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Home > Expression and Functional Analysis of Caveolar and Snare Proteins in Cancer Progression

Expression and Functional Analysis of Caveolar and Snare Proteins in Cancer Progression

Thesis Info

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Author

Raja, Sadaf Azad

Program

PhD

Institute

COMSATS University Islamabad

City

Islamabad

Province

Islamabad.

Country

Pakistan

Thesis Completing Year

2020

Thesis Completion Status

Completed

Subject

Bio sciences

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/12127/1/Sadaf%20Azad%20Raja%20biosci%202020%20comsats%20isb%20prr.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676726169220

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Membrane proteins are highly important players that help to maintain cellular integrity and control cell to cell interactions. One of the most intricate mechanisms of cell to cell communication is via vesicles formation and trafficking, carrying signaling proteins and molecules. Caveolar complex and SNARE (Soluble N-ethylmaleimidesensitive-factor attachment protein receptors) complex are among the membrane protein complexes that are functionally involved in vesicle trafficking and so are critical in cell to cell communication and development. Recent studies have observed an association of dysregulation of gene expression of CAV1and, DNM2 with cancer progression. Strategic components of SNARE machinery, Synaptobrevin2/Vesicle associated membrane protein-2 (v-SNARE) and Syntaxin 1A (t-SNARE) play a critical role in colon, lung and breast cancer progression and metastasis. In this study, we analyzed the relative expression of the STX1A, VAMP2, CAV1, and DNM2 for their possible association in the progression and metastasis of bladder cancer. The quantitative expression of the target genes was observed in human bladder cancer samples. The expression of STX1A, VAMP2, CAV1, and DNM2 were increased 5-fold, 2.9-fold, 8.88-folds and, 8.62-fold respectively. The overall expression of STX1A and VAMP2 found to be elevated significantly (P<0.0001) in high-grade tumors cells compared to normal and low-grade tumors. Similarly high-grade tumors had significantly higher expression of CAV1 (P<0.0001) and DNM2 (P<0.0001) compared to low-grade tumors. The correlation between the STX1A and VAMP2 was positive while between CAV1 and DNM2 the correlation was found strong positive. These data suggests that the increased expression of the key components of caveolar and SNARE complex might be responsible for the tumor grade and stage progression in bladder cancer. To further dissect the downstream signaling involved in vesicular trafficking we selected Cav-1 protein to provide an insight into its functional aspects. For this purpose we exploited a functional disruption imposed by a specific frame shift mutation at F160X resulting into truncated protein. Functional analyses of this Cav-1-mutant protein in MIA-PaCa-2, HT1376, HCT116, HT-29, and HCC827 showed that the Cav-1-mut cells have reduced growth, proliferation with improved mitochondrial respiration. The Cav-1-mut cells showed increased OCRs and reduced ECARs showing their reduced glycolytic behaviour compared to control cancer cells. The functional impact of this mutant Cav-1 might be the missing C-terminus including the important conserved phosphorylation site S168E, which is required for the translocation of the Cav-1 protein. As a structural component of caveolae, Cav-1 interacts with signaling molecules via a caveolin scaffolding domain (CSD) regulating cell signaling. Recent reports have shown that Cav-1 is a negative regulator in tumor metastasis. Therefore, we hypothesized that Cav-1 inhibits cell migration through its CSD. HeLa cells were engineered to overexpress Cav-1 (Cav-1 OE), Cav-1 without a functional CSD (∆CSD), or enhanced green fluorescent protein (EGFP) as a control. HeLa cell migration was suppressed in Cav-1 OE cells while ∆CSD showed increased migration. This altered migration corresponded to a decreased expression of zona occludens (ZO-1) with increased expression of vimentin. This shows that the CSD impacts the migratory phenotype in HeLa cells. Impact of Cav-1∆CSD on cellular migration was further confirmed in epithelial based colon cancer cell lines that have high (HCT116) and low (HT29) expression of this gene. To further elucidate the function of CSD we explored the JAK/STAT3 signaling account for the hypermigratory phenotype in the Cav-1∆CSD cells. Phosphorylated STAT3 was decreased in Cav-1 OE cells compared to control and ∆CSD cells; reducing STAT3 expression alone decreased cell migration. ∆CSD blunted HeLa proliferation by restricting the cells in G2/M phase of the cell cycle. Overexpressing the CSD peptide alone suppressed HeLa cell migration and inhibited phosphorylation of STAT3. These data suggests the importance of CSD as a negative regulator of STAT3 phosphorylation. Our functional investigations of Cav-1 specifically revealed that Cav-1 CSD may be critical in controlling the dynamic phenotype of cancer cells by facilitating the interactions of specific signal transduction pathways, regulating STAT3 and participating in a G2/M checkpoint. Modulating the CSD and targeting specific proteins may offer potential targets for novel therapies to control cancer metastasis.
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تعلیمی اداروں میں کھیلوں کی اہمیت

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تاریخ اقوام عالم پر نظر دوڑائیں تو کاروبار اور محنت و مشقت کے ساتھ ساتھ ایسے لمحات بھی نظر آتے ہیں جن میں تفریح اور کھیل کود کے سوا کچھ نہیں ہوتا، اپنے متعین کردہ اوقات میں وہ صرف اور صرف کھیل کود اورتفریحی پروگراموں سے لطف اندوز ہوتے ہیں۔ کھیل کود کا تصور جس طرح دیگر مذاہب میں موجود ہے اسی طرح وُہ اسلام میں بھی موجود ہے، محسنِ کائنات حضرت محمد صلی اللہ علیہ و آلہٖ وسلم نے بھی تیرا کی اور دوڑ کے مقابلوں میں حصہ لیا۔ کھیل اور تفر یحی پروگرام کردار پر بڑا اچھا اثر ڈالتے ہیں۔ یہی وجہ ہے کہ دیگر ممالک کی طرح پاکستان کے نظام تعلیم میں کھیل کا تصور کلیتاً موجود ہے۔ اوقاتِ تعلیم و تدریس میں با قاعدہ ڈرل کے نام سے کھیل کا ایک پیریڈ مقرر کر دیا جا تا ہے۔ جس میں طلباء کو فزیکل انسٹرکٹر مقررہ مشقیں کرواتا ہے۔ طلباء ذہنی آسودگی محسوس کرتے ہیں اور دن بھر کی رطب و یابس...

