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Home > Expression of Micro-Rnas and Brcai and Brca2 in Breast Cancer Patients and Their Association With the Grade and Stage of Tumor

Expression of Micro-Rnas and Brcai and Brca2 in Breast Cancer Patients and Their Association With the Grade and Stage of Tumor

Thesis Info

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Author

Nadeem, Farkhunda

Program

PhD

Institute

Isra University

City

Hyderabad

Province

Sindh

Country

Pakistan

Thesis Completing Year

2016

Thesis Completion Status

Completed

Subject

History & geography

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/9757/1/Farkhunda_Nadeem_Pathology_Isra_2016.docx

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676724706277

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Breast cancer (BC) is the most frequently reported and diagnosed cancer worldwide and is one of the leading causes of mortality in women. Pakistan has the highest breast cancer incidence rate in Asia and majority of the patients present with advanced stage disease with poor survival. This situation accentuates towards the need of a safe and reliable method for early diagnosis of BC to improve the survival rate. MicroRNAs (miRNAs) are small non-coding RNAs. Abnormal expression of miRNA is a sign of various diseases, including cancer. They are remarkably stable in serum and other body fluids and can serve as reliable marker for early diagnosis of BC. The present study has included miRNA 497 and 195 which have not been studied in detail and correlated with clinico-pathological parameters in breast cancer till date. Hereditary breast cancer occurs in 5–10% of women, but those carrying BRCA1 and BRCA2 gene mutations have 40 to 84% life time high risk. Therefore, evaluation of BRCA genes mutations is helpful in predicting BC development on contralateral side and in siblings. The objectives of the present study were to assess the level of expression of miRNAs 497 and 195 in BC patients and to detect mutations in BRCA1 and BRCA 2 genes in BC patients. Additionally, to determine the association of grade and stage of BC with expression status of miRNAs. The samples and relative information of the patients were collected from various hospitals of Hyderabad and Karachi. A total of 209 females including 139 diagnosed BC cases and 70 controls matched for age were studied. Peripheral blood samples were used for extraction of total RNA. cDNA for miR-497 and miR-195 were prepared by using miR-497 antisense primer /Oligo(dT)18 Primer and miR-195 stem loop RT primers / gene specificantisense primer. GAPDH and U6 IT were used as control. The levels of expression of miRNA were recorded through amplification by qPCR. For detection of any mutation in BRCA 1 & 2 genes, DNA bands were identified by gel electrophoresis and visualized under Ultravioletilluminator. The ages of patients ranged from 25 to 65 years and majority of them belonged to 5th decade of life (71.22%). Only 15 % of the patients had history of BC in first degree relative. All of these patients (100%) presented with unilateral breast lump and out of these, 91.3% had breast lump larger than 5.0 cm. Axillary lymph node involvement was present in 64% of these patients. More than half of the patients (66.1%) were in grade II followed by 19.4% in grade I differentiation. The majority of BC patients (55.39 %) were in stage III. In total, out of 139 cases, 76.97% of BC patients and 8.57% of the controls showed downregulation of miRNA 497, while 72.66% of the BC patients and 15.71% of the controls showed downregulation of miRNA 195. Out of 89 patients who were axillary lymph node positive, 76.4% and 71.91% showed downregulation of miRNA 497 and 195 respectively. In contrast, out of the rest of 50 cases, who were axillary lymph nodes negative, 78% and 74%showed downregulation of miRNA 497 and 195 respectively. The results were highly significant at p < 0.001 in both groups. Results for correlation of both miRNAs with histological grade and clinical stage revealed that out of 27 cases of grade I, 92 cases of grade II and 20 cases of grade III BC, 55.55% (p=0.01), 85.86% (p=0.0001) and 65% (p=0.001) showed downregulation of miRNA 497 respectively, while 62.96% (p=0.001), 75% (p=0.0001) and 75% (p=0.001) revealed downregulation of miRNA 195 respectively. Almost similar results were found when correlation of miRNA 497 and 195 were done with clinical staging. It showed that, out of 21 cases of stage I, 39 cases of stage II, 77 cases of stage III and 2 cases of stage IV BC, 85.71%(p=0.01), 79.48%(p=0.001), 72.72%(p=0.001) and 100%(p=0.00001) of the cases showed downregulation of miRNA 497 respectively, whereas 80.95%(p=0.001), 64.1%(p=0.002), 74%(p=0.0001) and 100%(p=0.00001) of the cases showed downregulation of miRNA 195 in the above mentioned stages, respectively. The miRNAs 497 and 195 have been reported to be downregulated in BC. Few studies have been done regarding the level of expression of these miRNAs and their correlation with axillary lymph node involvement, grade and stage of BC. The results of the present study are highly significant and showed that levels of expression of both of these miRNAs are markedly downregulated in BC patients irrespective of lymph node involvement and grades and stages of tumor. All of these BC cases were also evaluated for selected mutations in BRCA 1 and BRCA 2 genes and the results showed the appearance of bands for wild-type alleles in all cases of BC. None of the case showed presence of bands for mutant alleles in any of the three selected founder mutations. In conclusion, the present study reports a significant association of miR-497- and miR-195 with histological grading, lymph node involvement and clinical staging. Both of these miRNAs are significantly down regulated in all grades and stages of BC, even in lymph node negative patients hence, they may be used asreliable, non-invasive biomarkers for the diagnosis and early detection of BC.
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راحتِؐ کون ومکاں سے ہر خوشی کی آبرو

