First pages:

التقييم الجزيئي لنسخ جين المقاومة الدوائية MDR1 في بعض المرضى العراقيين البالغين المصابين بسرطان ابيضاض الدم الحاد == Molecullar Assessment of Multidrug Resistance Gene (MDR1) Transcript In Some Adult Iraqi Patients With Acute Leukemia

Author name: كفاح جبار شاكر اليعقوبي

Supervisor name: عبد الحسين الفيصل

General topic: Biology

Specific topic: Biotechnologies

Degree: Doctorate

University: University of Baghdad - Institute Of Genetic Engineering And Biotechnology - Department Of Biotechnology

Language: English

University location: Baghdad

First pages:

Abstract: The present study aims to shed light on the follow up of acute leukemic (AL) patients at initial diagnosis and after treatment to assess the response and early relapse through evaluating the gene expression level of one of the major multidrug resistance genes which is the multidrug resistance 1 (MDR1) to investigate the possible association between level of MDR1 gene expression and the clinical outcomes and this may be considered as a potential marker for response to chemotherapy of acute leukemic patients. Furthermore, the current study correlates between the MDR1 gene phenotype and MDR1 genotype in three important coding regions (C1236T, G2677T/A, and C3435T considering the potential influence of altering MDR1 activity and its effect on therapy outcome as well as susceptibility to develop cancer.White blood cells (WBCs) isolated from 106 blood sample of acute leukemic patients were provided by Iraqi hospitals in Medical City. These samples were distributed as follows : 46 newly diagnosed patients with acute leukemia who had not received chemotherapy and follow - up 25 after 1st induction, 17 after 2nd induction and 8 at consolidation, with 10 blood samples of healthy voluntaries. Two comparative groups were taken included 46 sample of peripheral blood (PB) and 26 sample of bone marrow biopsy (BMB) in paraffin blocks to validate the level of gene expression compare to WBCs. For genotyping analysis, 56 of blood sample were taken to study genetic variation of MDR1 gene polymorphism. The samples preservation with TRIzol was done. Samples subjected to total RNA and DNA extraction, then molecular study by using reverse transcription, Quantitative Real Time - polymerase chain reaction (qRT - PCR) and direct sequencing, at Molecular Oncology Unit in Guy´s Hospital - Kings College / London.The study reached at the following results : 1 - The results showed age groups (20 - 39 years) were associated with acute myeloid leukemia (AML), while (13 - 19 years) with acute lymphoblastic leukemia (ALL).2 - The level of MDR1 gene expression showed high significant differences with WBCs compared to PB and BMB.3 - The clinical outcomes indicated that the rate of complete response (CR) of newly diagnosed acute leukemic patients was 19(41%), while 27(58.7%) was non - responder (NR).4 - Statistical analysis showed significant differences with NR at initial diagnosis in acute myeloid leukemia, while appeared after 1st induction in lymphoid type.5 - The results of positivity MDR1 gene expression were 10(21.7%) out of 46 newly diagnosed in acute leukemia, while 36(78.3%) were MDR1 - negative depend on (1.1±0.03) cutoff value.6 - The positivity MDR1 gene expression appeared mainly in non - responders patients at initial diagnosis, and with early relapse patients, after complete remission, in consolidation.7 - The MDR1 mRNA expression showed significant differences with high level in NR compared to CR patients at initial diagnosis. During treatment follow up the increased level of MDR1 gene expression in CR patients and appeared non - significantly with NR.8 - The results of MDR1 C1236T genotype and allele frequency showed that 1236CC wild type genotype and C allele were significantly frequent in healthy control. While CT heterozygous genotype frequency was highly significant in AML and no significant difference in allele frequency. ALL showed non - significant difference in genotype and allele frequency of MDR1 C1236T.9 - Odds ratio and 95% confidence interval (ORs and 95%CI) analysis showed no evidence associated with risk factor in MDR1 C1236T ALL carriers. While risk factor observed in AML with MDR1 1236CT carriers.10 - The results of MDR1 phenotype - genotype association indicate that MDR1 1236CC wild type was significantly high expression among healthy and it was aprotective genotype. While the MDR1 1236CT showed significant differences with high level of MDR1 gene expression in AML patients. Whereas ALL revealed significant differences in high level of MDR1 gene expression with MDR1 1236TT genotype. Both CT and TT were affected genotypes.11 - The results of MDR1 G2677T genotype and allele frequency indicated that 2677GA genotype significantly appeared with low frequency in healthy control with no significant difference in allele frequency. Both ALL and AML showed high significant frequency in 2677GT genotype. G allele frequency was showed significant differences in AML while non - significant in with ALL.12 - Odds ratio and 95% confidence interval (ORs and 95%CI) analysis showed the MDR1 2677GT genotype was associated with risk factor to developing ALL and AML. Whereas the GG appeared associated with AML only.13 - MDR1 phenotype - genotype association, indicate that MDR1 2677GA genotype was significantly high expression in healthy individual. While AML patients showed significant differences with high level of MDR1gene expression in 2677GT genotype. ALL showed significant differences with high level of MDR1 gene expression in MDR1 2677TT genotype.14 - The results of MDR1 C3435T genotype and allele frequency showed significant difference in genotype and allele frequency with heterozygous CT in both control and AML patients and mutant T allele. Whereas non - significant genotype and allele frequency with ALL.15 - Odds ratio and 95% confidence interval (ORs and 95%CI) analysis showed that the MDR1 3435CC genotype carriers associated with risk to developing ALL. While no risk factor associate with MDR1 C3435T variants to develop AML.16 - MDR1 phenotype - genotype association, indicate that the wild type 3435CC genotype was significantly high expression in healthy control. The MDR1 3453CT genotype showed high significance with high level of MDR1 gene expression inAML. While ALL showed significantly high level of MDR1 gene expression in 3435TT genotype.17 - The results of MDR1 genotype - phenotype association showed similar impact of MDR1C1236T, G2677T/A and C3435T genotypes in AML clinical outcomes. The MDR1 CT/GT/TT genotypes were associated in NR AML with high level of expression at presentation, compared to significant low level in CC/GG genotype. In contrast, CR patients were observed non - significant with MDR1 gene expression at presentation and significant with low MDR1CC/GG genotypes in post treatment. In regards to ALL patients the MDR1 TT genotype showed significant differences with high level of MDR1 gene expression in NR and CR ALL at presentation and significant only with NR at post treatment. So there was no clear evidence between MDR1 genotypes and clinical outcome with ALL.18 - The haplotype results showed that the three MDR1 C1236T, G2677T/A and C3435T genotype were linkage disequilibrium significantly with heterozygous haplotype B (CT - GT - CT) compared to A(CGC) and C(TTT). Also B haplotype appeared significantly with high level of MDR1 gene expression compared to A and C. According to the clinical outcome, haplotype B was observed significant differences in NR AML patients while other haplotypes were non - significant

