Kemik paget hastalığı ve frontotemporal demans ile gözlenen inklüzyon cisimcik miyopati hastalığıyla (IBMPFD) ilişkili bazı vcp mutant proteinlerin rekombinant DNA teknolojisi ile saflaştırılması ve in vitro bağlanma modellerinin incelenmesi

Inclusion body myopathy associated with Paget disease of the bone and frontotemporal dementia (IBMPFD) is a rare autosomal dominant multi system disease caused by mutations in p97/VCP gene. p97/VCP is a hexameric type II AAA ATPase that functions in the variety of cellular processes such as endoplasmic reticulum associated degradation (ERAD), organelle biogenesis, DNA repair, regulation of transcription factors, autophagy and cell-cycle regulation. In this thesis, we analyzed the effect of IBMPFD associated p97/VCP mutant proteins on its interaction with ERAD substrates and its partners. In addition, we investigated the solubility profile, subcellular localization and oxidative stress response of p97/VCP mutant proteins. For this purpose, we examined the effect of 12 IBMPFD associated p97/VCP mutations on ERAD substrates CFTR?F508 and Tirozinaz C89R. Interaction of p97/VCP mutants with p47, ubiquitin and gp78 were examined by immunoprecipitation. Solubility profiles and subcelluler localizations of these mutants were determined using immunoblotting. Susceptibility to oxidative stress were examined via their effect on cell proliferation using WST-1 reagent. The colocalization of wt VCP, R155C and P137L mutants with CD3?, Ufd1, gp78 and ubikitin were analyzed using immunofluorescence studies. Moreover interaction of purified recombinant R155C and P137L mutants with Ufd1 and gp78 VIM proteins were also analyzed in vitro. Our results revealed that while all mutants of p97/VCP proteins cause ERAD substrate acumulation, P137L differs from other IBMPFD mutants by having a unique solubility profile and subcellular localization. Interestingly, even though almost all mutants exhibits enhanced p47 and ubiquitin binding, the P137L mutation completely abolishes p97/VCP interactions with p47 and ubiquitin, while retaining its ability to interact with gp78. Consistently, our results show that recombinant R155C protein maintains interaction with Ufd1 and gp78 VIM. On the other hand P137L mutant protein lost its ability to interact with Ufd1 in contrast to gp78 VIM. The differential impairments in p97/VCP interactions with its functional partners and function may increase our understanding of the molecular pathogenesis of IBMPFD.Kemik Paget hastalığı ve frontotemporal demans ile gözlenen inklüzyon cisimcik miyopati hastalığı (IBMPFD), p97/VCP genindeki mutasyonların neden olduğu nadir rastlanan,otozomal domainant kalıtım gösteren bir multisistem bozukluğudur. p97/VCP, endoplazmik retikulum ilişkili yıkım (ERAD), organel biyogenezi, DNA tamiri, transkripsiyon faktörlerinin regülasyonu, otofaji ve hücre döngüsü kontrolü gibi çeşitli hücresel süreçlerde rol alan hekzamerik tip II AAA ATPaz?dır. Bu tez çalışmasında IBMPFD ilişkili p97/VCP mutant proteinlerinin ERAD substratları ve p97/VCP?nin partnerleri ile olan ilişkileri incelendi. Ayrıca p97/VCP mutant proteinlerinin çözünürlükleri, subsellüler lokalizasyonları ve oksidatif sterese yanıtları araştırıldı. Bu amaçla, IBMPFD ile ilişkilendirilen 12 p97/VCP mutantının ERAD substratı olan CFTR?F508 ve Tirozinaz C89R üzerindeki etkilerine bakıldı. p97/VCP mutantlarının p47, ubikitin ve gp78 ile olan etkileşimleri immünopresipitasyon yöntemi ile incelendi. Bu mutantların subsellüler lokalizasyonları ve çözünürlükleri immünoblotlama ile belirlendi. Oksidatif strese karşı olan duyarlılıkları hücre proliferasyonuna etkisi üzerinden WST-1 reaktifi kullanılarak incelendi. İmmünofluoresans mikroskopi yöntemi ile yabanıl tip, R155C ve P137L p97/VCP mutantlarının CD3?, Ufd1, gp78, ubikitin ile olan kolokalizasyonları incelendi. Ayrıca saflaştırılan rekombinant R155C ve P137L mutantlarının Ufd1 ve gp78 VIM ile olan etkileşimleri in vitro olarak incelendi. Sonuçlarımıza göre, tüm mutantlar ERAD substrat akümülasyonuna neden olurken, p97/VCP mutantı P137L diğer IBMPFD ilişkili mutantlardan subsellüler lokalizasyonu ve çözünürlüğü açısından farklılıklar göstermiştir. İlginç olarak, neredeyse tüm mutantlar p47 ve ubikitin bağlanmasını kuvvetlendirirken, P137L mutasyonu ubikitin ve p47 ile etkileşimini tamamiyle kaybetmişken, gp78 ile bağlanmasını korumuştur. Benzer şekilde rekombinant R155C mutant proteini Ufd1 ve gp78 VIM ile etkileşimini sürdürürken, P137L mutant proteini Ufd1 ile bağlanma özelliğini kaybetmesine rağmen VIM ile sürdürmüştür. p97/VCP?nin fonksiyonel partnerleri ile etkileşimleri ve fonksiyonundaki bozulmalar, IBMPFD?nin moleküler patogenezinin anlamamıza yardımcı olacaktı

