Human Genetics And Molecular Medicine - Master Dissertation

Permanent URI for this collectionhttps://kr.cup.edu.in/handle/32116/104

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Generation of Rho-0 Cells using MDA-MB-231 Cell Line and Measurement of Drug Cytotoxicity
(Central University of Punjab, 2018) Sharma, Bharti; Singh,Sandeep
The ATP generation via Oxidative phosphorylation (OXPHOS) system located in the inner membrane of mitochondria, is regulated by the coordinated interaction between nucleus and mitochondria. In the same context, mitochondrial-depleted cell (Rho-0) can be a helpful approach to study the mitochondrial metabolism, mitochondrial role in various cellular processes such as apoptosis, mitochondrial role in various mitochondrial related disorders and cancer. To generate Rho-0 cells, EtBr mediated mtDNA depletion was done and verified by agarose gel electrophoresis. % cell viability, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) production was measured after 24 hr treatment with 3 drugs, ?-amanitin, Doxorubicin and DCA in both parental MDA-MB-231 and Rho-0 cells. Reduced cell death and ROS production was observed in Rho-0 cells indicating the resistance against apoptosis in Rho-0 cells and demonstrating the possible role of mitochondria in intrinsic pathway of apoptosis. MMP was observed to be maintained in Rho-0 cells indicating the role of nuclear genome in the maintenance of MMP.
Identification of Novel lncRNA in Breast Cancer Cell Line
(Central University of Punjab, 2018) Sharma, Divya; Singh,Sandeep
Long non-coding RNA(lncRNA) is RNA transcript having size of more 200 nucleotides and low number of exons are present due to which it expressed at lower level in specific tissues. Mainly the lncRNA is involved in gene regulations such as epigenetic regulation, chromatin remodeling and posttranscriptional regulations.The localization of lncrna is mainly in nucleus but there is considerable amount of lncrna present in cytoplasm. Lncrna can also target the mitochondrial genome. Identification of the targets of lncrna on mitochondrial genome helps to find the relationship between lncrna and mitochondrial functions which further helps in diagnosis and in therapy of the mitochondrial related diseases. One such lncRNA is MIR503HG, which act as a tumor suppressor in breast cancer cell line. Expression of lncRNA MIR503HG was analyzed in normoxic and hypoxic conditions upon the treatments of three different types of drugs on breast cancer cell line. The study of expression level of lncRNA MIR503HG in breast cancer cell line suggest a new cancer biomarker.
Evaluation Of Association Of Folic Acid Metabolism In Chronic Obstructive Pulmonary Disease
(Central University of Punjab, 2018) Chaudhary, Deepti; Senapati, Sabyasachi
COPD is characterized by increasing breathlessness. It is the fourth cause of death in the world and it is currently presenting a major global public health challenge, causing premature death from pathophysiological complications. It continues to be an important cause of morbidity, mortality, and health-care costs worldwide. It is a global health issue, with cigarette smoking being an important risk factor universally with several other factors, such as exposure to indoor and outdoor air pollution, occupational hazards, and infections. As the global population ages, the burden of COPD will increase in years to come. Folic acid or vitamin B9 is a water soluble vitamin plays a major role in metabolism. Since, COPD is a disease characterized by complex nutritional abnormalities and lower level of vitamin B9 and B12. Homocysteine or total homocysteine is a sulfur containing amino acid, acts as an intermediate in the metabolism of methionine in the folic acid or folate pathway. As observed among general population hyperhomocysteinaemia (mild to moderate), is mainly associated with vitamin B deficiencies, which are considered to be essential cofactors for the level and catabolism of hcy. Assessment of level of micronutrients is useful in establishing effects in COPD.
