Life Sciences Commons^™

Optimization Of Tyrosine Hydroxylase Antibody For Immunohistochemistry Fluorescence Detection In Zebrafish (Danio Rerio), Madison Thurber

Theses/Capstones/Creative Projects

Dopamine is an important neurotransmitter produced through the catecholamine synthesis pathway that affects brain activity. Unregulated dopamine levels can lead to various diseases such as Parkinson’s Disease or attention deficit hyperactivity disorder (ADHD). Optimization of an immunohistochemistry protocol will allow for the quantification of tyrosine hydroxylase antibody, which indirectly allows for dopamine quantification in dopaminergic regions within the brain. However, the antibody concentration to give the optimal signal-to-noise ratio in IHC varies across studies. Through this experiment, I determined the concentration of tyrosine hydroxylase (TyrH) antibody for immunohistochemistry that gave the best signal-to-background noise ratio within several known dopaminergic regions …

Intrinsically Disordered Proteins And Their Role In Biomolecular Condensates, Danielle Latham

All Dissertations

Proteins are biomacromolecules responsible for the functions of life. While classically proteins are thought to be well structured in order to perform a specific function, 50% of proteins within Eukaryotic cells contain intrinsically disordered regions (IDRs), regions with no well-defined structure. IDRs are often used for cell signaling, responding to external factors such as temperature changes or the presence of small molecules. To understand how IDRs can function without structure, it is important to understand the dynamics of such systems. Understanding IDR intramolecular and intermolecular interactions will shed light on IDR dynamics. Intramolecular interactions are first explored using fluorescence spectroscopy …

Synthesis And Biophysical Analysis Of Modified Cell-Penetrating Peptides, Joel Mitchell

Theses and Dissertations (Comprehensive)

Cell-penetrating peptides (CPPs) are a family of peptides that have the ability to penetrate biological membranes. They were discovered in the late 1980s and have been the topic of many studies. Much of the interest in CPPs has been due to their ability to translocate biological membranes, and the possibility that they could offer a novel drug delivery method by conjugation to biologically active molecules. Linear CPPs can be modified to form cyclic structures. This change in structure has been observed to enhance the stability and penetrative ability of the CPPs which have been studied. The current thesis focuses on …

Engineering Exosomes To Specifically Target The Mitochondria Of Brain Cells, Xin Yan, Xinqian Chen, Zhiying Shan, Lanrong Bi

Michigan Tech Publications, Part 2

Mitochondrial dysfunction is associated with various health conditions, including cardiovascular and neurodegenerative diseases. Mitochondrial-targeting therapy aims to restore or enhance mitochondrial function to treat or alleviate these conditions. Exosomes, small vesicles that cells secrete, containing a variety of biomolecules, are critical in cell-to-cell communication and have been studied as potential therapeutic agents. Exosome-based therapy has the potential to treat both cardiovascular and neurodegenerative diseases. Combining these two approaches involves using exosomes as carriers to transport mitochondrial-targeting agents to dysfunctional or damaged mitochondria within target cells. This article presents a new technique for engineering brain-derived exosomes that target mitochondria and has …

Engineering Exosomes To Specifically Target The Mitochondria Of Brain Cells, Xin Yan, Xinqian Chen, Zhiying Shan, Lanrong Bi

Michigan Tech Publications, Part 2

Mitochondrial dysfunction is associated with various health conditions, including cardiovascular and neurodegenerative diseases. Mitochondrial-targeting therapy aims to restore or enhance mitochondrial function to treat or alleviate these conditions. Exosomes, small vesicles that cells secrete, containing a variety of biomolecules, are critical in cell-to-cell communication and have been studied as potential therapeutic agents. Exosome-based therapy has the potential to treat both cardiovascular and neurodegenerative diseases. Combining these two approaches involves using exosomes as carriers to transport mitochondrial-targeting agents to dysfunctional or damaged mitochondria within target cells. This article presents a new technique for engineering brain-derived exosomes that target mitochondria and has …

Cellular Control Of Proteostasis, Cytoskeleton Organization, And Polarity Remodeling During Infection-Related Development By The Rice Blast Fungus, Audra Rogers

Graduate Theses and Dissertations

Understanding the intricate processes underlying fungal infection-related development is crucial for combating plant pathogens. In this study, we investigate the coordination of morphogenetic transitions during plant infection by Magnaporthe oryzae, the blast fungus. We focus on three key aspects: protein quality control, cytoplasmic microtubule organization, and cell-end marker proteins. M. oryzae forms a specialized infection cell known as an appressorium. The proper formation of this cell is crucial for the pathogen to infect a susceptible host. First, we investigate protein quality control mechanisms during infection-related development. Our findings demonstrate that aggregated proteins are sequestered into quality control compartments within conidia …