دراسة جزيئية عن جين المقاومة mecA في بكتريا العنقودية الذهبية المقاومة للمشيسلين والمعزولة من بعض مستشفيات بغداد == Molecular Study For Detection of Meca Gene In Methicillin - Resistant Staphylococcus Aureus Isolated From Some Hospital In Baghdad City

Author name: لمى ياسين موسى

Supervisor name: محمد ابراهيم نادر

General topic: Biology

Specific topic: Biotechnologies

Degree: Master

University: University of Baghdad - Institute Of Genetic Engineering And Biotechnology - Department Of Biotechnology

Language: English

University location: Baghdad

First pages:

Abstract: Two hundred and thirty clinical specimens were collected from two different hospitals in Baghdad during the period (December 2012 to April 2013). These specimens were collected from visitors, hospitalized patients and the health care workers in these hospitals. The specimens included nasal swab, wound swab, burn swab, abscess and pus, sputum, ear swab, urine and blood culture diagnostic results show that 150 out of the (230) specimens gave positive bacterial cultures and (100) isolates are characterized as Staphylococcus aureus depending on the cultural and biochemical examinations.the coagulase test was performed and the results showed that from total 150 isolates of Staphylococci, 100 isolates (61%) were coagulase - positive (COPS), while only 50 isolates (39%) were coagulase negative (CONS). In addition, the distribution of methicillin resistance among Staphylococcus spp. was investigated.The use of the antibiotic susceptibility profile for these isolates was examined against methicillin resistance. Using disk diffusion method revealed that (13) isolates were proved to be methicillin resistant Staphylococcus aureus (MRSA), While (87) isolates of S. aureus showed sensitivity to methicillin (MSSA) and there was no intermediate resistance among these isolates.The ability of MRSA isolates to produce some virulence factors were investigated and the results showed that MRSA isolates produce many enzymes and toxins that contributed in their virulence such as protease, urease, dnase and gelatinase, and also produce a beta types of haemolysins.The ability to produce slime layer by MRSA isolates was also investigated and the results showed that all isolates of MRSA were produced slime layer when theytested by Congo red agar method and the results showed that all of MRSA isolates produced strong slime layer.One of the aims of this study was to adopted a accurate diagnostic method to detect S.aureus by its genetic material contents through extracting of DNA and gel electrophoresis of the PCR product for the specific gene.Detection of methicilline - resistance gene represented by A confirmatory test was carried out for the selected isolates using Polymerase chain reaction (PCR) technique for further characterization up to the species level by the amplification of (mecA) gene.This is Staphylococcus aureus specific gene that encodes the extra Penicillin Binding Protein, which is unique to methicillin - resistant staphylococci. All the (13) positive isolates by disk diffusion test are found to be positive for the presence of (mecA) gene as their agarose gel revealed the presence of DNA band of mecA gene with a molecular size about (200 bp.).Results of the detecting (femB) gene showed that it was positive in all of MRSA isolates as they appear to have a band with a molecular size of about (651 bp). The genetic determinants of methicillin resistance mecA and femB genes were amplified using multiplex PCR technique in order to identify methicillin resistant (mecA+) and susceptible (lacking mecA) staphylococci and to differentiate S. aureus (femB+) from coagulase negative staphylococci (lacking femB). All of the S. aureus isolates (100%) were found to harbor femA gene, it is species specific marker for S. aureus.

التحري عن فيروس التهاب الكبد نمط B باستخدام تقنيات ELISA وReal Time PCR في دم ولعاب المرضى في محافظة الانبار == Detection of Hepatitis B Virus By Using Elisa And Real Time PCR Techniques In Patients' Blood And Saliva In Al - Anbar Governorate

Author name: ثائر عبد الله حسن الدليمي

Supervisor name: امين سليمان بدوي الجنابي

General topic: Biology

Specific topic: Biotechnologies

Degree: Master

University: Tikrit University - Faculty Of Education - Department Of Biology

Language: Arabic

University location: Salahaddin

First pages:

Abstract: اجريت هذه الدراسة في مختبرات قسم علوم الحياة في كلية التربية وفي مختبرات مستشفى الرمادي التعليمي العام ومستشفى الرمادي للنسائية والاطفال ومختبرات النظائر المشعة في بغداد للفترة من 1/11/2011 ولغاية 1/5/2012 والتي تضمنت التحري عن فيروس التهاب الكبد نمط B في | The present study has been carried out at the laboratories of the department of biology at the college of Education and the laboratories of the public - teaching Al - Ramadi hospital and Al - Ramadi hospital for women and children and laboratories of Al - Nathaer Al - Mushe'ah in Baghdad for the period from 1/11/2011 to 1/5/2012. The study consisted of the investigation about Hepatitis B virus in the saliva as compared with serum by using the ELISA and Real Time PCR techniques as well as the detection about antigens and antibody to this virus in serum. It also seeks about the liver functions enzymes and evaluating the extent of its importance for diagnosing liver disease resulted that virus. However, the obtained results showed that HBsAg antigen was found in 99.4% and (1.101± 0.335) mean ±SD in serum as compared with 48.6% in saliva with a (0.330±0.278) mean ±SD, that gave a noticeable increase when comparing it with the control group which was (0.0039± 0.0024) and (0.0017±0.0014 ) for serum and saliva respectively. The value of T test for serum and control group was (16.25) , while the value of T test for saliva and control group was (4.18). The proportion of correlation between saliva and serum in ELISA test was equal to (0.361) with high significance of test while the value of T test for them was (28.902) with high significance of test. The results have also stated that the age groups involved in the experiment contributed as being high proportion of infection in the serum, but in the saliva, these proportions were different and it has been found that the high proportion of infection was 53.3% in the group (10 - 20) yrs., While the lowest proportion was 38.4% in the (31 - 40) yrs. Moreover, it has been concluded that the number of males exceeded the number of females, but the number of females was the most infected with HBsAg in the experiment group. However, the total number of males was 91, It was found that 90 males have positive test to the HBsAg in serum with a proportion equals 98.9% whiles 42 males have a positive test to the HBsAg in saliva with a proportion equals 46.1%. on contrary, the number of female was 84 female and all of them had a positive test to the HBsAg in serum with a proportion 100% and 43 females have a positive test to the HBsAg in the saliva with 51.1 % as compared with control group which included 15 males and 10 female. All of them were negative to the HBsAg test in serum and saliva... The levels of HBV DNA were determined in the samples of positive saliva to ELISA test by using Real Time PCR. 95% from tested serum samples were positive to the viral load While in saliva 50% from the samples was HBV DNA positive. The results also showed that soluble antigen HBeAg has been detected in 24 patients with 13.7% from a total 175 patient as compared with the control group that showed a negative result to this antigen. As for the antibodies, it has been found that anti - HBc IgM was found in only 5 patients with 2.5% while anti - HBc IgG was found in 170 patients with 97.2% from the total number of patients. The control group revealed negative result for these two antibodies. The anti - HBeAg was in 150 patients with 85.7% from the total number of patients. There were no positive patients to this antibody in control group. The anti - HBs wasn’t found in any patient but it was found in the half of control group. This emphasizes to immunize against HBV in those members. The results also exposed that there was a noticeable increase in liver enzymes that it was found that ALT the elevated levels of were found in 76% of patients. As compared with the control group which has normal values 100%. As for AST enzyme was elevated in 60.6%, As compared with the control group which