Association of UGT1A6 2 (Ser7Ala) polymorphism with therapeutic response to aspirin in ischemic stroke patients
(Central University of Punjab, 2018) Kumar, Dharmendra; Munshi,Anjana
Ischemic stroke occurs due to the formation of thrombus or embolism within the arteries due to platelet aggregation. Aspirin therapy is used for the prevention of secondary stroke. The variant of UGT1A6 (Ser7Ala) gene has been found to be associated with ischemic stroke as well as aspirin resistance. We aim to study the demographic profile of ischemic stroke patients from Malwa region of Punjab and to evaluate the frequency of UGT1A6*2 Ser7Ala polymorphism and correlate it with aspirin resistance and ADRs (if any). We collected 30 samples from confirmed ischemic stroke patients from Guru Gobind Singh Medical College and Hospital in Malwa region of Punjab. DNA was isolated from blood and PCR- RFLP technique was used to evaluate the UGT1A6 gene variant in the patients. mRS value was used to classify patients as responders or nonresponders. 24 patients had TT genotype and 6 patients were found to bear TG genotype. 90% of patients were aspirin responders and 10% were aspirin nonresponders. Since the sample size was too low to identify significant associations, a large number of samples should be screened before coming to a conclusion. However, this preliminary study indicates that UGT1A6*2 (Ser7Ala) variant of UGT1A6 gene might be a risk factor for aspirin resistance in the studied group.
Meta-analysis and estimation of gene expression to establish the role of SFTPD in COPD
(Central University of Punjab, 2018) Nandy, Debparna; Senapati, Sabyasachi
COPD is considered to be third leading cause of death by 2020. So far, there is no proper biomarker available for the diagnosis of all the different sub-phenotypes. Multiple GWAS studies have reported the role of SFTPD (both genetic and serum protein) as a diagnosis biomarker. This current study aims to systemic - review and meta-analyse the association of anthropometric parameter (Smoking status, gender), Protein biomarker (serum SFTPD) and genetic biomarker (SFTPD rs721917) with COPD and its other phenotypes. To support the secondary findings, this study also aims to analyse the expression of SFTPD gene among COPD cases and healthy control from North Indian population. So far bronchodilators therapy are available for providing temporary relief to the patients. But bronchodilators are found to be associated with multiple number of side-effects. Indian Traditional medicine options like Ayurveda and yoga has lot to offer in this case. Many local Indian herbs are found to be significantly effective against COPD condition. Most importantly this herbs don't have any side effects. Pranayama and Dhyana have also been found to be improving Lung functions in multiple studies.
Expression of CHAC1 in Breast Cancer Cell Lines
(Central University of Punjab, 2018) Sharma, Ankita; Chander, Harish
Breast cancer is the commonly diagnosed type of cancer in women and is a major cause of deaths in women. Unfolded Protein Response Pathway is a signaling pathway induced in endoplasmic reticulum as a stress response. This type of stress signaling has been seen to be activated in many tumors including breast cancer. CHAC1 (Glutathione specific gamma- glutamylcyclotransferas-1) is a member of UPR Pathway. It was first discovered as a component of the ATF4 arm of the UPR pathway in a co-regulated group of genes. CHAC1 expression is necessary and sufficient to induce well-characterized markers of apoptosis. CHAC1 is involved in the inhibition of TNFRS6B via ATF4-ATF3-CHOP signaling. This sensitizes cells to commit to apoptosis following induction of UPR pathway. Although CHAC1 is a pro-apoptotic component of the UPR pathway, its expression in breast cancers have been noted to be remarkably high. To study the role of CHAC1 in breast cancer, we analyzed the expression of CHAC1 at mRNA level by using Real-Time PCR and further checked its' protein expression by Western-blotting. Its expression was found to be higher in ER positive cells as compared to the ER negative cells. Further investigations were performed by transfecting MDA-MB-231 cells by ER-alpha, which confirmed that presence of ER-alpha leads to the higher expression cHAC1 in breast cancer cells.
Potential Mitochondrial-Specific Function Of piRNAs
(Central University of Punjab, 2018) Paul, Shouvik; Singh, Sandeep
Piwi-interacting RNAs (piRNAs) are (26-31 nt) small noncoding RNAs processed from their longer precursor transcripts with the help of Piwi proteins. There are more than 30,000 piRNA genes present in the human genome which now turns out to be emerging player in both homeostasis and diseases. Localization of piRNA and PIWI in the repeat region of the mammalian nuclear genome in germ cells has been reported, although localization and potential functional role of piRNA in the mammalian mitochondrial genome are largely unknown. We have taken 111 piRNA sequences found in the MCF-7 mitochondrial genome, which is obtained by NGS analysis for alignment study. Resulting piRNA have been aligned with DQ112870 North American Homo sapiens mitochondrion genome for studying post- transcriptional roles of piRNA.