Fluorescent Molecular Rotors As Versatile In Situ Sensors For Protein Quantitation, Kevin Daus, Sorachat Tharamak, Wanchai Pluempanupat, Peter Galie, Maria A Theodoraki, Emmanuel A Theodorakis, Mary Alpaugh

Faculty Scholarship for the College of Science & Mathematics

Accurate protein quantitation is essential for many cellular mechanistic studies. Existing technology relies on extrinsic sample evaluation that requires significant volumes of sample as well as addition of assay-specific reagents and importantly, is a terminal analysis. This study exploits the unique chemical features of a fluorescent molecular rotor that fluctuates between twisted-to-untwisted states, with a subsequent intensity increase in fluorescence depending on environmental conditions (e.g., viscosity). Here we report the development of a rapid, sensitive in situ protein quantitation method using ARCAM-1, a representative fluorescent molecular rotor that can be employed in both non-terminal and terminal assays.

Binding Interactions Of Biologically Relevant Molecules Studied Using Surface-Modified And Nanostructured Surfaces, Palak Sondhi

Dissertations

This research focuses on the field of surface nanobioscience, wherein different nanosurfaces that will be used as working electrodes in the electrochemical cell are manufactured and surface modified to understand the critical binding interactions between biologically significant molecules like proteins, carbohydrates, small drug molecules, and glycoproteins. This research is essential if we are to determine whether a synthetic molecule can serve as a therapeutic candidate or diagnose a disease in its early stages. In order to fully understand the binding interactions, the study begins with defining some of the fundamental concepts, principles, and analytical tools for biosensing.

Afterwards, we addressed …

A Natural Near-Infrared Fluorescent Probe For Cancer Cell Imaging, Benilde E. Adriano, Nycol M. Cotto, Neeraj Chauhan, Meena Jaggi, Subhash C. Chauhan, Murali M. Yallapu

Research Colloquium

Introduction: Near-Infrared (NIR) fluorescence-based imaging is a noteworthy and safer strategy for cancer cells/tissues imaging compared to radiological imaging. NIR fluorescence offers deep tissue penetration and have minimal obstruction by autofluorescence and photon scattering [1]. There are several NIR dyes including indocyanine green (ICG) [2] and IR-1061 [3] that allow high-resolution tissue imaging. However, these dyes possess some low-quality characteristics which limit their use, namely photo instability, toxicity, poor water solubility, and short half-lives [4]. Therefore, more efficient and effective alternatives are urgently required to provide the desired clinical outcomes. Chlorophyll (Chl) is a natural dietary NIR fluorescence emitting …

The Effects Of Water Availability On Plant Growth In Sesleria Albicans - Dominated Grasslands In The Burren, Co. Clare, Patrick Moran, S. Ryal, B. Osborne

IGC Proceedings (1997-2023)

The Burren is a karstic region in the west of Ireland characterised by large areas of exposed limestone pavement with sparse vegetation. Despite the prevailing oceanic climate and high rainfall, substrate volumetric water content values are similar to those of semi-arid habitats due to high run-off. As a consequence, plants growing on the pavement regularly experience water deficit during the summer months. S. albicans, a species reported to be tolerant of water deficits, is one of the most abundant species growing on the limestone pavement. The objective of this study was to determine the impact of water availability on …

Examining Protein Localization Via Fluorescence Microscopy In Saccharomyces Cerevisiae, Lewis Barr, Richard H. Adams, Ellen France Phd

Biology Theses

Twenty-seven years ago, Saccharomyces cerevisiae became the first eukaryote to have its full genome sequenced, thus leading to the discovery that 30% of genes related to human diseases are orthologous to those in S. cerevisiae. Since then, 75% of the proteome has had its localization classified, and we sought to fill the remaining gaps of knowledge by identifying the localization of three proteins: Fsh3, Gid10, and Ade13, which function as a serine hydrolase, ubiquitin ligase, and adenylosuccinate lyase, respectively. To visualize cellular localization, we used a C-terminal GFP tagging strategy and subsequent fluorescence microscopy. Through colocalization analyses, we identified …

Examination Of Microplastics And Organic Contaminants In Tampa Bay’S Eastern Oyster (Crassostrea Virginica) Through Visual And Chemical Analysis, Andrea Murray