was normal 100%.It has been observed that ALP enzyme was elevated with a 38.4% in the experiment patients as compared with the control group which was normal 100%. Finally, the total values of TSB was elevated in 35.5%, As compared with control group which was normal 100% with (0.52±0.19). Nevertheless, it has been noticed that it cannot be judged about the person's blood on the basis that it is healthy in case of absence of surface antigen unless it is certain that his/her blood is free from HBV DNA by using the technique of Real Time PCR - since many patients who heal from past infection or their infection in a time of passive and amounts of HBV DNA can't be diagnosed by using the ELISA technique

عزل وتشخيص الجين lipA المنتج من بكتريا الزائفة الزنجارية من مياه الصرف الصناعي == Isolation And Identification of lipA Gene Producing Pseudomonas Aeruginosa From Industrial Wastewater

Author name: انتصار طه لفتة

Supervisor name: واثق عباس الدراغي

General topic: Biology

Specific topic: Biotechnologies

Degree: Master

University: University of Baghdad

Language: Arabic

University location: Baghdad

First pages:

Abstract: توضح هذه الدراسة عزل lipA gene من الزوائف الزنجارية من مياه الصرف الصناعية للزيوت النباتية وتشخيصها اعتمادا على تفاعل البلمرة المتسلسل (PCR). استغرقت هذه الدراسة تسعة اشهر, من شهر شرين الاول 2014 لغاية حزيران 2015. تم جمع خمسين عينة من مياه الصرف الصناع | This study clarify the isolation and identification of lipA gene in Pseudomonas aeruginosa from industerial wastewater of vegetable oils factories depending on the polymerase chain reaction (PCR) technique. The present study had taken nine monthes starting from October 2014, till the mid of June 2015. Fifty samples of industrial wastewater were collected from the factories of the general company of vegetable oils, fourty from AL Rasheed factory and ten from AL ameen factory, the samples collected from the physical, chemical and biological treatment units, and other different wastewater tanks departments. While the four sewage samples were collected from sewer service Baghdad /Alrustumaiya. For Identification of Pseudomonas aeruginosa, two types of tests were used in this study. The first type was the routine tests, which include selective cultures, bacteriological and biochemical tests. Thirty four of fifty samples (68%) gave a positive growth and results for tests that were used to confirm the presence of P. aeruginosa. In addition to diagnosis P. aeruginosa in sewage sample which prepare the biological treatment units in vegetable oils factories as active sludge, other bacterial types have been diagnosed by using traditional methods and API 20 E system. For detection of bacterial lipolytic activity, two methods were used for this purpose, the first was the screening of bacterial lipolytic activity which was based on values of clear zones diameter around bacterial colony. The best values were between (1.9 _ 2.7) cm. The second test was carried by the Gas - Chromatography - Flame ionization detector, fatty acids solutions that were produced from hydrolysis by lipase enzyme were extracted by Petrolium ether solvent then analyzed by gas chromatograph apparatus. The second type of test was the Molecular diagnosis by using the Polymerase Chain Reaction (PCR) to detect lipA gene in Pseudomonas aeruginosa by two primers. Through two primers used, lipA 948 was the best and more specialized primer to isolation lipA gene of P. aeruginosa. It gave (100%) a positive result. While the second primer lipA 558, gave (66.66%) a positive result and that may be due to the design of this oligonucleotides was not specific for lipA of P.aeruginosa. This is so as was based on highly preserved region of 12 bacterial lipA - homologous genes for many genus and many species belong to Pseudomonas. DNA sequencing done for amplicon generated using lipA 948, this sequence aligned by using BLASTn software against NCBI database to validate the results and investigate the similarity degree with other corresponding strains.