Bioinformatic Analysis of Whole Exome Data
(Central University of Punjab, 2018) Md Momin Ali; Khetarpal, Preeti
Whole Exome Sequencing (WES) is a capture-based method developed to sequence exome and to identify variants in the coding region of genes that affect protein function. Understanding the exomes of individuals at single base resolution allows the identification of pathogenic variants responsible for causing genetic disease. In this paper we mentioned all the object of the project steps and bioinformatic computational tools involved step by step. The objective of the study was to find all the disease causing heterozygous variants in the proband from the WES data. Using in silico tools SIFT, PolyPhen and ANNOVAR. All the annotated non-synonymous SNPs was arranged and filtered according to the pathogenicity of variants. All the variants were narrowed down to five matching variants. associated with clinical phenotype of the proband. As a conclusion Bioinformatic analysis of WES data proved to be a good tool for finding disease causing variants. Majority of the tools used in the analysis are generally linux based with poor user interface which makes it a challenging experience for a non-computational individual. Future of this field is dependent on software developers, so that more people can understand and help in the progress.
Investigation for Maternal Uniparental Disomy of Chromosome 7 in a Silver Russell Syndrome patient
(Central University of Punjab, 2018) Gupta, Swati; Khetarpal, Preeti
Silver-Russell syndrome (SRS) is a genetically heterogeneous disorder. Individuals with SRS show clinical features with varying severity. The major criteria for clinical diagnosis of SRS are intrauterine growth retardation (IUGR) accompanied with post natal growth retardation (PNGR) and relative macrocephaly, triangular face, feeding difficulties, fifth finger clinodactyly. Maternal Uniparental disomy of chromosome 7 had been implicated in 10% of SRS cases; in about 1% cases, structural chromosomal aberrations has been reported and in about 45% cases, epimutation have been detected in Imprinting control region (ICR1) of 11p region. Aetiology of remaining cases unknown. To investigate Maternal Uniparental Disomy of chromosome 7 in a Silver Russell Syndrome patient. The sample was collected of female patient suggested of SRS, clinically diagnosed with relative macrocephaly, mild facial asymmetry with postnatal growth retardation. The present study had been undertaken with an objective to detect maternal Uniparental disomy of chromosome 7 using locus specific primers to amplify STR loci by PCR. PCR products were visualized on 7.5% native Polyacrylamide Gel Electrophoresis (PAGE). On analysing the gel, matUPD7 was ruled out. Patient recruited in this study was an isolated cases with no previous incidence in the family.
Study the effect of phytochemicals phenethyl isothiocyanate (PEITC) and Quercetin on mitochondrial biogenesis in cancer and normal cell lines
(Central University of Punjab, 2018) Thakur, Anchal; Chander, Harish
Phytochemicals are plant-derived chemicals generally are biologically active compounds and mediate positive health benefits by targeting genes or metabolic pathways of a cell. The phytochemicals examined can be classified into main categories, such as carotenoids and polyphenols, which include phenolic acids, flavonoids and stilbenes / lignans. Quercetin is isolated from the Tridax procumbens (Linn.). Its anti-cancer activity has been well documented in vitro and in vivo. It could be pro-apoptotic as well as anti-apoptotic depending upon its concentration of it and time of exposure. Isothiocyanates are cruciferous derived phytochemicals. PEITC majorly isolated from Nasturtium officinale (watercress) has shown to mediate its anti-cancer activity through ROS-mediated pathway. It is a basic leucine zipper protein involved in protection against oxidative damage triggered by stress like injury or inflammation through regulation of the expression of anti- oxidant proteins. Under oxidative stress, inactivation of Kelch-like ECH-associated protein 1 (Keap1) occurs which is a cytosolic repressor protein that binds to Nrf2. This results in Nrf2-Keap1 complex dissociation, and hence, promoting the translocation of Nrf2 to the v nucleus where it binds to ARE (anti-oxidant response element), and induce the transcription of anti-oxidative proteins. Quercetin and PEITC treatment to the cancer cells led to decreased mitochondrial biogenesis as the NRF-2 levels diminishes as the concentration of the drug increases. The anti-oxidant levels are getting down in the cancer cells leading to ROS accumulation in the cancer cells leading ultimately to the death. Quercetin and PEITC treatment to the normal HBL-100 cells induced the mitochondrial biogenesis by increasing NRF-2 levels as the concentration of the drug increases. Confocal microscopy results also proved that treatment of quercetin or PEITC or the combination of both drugs was found to be effective in cancer cells as the mitochondria size and shape got decreased interpreted through the intensity of green dye. To conclude our study, it has been shown that quercetin and PEITC lead to increased mitochondrial biogenesis in normal cells whereas decreased mitochondrial biogenesis in cancer cells.
