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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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جو مہرباں تھے مرے اب وہ مہرباں نہ رہے

جو مہرباں تھے مرے اب وہ مہرباں نہ رہے
خفا کسی سے کبھی ایسے آسماں نہ رہے

جو وقت بدلا تو پھر لوگ بھی بدلنے لگے
جو راز داں تھے کبھی پھر وہ رازداں نہ رہے

ہر ایک شخص گیا ہٹ مقام سے اپنے
نہ پیر پیر رہے وہ جواں جواں نہ رہے

ستم ظریف نے کہہ کر یہ آگ لگوائی
کسی بھی طور مرا باقی آشیاں نہ رہے

وہ خوش نصیب ہے خوش بخت جس کا تائب جی
زمانہ لاکھ رہے یار بدگماں نہ رہے

EFFECT OF STATIC STRETCHING OF HAMSTRING ON NON-SPECIFIC LOW BACK PAIN

Objective: To determine the effect of static stretching of hamstring muscle on the non-specific low back pain. Methodology: A Quasi-Experimental study was conducted in Rabia Moon Institute of Neurology which total 30 participants were included through non-probability purposive sampling. Thirty participants were selected who fulfilled our inclusion criteria, they were divided into two groups; group A or treatment group received conventional physiotherapy treatment as well a static stretching exercise protocol for 5 days. Group B or control group received conventional physiotherapy treatment only. VAS (Visual analog scale) and Oswestry Disability questionnaire, SFGD (Standing Finger to ground Distance,) PSLR (passive straight leg raise) for both legs were measured pre- and post-treatment. Result: A total of 30 patients aged 20-55 were included in the study. Mean age of the participants was found to be 37.88 years.  The difference in means of all the assessment parameters pre and post-treatment for both groups were analyzed through paired t-test. There was a significant improvement in VAS, SFGD, Passive Straight leg Raise PSLR (right leg), PSLR (left leg) and level of disability pre- and post-treatment in the treatment group. Conclusion: This present study concluded that static stretching of hamstrings is effective in decreasing non-specific low back pain.

Pakistan-Us Security Relationship 1947-2006 Analysis of Areas of Convergence and Divergence

The US – Pakistan Security Relationship is almost as old as Pakistan itself. Its origin dates back to the initial days of partition, when a nascent, weak nation, born with a major birth defect “The Kashmir Issue”, looked for external help and support against a hegemonic neighbour. Since then, the US-Pakistan security relationship has been a roller – coaster ride of intimate interaction and a pariah state status during Pakistan’s 63 years existence. The world’s major military and economic power has maintained, what has been termed by many as “transactional” relationship, with its much smaller South Asian ally. During the height of the cold war struggle and the heydays of pacts and alliances, as US looked to form an anti - communist ring around the Soviet Union, Pakistan emerged as a staunch ally in the South – Asia region. It was called by many as the “Most Allied Ally” of the United States. In the ardent quest for gaining military and economic benefits from the US, it invited the wrath of the Soviet Union, hoping to use the US support in resolution of the long – standing dispute with India over Kashmir, and bolster its security against Indian military threat. The seismic changes brought about by the Indo – China War of 1962, radically altered this cozy equation for Pakistan. Its rapprochement with China became a major stumbling block in the security relationship and resulted in estrangement with the US. Pakistan’s military gamble in 1965 to settle the Kashmir dispute resulted in disappointment. Not only did the country fail to achieve anything tangible in Kashmir but the war also exposed all the inconsistencies and differences in the strategic perception of US and Pakistan, ultimately ending the security partnership. Though the alliances continued to exist on paper, yet they did not carry any real weight. President Nixon’s quest for rapprochement with China in 1969-1970 ironically brought Pakistan to the centre stage once again. Now the same issue on China, which had been a source of discord, revitalized the mutual relationship. While General Yahya acted as an intermediary in the US – China dialogues, the country drifted towards anarchy and breakup, owing to years of mismanagement and neglect towards the Eastern Wing. India’s invasion of East Pakistan, resulted in dismemberment of the country and a humiliating defeat, which owed much to the problems of successive regimes in West Pakistan. Yet the US response during crisis once again came under sharp criticism, as it was considered to be Pakistan’s patron and ally, and expected to support the country in the time of need. The thesis tries to identify and analyze the differing strategic perspectives during the two Indo-Pakistan Wars, as well as perceptions that resulted in disappointment and disenchantment in Pakistan. In the aftermath of the 1971 debacle, Prime Minister Bhutto looked towards nuclear deterrence as a security against Indian hegemony, because of failure of the security relationship with US to deliver at the critical time. This led to a new source of divergence in the security relationship, as the US non – proliferation goals clashed with Pakistan’s security imperatives. The Soviet invasion of Afghanistan led to a period of close security relationship, which was terminated abruptly by the US, in the wake of Soviet withdrawal from Afghanistan. This decision reinforced perceptions in Pakistan that US is an unreliable partner. The coming decade was that of Pakistan being put under pressure for its nuclear and missile programs. It also pursued an Afghan policy according to its own national interests that further estranged it from its closest ally, with every passing year. Pakistan’s support to the obscurantist Taliban regime resulted in gradual estrangement of the US Government. Even more sinister for Pakistan’s interests, was the nexus between Taliban and Al-Qaeeda. This was however glossed over by Pakistan, till the fateful events of September 11, 2001. The September 11 attack on the World Trade Center in 2001, was a defining moment in the security relationship, as President Musharraf was asked to make a tough policy decision. This time Pakistan was threatened with military action, if it decided to continue support for the Taliban. Pakistan’s decision to join the war on terrorism has resulted in a renewed security relationship with added economic and political benefits. Yet the tough decisions have resulted in internal discontent and unrest, which Pakistan is still grappling with. Now, as the Obama Administration looks towards an exit strategy from Afghanistan, Pakistan figures out as an essential component of any successful US disengagement. Will the security relationship last after the present partnership of war on terror, or wither away as the historical precedent indicates? What are the ideal building blocks of a stable, sustainable and mutually rewarding partnership? These are the questions which this thesis attempts to answer, in the light of the analysis of area of convergence and divergence over the last 63 years.