USF Tampa Graduate Theses and Dissertations

Microplastic pollution is an increasingly alarming concern with widespread global distribution in aquatic environments. Filter feeding organisms such as bivalves, have a heightened risk of microplastic ingestion through free floating particulates in the water. This can lead to bioaccumulation moving up through trophic levels to apex predators and humans. Spatial and temporal differences in microplastic abundance were evaluated in the Eastern Oyster, Crassostrea virginica, at six sites within Tampa Bay. Oyster tissue was digested using 30% hydrogen peroxide (H2O2) and microplastics were quantified using Nile Red dye and fluorescent particle excitement. A total of 3025 microplastics were found throughout six …

Structural Integrity And Stability Of Dna In Ionic Liquid And Near-Infrared Indolizine Squaraine Dye, Ember Yeji Suh

Honors Theses

Luminol, the most common presumptive test for blood at a crime scene, has multiple issues, such as false positive results with chemical agents, no luminescence due to “active oxygen” cleaning agents on bloodstains, and inability to penetrate textile materials. A combination of indolizine squaraine dye and ionic liquid (IL), or Dye Enhanced Textile Emission for Crime Tracking (DETECT), have shown potential to address these issues. The purpose of this study was to assess the binding mechanism of CG (1:1) and SO₃SQ dye to HSA and how the mechanism can explain the W214 fluorescence quenching effect and to determine …

Repurposing The Calcium Biosensor Twitch-2b: Creating Transition Metal Detection Proteins Through Fluorescent Analysis, Sarah Mateer

Honors Projects and Presentations: Undergraduate

Metal ions are key in regulating many biological functions, but their accumulation is associated with neurological diseases. Cellular metal ion concentrations are often detected through exogenous fluorescent dyes, but less invasive and more specific detection can be done using genetically encoded FRET-enabled metal-binding proteins, such as Twitch-2B. Twitch-2B and other ratiometric proteins change conformation when a calcium ion is bound to the active site, initiating an energy transfer between two fluorescent peptides that can be quantified through fluorescent spectroscopy. Previous analyses have demonstrated that wild-type calcium binding can be accurately detected via this method for concentrations from 10 uM to …

Genetic Stop Signs: Nontraditional Sequences That Terminate Rna Production, Christina Iverson, Samuel Scott

Library/Life Sciences Undergraduate Poster Competition 2023

3rd place open house

Gene expression can change everything about the identity of a bacterial cell. This process begins when the protein RNA polymerase reads a DNA strand and produces, or transcribes, a matching messenger RNA sequence. This RNA sequence will then be read by a protein-building complex which builds a protein encoded by the RNA. When RNA polymerase finishes producing the RNA sequence, the RNA polymerase will fall off the DNA strand, known as transcription termination. Currently, there are two known mechanisms for transcription termination. We believe that there may be more unknown mechanisms for termination.

Psf Sampling In Fluorescence Image Deconvolution, Eric A. Inman

Master's Theses

All microscope imaging is largely affected by inherent resolution limitations because of out-of-focus light and diffraction effects. The traditional approach to restoring the image resolution is to use a deconvolution algorithm to “invert” the effect of convolving the volume with the point spread function. However, these algorithms fall short in several areas such as noise amplification and stopping criterion. In this paper, we try to reconstruct an explicit volumetric representation of the fluorescence density in the sample and fit a neural network to the target z-stack to properly minimize a reconstruction cost function for an optimal result. Additionally, we do …

Comparison Of In-Vitro 3d Human Embryoids With Current Models For Gastrulation, Jin Park

McKelvey School of Engineering Theses & Dissertations

Gastrulation is an early morphogenetic process that is conserved across most metazoans and lays out the future body plan through the formation and shaping of the three germ layers: endoderm, mesoderm, and ectoderm. Despite its importance, not much is known about the events surrounding human gastrulation that occurs in utero due to ethical and technical limitations on studying human embryos. Therefore, many researchers have devised protocols for creating in vitro models of gastrulation using embryonic stem cells. Initially starting with mouse embryonic stem cells, the field of in vitro embryo models has advanced rapidly, with protocols using human embryonic stem …

Novel Mechanistic Insight Into Ciliary Regulation: Old Pathways Yield New Mechanisms, Larissa L. Dougherty

Dartmouth College Ph.D Dissertations

Cilia are structures present on most eukaryotic cells which provide important signaling and motile components to cells from early development to fully differentiated and matured cells. Regulation of these structures is critical to proper functioning of the cell and is known to be tied to the cell cycle. Preparation for ciliary assembly following cell cycle exit and ciliary disassembly following cell cycle reentry requires components throughout the cell body and within the cilium to facilitate this process. Here I identify how the cell adapts to ensure modifications to cilia occur for assembly or disassembly using the model organism Chlamydomonas reinhardtii. …