Analysis of exonic region of PCNT gene in Microcephalic Osteodysplastic Primordial Dwarfism Type II subjects
(Central University of Punjab, 2018) Gupta, Neha; Khetarpal, Preeti
MOPD II is an autosomal recessive disorder. It is characterised by the presence of intra uterine growth retardation as well as post natal growth retardation. The adult height is not more than 100 cm. It has been found that mutation in PCNT gene is associated with MOPD II. The cytogenetic location of this gene is 21q22.3 and it contains 47 exons. It encodes for PCNT protein which is a very large coiled scaffold protein and helps in microtubule polymerisation ensuring proper cell division. Till date 74 mutations have been identified this includes deletion, stop, frame shift and non sense mutation. The present study was carried out to analyse the exonic region of PCNT gene in Microcephalic Osteodysplastic Primordial Dwarfism Type II subjects. As it is an autosomal rescessive disorder both male and female were equally affected. The study included three subjects diagnosed with MOPD II .The DNA was extracted from whole blood and was amplified using locus specific primers. The products were sequenced using Sanger sequencing and were analysed. Total 12 variants were detected and 2 of which were pathogenic and 2 were synonymous and remaining 8 were polymorphic variants. 3 were present in exon 44 and 1 in exon 31 .These 3 variants were found to be present in all four subjects while 1 was present in only one subject. Change in nucleotide sequence may produce deleterious affect which is needed to be explored along with the complete structure of PCNT protein.
Literature survey of HDACs in cancer and in-vitro analysis of HDAC inhibitor mediated chemo-sensitization
(Central University of Punjab, 2018) Singh, Tashvinder; Singh, Sandeep
HDACs are chromatin modifying enzymes and post translational modifiers regulating transcription and activity of various proteins such as tubulin, Hsp90 and cortactin respectively. HDAC is divided into four classes (Class 1, Class 2(a, b), Class 3 and Class 4) and 2 families (based on homology) which are Rdp3/Hda1 family and Sirtuin family (NAD+ Dependent Sir2). Tumor cells having no HDACs expression or loss of enzymatic activity due to mutation make them more resistant to HDACi than cells having higher HDAC expression. The objectives of this study were to survey the literature in role of HDACs expression during cancer progression and we found that HDACs are associated with specific cancer types but their appropriate correlation has not been found till now. To analyze the HDAC inhibitor (TSA) mediated chemo-sensitization in doxorubicin treated MDA-MB-231 cells, MTT assay was used to study the chemo-sensitization effect of TSA on cells treated with doxorubicin at different concentrations. We found out that TSA and Doxorubicin alone had higher anti-proliferative effect on MDA-MB-231 cells than other combinatorial doses dependent fashion. None of the above given doses had reduced viability less than 50%, so IC50 value of these given doses are undetermined. 16hrs 100nM TSA treatment followed by 10nM doxorubicin combined had higher anti proliferative effect as compared to other concentration. 16hrs 100nM TSA followed by v 24hrs 10nM doxorubicin treatment results in 30% cell death while for similar experiment at higher concentration of TSA and Doxorubicin, viability is regained in MDA-MB-231 treated cells. This may suggest that TSA in-sensitize/nullified the Doxorubicin induced mitochondrial mediated apoptosis. Combinatorial effect might not be effective in comparison to dual or multi-target inhibitors. HDAC inhibitor mediated chemo-sensitization could be analyzed on other drug treated cancer cell lines. Use of multi-target inhibitor could be seen as effective therapeutic agent against tumor cells.
