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polymerase-chain-reaction
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Design, Simulation and Construction of a Rapid Gene Amplification Microfluidic Device Using Polymerase Chain Reaction (PCR) Method
, M.Sc. Thesis Sharif University of Technology ; Shamloo, Amir (Supervisor)
Abstract
Polymerase chain reaction method is a conventional method for obtaining multiple copies of a specified segment in the DNA molecule and to amplify the DNA molecule itself. Therefore, this method has been of paramount importance in different fields of research and has been applied for different applications. PCR requires thermal cycling, or repeated temperature changes between two or three discrete temperatures to amplify specific nucleic acid target sequences. To achieve such thermal cycling, conventional bench-top thermal cyclers generally use a metal heating block powered by Peltier elements or benefit from forced convection heat transfer. Although efforts have been made in order to reduce...
Design and Fabrication of a Microfluidic Digital PCR
, M.Sc. Thesis Sharif University of Technology ; Saeedi, Mohammad Saeed (Supervisor) ; Firouzabadi, Bahar (Supervisor)
Abstract
Polymerase chain reaction, abbreviated as PCR, is a method of amplifying the number of copies of a desired DNA sequence through special protocols. The rapid advance of microfluidic devices and the emerging concept of digital microfluidics has resulted into the development of digital droplet PCR (ddPCR) systems with their significant uses in the detection of rare mutations, cancer diagnosis, and surveillance. In this study, an integrated ddPCR microfluidic chip alongside its thermoelectric thermal cycler and optical analysis systems have been designed, fabricated and their performance has been tested and evaluated. It was shown that the effect of master mold’s surface quality, the material of...
Design of a Microfluidic Digital Droplet PCR
, M.Sc. Thesis Sharif University of Technology ; Saeedi, Mohammad Saeed (Supervisor)
Abstract
Polymerase chain reaction, abbreviated as PCR, is a method of amplifying the number of copies of a desired DNA sequence through special protocols. The rapid advance of microfluidic devices and the emerging concept of digital microfluidics has resulted into the development of digital droplet PCR (ddPCR) systems with their significant uses in the detection of rare mutations, cancer diagnosis, and surveillance. A large number of micron-sized droplets are required to perform ddPCR. in this study, To investigate the gradient of confinements induced droplet self-breakup mechanism, we carried out the computational fluid dynamics (CFD) simulations for the two-phase flow using a commercial software...
Cloning and Production of the Flavin Reductase Enzyme in Order to Perform Organic Reaction and Synthesis of Cation Exchange Column for Protein Purification
, M.Sc. Thesis Sharif University of Technology ; Kalhor, Hamid Reza (Supervisor)
Abstract
The reductase enzymes perform an important function in the cell. These enzymes are usually coupled with other enzymes and participate in reactions such as halogenation, reduction of alkene, and carboxylic acids. These reactions are carried out by reproducing cofactors such as FADH2 and NAD+; however, there is no evidence that this enzyme alone can perform an organic reaction. In this work, we aim to examine whether recombinant flavin reductase can carry out organic reactions. In order to amplify the flavin reductase enzyme gene from the E. coli genome by PCR (Polymerase Chain Reaction) method, two synthetic primers were designed; The forward primer of the gene contained a cleavage site for...
Study, Optimization and Construction of a Microfluidic Gene Amplification Device by Using Thin Film Layer Method
, M.Sc. Thesis Sharif University of Technology ; Vosoughi, Manouchehr (Supervisor) ; Alemzadeh, Iran (Co-Supervisor) ; Shamloo, Amir (Co-Supervisor)
Abstract
Polymerase Chain Reaction (PCR) is a process in which a special piece of a gene is amplified millions of times over a short period. This method has been of paramount importance in different fields of research and has been applied for different applications. PCR requires thermal cycling, or repeated temperature changes between two or three discrete temperatures to amplify specific nucleic acid target sequences. To achieve such thermal cycling, conventional bench-top thermal cyclers generally use a metal heating block powered by Peltier elements or benefit from forced convection heat transfer. Due to the fact that these methods are time consuming, it seems that design and fabrication of a fast...
Creating Random Mutations on The Human Transglutaminase 2 Gene and Lysozyme Gene to Increase The Solubility of the Enzymes in order to Perform Promiscuous Organic Reactions Using The Recombinant Enzymes
, M.Sc. Thesis Sharif University of Technology ; Kalhor, Hamid Reza (Supervisor)
Abstract
Transglutaminase 2 enzyme is expressed in various parts of the human body and is present in almost all tissue cells. The function of this enzyme is multiple and it catalyzes different reactions depending on where it is located in the cell. The most important activities of this enzyme are: isopeptide bond formation between the side chains of two proteins (transamidation reaction), diamidation reaction (hydrolysis of amide bond), esterification, kinase activity. The hen egg white lysozyme protein is another natural antimicrobial protein that has been extensively studied. The muramidase activity of egg white lysozyme against highly gram-positive bacteria is well known, and this property has...