Carbon Dots: An Updated Overview On Their Synthesis, Properties, And Applications In Pharmaceutical And Biomedical Sciences, Earle Radha Rani, Majji Usharani, A. Lakshmi Usha

The Thai Journal of Pharmaceutical Sciences

Nowadays, carbon-based materials have acquired more attention in numerous fields including engineering, medicine, and pharmaceutics. As a new class of small carbon nanomaterials, carbon dots are developed due to their excellent physicochemical properties such as spherical shape, non-toxicity, cost effectiveness, facile, biocompatibility, and discrete particles <10 nm in diameter. In 2004, these carbon dots were first discovered accidentally during processing of single walled carbon nanotubes. Carbon dots have created huge excitement due to their advantages such as high water solubility, excellent biocompatibility, and resistance to photo bleaching. Carbon dots show unique properties such as photo stability, low toxicity, fluorescence, photo chemiluminescence, and electrochemiluminescence. Due to these properties, they have a wide range of applications in the field of bioimaging and drug delivery. Carbon dots are classified into two groups: Amorphous based carbon dots and graphene-based quantum dots with sp2 hybridization. The synthesis methods of carbon dots were reviewed along with their advantages and disadvantages. The applications of carbon dots in drug delivery, gene delivery, targeted drug delivery, crossing blood–brain barrier, and bio imaging were discussed.

Continuous Fluorescence-Based Endonuclease-Coupled Dna Methylation Assay To Screen For Dna Methyltransferase Inhibitors, Rebecca Switzer, Katie A. Ward, Jessica Medrano

Faculty Journal Articles

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential …

Mechanism Of Sila- And Germafluorenes For Biological Applications, Shelby Jarrett

Dissertations

2,7-disubstituted silafluorenes and germafluorenes, originally designed for OLED applications, are a class of fluorescent dyes that have gained recent interest as probes for bioimaging and as biosensors to monitor cellular dynamics and interactions. Desirable biological probes absorb in the visible region, have high extinction coefficients, high quantum yield and excellent photostability. Here, their spectral properties are investigated under aqueous conditions for relevant biological applications. These molecules display intense blue fluorescence in the solid state and in solution, have high extinction coefficients, and exhibit appreciable solubility in aqueous solution. To better understand potential applications, the mechanism of fluorescence was investigated. It …

The Synthesis And Characterization Of Ionic Liquids Using Nitrogen-Based Cations For Transdermal Delivery, Jo Galloway

Honors Theses

The purpose of this research was to explore the synthesis mechanisms of water-soluble ionic liquids with nitrogen-containing cationic bases for future use in transdermal drug delivery and forensic science applications. Ionic liquids are salts with an organic cation and either an organic or inorganic anion. They have asymmetric structures, which means that the molecules don’t pack together as neatly as other salts do, therefore, they don’t crystallize as easily, and their melting points are lower. Ionic liquids have melting points below 100°C, and many are liquid at room temperature. Mechanistic studies reveal that the potency of ILs in enhancing transdermal …

Investigation Of The Binding Domain Interfaces Of The C-Terminus Of The Albino3 Insertase And The 43kda Chloroplast Signal Recognition Particle Subunit Via Single Molecule Förster Resonance Energy Transfer, Amanda Tomanek

Chemistry & Biochemistry Undergraduate Honors Theses

Fluorescent labeling is a technique used for visualizing functional groups contained in biomolecules by fluorescence imaging. This technique was used in this project to analyze post-translational targeting of light-harvesting chlorophyll-binding proteins (LHCP), which are the core complexes that harvest sunlight to drive photosynthetic electron transfer. This protein is synthesized in the cytosol and post-translationally targeted to the stroma of chloroplasts. CpSRP43 is a signal recognition particle (SRP) subunit unique to chloroplasts, which has been shown to interact with the stroma-soluble C-terminus of the thylakoid-bound Albino3 insertase (Alb3-Cterm). In the chloroplast stroma, targeting to thylakoids is performed via the cpSRP pathway …

Investigating The Impact Of Hypoxia On Reactive Oxygen Species Generation Within Murine Breast Cancer Cells, Jared Mcpeake

Biomedical Engineering Undergraduate Honors Theses

When cancer metastasizes from a primary tumor site to secondary site through the bloodstream or lymph, the cancer becomes more difficult to treat. For this reason, it is vital to study what indicates the metastatic potential of a tumor. Current research has shown that cell lines with high metastatic potential display increased levels of metabolic adaptability over their nonmetastatic counterparts after undergoing hypoxic conditions. One method of assessing this adaptability is to measure the concentration of reactive oxygen species (ROS) produced by the cells while undergoing oxidative stress. In highly adaptable metastatic cells, an increase of ROS buildup within the …

