Cell and Developmental Biology Commons^™

Early Onset Alzheimer’S Disease Markers In Mouse Hippocampus Unveiled By Single-Cell Transcriptomic Analysis Following Cranial Radiotherapy, Tuba Aksoy

Dissertations & Theses (Open Access)

Cranial radiation therapy plays an integral role in the treatment of brain tumors but can lead to progressive cognitive deficits in survivors by mechanisms that are poorly understood. To develop preventive or mitigative strategies, it is crucial to better understand the underlying pathogenesis of radiation-induced cognitive impairments. The study investigated single-cell transcriptomics and DNA methylation changes as potential drivers of persistent cellular dysfunction after radiation exposure, specifically concentrating on the CA1-3 regions of the hippocampus and the prefrontal cortex due to their role in cognitive functions. Thirteen-week-old mice underwent whole-brain radiation at clinically relevant doses. Following whole-brain radiation, an assessment …

Deciphering The Contribution Of Microglia To Neurodegeneration In Friedreich's Ataxia, Sydney N. Gillette

Master's Theses

Friedreich's ataxia (FRDA) is the most prevalent inherited ataxia, affecting one in every 50,000 individuals in the United States. This hereditary condition is caused by an abnormal GAA trinucleotide repeat expansion within the first intron of the frataxin gene resulting in decreased levels of the frataxin protein (FXN). Insufficient cellular frataxin levels results in iron accumulation, increased reactive oxygen species production and mitochondrial dysfunction. Tissues most heavily impacted are those most dependent on oxidative phosphorylation as an energy source and include the nervous system and muscle tissue. This is evident in the clinical phenotype which includes muscle weakness, ataxia, neurodegeneration …

Cell-Based Screening Of Antistress Activity Of Some Phytochemicals: Identification, Validation, And Relevance To Old-Age Related Pathologies, Huayue Zhang, Sunil Kaul, Renu Wadhwa

Research Symposium

Background: A variety of environmental stresses have been shown to contribute to poor quality of life, tissue dysfunctions and ailments including metabolic disorders, cognitive impairment, and accelerated aging. Oxidative stress (an imbalance between the production and processing of highly reactive oxygen species) is largely associated with these phenotypes. Whereas drug development and disease therapeutics have advanced remarkably in last three decades, there are still limited options for stress management. Since the later can effectively decrease the disease burden, we aimed to screen phytochemicals with anti-oxidative stress activity using cell-based assays.

Methods: Brain-derived cells were subjected to chemical models of oxidative …

Modeling Developmental, Molecular, And Behavioral Effects Of An Apolipoprotein-E4 Fragment On The Embryogenesis Of Zebrafish, Madyson Mccarthy

Boise State University Theses and Dissertations

Although the increased risk of developing sporadic Alzheimer’s disease (AD) associated with the inheritance of the apolipoprotein E4 (APOE4) allele is well characterized, the molecular underpinnings of how ApoE4 imparts risk remains unknown. Enhanced proteolysis of the ApoE4 protein with a toxic-gain of function has been suggested and a 17 kDa amino-terminal ApoE4 fragment (nApoE4_1-151) has been identified in post-mortem human AD frontal cortex sections. Recently, we demonstrated in vitro, exogenous treatment of nApoE4_1-151 in BV2 microglial cells leads to uptake, trafficking to the nucleus and increased expression of genes associated with cell toxicity …

Impact Of Arginine Metabolism And Sensing In Mouse Models Of Alzheimer’S Disease, Chao Ma

USF Tampa Graduate Theses and Dissertations

Alzheimer’s disease (AD) remains the most common neurodegenerative disease in the central nervous system (CNS), with amyloidosis and tauopathy as their two main hallmarks. Typical AD pathologies include cerebral plaques deposited by amyloid-β, neurofibrillary tangles aggregated by tau, and neuroinflammation caused by activated brain myeloid cells. A critical theme is centered on impaired brain metabolism. Emerging evidence showed that impaired arginine metabolism was a novel biomarker pathway for AD. The manipulation of arginine metabolism by a critical enzyme arginase 1 (ARG1) in neurons indicated therapeutic benefits in alleviating tau pathology. Balanced cellular proteostasis was governed by the mechanistic target of …