PCR Amplification Prediction using Machine Learning
, M.Sc. Thesis Sharif University of Technology ; Hossein Khalaj, Babak (Supervisor)
Abstract
Polymerase Chain Reaction (PCR) is a laboratory method for amplifying a part of DNA. This method is used in determining the sequence of genes, detecting pathogenic agents in epidemics, creating genetic changes in bacteria, diseases, plants and even animals. Many factors affect the quality of the reaction. Each of these factors can be effective in amplifying the target in DNA. If we can predict the result of PCR using the factors involved in the reaction, it will save a lot of money and time. The aim of this research is to predict the result of PCR amplification using machine learning methods. For this purpose, two methods are proposed: feature-based method and string-based method. In the...
Engineering of mature human induced pluripotent stem cell-derived cardiomyocytes using substrates with multiscale topography
, Article Advanced Functional Materials ; Volume 28, Issue 19 , 2018 ; 1616301X (ISSN) ; Garbern, J. C ; Behzadi, S ; Hill, M. J ; Tresback, J. S ; Heydari, T ; Ejtehadi, M. R ; Ahmed, N ; Copley, E ; Aghaverdi, H ; Lee, R. T ; Farokhzad, O. C ; Mahmoudi, M ; Sharif University of Technology
Wiley-VCH Verlag
2018
Abstract
Producing mature and functional cardiomyocytes (CMs) by in vitro differentiation of induced pluripotent stem cells (iPSCs) using only biochemical cues is challenging. To mimic the biophysical and biomechanical complexity of the native in vivo environment during the differentiation and maturation process, polydimethylsiloxane substrates with 3D topography at the micrometer and sub-micrometer levels are developed and used as cell-culture substrates. The results show that while cylindrical patterns on the substrates resembling mature CMs enhance the maturation of iPSC-derived CMs, sub-micrometer-level topographical features derived by imprinting primary human CMs further accelerate both the...
Magnetoelectric nanocomposite scaffold for high yield differentiation of mesenchymal stem cells to neural-like cells
, Article Journal of Cellular Physiology ; Volume 234, Issue 8 , 2019 , Pages 13617-13628 ; 00219541 (ISSN) ; Soleimani, M ; Ghiass, M. A ; Hatamie, S ; Vakilian, S ; Zomorrod, M. S ; Sadeghzadeh, N ; Vossoughi, M ; Hosseinzadeh, S ; Sharif University of Technology
Wiley-Liss Inc
2019
Abstract
While the differentiation factors have been widely used to differentiate mesenchymal stem cells (MSCs) into various cell types, they can cause harm at the same time. Therefore, it is beneficial to propose methods to differentiate MSCs without factors. Herein, magnetoelectric (ME) nanofibers were synthesized as the scaffold for the growth of MSCs and their differentiation into neural cells without factors. This nanocomposite takes the advantage of the synergies of the magnetostrictive filler, CoFe 2 O 4 nanoparticles (CFO), and piezoelectric polymer, polyvinylidene difluoride (PVDF). Graphene oxide nanosheets were decorated with CFO nanoparticles for a proper dispersion in the polymer through...
Comparing the effects of endurance and resistance trainings on gene expression involved in protein synthesis and degradation signaling pathways of Wistar rat soleus muscle
, Article Tehran University Medical Journal ; Volume 77, Issue 11 , 2020 , Pages 668-677 ; Seifabad, M ; Asad, M. R ; Sharif University of Technology
Tehran University of Medical Sciences
2020
Abstract
Background: Skeletal muscle mass, which is regulated by a balance between muscle protein synthesis and degradation, is an important factor for movement to meet everyday needs, especially in pathological conditions and aging. The purpose of the present investigation was to compare the alterations of the gene expression involved in muscle protein synthesis and degradation signaling pathways induced by two exercise training protocols. Methods: Eight weeks old Wistar rats have been assigned to the present experimental study, which was conducted from August 2018 to October 2018 at the animal laboratory of Tehran University. They were randomly divided into two resistance and endurance training...
Niosomal delivery of simvastatin to MDA-MB-231 cancer cells
, Article Drug Development and Industrial Pharmacy ; Volume 46, Issue 9 , 2020 , Pages 1535-1549 ; Saremi Poor, A ; Yaghmaei, S ; Norouzian, D ; Noorbazargan, H ; Saffar, S ; Ahangari Cohan, R ; Bakhshandeh, H ; Sharif University of Technology
Taylor and Francis Ltd
2020
Abstract
Objective: The objective of this study was to use nano-niosomal formulations to deliver simvastatin as a poor-water soluble drug into breast cancer cells. Significance: Our study focused on the problem associated with poor water-soluble drugs which have significant biological activity in vivo. Methods: Different niosomal formulations of simvastatin were prepared and characterized in terms of morphology, size, encapsulation efficiency (EE), and release kinetic. Antiproliferative activity and the mechanism were assessed by quantitative real-time PCR and flow cytometry. Moreover, confocal microscopy was employed to analyze the cell uptake of simvastatin loaded niosomes to the cancerous cells....