In Silico Design Of Compounds As Sglt2 Inhibitors
(Central University of Punjab, 2018) Chakravorty, Kamaljyoti; Munshi,Anjana
SGLT2 inhibitors (SGLT2i) are the current novel therapeutic approach expected to control the growing threat of type 2 diabetes mellitus (T2DM). With comparatively least reported side effects, SGLT2i (gliflozins) have been identified as promising tools to tackle the threat of T2DM. However, studies have also reported these drugs to cause renal impairments and urinogenital infections among certain T2DM patients. The efficacy of an inhibitor is fundamentally determined by the stability of protein-inhibitor complex. Therefore, it is essential to study the binding site residues of SGLT2 in the light of inhibitors' interactions. Structural insights of SGLT2 suggested a competitive inhibition of the ligand glucose (agonist) by the gliflozins. The inhibitory effect of SGLT2i reduces the renal reabsorption of glucose and promotes glycosuria. Consequently, a reduced plasma glucose level prevents the risk of hyperglycemia and further T2DM. In order to design potent inhibitors the structures of the available gliflozins (empagliflozin, canagliflozin, dapagliflozin and ertugliflozin) were analysed. Accordingly, a library of 44 fragment molecules was generated for interactive enumeration. A core ligand structure was designed based on the structural analysis of the gliflozin. A total of 3250 ligand molecules were obtained using schrodinger maestro v11.3. Docking results have shown ligand molecules exhibiting two different modes of inhibition. Set A molecules exhibited glyconic mode of interaction while set B molecules displayed aglyconic mode of interaction at the glucose binding site. The interactions of set B molecules are similar to that of the standard gliflozins and are expected to be less stable. Lesser stability of the protein- iv ligand complex perhaps lead to the reported side effects. On the other hand, set A molecules (with lower docking score) are expected to be much stable in terms of their interactions which differ significantly from that of the standard gliflozins. Therefore, set A molecules are the anticipated potent SGLT2 inhibitors expected to show reduced side effects.
Eeffect of Natural Compounds on Her2 Expression
(Central University of Punjab, 2018) Uradanda, Praveen; Chander, Harish
Breast cancer is a heterogeneous disease that is challenging to treat.we examine potential natural compounds which may help in treating her2 positive breast cancer. The main objectives of this study are to determine the mRNA level and protein levels on exposing MDAMB453 cell lines with Quercetin and PEITC at 40µm,44hrs.we have found in result analysis that her2 expression is downregulated on treatment with PEITC.where cell line treated with quercetin as no effect to downregulate her2 expression in MDAMB453.
Identifying novel miRNA targets of anticancer drugs in normoxic and hypoxic condition
(Central University of Punjab, 2018) Goyal, Sapna; Singh, Sandeep
Breast cancer has become the prevailing cause of death, so the research for their treatment has become advanced day by day. Breast cancer behaves differently in normoxic and hypoxic condition so does the anticancer drugs in both the condition in order to treat them. These drugs might have different effect on the expression of miRNAs in both normoxic and hypoxic conditions. In the present study, we evaluated the IC50 of three anticancer drugs-doxorubicin, ?-Amanitin, DCA on MDA-MB-231 and after treatment with these drugs, have checked the expression of miRNAs- let-7b and miR1275 in both hypoxic and normoxic condition by RT-PCR and results were analyzed by native PAGE
Expression of KIBRA in Breast Cancer Cell Lines
(Central University of Punjab, 2018) Singh, Garima; Chander,Harish
Breast cancer the most frequently diagnosed cancer and its metastasis to distant organs accounts the majority of deaths. Numerous genes and proteins are involved in the cause of metastasis. Though KIBRA is one of the component of Hippo Pathway and is reported as tumor suppressor but this scaffolding protein has also been found to be an emerging and important player in the process of metastasis. It has been reported that KIBRA protein interacts with various proteins through its domain and leads cytoskeleton arrangement, cell polarity and migration. N terminal and C terminal of the protein contains the WW, Internal C2 & putative class III PDZ domain that interacts with DDR1, DLC1 & PKC? and helps the breast cancer cells to metastasize. To study whether KIBRA is involved in breast cancer metastasis, we checked its expression at both protein and mRNA level by Immunoblotting and Real Time PCR which showed increased KIBRA expression in ER positive cells. Further investigation to elucidate the role of KIBRA in ER positive cells, ER transfection and immunoblotting in triple negative breast cancer cell lines were performed, which indicated that ER leads to enhanced KIBRA expression in breast cancer cells.