Investigating The Main Protease (Mpro) Of Sars-Cov-2 As A Potential Drug Target, Valerie Giovina Pascetta

Honors Theses and Capstones

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 19 (COVID-19) pandemic has claimed the lives of roughly 6.2 million people worldwide as of May 2022. The virus’s main protease (M^pro ) has been identified as an attractive drug target due to the critical role it plays in the viral life cycle. The roughly 34 kDa M^pro cleaves functional viral polypeptides out of two long polyproteins at conserved cut sites, allowing them to fulfill their role in processes like transcription and replication. Here, we have studied the enzymatic activity …

The Design And Testing Of Fluorescence-Based Sensors For Phosphate And Phosphonate, Sara Hassanzadehroknabadi

Electronic Theses and Dissertations

Inorganic phosphate (Pi) is an essential compound for all living things, as it serves as a component of nucleic acids and ATP, which are required for plant growth and proliferation. However, when excess Pi enters the aquatic environment, it has severe detrimental effects through the induction of harmful algal blooms (HAB), which negatively impact aquatic life, the quality of drinking water as well as recreational activities. It is therefore critical to have fast, sensitive, and inexpensive sensors for Pi that quickly and quantitively measure its concentration in a field setting.

Phosphonates or organophosphorus compounds are phosphoric acids possessing alkyl or …

Part I: Development Of Small-Molecule-Based Probes For The Vitamin D Receptor; Part Ii: Development Of A Scalable Manufacturing Process For Orcein Dye, Tania Roseann Mutchie

Theses and Dissertations

PART I:The vitamin D receptor (VDR) is a ligand-dependent transcription factor and member of the nuclear hormone receptor superfamily. VDR is expressed in the epithelia of endocrine organs, digestive system, bronchi, kidneys, and thymus, as well as being present in leukocytes and bone cells. Cell proliferation, cell differentiation, and immunomodulation, along with calcium and phosphate homeostasis, are all processes regulated by the receptor. Within the cell, VDR can be membrane-bound or located in the nucleus. Nuclear localization of VDR transpires following the binding of vitamin D metabolites, the most active of which is 1α,25-dihydroxyvitamin D3 (calcitriol). Within the nucleus, interactions …

Investigation Of G Protein-Coupled Receptor Quaternary Structure Through Fluorescence Micro-Spectroscopy And Theoretical Modeling: Interdependence Between Receptor-Receptor And Receptor-Ligand Interactions, Joel David Paprocki

Theses and Dissertations

Proteins are of high interest in biophysics research due to the important roles they play within cells, such as sensing of chemical (ions and small molecules) and physical (e.g., light) stimuli, providing structure, transporting ions/molecules, signaling, and intercellular communication. The studies described in this dissertation focus on a particular type of membrane proteins known as G protein-coupled receptors (GPCR), which play a key role in cellular response to external stimuli. We used the sterile 2 α-factor mating pheromone receptor (Ste2), a prototypical class D GPCR present within Saccharomyces cerevisiae (baker’s yeast). Ste2 is responsible for initiating the second messenger signal …

Biomedical Applications And Syntheses Of Selected Anthraquinone Dyes, Richard Sirard

Senior Honors Theses

Anthraquinones are aromatic organic compounds that have multiple applications in the biomedical field. Some anthraquinone-based compounds are used as fluorophores to contrast cell nuclei while others act as chemotherapeutic agents. However, there are not many fluorescent anthraquinone cell stains currently available. In this study, commercially available anthraquinone dyes, in addition to other dye families and compounds, were reviewed for their unique properties, advantages, and drawbacks. The development and characterization of three novel anthraquinone fluorophores revealed promising photophysical characteristics, like large Stokes shifts. One of the compounds, RBS3, was chosen for fixed and live cell staining and exhibited desirable biomedical properties. …

Indolizine Donor-Based Dyes For Applications In Fluorescence Biological Imaging, William Meador

Honors Theses

NIR emissive fluorophores are intensely researched due to their potential to replace modern imaging procedures. Many molecular strategies have been employed in the literature to optimize fluorophores for deeper NIR absorption and emission, biocompatibility, and higher fluorescence quantum yields. Amongst the fluorophores studied to date, proaromatic indolizine donors are attractive alternatives to traditional alkyl amine and indoline based donors due to their 1) lower energy absorption and emission facilitated by proaromaticity, 2) large Stokes shifts due to increased dihedral angles about the π-system, 3) ease of functionalization and capacity for bioconjugation at the phenyl ring, and 4) potential for further …