تشبہ کی حقیقت، اقسام اور احکام کا فقہاء کی آراء کی روشنی میں تحلیلی مطالعہ

Today, majority of the Muslims' lives have turned out to be colored with a similar shade of the western civilization in every sphere of life. We watch that the matter of imitation achieved its most exceedingly awful pinnacle. In this era of ecstasy, the imitation of infidels and penitence of the Sunnah is growing rapidly due to the fact that Muslims have made it their habit. A particular group considers it enlightenment and ideological freedom and feels ashamed to follow Islam and Sunnah. The main reason for this attitude is that Muslims are unaware of the disadvantages of the imitation of infidels. In this article, the nuisances and flaws of imitation have been discussed, so that the Muslims could know about its imperfections and follow the Qur’ān and Sunnah.

An Analysis of Relevance of B. Ed. Curriculum With the Learning in Class Ix & Class X on the Basis of Feedback from the Stakeholders

The purpose of this study was to analyze the relevance of existing B.Ed. (one year) curriculum of formal colleges of education and universities with the learning in class IX & class X by taking views from the principals and the SSTs (senior school teachers) who were taken as stakeholders and to provide a set of recommendations for improving it on the basis of their feedback. B.Ed. (one year) curriculum in Pakistan has continuously been under criticism. Recognizing the declining quality of education at secondary school level, the present study was conducted to analyze the relevance of existing B.Ed. (one year) curriculum with the learning in class IX & class X. The objectives of study were to: (a) get the opinions of the stakeholders on the existing B.Ed. (one year) curriculum, (b) critically analyze the expectations of the stakeholders about B.Ed. graduates, and (c) provide a set of recommendations for improving B.Ed. curriculum according to the expectations of the stakeholders. The population of the study comprised the principals and the SSTs of all secondary schools (2996 male and 1733 female=4729) situated in the province of Punjab. A sample of nine hundred was selected by using three-staged cluster sampling technique. At the first stage, nine districts, out of thirty six districts of Punjab, (three districts each from upper, central and lower Punjab) were randomly selected. At the second stage, twenty secondary schools (ten males & ten females) from each district (total 180) were randomly selected. At the third stage, four SSTs from each sample school were randomly selected and the principals of the selected schools were also taken for data collection. Thus the total sample size of the study was 900 (180 principals & 720 SSTs). Research instrument for both stakeholders was used to collect the required data. A pilot study was conducted on a small sample to confirm the validity of the research instrument before administering it on the study sample. Having collected data, the reliability of the research instrument was confirmed with the use of Cronbach’s Alpha on SPSS version 16 which established it at 0.844. Collected data was arranged, tabulated, analyzed (phase-wise and item-wise) by applying t-test, F-test and Chi-Square through SPSS 16 version and interpreted in the light of objectives of the study. The results concluded that the existing B.Ed. (one year) curriculum did not reflect representative relationship with the learning in class IX and class X. It did not proficiently prepare B.Ed. graduates according to the aspirations, needs and demands of the secondary schools and concerned society. It did not prepare them to work with others to expand their content knowledge, sharpen their instructional skills, and build up their capacity to use data for meaningful decision making. The practicum component did not appropriately create association between theoretical and practical work. It did not enable B.Ed. graduates to apply diverse methods for standard assessment, to create leadership and collaboration abilities, to understand the individual differences of students and to manage classroom procedure appropriately for better teaching learning processes. On the basis of results and feedback from the stakeholders, school-based B.Ed. curriculum having appropriate relationship to the learning need of class IX and class X was suggested to be undertaken by the concerned authorities. Extension in the duration of B.Ed. training program was suggested in order to come up to the expectations of society. Integration of teaching practice with course work at every stage was suggested to be undertaken by the supervisors in formal institutions of teacher training and by the teachers of class IX and class X. Additional assessment techniques were recommended to be incorporated in B.Ed. (one year) curriculum for practical training in the preparation, conduct and scoring of the tests to the B.Ed. graduates. Additional techniques to create leadership and collaboration ability, ability to understand learning differences and ability to create proper environment in the classroom were recommended to be incorporated in B.Ed. curriculum as these techniques might be enable B.Ed. graduates to appropriately collaborate with concerned persons and manage classroom procedure appropriately for improved teaching learning processes. New strategies i.e., concept formation, brainstorming, problem solving, concept attainment, etc. were recommended to be incorporated in B.Ed. curriculum for developing effective ways of teaching and evaluating the thinking and learning processes of the students in B.Ed. graduates. Contribution of the secondary school principals and senior school teachers in the revision of B.Ed. curriculum was recommended as they were fundamental source for making it relevant to the learning need in class IX & class X. These recommendations are expected to go a long way to improve the B.Ed. curriculum, to ensure the quality of teacher education at B.Ed. level and correspondingly at school level for raising the standards of teaching at secondary school level.