The role of hippo signaling pathway in physiological cardiac hypertrophy
, Article BioImpacts ; Volume 10, Issue 4 , 2020 , Pages 251-257 ; Tabrizi, A ; Sharif University of Technology
Tabriz University of Medical Sciences
2020
Abstract
Introduction: The role of Hippo signaling pathway, which was identified by genetic studies as a key regulator for tissue growth and organ size, in promoting physiological cardiac hypertrophy has not been investigated. Methods: Fourteen male Wistar rats were randomly assigned to the exercise and control groups. The exercise group ran 1 hour per day, 5 days/week, at about 65%-75% VO2max on the motor-driven treadmill with 15ºslope, and the control group ran 15 min/d, 2 days/ week at 9 m/min (0ºinclination), throughout the eight-week experimental period. Forty-eight hours after the last session, hearts were dissected and left ventricles were weighed and stored for subsequent RT-PCR analysis....
CRISPRi-mediated knock-down of PRDM1/BLIMP1 programs central memory differentiation in ex vivo-expanded human T cells
, Article BioImpacts ; Volume 12, Issue 4 , 2022 , Pages 337-347 ; 22285652 (ISSN) ; Sayadmanesh, A ; Nazer, N ; Ahmadi, A ; Hemmati, S ; Mohammadzade, H ; Ebrahimi, M ; Baharvand, H ; Khalaj, B ; Aghamaali, M. R ; Basiri, M ; Sharif University of Technology
Tabriz University of Medical Sciences
2022
Abstract
Introduction: B lymphocyte-induced maturation protein 1 (BLIMP1) encoded by the positive regulatory domain 1 gene (PRDM1), is a key regulator in T cell differentiation in mouse models. BLIMP1-deficiency results in a lower effector phenotype and a higher memory phenotype. Methods: In this study, we aimed to determine the role of transcription factor BLIMP1 in human T cell differentiation. Specifically, we investigated the role of BLIMP1 in memory differentiation and exhaustion of human T cells. We used CRISPR interference (CRISPRi) to knock-down BLIMP1 and investigated the differential expressions of T cell memory and exhaustion markers in BLIMP1-deficient T cells in comparison with...
Expression of functional recombinant human tissue transglutaminase (TG2) using the bac-to-bac baculovirus expression system
, Article Advanced Pharmaceutical Bulletin ; Volume 6, Issue 1 , 2016 , Pages 49-56 ; 22285881 (ISSN) ; Azari, S ; Kalhor, H. R ; Sharif University of Technology
Tabriz University of Medical Sciences
Abstract
Tissue transglutaminase (TG2) is a unique multifunctional enzyme. The enzyme possesses enzymatic activities such as transamidation/crosslinking and non-enzymatic functions such as cell migration and signal transduction. TG2 has been shown to be involved in molecular mechanisms of cancers and several neurodegenerative diseases such as Alzheimer's disease. The present study aimed at cloning and expression of full length human TG2 in Bac-to-Bac baculovirus expression system and evaluation of its activity. Methods: pFastBac HTA donor vector containing coding sequence of human TG2 was constructed. The construct was transformed to DH10Bac for generating recombinant bacmid. The verified bacmid was...
The nanofibrous PAN-PANi scaffold as an efficient substrate for skeletal muscle differentiation using satellite cells
, Article Bioprocess and Biosystems Engineering ; Volume 39, Issue 7 , 2016 , Pages 1163-1172 ; 16157591 (ISSN) ; Mahmoudifard, M ; Mohamadyar Toupkanlou, F ; Dodel, M ; Hajarizadeh, A ; Adabi, M ; Soleimani, M ; Sharif University of Technology
Springer Verlag
Abstract
Among polymers, polyaniline (PANi) has been introduced as a good candidate for muscle regeneration due to high conductivity and also biocompatibility. Herein, for the first time, we report the use of electrospun nanofibrous membrane of PAN-PANi as efficient scaffold for muscle regeneration. The prepared PAN-PANi electrospun nanofibrous membrane was characterized by scanning electron microscopy (SEM), Attenuated total reflectance fourier transform infrared spectroscopy (ATR-FTIR) and tensile examination. The softer scaffolds of non-composite electrospun nanofibrous PAN govern a higher rate of cell growth in spite of lower differentiation value. On the other hand, PAN-PANi electrospun...