Analysis of PCNT gene coding sequence in subjects with Microcephalic Osteodysplastic Primordial Dwarfism Type II
(Central University of Punjab, 2018) Gautam, Saksham; Khetarpal,Preeti
Pericentrin (PCNT) is a main scaffold protein of Centrosome. It is encoded by PCNT gene which comprises of 47 exons and its cytogenetic location is 21q22.3. PCNT is a large protein containing 3336 amino acids. In PCNT protein two coiled-coil domains are bounded by a non-coiled region. Various mutations like non-sense, stop and deletion in PCNT are linked with human disorder Microcephalic Osteodysplastic Primordial Dwarfism Type II (MOPDII). The current project was carried out with an objective to analyze the coding region of PCNT gene among MOPDII patients. The DNA extracted from blood was amplified using locus specific primers for 30 exons of PCNT gene. Amplified PCR products were sequenced using chain termination method and obtained sequence contigs were then analyzed by comparing with reference sequence. After analyzing - exon sequence contigs in 3 subjects, 17 variants were identified. There is need to amplify remaining 17 exons of PCNT gene for the identification of novel mutation in subjects with MOPDII. Homozygous or compound heterozygous PCNT mutation could not be identified in our study in the PCNT coding region covered
Association of COX-2 (rs20417) polymorphism with aspirin resistance and adverse drug reactions (if any) in ischemic stroke patients in Malwa region of Punjab.
(Central University of Punjab, 2018) Kaur, Sukhvir; Munshi, Anjana
Ischemic stroke pathology involves the formation of a thrombus over an unstable atherosclerotic plaque through various inflammatory and clotting cascades. Aspirin is the oldest antiplatelet agent with well-established efficacy in stroke prevention. COX2 gene with rs20417 polymorphism has been found to be associated with ischemic stroke as well as aspirin resistance. We aimed to study the demographic profile of ischemic stroke patients from Malwa region of Punjab and check the frequency of rs20417 variant of COX 2 gene and correlate it with aspirin resistance and ADRs (if any) in ischemic stroke patients. We collected 30 samples from confirmed stroke patients from Malwa region of Punjab. DNA was isolated from blood and subjected to PCR and RFLP to evaluate rs20417 in COX 2 gene in the patients. Their mRS score was used to classify patients as aspirin responders or aspirin non-responders. 96.66% patients were aspirin responders and 3.33% were non-responders. Twenty-nine patients were carriers of CC genotype, of which 27 were responders and 2 were non-responders. Only 1 patient with CG genotype was a non-responder. A larger number of samples need to be screened for the COX-2 G765C polymorphism before coming to a conclusion. This preliminary study indicates that COX-2 G765C variant of COX-2 gene may not be a risk factor for aspirin resistance in ischemic stroke patients from Malwa region of Punjab
Psychological Burden Faced By Vitiligo Patients: A Comparative Study In Kerala And Punjab
(Central University of Punjab, 2018) G, Ameetha; Chander, Harish
Vitiligo is a common chronic skin disease having unknown aetiology, which causes a disfigurement and variable amount of skin and hair depigmentation and may affect a patient?s quality of life. To assess the psychological burden and epidemiologic profile of various age groups of patients affected by vitiligo in the southernmost district of the coastal state of Kerala and southern part of Punjab. All were investigated in a door- to- door survey. The questionnaire consisted of two sections, including psychological burden and their epidemiologic profile of various age groups. The questionnaire assigned contained questions about vitiligo characteristics such as the body surface area affected, skin tone, affected by genital vitiligo or not, marriage life and their sexual relationship, stigmatised conditions facing, whether or not affected areas were covered by dresses. 40% of males and 60 % of females were affected by vitiligo in Kerala but in Punjab males were more affected than females i.e. 60% and 40% respectively. The present study revealed that in Kerala and Punjab 25% of vitiligo patients were reported within the age group of 51-60 years and 20% within the age group of 21-30 years. More than 25% patients in Kerala and Punjab were fully affected with the vitiligo and the quality of life was impaired more in those patients and to a greater extent in women is more seen. About 76% of patients admitted of feeling self-conscious about their skin in Kerala whereas in Punjab 24% were affected.