implemented in LabVIEW with a Keithley picoammeter/voltage supply to confirm electric coercivity properties. Further characterization using scanning electron microscopy and atomic force microscopy confirmed the improvement in thin film fabrication over previous methods.
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    The Effect of Acyl Chain Unsaturation on Phospholipid Bilayer
    (2010-02-26T17:51:02Z) Soni, Smita Pravin; Wassall, Stephen R.; Petrache, Horia; Kemple, Marvin D.; Rader, Andrew J.
    Each biological cell is surrounded by a membrane that consists of many different kinds of lipids. The lipids are mainly composed of phospholipids, which form a fluid bilayer that serves as the platform for the function of membrane bound proteins regulating cellular activity. In the research described in this thesis we employed solid state 2H NMR, complemented by DSC (differential scanning calorimetry) and MD (molecular dynamics) simulations, to study the effect of PUFA (polyunsaturated fatty acids) and TFA (trans fatty acids) on molecular organization in protein-free model membranes of controlled composition. These two classes of unsaturated fatty acid incorporate into membrane lipids and have, respectively, a beneficial and harmful impact on health. The aim is to gain insight into the molecular origin of this behavior. DHA (docosahexaenoic acid), which with 6 "natural" cis double bonds is the most highly unsaturated PUFA found in fish oils, and EA (elaidic acid), which with only a single "unnatural" trans double bond is the simplest manmade TFA often found in commercially produced food, were the focus. 2H NMR spectra for [2H31]-N-palmitoylsphingomyelin ([2H31]16:0SM) in SM/16:0-22:6PE (1-palmitoyl-2-docosahexaenoylphosphatidylethanolamine)/cholesterol (1:1:1 mol) mixed membranes were recorded. This system served as our PUFA-containing model. The spectra are consistent with lateral separation into nano-sized (< 20 nm) domains that are SM-rich/cholesterol-rich (raft), characterized by higher chain order, and DHA-rich/cholesterol-poor (non-raft), characterized by lower chain order. The aversion cholesterol has for DHA, as opposed to the affinity cholesterol has for predominantly saturated SM, excludes the sterol from DHA-containing PE-rich domains and DHA from SM-rich/cholesterol-rich domains. It is the formation of highly disordered membrane domains that we hypothesize is responsible, in part, for the diverse health benefits associated with dietary consumption of DHA. 2H NMR spectra for 1-elaidoyl-2-[2H35]stearoylphosphatidylcholine (t18:1-[2H35]18:0PC) and 1-oleoyl-2-[2H35]stearoylphosphatidylcholine (c18:1-[2H35]18:0PC) were recorded to compare membranes with respect to a trans vs. cis ("natural") double bond. The spectra indicate that while a trans double bond produces a smaller deviation from linear conformation than a cis double bond, membrane order is decreased by a comparable amount because the energy barrier to rotation about the C-C single bonds either side of a trans or cis double bond is reduced. Although EA adopts a conformation somewhat resembling a saturated fatty acid, the TFA is almost as disordered as its cis counterpart oleic acid (OA). We speculate that EA could be mistaken for a saturated fatty acid and infiltrate lipid rafts to disrupt the high order therein that is necessary for the function of signaling proteins.
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    Effects of carbon nanotubes on airway epithelial cells and model lipid bilayers : proteomic and biophysical studies
    (2014) Li, Pin; Blazer-Yost, Bonnie; Witzmann, F. A. (Frank A.); Randall, Stephen Karl, 1953-; Petrache, Horia; Wang, Xianzhong
    Carbon nanomaterials are widely produced and used in industry, medicine and scientific research. To examine the impact of exposure to nanoparticles on human health, the human airway epithelial cell line, Calu-3, was used to evaluate changes in the cellular proteome that could account for alterations in cellular function of airway epithelia after 24 h exposure to 10 μg/mL and 100 ng/mL of two common carbon nanoparticles, singleand multi-wall carbon nanotubes (SWCNT, MWCNT). After exposure to the nanoparticles, label-free quantitative mass spectrometry (LFQMS) was used to study differential protein expression. Ingenuity Pathway Analysis (IPA) was used to conduct a bioinformatics analysis of proteins identified by LFQMS. Interestingly, after exposure to a high concentration (10 μg/mL; 0.4 μg/cm2) of MWCNT or SWCNT, only 8 and 13 proteins, respectively, exhibited changes in abundance. In contrast, the abundance of hundreds of proteins was altered in response to a low concentration (100 ng/mL; 4 ng/cm2) of either CNT. Of the 281 and 282 proteins that were significantly altered in response to MWCNT or SWCNT, respectively, 231 proteins were the same. Bioinformatic analyses found that the proteins common to both kinds of nanotubes are associated with the cellular functions of cell death and survival, cell-to-cell signaling and interaction, cellular assembly and organization, cellular growth and proliferation, infectious disease, molecular transport and protein synthesis. The decrease in expression of the majority proteins suggests a general stress response to protect cells. The STRING database was used to analyze the various functional protein networks. Interestingly, some proteins like cadherin 1 (CDH1), signal transducer and activator of transcription 1 (STAT1), junction plakoglobin (JUP), and apoptosis-associated speck-like protein containing a CARD (PYCARD), appear in several functional categories and tend to be in the center of the networks. This central positioning suggests they may play important roles in multiple cellular functions and activities that are altered in response to carbon nanotube exposure. To examine the effect of nanotubes on the plasma membrane, we investigated the interaction of short purified MWCNT with model lipid membranes using a planar bilayer workstation. Bilayer lipid membranes were synthesized using neutral 1, 2-diphytanoylsn-glycero-3-phosphocholine (DPhPC) in 1 M KCl. The ion channel model protein, Gramicidin A (gA), was incorporated into the bilayers and used to measure the effect of MWCNT on ion transport. The opening and closing of ion channels, amplitude of current, and open probability and lifetime of ion channels were measured and analyzed by Clampfit. The presence of an intermediate concentration of MWCNT (2 μg/ml) could be related to a statistically significant decrease of the open probability and lifetime of gA channels. The proteomic studies revealed changes in response to CNT exposure. An analysis of the changes using multiple databases revealed alterations in pathways, which were consistent with the physiological changes that were observed in cultured cells exposed to very low concentrations of CNT. The physiological changes included the break down of the barrier function and the inhibition of the mucocillary clearance, both of which could increase the risk of CNT’s toxicity to human health. The biophysical studies indicate MWCNTs have an effect on single channel kinetics of Gramicidin A model cation channel. These changes are consistent with the inhibitory effect of nanoparticles on hormone stimulated transepithelial ion flux, but additional experiments will be necessary to substantiate this correlation.
