Biochemistry, Biophysics, and Structural Biology Commons^™

Novel Roles Of The Protein Tyrosine Phosphatase Shp2 In Non-Small Cell Lung Cancer, Valentina Schneeberger

USF Tampa Graduate Theses and Dissertations

The gene PTPN11 was identified in the early 1990s, and encodes the non-transmembrane protein tyrosine phosphatase SHP2. SHP2 is expressed ubiquitously in cells, and plays an important role in cancer. Unlike most phosphatases, SHP2 positively regulates several signaling pathways including the Ras/MAPK and Src signaling pathways and acts as a proto-oncogene. SHP2 is also a cancer essential gene in certain types of carcinomas, and promotes growth, survival, and epithelial to mesenchymal transformation. Gain of function (GOF) SHP2 mutations are known leukemic oncogenes, and have been identified to a smaller extent in solid tumors as well. Currently, the roles of SHP2 …

Modeling The Adaptive Immune Response To Mutation-Generated Antigens, Rory J. Geyer

University Scholar Projects

Somatic mutations may drive tumorigenesis or lead to new, immunogenic epitopes (neoantigens). The immune system is thought to represses neoplastic growths through the recognition of neoantigens presented only by tumor cells. To study mutations as well as the immune response to mutation-generated antigens, we have created a conditional knockin mouse line with a gene encoding, 5’ to 3’, yellow fluorescent protein (YFP), ovalbumin (which is processed to the immunologically recognizable peptide, SIINFEKL), and cyan fluorescent protein (CFP), or, YFP-ovalbumin-CFP. A frame shift mutation has been created at the 5’ end of the ovalbumin gene, hence YFP should always be expressed, …

Modeling The Adaptive Immune Response To Mutation-Generated Antigens, Rory J. Geyer

Honors Scholar Theses

Somatic mutations may drive tumorigenesis or lead to new, immunogenic epitopes (neoantigens). The immune system is thought to represses neoplastic growths through the recognition of neoantigens presented only by tumor cells. To study mutations as well as the immune response to mutation-generated antigens, we have created a conditional knockin mouse line with a gene encoding, 5’ to 3’, yellow fluorescent protein (YFP), ovalbumin (which is processed to the immunologically recognizable peptide, SIINFEKL), and cyan fluorescent protein (CFP), or, YFP-ovalbumin-CFP. A frame shift mutation has been created at the 5’ end of the ovalbumin gene, hence YFP should always be expressed, …

Localization And Expression Level Of Vascular Endothelial Growth Factor After Partial Hepatectomy Of Wky Rats, Adam Kronebusch, Tyler Koonst

Undergraduate Research Symposium

Inflammation and fibrosis are indicative of liver regeneration following injury and chronic liver diseases, such as cirrhosis and hepatocellular carcinoma. The formation of new vasculature via the process of angiogenesis is vital to the pathological progression of liver regeneration and these diseases. Thus, inhibiting the process of angiogenesis could have the potential to stop or slow the progression of chronic liver diseases. Vascular endothelial growth factor (VEGF) is the most potent and specific growth factor for initiating the process of angiogenesis, making it a prime target to inhibit angiogenesis. Bevacizumab is an antibody that binds to VEGF, inhibiting it from …

Pharmaceuticals And Personal Care Products: Emerging Contaminants In Aquatic Ecosystems, Jenny E. Zenobio

Open Access Theses

In recent years, the presence of pharmaceuticals and personal care products (PPCPs) in aquatic systems has led to research on their fate and effects. PPCPs have been found in mixture in wastewater effluents, surface, ground, and drinking water at low concentrations from areas of intense urbanization. Although adverse effects to human health from the current environmental concentrations are unlikely, the impacts to ecological receptors are not clear. We performed field and laboratory studies to quantify and evaluate effects of PPCPs on fish. First, a field study was conducted at the Baca National Wildlife Refuge, Colorado (2010-2012) because a portion of …

Gossypiboma Diagnosed Fifteen Years After A Cesarean Section: A Case Report, Abdul Rehman, Noor Ul-Ain Baloch, Muhammad Awais

Department of Biological & Biomedical Sciences

Gossypiboma, a retained surgical sponge, is a rare complication following any surgical procedure and is primarily a result of human error. Such patients often have vague clinical presentations and the diagnosis often comes as a surprise. We present the case of a 40-year-old woman who had a long-standing history of lower abdominal discomfort. She was diagnosed with a gossypiboma fifteen years after a cesarean section. All general physicians, surgeons and radiologists must be aware of this rare but clinically significant entity.