Unbiased Automated Quantitation Of Ros Signals In Live Retinal Neurons Of Drosophila Using Fiji/Imagej, Prajakta Deshpande, Neha Gogia, Anuradha Venkatakrishnan Chimata, Amit Singh

Biology Faculty Publications

Numerous imaging modules are utilized to study changes that occur during cellular processes. Besides qualitative (immunohistochemical) or semiquantitative (Western blot) approaches, direct quantitation method(s) for detecting and analyzing signal intensities for disease(s) biomarkers are lacking. Thus, there is a need to develop method(s) to quantitate specific signals and eliminate noise during live tissue imaging. An increase in reactive oxygen species (ROS) such as superoxide (O2•-) radicals results in oxidative damage of biomolecules, which leads to oxidative stress. This can be detected by dihydroethidium staining in live tissue(s), which does not rely on fixation and helps prevent stress on tissues. However, …

Translational Fidelity And Its Role In Neuronal Homeostasis, Markus Terrey

Electronic Theses and Dissertations

The process of translation, which refers to decoding genetic information from mRNA to protein, is vital for all cellular function. Translational fidelity starts at the level of aminoacylation of transfer RNAs (tRNA). This reaction is catalyzed by aminoacyl tRNA synthetases where each amino acid is transferred to its corresponding cognate tRNA. Because tRNAs harbor the anticodon sequence to decodes a particular mRNA codon, the specific aminoacylation of the tRNA with a cognate amino acid establishes the rules of decoding genetic code into proteins. Aminoacylated tRNAs are then delivered to ribosomes, where ribosomes in a highly organized manner need to accurately …

Characterizing Ferroptosis Pathways In In Vitro Motor Neuron Models, Alejandra Martinez

Theses and Dissertations

Ferroptosis is a non-apoptotic regulated form of cell death driven by the toxic accumulation of lipid peroxides and the presence of iron. The mechanism by which ferroptosis operates requires further investigation; here I will discuss the findings regarding the role of ferroptosis in different cellular models as well as the genes that may be involved with either promoting or hindering the ferroptotic pathway. NSC-34 is a hybrid cell line of neuroblastoma and primary spinal cord cells that can be differentiated into a motor neuron-like state allowing the characterization of ferroptosis in two distinct cellular conditions. Additionally, iNIL cells are a …

Mitochondrial Aspects Of Neuronal Pathology In Triple-Transgenic Alzheimer’S Disease Mice, John Zachary Cavendish

Graduate Theses, Dissertations, and Problem Reports

Alzheimer’s disease (AD) is a fatal, progressive neurodegenerative disease afflicting millions of people in the United States alone and is the only one of the top leading causes of morbidity and mortality with no effective disease-modifying therapies. It is the most common form of dementia, affecting one in three people over the age of 85. While the hallmarks of the disease include accumulation of beta-amyloid-based extracellular plaques and hyperphosphorylated tau-based intracellular neurofibrillary tangles, treatment strategies centered on removing or mitigating these components of AD have all failed in humans. Mitochondrial dysfunction has been increasingly recognized as an early and consistent …

Calcium Dyshomeostasis In Neurodegeneration, Nicholas Emanuel Karagas

Dissertations & Theses (Open Access)

Neurodegenerative diseases, despite constituting a major and growing cause of mortality globally, have few effective treatments. In order to develop novel therapeutics to combat neurodegeneration, a better understanding of the molecular mechanisms underlying these diseases is needed. Neurons rely on Ca²⁺ to mediate many of their unique functions, and aberrant Ca²⁺ signaling has been broadly implicated in neurodegeneration. The goal of this dissertation is to delineate specific examples of Ca²⁺ dyshomeostasis that I have uncovered in Drosophila models of neurodegeneration.