راحتِ کون و مکاںؐ سے ہر خوشی کی آبرو
َ’’زندگی نے اُنؐ سے پائی زندگی کی آبرو‘‘

تاجدارِ دو جہاںؐ کے’’ فَقْرُ فَخْرِیْ‘‘ کے طفیل
ہے جہانِ رنگ و بُو میں سادگی کی آبرو

اتّباع رہبرِ جنّ و بشرؐ کے فیض سے
سوئے کعبہ ہر جبینِ بندگی کی آبرو

ہے شفیعِ المذنبیںؐ سے سارے عاصی آس مند
رب ھبلی کی دعا سے اُمّتی کی آبرو

ابنِ آدم کو عطا کی مسندِ انسانیت
فخرِ آدمؐ ہی کے دم سے آدمی کی آبرو

طائرِ حسنِ تخیل کی بھلا پرواز کیا
مدحتِ ممدوحِ رب سے شاعری کی آبرو

حبِّ محبوبِ خداؐ عرفانؔ کے دل کی طلب
آرزوئے دل یہی ہے دلبری کی آبرو

Creation of Pakistan and the Political Orientation of Bengali Muslims: 1940-47

, Officer Incharge/Senior Research Fellow, National Institute of Historical and Cultural Research, Centre of Excellence, Quaid-i-Azam University, Islamabad. Dr , Research Fellow, National Institute of Historical and Cultural Research, Centre of Excellence, Quaid-i-Azam University, Islamabad.

Soil Characterization of Rod Kohi Areas of D. I. Khan Division and Strategies for Integrated Soiland Water Resources Management for Sustainable Productivity