Molecular Actions Of The Novel Histone Deacetylase Inhibitor Cg-1521 In Models Of Inflammatory Breast Cancer, Namita Chatterjee

Legacy Theses & Dissertations (2009 - 2024)

Inflammatory breast cancer (IBC) is a rare and particularly lethal form of breast cancer. Despite aggressive therapeutic approaches, the 5-year survival rate is only 34%. As this disease is severely understudied, we have investigated the therapeutic potential of the novel hydroxamic acid-derived histone deacetylase inhibitor (HDACi), CG-1521 in comparison to a structurally similar compound, Trichostatin A (TSA) in two IBC cell lines: SUM149PT and SUM190PT. In these cells, CG-1521 and TSA induce dose- and time-dependent induction of cell cycle arrest and apoptosis regardless of the presence of 17β-estradiol (E2). Interestingly, the cell lines have considerably different sensitivities to these treatments …

Tmprss2-Erg Regulation Of Androgen Biosynthetic Enzyme Expression, Dht Synthesis, And Androgen Receptor Activation In Prostate Cancer, Katelyn Ann Powell

Wayne State University Dissertations

Intratumoral androgen synthesis in prostate cancer (PCa) contributes to the development of castration-resistant prostate cancer (CRPC). Several enzymes responsible for androgen biosynthesis have been shown to be overexpressed in CRPC, thus, contributing to CRPC in a castrated environment. Although intratumoral androgen synthesis is thought to contribute to the development and progression of CRPC, currently little is known regarding the regulation of androgen biosynthetic enzyme gene expression in PCa. The TMPRSS2-ERG transcription factor has been shown to be present in primary PCa tumors as well as CRPC tumors. The hypothesis was investigated that TMPRSS2-ERG fusions regulate androgen biosynthetic enzyme (ABE) gene …

Molecular Studies On The Anti-Tumor Effects Of Metal-Based Complexes: Involvement Of The Ubiquitin-Proteasome And Apoptotic Pathways, Sara M. Schmitt

Wayne State University Dissertations

The ubiquitin-proteasome pathway is crucial to normal cellular function, and as such, has been extensively investigated as a potential target for cancer therapeutics. Many compounds have been tested for their proteasome inhibitory ability, including various small peptide aldehydes, and, following the success of cisplatin, several metal-containing complexes. The efficacy of these compounds in preclinical studies ultimately resulted in the development and approval of the first-in-class proteasome inhibitor bortezomib, the use of which, unfortunately, has been hindered by toxicity and resistance. These limitations have led to a massive push toward designing and developing new, less toxic proteasome inhibitors for clinical use. …

Culturable Aerobic And Facultative Anaerobic Intestinal Bacterial Flora Of Black Cobra (Naja Naja Karachiensis) In Southern Pakistan, Junaid Iqbal, Mehwish Sagheer, Nazneen Tabassum, Ruqaiyyah Siddiqui, Naveed Ahmed Khan

Department of Biological & Biomedical Sciences

Using morphological analysis and biochemical testing, here for the first time, we determined the culturable gut bacterial flora (aerobes and facultative anaerobes) in the venomous Black Cobra (Naja naja karachiensis) from South Asia. The findings revealed that these snakes inhabit potentially pathogenic bacteria including Serratia marcescens, Pseudomonas aeruginosa, Shewanella putrefaciens, Aeromonas hydrophila, Salmonella sp., Moraxella sp., Bacillus sp., Ochrobactrum anthropi, and Providencia rettgeri. These findings are of concern, as injury from snake bite can result in wound infections and tissue necrosis leading to sepsis/necrotizing fasciitis and/or expose consumers of snake meat/medicine in the community to infections.