I first define the role a neurodegeneration-associated mutation plays in perturbing presynaptic [Ca²⁺], which is …

Ceramide Analog [18F]F-Hpa-12 Detects Sphingolipid Disbalance In The Brain Of Alzheimer’S Disease Transgenic Mice By Functioning As A Metabolic Probe, Simone M. Crivelli, Daan Van Kruining, Qian Luo, Jo A. A. Stevens, Caterina Giovagnoni, Andreas Paulus, Matthias Bauwens, Dusan Berkes, Helga E. De Vries, Monique T. Mulder, Jochen Walter, Etienne Waelkens, Rita Derua, Johannes V. Swinnen, Jonas Dehairs, Felix M. Mottaghy, Mario Losen, Erhard Bieberich, Pilar Martinez-Martinez

Physiology Faculty Publications

The metabolism of ceramides is deregulated in the brain of Alzheimer’s disease (AD) patients and is associated with apolipoprotein (APO) APOE4 and amyloid-β pathology. However, how the ceramide metabolism changes over time in AD, in vivo, remains unknown. Distribution and metabolism of [¹⁸F]F-HPA-12, a radio-fluorinated version of the ceramide analog N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl) dodecanamide, was investigated in the brain of AD transgenic mouse models (FAD) on an APOE4 or APOE3 genetic background, by positron emission tomography and by gamma counter. We found that FAD mice displayed a higher uptake of [¹⁸F]F-HPA-12 in the brain, independently from the APOE4 …

Acute Systemic Inflammatory Response Alters Transcription Profile Of Genes Related To Immune Response And Ca 2+ Homeostasis In Hippocampus; Relevance To Neurodegenerative Disorders, Grzegorz A. Czapski, Yuhai Zhao, Walter J. Lukiw, Joanna B. Strosznajder

School of Medicine Faculty Publications

Acute systemic inflammatory response (SIR) triggers an alteration in the transcription of brain genes related to neuroinflammation, oxidative stress and cells death. These changes are also characteristic for Alzheimer’s disease (AD) neuropathology. Our aim was to evaluate gene expression patterns in the mouse hippocampus (MH) by using microarray technology 12 and 96 h after SIR evoked by lipopolysaccharide (LPS). The results were compared with microarray analysis of human postmortem hippocampal AD tissues. It was found that 12 h after LPS administration the expression of 231 genes in MH was significantly altered (FC > 2.0); however, after 96 h only the S100a8 …

Tdp-43 Mediated Blood-Brain Barrier Permeability And Leukocyte Infiltration Promote Neurodegeneration In A Low-Grade Systemic Inflammation Mouse Model, Frank Zamudio, Anjanet R. Loon, Shayna Smeltzer, Khawla Benyamine, Nanda K. Navalpur Shanmugam, Nicholas J. F. Stewart, Daniel C. Lee, Kevin Nash, Maj-Linda B. Selenica

Sanders-Brown Center on Aging Faculty Publications

BACKGROUND: Neuronal cytoplasmic inclusions containing TAR DNA-binding protein 43 (TDP-43) are a neuropathological feature of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's Disease (AD). Emerging evidence also indicates that systemic inflammation may be a contributor to the pathology progression of these neurodegenerative diseases.

METHODS: To investigate the role of systemic inflammation in the progression of neuronal TDP-43 pathology, AAV9 particles driven by the UCHL1 promoter were delivered to the frontal cortex of wild-type aged mice via intracranial injections to overexpress TDP-43 or green fluorescent protein (GFP) in corticospinal motor neurons. Animals were then subjected …

Inactivation Of Hippo And Cjun-N-Terminal Kinase (Jnk) Signaling Mitigate Fus Mediated Neurodegeneration In-Vivo, Ankita Sarkar, Abijeet Singh Mehta, Prajakta Deshpande, Madhuri Kango-Singh, Udai Bhan Pandey, Amit Singh

Biology Faculty Publications

Amyotrophic Lateral Sclerosis (ALS), a late-onset neurodegenerative disorder characterized by the loss of motor neurons in the central nervous system, has no known cure to-date. Disease causing mutations in human Fused in Sarcoma (FUS) leads to aggressive and juvenile onset of ALS. FUS is a well-conserved protein across different species, which plays a crucial role in regulating different aspects of RNA metabolism. Targeted misexpression of FUS in Drosophila model recapitulates several interesting phenotypes relevant to ALS including cytoplasmic mislocalization, defects at the neuromuscular junction and motor dysfunction. We screened for the genetic modifiers of human FUS-mediated neurodegenerative phenotype using molecularly …

Neurodegenerative Modeling: Tau Protein, Degradative Pathways, And Gene Expression Profiling Of Human Ipsc-Derived Neural Precursors And Differentiated 3-D Neural Sphere Versus 2-D Monolayer Cultures, Kyle H. Anthoney