Laboratory detection methods for the human coronaviruses
, Article European Journal of Clinical Microbiology and Infectious Diseases ; Volume 40, Issue 2 , 2021 , Pages 225-246 ; 09349723 (ISSN) ; Dowlatshahi, S ; Abdekhodaie, M. J ; Sharif University of Technology
Springer Science and Business Media Deutschland GmbH
2021
Abstract
Coronaviruses are a group of envelop viruses which lead to diseases in birds and mammals as well as human. Seven coronaviruses have been discovered in humans that can cause mild to lethal respiratory tract infections. HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1 are the low-risk members of this family and the reason for some common colds. Besides, SARS-CoV, MERS-CoV, and newly identified SARS-CoV-2, which is also known as 2019-nCoV, are the more dangerous viruses. Due to the rapid spread of this novel coronavirus and its related disease, COVID-19, a reliable, simple, fast, and low-cost detection method is necessary for patient diagnosis and tracking worldwide. Human coronaviruses detection...
Unraveling cancer metastatic cascade using microfluidics-based technologies
, Article Biophysical Reviews ; Volume 14, Issue 2 , 2022 , Pages 517-543 ; 18672450 (ISSN) ; Kermanshah, L ; Abouali, H ; Hashemi, H. M ; Yari, A ; Khorasheh, F ; Alemzadeh, I ; Vossoughi, M ; Sharif University of Technology
Springer Science and Business Media Deutschland GmbH
2022
Abstract
Cancer has long been a leading cause of death. The primary tumor, however, is not the main cause of death in more than 90% of cases. It is the complex process of metastasis that makes cancer deadly. The invasion metastasis cascade is the multi-step biological process of cancer cell dissemination to distant organ sites and adaptation to the new microenvironment site. Unraveling the metastasis process can provide great insight into cancer death prevention or even treatment. Microfluidics is a promising platform, that provides a wide range of applications in metastasis-related investigations. Cell culture microfluidic technologies for in vitro modeling of cancer tissues with fluid flow and the...
MiR-361-5p as a promising qRT-PCR internal control for tumor and normal breast tissues
, Article PLoS ONE ; Volume 16, Issue 6 June , 2021 ; 19326203 (ISSN) ; Salimi, A ; Rahmani, S ; Nafissi, N ; Sharifi Zarchi, A ; Mowla, S. J ; Sharif University of Technology
Public Library of Science
2021
Abstract
Background: One of the most widely used evaluation methods in miRNA experiments is qRT-PCR. However, selecting suitable internal controls (IC) is crucial for qRT-PCR experiments. Currently, there is no consensus on the ICs for miRNA qRT-PCR experiments in breast cancer. To this end, we tried to identify the most stable (the least expression alteration) and promising miRNAs in normal and tumor breast tissues by employing TCGA miRNA-Seq data and then experimentally validated them on fresh clinical samples. Methods: A multi-component scoring system was used which takes into account multiple expression stability criteria as well as correlation with clinical characteristics. Furthermore, we...
Development of simple and efficient Lab-on-a-Disc platforms for automated chemical cell lysis
, Article Scientific Reports ; Volume 10, Issue 1 , 6 July , 2020 ; Saadatmand, M ; Eghbal, M ; Parsa Yeganeh, L ; Sharif University of Technology
Nature Research
2020
Abstract
Cell lysis is the most important first step for molecular biology and diagnostic testing. Recently, microfluidic systems have attracted considerable attention due to advantages associated with automation, integration and miniaturization, especially in resource-limited settings. In this work, novel centrifugal microfluidic platforms with new configurations for chemical cell lysis are presented. The developed systems employ passive form of pneumatic and inertial forces for effective mixing of lysis reagents and cell samples as well as precise fluidic control. Characterizations of the developed Lab-on-a-Discs (LoaDs) have been conducted with dyed deionized (DI) waters and white blood cells...
A plasmonic gold nanofilm-based microfluidic chip for rapid and inexpensive droplet-based photonic PCR
, Article Scientific Reports ; Volume 11, Issue 1 , December , 2021 ; 20452322 (ISSN) ; Bagheri, M ; Shamloo, A ; Kazemipour Ashkezari, A. H ; Sharif University of Technology
Nature Research
2021
Abstract
Polymerase chain reaction (PCR) is a powerful tool for nucleic acid amplification and quantification. However, long thermocycling time is a major limitation of the commercial PCR devices in the point-of-care (POC). Herein, we have developed a rapid droplet-based photonic PCR (dpPCR) system, including a gold (Au) nanofilm-based microfluidic chip and a plasmonic photothermal cycler. The chip is fabricated by adding mineral oil to uncured polydimethylsiloxane (PDMS) to suppress droplet evaporation in PDMS microfluidic chips during PCR thermocycling. A PDMS to gold bonding technique using a double-sided adhesive tape is applied to enhance the bonding strength between the oil-added PDMS and the...