Using Expression Profiling To Understand The Effects Of Chronic Cadmium Exposure On Mcf-7 Breast Cancer Cells, Zelmina Lubovac-Pilav, Daniel M. Borras, Esmeralda Ponce, Maggie Louie

Collected Faculty Scholarship

Cadmium is a metalloestrogen known to activate the estrogen receptor and promote breast cancer cell growth. Previous studies have implicated cadmium in the development of more malignant tumors; however the molecular mechanisms behind this cadmium-induced malignancy remain elusive. Using clonal cell lines derived from exposing breast cancer cells to cadmium for over 6 months (MCF-7-Cd4, -Cd6, -Cd7, -Cd8 and -Cd12), this study aims to identify gene expression signatures associated with chronic cadmium exposure. Our results demonstrate that prolonged cadmium exposure does not merely result in the deregulation of genes but actually leads to a distinctive expression profile. The genes deregulated …

Development Of A Conditional Mesd (Mesoderm Development) Allele For Functional Analysis Of The Low-Density Lipoprotein Receptor-Related Family In Defined Tissues, Andrew V. Taibi, Janet K. Lighthouse, Richard C. Grady, Kenneth R. Shroyer, Bernadette Holdener

Department of Biomedical Engineering Faculty Publications

The Low-density lipoprotein receptor-Related Protein (LRP) family members are essential for diverse processes ranging from the regulation of gastrulation to the modulation of lipid homeostasis. Receptors in this family bind and internalize a diverse array of ligands in the extracellular matrix (ECM). As a consequence, LRPs regulate a wide variety of cellular functions including, but not limited to lipid metabolism, membrane composition, cell motility, and cell signaling. Not surprisingly, mutations in single human LRPs are associated with defects in cholesterol metabolism and development of atherosclerosis, abnormalities in bone density, or aberrant eye vasculature, and may be a contributing factor in …

Chronic Cadmium Exposure Stimulates Sdf-1 Expression In An Erα Dependent Manner, Esmeralda Ponce, Natalie B. Aquino, Maggie Louie

Collected Faculty Scholarship

Cadmium is an omnipotent environmental contaminant associated with the development of breast cancer. Studies suggest that cadmium functions as an endocrine disruptor, mimicking the actions of estrogen in breast cancer cells and activating the receptor to promote cell growth. Although acute cadmium exposure is known to promote estrogen receptor-mediated gene expression associated with growth, the consequence of chronic cadmium exposure is unclear. Since heavy metals are known to bioaccumulate, it is necessary to understand the effects of prolonged cadmium exposure. This study aims to investigate the effects of chronic cadmium exposure on breast cancer progression. A MCF7 breast cancer cell …

Efficacy Of Gold Silica Nanoshells And Gold Nanorods For Photothermal Therapy Of Human Glioma Spheroids, Suyog Jung Chhetri

UNLV Theses, Dissertations, Professional Papers, and Capstones

Gold-based nanoparticles including gold-silica nano-spheres and gold nano-rods have been investigated for a number of therapeutic and diagnostic applications. The ability of these nanoparticles to convert light into heat energy makes them particularly appealing for photothermal therapy in which cancer cells are destroyed via light-induced heat generation. The overall objective of the study is to compare the efficacy of gold-silica nano-spheres and gold nano-rods in an in vitro system consisting of human brain tumor (glioma) spheroids.

Delivery of the nanoparticles to the spheroids was accomplished using murine macrophages. Nanoparticles (spheres or rods) were incubated with macrophages for 24 hours. Thereafter, …

Gender Differences In The Development Of Uremic Cardiomyopathy Following Partial Nephrectomy: Role Of Progesterone, Christopher A. Drummond, George Buddny, Steven T. Haller, Jiang Liu, Yanling Yan, Zijian Xie, Deepak Malhotra, Joseph I. Shapiro Md, Jiang Tian

Biochemistry and Microbiology

Gender difference has been suggested as a risk factor for developing cardiovascular and renal diseases in humans and experimental animals. As a major sex hormone, progesterone was reported to compete with cardiotonic steroid binding to Na/K-ATPase. Our previous publication demonstrated that cardiotonic steroids (e.g., marinobufagenin) play an important role in the development of experimental uremic cardiomyopathy. We also observed that the putative mineralocorticoid antagonists, spironolactone and its major metabolite canrenone, antagonize binding of cardiotonic steroids to Na/K-ATPase in a competitive manner and also ameliorate experimental uremic cardiomyopathy induced by partial nephrectomy. In the following studies, we noted that progesterone displayed …

Aβ Alters The Dna Methylation Status Of Cell-Fate Genes In An Alzheimer’S Disease Model, Gary D. Isaacs, Noor Taher, Courtney Mckenzie, Rebecca Garrett, Matthew Baker, Nena Fox