Cal Poly Humboldt theses and projects

Human induced pluripotent stem cells offer a model for human brain development and disease by differentiation into brain organoids; however, current neural culture systems lack the microenvironment, neuronal circuits and connectivity, vascular circulation, and immune system that exist in vivo. After differentiation and development of neuronal and non-neuronal cell types within two formats of cell cultures, we can visualize and recapitulate in vivo protein accumulation, gene expression, and degradative processes such as autophagy. Using RNA extraction, purification methods and reverse transcription I compared traditional monolayer cultures and novel 3-D neural sphere cultures via gene expression analysis. This analysis indicated …

Alzheimer's And Amyloid Beta: Amyloidogenicity And Tauopathy Via Dyshomeostatic Interactions Of Amyloid Beta, Jordan Tillinghast

Senior Honors Theses

This paper reviews functions of Amyloid-β (Aβ) in healthy individuals compared to the consequences of aberrant Aβ in Alzheimer’s disease (AD). As extraneuronal Aβ accumulation and plaque formation are characteristics of AD, it is reasonable to infer a pivotal role for Aβ in AD pathogenesis. Establishing progress of the disease as well as the mechanism of neurodegeneration from AD have proven difficult (Selkoe, 1994). This thesis provides evidence suggesting the pathogenesis of AD is due to dysfunctional neuronal processes involving Aβ’s synaptic malfunction, abnormal interaction with tau, and disruption of neuronal homeostasis. Significant evidence demonstrates that AD symptoms are partially …

Alpha-Synuclein Toxicity Is Caused By Mitochondrial Dysfunction, Michael G. Tauro

Electronic Thesis and Dissertation Repository

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, affecting roughly 1% of the population over the age of sixty years. Alpha-synuclein (aSyn) is a protein implicated in both familial and idiopathic forms of PD, yet despite the wealth of data implicating aSyn as a causative agent in PD, the mechanisms underlying its toxicity remain mostly unknown. Mitochondrial dysfunction is a major hallmark of PD, yet there is only limited evidence linking aSyn toxicity to mitochondrial dysfunction. My study establishes a novel aSyn model in respiring yeast cells, which allows me to explore how aSyn affects mitochondrial homeostasis and …

Molecular Mechanism Of Neurodegeneration Induced By 4-Nonylphenol, Michelle Alejandra Aranda

Open Access Theses & Dissertations

Neurodegeneration, a progressive loss of nerve cells, occurs in many neurological disorders, including Alzheimer's disease (AD) and Parkinson's diseases (PD), as well as in dementia. The pathogenesis of these diseases is unknown, and recent evidence suggests that environmental factors, which act as endocrine-disrupting compounds (EDCs) could play a significant role in developing the disease process. 4-Nonylphenol (4-NP), an EDC, and a ubiquitous environmental toxin has been shown to affect brain development and may cause neurodegeneration. 4-NP is produced in large quantities in the U.S. and used as raw materials for making detergents, pesticides, plastics, paints, cosmetics, rubber, and other industrial/household …

Investigating Autophagy Dysfunction Induced By A Parkinson's Disease-Causing Mutation In Vps35, Abir Ashfakur Rahman

Boise State University Theses and Dissertations

Parkinson’s Disease (PD) is an idiopathic disorder with no known cure. With number of cases steadily rising around the world, it is imperative to turn to the underlying cellular and molecular mechanisms of the disease manifestation and neurodegeneration to craft novel modes of therapy. VPS35 is one of the few genes that have identified and definitively linked to familial PD. The particular mutation that has been associated is known to cause dysfunction of a key cellular process known as autophagy. This process is primarily responsible for clearance of unwanted, damaged or misfolded proteins, among other things. Our study reveals an …

The Regulation Of Extracellular Amyloid-Β Levels By Ionotropic Glutamatergic Transmission In An Alzheimer’S Disease Mouse Model, Jane Cecelia Hettinger