Rod kohi irrigation farming system is a peculiar system of farming being practiced in Piedmont plains of D.I.Khan division (NWFP) Pakistan covering almost 60% of cultivated area. The major constraint is the use of Rod kohi water which is unpredictable and highly variable in quantity and distribution both in time and space. Rod kohi soils are particularly confronted with problems of soil productivity, soil surface sealing, amount of irrigation water application and nutritional requirement of crops. In this regard, a series of experiments were conducted to understand the problems related to Rod kohi soils of D.I.Khan division and develop strategies for addressing them. The first three chapters deal with general introduction of Rod kohi area, review of literature and methodologies used in different studies. Chapter IV relates to the characterization of soils of Rod kohi area. Eighty seven soil samples from different mozas (villages) were collected to make inferences regarding the nature of the soils, salinity/ sodicity problems and productivity status. It was found that 50.57% of soils were medium textured while 45.98% samples were fine textured and only 3.45% soils were moderately coarse textured. All soils were alkaline in nature with pH ranging from 7.7 to 8.6. The ECe varied from 0.75 to 8.00 dS m-1 with a mean value of 3.156 dS m-1 and SAR was in the range of 3.00 to 12.78. Almost 74.71% soils were normal, 13.79% of soils saline while 5.75% soils were sodic and another 5.75% of samples were saline sodic. Similarly 74.71% of samples were strongly calcareous while 25.29% samples were moderately calcareous. Almost 83.53% samples were deficient in organic matter while 16.47% samples contained marginal amount of organic matter. All samples were deficient in mineral N, while 89.41% samples were deficient in P, however, 70.59% samples contained adequate amount of K. Agriculture in Rod kohi areas totally depends on hill torrent flow that is un-predictable in terms of timing, magnitude and frequency thus making scheduled irrigations impossible. Farmers in Rod kohi area usually over irrigate their fields which results wastage of water. Experiment regarding determining the optimum amount of water required for maximum production in Rod kohi areas has been discussed in Chapter V. Different quantities of Rod kohi water viz 15-25 cm, 25-35 cm, 35-45 cm, and 45-55 cm depth were applied to fields of variable textures i.e. silty clay loam, silt loam and loam, to see their effect on the yield of wheat, application efficiency (Ea), water use efficiency (WUE), moisture and bulk density (BD) of soils. It was revealed that grain yield of wheat increased with the increment of irrigation water in all types of soil and maximum grain yield was obtained after the application of 35-45 cm irrigation depth; beyond this the yield declined suggesting that 35-45 cm depth of irrigation is the optimum water requirement for wheat in Rod kohi areas of D.I.Khan. Amongst textural classes, the highest grain yield was achieved from loamy soil followed by silty loam and silty clay loam soil. The application efficiency of irrigation water differed significantly among different textured soils as well as depth of water applied and their interaction. It decreased as the depth of irrigation water increased. The maximum Ea was recorded with the application of 15-25 cm irrigation. Hence, it is suggested that water should be applied in such amount that is efficiently utilized and the crop yield is also not affected. The silty clay loam soil had the highest Ea (63.75%) while loamy soil has the lowest (42.24%) and silt loam was at intermediate position (50.43%). In water-deficient environments, crop productivity is determined by the amount of water available and water use efficiency (WUE) of crop. The WUE was determined on grain yield basis per mm water application. The results revealed that WUE was significantly influenced by the volume of water applied as well as soil texture and their interactions. It ranges from 5.917-13.627 kg ha-1 mm-1 being the minimum for 45-55 cm depth of irrigation and the maximum for 15-25 cm depth suggested that the water use efficiency (WUE) was inversely related to irrigation water volumes. It decreased linearly as irrigated water volumes increased. The loamy soils had higher (10.238 kg ha-1 mm-1) while silty clay loam soils had lower (8.914 kg ha-1 mm-1) WUE and intermediate values (9.244 kg ha-1 mm-1) of WUE were recorded for silt loam soils. The soil moisture content determines the success or failure of crop production in Rod kohi agriculture where yield of crop is significantly affected by the availability of moisture in the root zone during growing season. The soil moisture content was determined at three stages viz before flood, at the time of sowing and after harvest of crop at different sites having variable textured soils and amount of water applied. The silty clay loam soil retained highest moisture content followed by silt loam and loam. The highest soil moisture content was found at the time of sowing at all textured soils this caused the decreased at the time of harvest of the crop, while the lowest moisture content was recorded in samples collected before flood. The moisture content in the soils increased with the increase in irrigation depths. The bulk density (BD) of different textured soils which were applied different volumes of water was also determined at three stages i.e. before flood, at the time of sowing and after harvest of crop. It varied in different textured soils but the differences were not significant. The loamy soils had the highest BD, while silty clay loam soils gives the lowest but with no statistical difference. Soil surface sealing is a major problem in Rod kohi soils when rainfall occurs after sowing of seed and before germination. In this work, different textured soils viz silty clay loam, silt loam and loam were subjected to variable simulated rainfall intensities (5.0 mm, 10.0 mm and 15.0 mm) after sowing of the wheat seed. Different soil crust management practices i.e. hand hoeing, crop residue cover and application of FYM were applied to reduce crust development and increase seedling emergence and yield of wheat. The results of our observations are presented in chapter VI. It was inferred that the soils of Rod Kohi area are prone to crust development irrespective of texture and rainfall intensities which retarded seedlings emergence and consequently reduced grain yield of wheat. Soil crusting reduced 35.86 to 52.94% seedlings germination. The silty clay loam soil was more susceptible to crusting as compared to silty loam and loamy soil. Amongst rainfall intensities, the 5.0 mm rainfall intensity significantly reduced seedlings emergence at all textured soils, reflecting greater soil crust strength as compared to 10.0 mm and 15.0 mm rainfall. Different soil crust management practices improved seedlings emergence significantly at all types of soils. The significantly highest germination count was recorded in manual hoeing followed by crop residue (CR) and FYM. Similarly, the grain yield of wheat was significantly affected by different rainfall intensities and soil crust management practices in all soils. The significantly lowest grain yields were noted with 5.0 mm rainfall intensity at all the three locations, whereas the highest yield was found in 15.0 mm rainfall intensity. The significantly highest grain yield was achieved in loamy soil followed by silty loam soil while the lowest in silty clay loam soil. The results indicated that manual hoeing proved most effective in enhancing seedlings emergence and increasing the grain yield followed by CR and FYM. The combined data of three locations showed that increase in grain yield from FYM, CR and manual hoeing over control was 48.84%, 60.46% and 73.33% respectively. Studies pertaining to nutritional requirement of wheat under Rod Kohi conditions are reported in chapter VII. Field trials were conducted on silty clay loam and loamy soils for two consecutive years during 2006-07 and 2007-08. Fertilizers rates included NPK @ (i) 0-0-0, (ii) 20-0-0, (iii) 40-20-0, (iv) 40-20-10, (v) 60-30-20, (vi) 80-40-30 and (vii) 100-50-40 kg ha-1. Amongst different yield contributing parameters, plant height, number of productive tillers m-2, ear length and grain yield were significantly affected by application of different rates of NPK at both locations. All parameters increased with the increment of fertilizers rates. The maximum plant height (78.88 cm), number of productive tiller m-2 (378.40) and longest spikes (9.265 cm) were recorded in plots receiving NPK @ 100-50-40 kg ha-1 followed by NPK@ 80- 40-30 kg ha-1. Similarly the highest grain yield was achieved by the application of NPK @ 100-50-40 kg ha-1 closely followed by NPK@ 80-40-30 kg ha-1 and both were significantly similar. Computing the economic of fertilizers application revealed that the most economical dose of fertilizers for achieving maximum grain yield of wheat under Rod Kohi conditions of D.I.Khan is NPK @ 80-40-30 kg ha-1 it may be attributed to the better growing conditions and residual effects of NPK. In silty clay loam soil 8.33 to 27.91% increase in grain yield was noted due to fertilizers application while in loamy soil, the increase was 8.43 to 38.62%. All parameters had higher values at loamy soil against silty clay loam soil." xml:lang="en_US