Faculty Publications and Presentations

Alzheimer’s disease (AD) is characterized by neurofibrillary tangles and extracellular amyloid-β plaques (Aβ). Despite ongoing research, some ambiguity remains surrounding the role of Aβ in the pathogenesis of this neurodegenerative disease. While several studies have focused on the mutations associated with AD, our understanding of the epigenetic contributions to the disease remains less clear. To that end, we determined the changes in DNA methylation in differentiated human neurons with and without Aβ treatment. We isolated the DNA from neurons treated with Aβ or vehicle, and digested the two samples with either a methylation-sensitive (HpaII) or a methylation-insensitive (MspI) restriction endonuclease. …

Doxorubicin-Induced, Tnf-Α-Mediated Brain Oxidative Stress, Neurochemical Alterations, And Cognitive Decline: Insights Into Mechanisms Of Chemotherapy Induced Cognitive Impairment And Its Prevention, Jeriel T. Keeney

Theses and Dissertations--Chemistry

The works presented in this dissertation provide insights into the mechanisms of chemotherapy-induced cognitive impairment (CICI or “ChemoBrain”) and take steps toward outlining a preventive strategy. CICI is now widely recognized as a complication of cancer chemotherapy experienced by a large percentage of cancer survivors. Approximately fifty percent of existing FDA-approved anti-cancer drugs generate reactive oxygen species (ROS). Doxorubicin (Dox), a prototypical ROS-generating chemotherapeutic agent, produces the reactive superoxide radical anion (O₂^-•) in vivo. Dox treatment results in oxidation of plasma proteins, including ApoA-I, leading to TNF-α-mediated oxidative stress in plasma and brain. TNF-α elevation in brain …

Characterization Of Beryllium As A Novel Agent To Study Cell Cycle Arrest And Cellular Senescence, Priyatham Gorjala

UNLV Theses, Dissertations, Professional Papers, and Capstones

Cancer cells evade senescence, apoptosis, and other constraints on proliferation, often via mutation of the p53 tumor suppressor gene (TP53). Normal human lung fibroblasts have been shown to enter premature senescence upon exposure to beryllium. In these cells, BeSO₄ stabilizes p53 protein, increases p21 gene expression, induces senescence-associated β-galactosidase activity and causes cell proliferation arrest. In the present study, we have investigated whether BeSO₄ is able to induce similar effects in cancer cells that have wildtype p53. We have demonstrated that beryllium salt at low concentration can induce molecular changes in the p53 signaling pathway leading to cell …

Regulation Of Protein Degradation In The Heart By Amp-Activated Protein Kinase, Kedryn K. Baskin

Dissertations and Theses (Open Access)

The degradation of proteins by the ubiquitin proteasome system is essential for cellular homeostasis in the heart. An important regulator of metabolic homeostasis is AMP-activated protein kinase (AMPK). During nutrient deprivation, AMPK is activated and intracellular proteolysis is enhanced through the ubiquitin proteasome system (UPS). Whether AMPK plays a role in protein degradation through the UPS in the heart is not known. Here I present data in support of the hypothesis that AMPK transcriptionally regulates key players in the UPS, which, under extreme conditions can be detrimental to the heart. The ubiquitin ligases MAFbx /Atrogin-1 and MuRF1, key regulators of …

Chemosensitization Of Hepatocellular Carcinoma To Gemcitabine By Non-Invasive Radiofrequency Field-Induced Hyperthermia, Mustafa Raoof

Dissertations and Theses (Open Access)

Gemcitabine is a potent nucleoside analogue against solid tumors however drug resistance rapidly emerges. Removal of gemcitabine incorporated in the DNA by repair mechanisms could potentially contribute to resistance in chemo-refractory solid tumors. In this study, we evaluated homologous recombination repair of gemcitabine-stalled replication forks as a potential mechanism contributing to resistance. We also studied the effect of hyperthermia on homologous recombination pathway to explain the previously reported synergy between gemcitabine and hyperthermia. We found that hyperthermia degrades and inhibits localization of Mre11 to gemcitabine-stalled replication forks. Furthermore, gemcitabine-treated cells that were also treated with hyperthermia demonstrate a prolonged passage …