Arts & Sciences Electronic Theses and Dissertations

Brain extracellular concentration of the peptide amyloid-β (Aβ) is a major contributor to Alzheimer’s disease (AD) pathogenesis. High Aβ levels in the extracellular space precipitate aggregation of the peptide into soluble and insoluble toxic species. This process begins decades before cognitive impairment and triggers the cascade of pathology that eventually leads to AD. Synaptic activity is key to the regulation of extracellular Aβ levels. Presynaptic activity drives the production of Aβ, while postsynaptic receptor activation exhibits more nuanced regulation. For example, high levels of NMDA receptor (NMDA-R) activation have been shown to decrease Aβ production through the extracellular signal-regulated kinase …

Characterization Of Neuronal Specific Responses To Induced Misfolded Protein Stress In Caenorhabditis Elegans, Claire Gormley

Senior Honors Projects, 2010-2019

Abstract

Misfolded protein stress has been associated with many types of disease,

including neurodegenerative disorders like Alzheimer’s, Parkinson’s and Huntington’s

disease. When a cell accumulates misfolded proteins in the endoplasmic reticulum,

misfolded protein stress occurs and the unfolded protein response (UPR) is triggered to

induce mechanisms that will allow the cell to either survive or undergo cell death. The

nascent polypeptide associated complex (NAC) is a co-translational chaperone and α/β

heterodimer that manages protein folding and localization, and protects against misfolded

protein stress; changes in NAC function have been linked to both neurodegeneration and

cancer. In these studies, I depleted …

Thiamine Deficiency Induces Endoplasmic Reticulum Stress And Oxidative Stress In Human Neurons Derived From Induced Pluripotent Stem Cells, Xin Wang, Mei Xu, Jacqueline A. Frank, Zun-Ji Ke, Jia Luo

Pharmacology and Nutritional Sciences Faculty Publications

Thiamine (vitamin B1) deficiency (TD) plays a major role in the etiology of Wernicke's encephalopathy (WE) which is a severe neurological disorder. TD induces selective neuronal cell death, neuroinflammation, endoplasmic reticulum (ER) stress and oxidative stress in the brain which are commonly observed in many aging-related neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and progressive supranuclear palsy (PSP). However, the underlying cellular and molecular mechanisms remain unclear. The progress in this line of research is hindered due to the lack of appropriate in vitro models. The neurons derived for the human induced pluripotent stem …

4-Nonylphenol Induces Neurodegeneration By Altering Cytoskeleton, Cynthia Carreon

Open Access Theses & Dissertations

4-nonylphenol (4-NP), an endocrine-disrupting compound (EDC), has been shown to affect brain development and may cause neurodegeneration. In the environment, 4-NP arises as a degradation product of alkylphenol polyethoxylates, compounds widely used as nonionic surfactants in commercial production, as well as in herbicides, pesticides, polystyrene plastics, and paints and has been shown to undergo a high level of accumulation in biological tissues. However, the mechanism by which 4-NP exerts its effect is not understood. Recent results from our laboratory indicate that Gβγ, an important component of the G protein-signaling pathway, induces neuronal outgrowth and differentiation by modulating microtubule (MT) assembly, …

Mitogen And Morphogen Signaling Dysregulation: Pathophysiological Influence In Pancreatic Cancer And Alzheimer’S Disease, Eric Cruz

Theses & Dissertations

Although the etiology of a particular disease will vary, there are genetic and epigenetic bottlenecks that frequently converge resulting in dysregulation of mitogenic and morphogenetic signaling. This propensity is acutely experienced in malignancy and neurodegenerative disease.

Here, we have first investigated the role of dysregulated signaling in the context of pancreatic cancer (PC). Morphogenetic signaling has been regarded as a pleiotropic pathway with the potential to promote and inhibit metastatic features. Our investigation of bone morphogenetic protein 2 (BMP-2), an archetypical member of the BMP superfamily, has revealed the presence of extracellular, intracellular, and long non-coding RNA products. Our findings …

The Role Of Daf-19 In Non-Ciliated Neurons: How Is Neural Development Regulated By Different Daf-19 Isoforms?, Zabdiel Ek Vazquez

Lawrence University Honors Projects

A degenerative disease-like phenotype, specifically reduction in synaptic protein levels in adult worms, is correlated with loss-of-function of the only RFX transcription factor gene, daf-19, in C. elegans. This gene encodes four known transcription factor isoforms, two of which are correlated with particular functions. The DAF-19C isoform activates genes responsible for cilia development, while DAF-19M is needed for cilia specification in males. A comparison of the transcriptome of daf-19 null and isogenic wild type adult worms suggests both positive and negative regulation of gene expression is correlated with the presence of DAF-19 proteins. We have assessed DAF-19 regulation …