Author name: استبرق اكرم بيرام الحسيني

Supervisor name: عصام فاضل علوان الجمیلي

General topic: Biology

Specific topic: Biotechnologies

Degree: Master

University: University of Baghdad - Institute Of Genetic Engineering And Biotechnology - Department Of Biotechnology

Language: English

University location: Baghdad

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Abstract: يعد مرض ابيضاض الدم النخاعي المزمن (CML) من الاورام النخاعية التكاثرية، وينشا نتيجة تكون جين Bcr - Abl (الذي يسمى كروموسوم فيلادلفيا) داخل الخلايا الجذعية المكونة للدم. يؤدي هذا الشذوذ الجيني الى تفعيل متواصل لانزيم التايروسين كاينيز وبالتالي نمو وتكاثر غ | Chronic myelogenous leukemia (CML) is a myeloproliferative neoplasm arises from Bcr - Abl gene translocation (called Ph chromosome) in hematopoietic stem cells (HSCs). This genetic abnormality results in constitutive activation of tyrosine kinase and subsequent uncontrol growth and multiplication of granulocytes. The cornerstone in treatment of CML are tyrosine kinase inhibitors, of which imatinib is the most effectively used. JAK2V617F mutation is an acquired single nucleotide polymorphism (SNP) occurs in JAK2 gene and is associated with many hematological malignancy other than CML. It was thought that the two genetic abnormalities (Bcr - Abl and JAK2V617F) occur mutually; however, growing body of evidences suggested the reverse. This study aimed to investigate the prevalence of JAK2V617F mutation associated with serum levels of alkaline phophatase (ALP) and lactate dehydrogenase (LDH) in Ph+ CML Iraqi patients treated with imatinib. A total of 43 Ph+ CML patients (25 males and 18 females, age range 16 - 80 years) who attend Iraqi National Center of Hematology for Research and Treatment/Baghdad were enrolled in this study. Each patient has been received at least six month therapy with imatinib. A consent form involving age, gender, height, weight, smoking status, residency and first family relative history of leukemia was obtained from each patient. Besides, blood samples were collected, from which the granulocytes were separated and then DNA was extracted using a ready kit. Two assays were used for detection of JAK2V617F mutation; real time polymerase chain reaction (qPCR) using specific primers and probe, and allele specific PCR (AS - PCR) using specific primers. Total white blood corpuscles (WBC) as well as serum levels of ALP and LDH were measured. qPCR assay revealed 5 patients out of 43 (11.62%) were heterozygous for the muatant allele of JAK2V617F mutation (genotype GT). The concentration of this allele ranged from 0.01% to 0.12%. None of blood sample gave positive result for AS - PCR assay. From the all risk factors, only gender had significant association with the incidence of JAK2V617F mutation. Average total WBC count, and serum levels of ALP and LDH were higher in JAK2V617F - positive patients (9042±1512.55, 146.05±8.028 IU/L and 204±10.85 IU/L respectively) than that of JAK2V617F - negative patients (6039±1772.239, 64.45±40.15 IU/L and 178.33±13.693 IU/L respectively) with significant differences. These results indicate that JAK2V617F mutation can occur simultaneously with Ph chromosome in CML patients, and qPCR is a highly sensitive method for the detection of this mutation. Furthermore, serum activity of ALP can be used as an indicator for the presence of JAK2V617F mutation in CML patients.