Matrix Metalloproteinase Genes Are Transcriptionally Regulated By E2f Transcription Factors: A Link Between Cell Cycle Control And Metastatic Progression, Jacqueline Lea Johnson

USF Tampa Graduate Theses and Dissertations

The RbµE2F transcriptional regulatory pathway plays a critical role in the cell cycle. Rb is inactivated through multiple waves of phosphorylation, mediated mainly by cyclin D and cyclin E associated kinases. Once Rb is inactivated, cells can enter Sµphase. Collectively, three Rb family members and ten E2F proteins coordinate every additional stage of the cell cycle, from quiescence to mitosis. However the RbµE2F pathway is frequently altered in cancer. Aside from cell proliferation, the RbµE2F pathway regulates other essential cellular processes including apoptosis, cell differentiation, angiogenesis and DNA damage repair pathways, but its role in invasion and cancer progression is …

The Role Of Ezh2 In The Regulation Of The Activity Of Matrix Metalloproteinases In Prostate Cancer Cells., Yong Jae Shin, Jeong-Ho Kim

Biochemistry and Molecular Medicine Faculty Publications

Degradation of the extracellular matrix (ECM), a critical step in cancer metastasis, is determined by the balance between MMPs (matrix metalloproteinases) and their inhibitors TIMPs (tissue inhibitors of metalloproteinases). In cancer cells, this balance is shifted towards MMPs, promoting ECM degradation. Here, we show that EZH2 plays an active role in this process by repressing the expression of TIMP2 and TIMP3 in prostate cancer cells. The TIMP genes are derepressed by knockdown of EZH2 expression in human prostate cancer cells but repressed by overexpression of EZH2 in benign human prostate epithelial cells. EZH2 catalyzes H3K27 trimethylation and subsequent DNA methylation …

Consumption Of High Ω-3 Fatty Acid Diet Suppressed Prostate Tumorigenesis In C3(1) Tag Mice, Juliana A. Akinsete, Gabriela Ion, Theodore R. Witte, W. Elaine Hardman

Biochemistry and Microbiology

Prostate cancer incidence and mortality are high in the Western world and high ω-6/ω-3 PUFA in the Western diet may be a contributing factor. We investigated whether changing from a diet that approximates ω-6 fat content of the Western diet to a high ω-3 fat diet at adulthood might reduce prostate cancer risk. Female SV 129 mice that had consumed a high ω-6 diet containing corn oil for 2 weeks were bred with homozygous C3(1)Tag transgenic male mice. All male offspring were weaned to the corn oil diet (CO) until postpuberty when half of the male offspring were transferred to …

The Role Of Cadmium And Nickel In Estrogen Receptor Signaling And Breast Cancer: Metalloestrogens Or Not?, Natalie B. Aquino, Mary B. Sevigny, Jackielyn Sabangan, Maggie C. Louie

Collected Faculty Scholarship

During the last half-century, incidences of breast cancer have increased globally. Various factors —genetic and environmental— have been implicated in the initiation and progression of this disease. One potential environmental risk factor that has not received a lot of attention is the exposure to heavy metals. While several mechanisms have been put forth describing how high concentrations of heavy metals play a role in carcinogenesis, it is unclear whether chronic, lowlevel exposure to certain heavy metals (i.e. cadmium and nickel), can directly result in the development and progression of cancer. Cadmium and nickel have been hypothesized to play a role …

Modulation Of Anti-Tumor Immune Response By Tgf-Β-Inducible Early Gene 1 (Tieg1), Andi Cani

Wayne State University Theses

Cancer immunotherapy has had limited clinical efficacy partly because regulatory T cells (Treg) suppress the immune response to tumor-associated antigens. Inducible regulatory T cells (iTreg), which are converted from naïve CD4 T cells by TGF-β, an abundant cytokine in the tumor microenvironment, may contribute to this immune suppression. Induction of Foxp3 by TGF-β is mediated by the transcription factor TIEG1 and abrogation of this protein prevents Foxp3 expression. We are testing the hypothesis that blockade of TIEG1 to prevent iTreg conversion will enhance immune response in DNA vaccination to the tumor associated antigen Her-2. Wild type and TIEG1 knockout mice …