Potential Role Of Pctaire-2, Pctaire-3 And P-Histone H4 In Amyloid Precursor Protein-Dependent Alzheimer Pathology, Dale Chaput, Lisa Kirouac, Stanley M. Stevens Jr., Jaya Padmanabhan

Molecular Biosciences Faculty Publications

Amyloid Precursor Protein (APP) is regulated in a mitosis-specific manner and plays a role in proliferative signaling in cells. Though APP-derived Aβ generation has a well-established role in neurodegeneration, the mechanistic role of APP in this process is not fully understood. Here, we performed an unbiased, comprehensive analysis of the phosphoproteome signature in APP-null neuroblastoma cells (B103) compared to those expressing APP-695 isoform (B103-695) to determine if APP expression affects protein phosphorylation. Stable isotope labeling by amino acids in cell culture (SILAC) followed by mass spectrometry-based phosphoproteomic analysis with PolyMAC identified a total of 2,478 phosphopeptides in the B103 and …

Dna Repair Deficiency In Huntington's Disease Fibroblasts And Induced Pluripotent Stem Cells, Peter Anthony Mollica

Biological Sciences Theses & Dissertations

Mutant huntingtin protein (mhtt)– the protein responsible for cellular dysfunction in Huntington’s disease (HD) –is a product of an expanded trinucleotide repeat (TNR) cytosine-adenine-guanine (CAG) sequence in exon 1 of the huntingtin (HTT) gene. The pathology of HD has been extensively researched; however, the mechanism by which the disease-causing TNR expansions occur in somatic cells remains elusive. Interestingly, HD has often been referred to a ‘DNA repair disease’, even though DNA repair dysfunction in situ has not been identified. We hypothesized that presence of the mhtt protein affects the expression of DNA repair genes used to address DNA repair, ultimately …

Analyzing A-Series Gangliosides In Neurons Following Exposure To Glutamate, Dae Hee Park

Electronic Thesis and Dissertation Repository

Neurons within different brain regions have varying levels of vulnerability to external stress and therefore respond differently to injury. A potential reason to explain this may lie within a key lipid class of the cell’s plasma membrane called gangliosides. These glycosphingolipid species have been shown to play various roles in the maintenance of neuronal viability. The purpose of this study is to use electrospray ionization mass spectrometry (ESI-MS) technique and immunohistochemistry to evaluate the temporal changes in the expression profiles of various ganglioside species during the course of neurodegeneration in rat primary cortical neurons exposed to glutamate toxicity. Primary embryonic …

Gbetagamma-Microtubule Mediated Mechanism Of Neuronal Differentiation, Jorge Anibal Sierra Fonseca

Open Access Theses & Dissertations

Neurodegeneration, a progressive loss of nerve cells (neurons), occurs in many neurological disorders, including Alzheimer's and Parkinson's diseases, as well as in the aging brain. Disruption of microtubules in neurons and the aggregation of proteins associated with them is the hallmark of neurodegeneration. Nevertheless, the cause of this disorder is largely unknown, and no effective drugs are available to treat the disease processes. Therefore, there is a need to understand the molecular mechanisms that drive the assembly and disassembly of microtubules during neurite outgrowth and differentiation. Evidence suggests that nerve growth factor (NGF) induces neurite outgrowth from PC12 cells by …

Screen For Suppressors And Enhancers Of Excitotoxic Neurodegeneration, Anthony Omorodion Edokpolo

Dissertations and Theses

Excitotoxicity is an important and frequently observed neurodegenerative process. Excitotoxicity mediates brain damage in a range of diseases and conditions including stroke, and is triggered by excessive stimulation of glutamatergic synapses. In spite of extensive studies, the molecular mechanisms involved in excitotoxicity following the over-activation of postsynaptic glutamate receptors are not well understood, and clinical trials based on our partial understanding of the process ended with disappointment. Genetic screens in simple animal models offer a powerful alternative approach, since screens are unbiased, analysis is facilitated by strong research tools, and cellular mechanisms are highly conserved through evolution. We produced a …