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    Effects of carbon nanotubes on barrier epithelial cells via effects on lipid bilayers
    (2013) Lewis, Shanta; Blazer-Yost, Bonnie; Petrache, Horia; Witzmann, F. A. (Frank A.); Atkinson, Simon
    Carbon nanotubes (CNTs) are one of the most common nanoparticles (NP) found in workplace air. Therefore, there is a strong chance that these NP will enter the human body. They have similar physical properties to asbestos, a known toxic material, yet there is limited evidence showing that CNTs may be hazardous to human barrier epithelia. In previous studies done in our laboratory, the effects of CNTs on the barrier function in the human airway epithelial cell line (Calu-3) were measured. Measurements were done using electrophysiology, a technique which measures both transepithelial electrical resistance (TEER), a measure of monolayer integrity, and short circuit current (SCC) which is a measure of vectorial ion transport across the cell monolayer. The research findings showed that select physiologically relevant concentrations of long single-wall (SW) and multi-wall (MW) CNTs significantly decreased the stimulated SCC of the Calu-3 cells compared to untreated cultures. Calu-3 cells showed decreases in TEER when incubated for 48 hours (h) with concentrations of MWCNT ranging from 4µg/cm2 to 0.4ng/cm2 and SWCNT ranging from 4µg/cm2 to 0.04ng/cm2. The impaired cellular function, despite sustained cell viability, led us to investigate the mechanism by which the CNTs were affecting the cell membrane. We investigated the interaction of short MWCNTs with model lipid membranes using an ion channel amplifier, Planar Bilayer Workstation. Membranes were synthesized using neutral diphytanoylphosphatidylcholine (DPhPC) and negatively charged diphytanoylphosphatidylserine (DPhPS) lipids. Gramicidin A (GA), an ion channel reporter protein, was used to measure changes in ion channel conductance due to CNT exposures. Synthetic membranes exposed to CNTs allowed bursts of currents to cross the membrane when they were added to the membrane buffer system. When added to the membrane in the presence of GA, they distorted channel formation and reduced membrane stability.
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    Elastic Properties of Polyunsaturated Phosphatidylethanolamines Influence Rhodopsin Function
    (Royal Society of Chemistry, 2013) Teague, Walter E., Jr.; Soubias, Olivier; Petrache, Horia; Fuller, Nola; Hines, Kirk G.; Rand, R. Peter; Gawrisch, Klaus; Physics, School of Science
    Membranes with a high content of polyunsaturated phosphatidylethanolamines (PE) facilitate formation of metarhodopsin-II (M(II)), the photointermediate of bovine rhodopsin that activates the G protein transducin. We determined whether M(II)-formation is quantitatively linked to the elastic properties of PEs. Curvature elasticity of monolayers of the polyunsaturated lipids 18 : 0-22 : 6(n - 3)PE, 18 : 0-22 : 5(n)- 6PE and the model lipid 18 : 1(n - 9)-18 : 1,(n- 9)PE were investigated in the inverse hexagonal phase. All three lipids form lipid monolayers with rather low spontaneous radii of curvature of 26-28 angstroms. In membranes, all three PEs generate high negative curvature elastic stress that shifts the equilibrium of MI(I)/M(II) photointermediates of rhodopsin towards M(II) formation.
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    Imaging the Dynamics of Chromatin at Single-Nucleosome Resolution
    (2024-12) Iqbal, Mohamed Fadil; Vemuri, Gautam; Liu, Jing; Petrache, Horia; Decca, Ricardo; Wassall, Stephen
    DNA is organized into chromatin – a complex polymeric structure which stores information and controls gene expressions. Advancements in microscopy have enabled us to see chromatin in motion – which was previously thought to be static, and these motions contribute to various cellular functions. In my thesis I will demonstrate the molecular tools and biophysical approaches our lab has developed to uncover the mysteries of chromatin dynamics and structures at the single nucleosome resolution; I will also discuss how these new discoveries in chromatin enable us to explore its role in cell functions. This dissertation will first describe the technology advancement of live-cell image analysis; particularly, I will discuss the utilization of AI to improve the spatial and temporal resolution of chromatin imaging. Then I will show complex nature of chromatin where depending on the temporal scale of observation we see a different behavior and how computer simulations can see these differences. Following that, I will introduce our investigation on the role of chromatin motion in DNA damage and repair. Afterwards, I will discuss how the cell regulates its chromatin dynamics in response to the metabolism indicators AMPK (AMP-activated protein kinase). I will also show how chromatin motion and structure behave without the presence of key proteins such as RAD51 that aid in DNA damage. Finally I will go over future directions and improvements we can do to our current techniques to improve our understanding of chromatin’s role is various biological functions. We expect that the exploration of the spatiotemporal dynamics in live cells will facilitate the diagnosis, treatment, and prevention of cancers.