Identifying Sm22 As A Key Player In Arterial Diseases, Jianbin Shen

Wayne State University Dissertations

Background : Expression of vascular smooth muscle cell (VSMC) cytoskeleton markers including SM22 is down-regulated in arterial diseases including atherosclerosis where inflammation and osteochondrogenesis are present. However, the role of this downregulation in arterial pathogenesis is unknown. Hypothesis : Downregulation of SM22 may actively contribute to arterial pathogenesis. Methods : Five Sm22 knockout (Sm22^-/-) mice and their wild type littermates were subjected to carotid artery denudation, an artery injury model. Analyses were conducted on carotid arteries 2 weeks after injury. Primary VSMCs were isolated from mouse aortas and investigated individually at passage 2 to 4. Sm22 knockdown was …

Hdm2 Small-Molecule Inhibitors For Therapeutic Intervention In B-Cell Lymphoma, Angela Sosin

Wayne State University Dissertations

Lymphomas frequently retain wild-type (wt) p53 function but overexpress HDM2, compromising p53 activity. Therefore, lymphoma is a suitable model for studying therapeutic value of disrupting HDM2-p53 association by small-molecule inhibitors (SMIs). HDM2 SMIs have been developed and are currently under various stages of preclinical and clinical investigation. This study examined various molecular mechanisms associated and biological effects of two different classes of HDM2 SMIs: the spiro-oxindoles (MI-219) and cis-imidazoline (Nutlin-3) in lymphoma cell lines and patient-derived B-lymphoma cells. Surprisingly, results revealed significant quantitative and qualitative differences between these two agents. At the molecular level, effect of Nutlin-3 was generally more …

Rna Oxidation Adducts 8-Ohg And 8-Oha Change With Aβ42 Levels In Late-Stage Alzheimer's Disease, Adam M. Weidner, Melissa A. Bradley, Tina L. Beckett, Dana M. Niedowicz, Amy L.S. Dowling, Sergey V. Matveev, Harry Levine, Mark A. Lovell, M. Paul Murphy

Sanders-Brown Center on Aging Faculty Publications

While research supports amyloid-β (Aβ) as the etiologic agent of Alzheimer's disease (AD), the mechanism of action remains unclear. Evidence indicates that adducts of RNA caused by oxidation also represent an early phenomenon in AD. It is currently unknown what type of influence these two observations have on each other, if any. We quantified five RNA adducts by gas chromatography/mass spectroscopy across five brain regions from AD cases and age-matched controls. We then used a reductive directed analysis to compare the RNA adducts to common indices of AD neuropathology and various pools of Aβ. Using data from four disease-affected brain …

Cell Migration Dynamics After Alteration Of Cell-Cell Contacts In Fibrosarcoma And Glioblastoma Cell Lines, Hassan S. Rizvi, Ronald K. Gary

Undergraduate Research Opportunities Program (UROP)

Cell migration is a vital component of metastasis. In this study, our intent was to study cell migration by alteration of the Wnt/GSK-3 Pathway. Since BeSO4 is a known GSK-3 kinase inhibitor, we hypothesized that this agent would cause cell migration to decrease as a result of β-catenin stabilization. Two human cell lines, HT-1080 (fibrosarcoma) and A172 (glioblastoma), were used to observe migration levels in the presence and absence of BeSO4. Our results show that cell migration is diminished for cells that were pre-treated with BeSO4, in comparison to the untreated (control) cells.

Sonic Hedgehog Dependent Phosphorylation By Ck1Α And Grk2 Is Required For Ciliary Accumulation And Activation Of Smoothened, Yongbin Chen, Noriaki Sasai, Guoqiang Ma, Tao Yue, Jianhang Jia, James Briscoe, Jin Jiang

Markey Cancer Center Faculty Publications

Hedgehog (Hh) signaling regulates embryonic development and adult tissue homeostasis through the GPCR-like protein Smoothened (Smo), but how vertebrate Smo is activated remains poorly understood. In Drosophila, Hh dependent phosphorylation activates Smo. Whether this is also the case in vertebrates is unclear, owing to the marked sequence divergence between vertebrate and Drosophila Smo (dSmo) and the involvement of primary cilia in vertebrate Hh signaling. Here we demonstrate that mammalian Smo (mSmo) is activated through multi-site phosphorylation of its carboxyl-terminal tail by CK1α and GRK2. Phosphorylation of mSmo induces its active conformation and simultaneously promotes its ciliary accumulation. We demonstrate that …