Laparoscopic cholecystectomy results in patients with different age groups

Amaç Laparoskopik kolesistektomi benign safra kesesi hastalıklarında en çok tercih edilen metod olmuştur. İleri yaş ,mortalite ve morbiditenin artmasına neden olur. Bu çalışmanın amacı farklı yaşlardaki hastalarda laparoskopik kolesistektominin sonuçlarını karşılaştırmakır (yaş yaş). Gereç ve Yöntemler Kasım 2001-Mayıs 2009 tarihleri arasında OMÜ Tıp Fakültesi ve Mustafa Kemal Üniversitesi Tıp Fakültesinde benign safra kesesi hastalığı nedeniyle laparoskopik kolesistektomi uygulanan toplam 511 hasta retrospektif olarak analize edildi. Hastalar yaşlarına göre 3 gruba bölündü. Grup A (,n=96). Bulgular Tüm gruplarda laparoskopik kolesistektomi için en sık endikasyon semtomatik kolelitiazisdir. Ko-morbit hastalılar Grup C’de, Grup A ve B’ye göre anlamlı olarak daha yüksekti.İnsidental bilier patolojiler ve abdominal operasyon öyküsü tüm gruplarda benzer şekildeydi. Bununla birlikte bilier kanal ve sistik arter anomalileri Grup A’da,Grup B ve C’ ye göre anlamlı olarak yüksek bulundu. Bu çalışmada 26 hastada (%5.08) açık kolesistektomiye dönmek gerekti. Açığa dönüşün en büyük nedeni Callot üçgeni zor diseksiyonu idi( 21 hastada ;%80). Gruplar arasında morbidite açısından farklılık yoktu. Sonuç Sonuç olarak, genç hastalarda safra yolu anomalilerinin ve yaşlı hastalarda ko-morbid bozuklukların daha yaygın olduğu düşünülmelidir. Bu faktörler hastanın preoperatif iyi değerlendirilmesi sonucu etkilemez. Bununla birlikte cerrah bu durumun farkında olmalı ve intraoperatif ve postoperatif komplikasyonlar nedeniyle dikkatli olmalıdır.Aim Laparoscopic cholecystectomy (LC) has been most preferable method for benign gallbladder disease. Advanced age may be increased morbidity and mortality. The aim of this study was to compare the results of LC in patients according to different ages (age ≤ 30, 31 – 64 years, and age ≥ 65). Material and Methods A retrospective analysis was performed including overall 511 patients who underwent LC for benign disease of gallbladder at Ondokuzmayis University Medical Faculty and Mustafa Kemal University, Medical Faculty between November 2001 and November 2009. The patients are divided into three groups according to ages: Group A (age ≤ 30 years, n = 47), Group B (age = 31 – 64 years, n = 368), Group C (age ≥ 65, n = 96). Results Symptomatic cholelithiasis was the most common indication for LC in all the groups (p > 0.05). Co-morbid diseases were significantly higher in the Group C (≥ 65 years) than in the Group A and B (≤ 30 years, 31 - 64 years) (p=0.001). Co-incidental biliary pathologies, and history of abdominal operation were similar in all the groups. However biliary duct and cystic artery anomalies were significantly more common in the Group A than in the Group B and C (p=0.001). Conversion to OC was required in 26 (5.08 %) patients in this study. The major reason for the (21 cases, 80.76 %) was difficult dissection of the Calot’s triangle. There was no difference in morbidity among the groups (p>0.05). Conclusions As a conclusion, it’s thought that biliary anomalies in young patients and co-morbid diseases disorders in elderly patients are more common. These factors do not affect the results of patient that preoperatively well evaluated. However surgeon should be aware of this condition and be careful for intraoperative and postoperative complication

A unique IBMPFD-related P97/VCP mutation with differential binding pattern and subcellular localization

p97/VCP is a hexameric AAA type ATPase that functions in a variety of cellular processes such as endoplasmic reticulum associated degradation (ERAD), organelle biogenesis, autophagy and cell-cycle regulation. Inclusion body myopathy associated with Paget disease of the bone and frontotemporal dementia (IBMPFD) is an autosomal dominant disorder which has been attributed to mutations in p97/VCP. SeveraL missense mutations affecting twelve different amino acids have been identified in IBMPFD patients and some of them were suggested to be involved in the observed pathology. Here, we analyzed the effect of all twelve p97/VCP variants on ERAD substrates and their cofactor binding abilities. While all mutants cause ERAD substrate accumulation, P137L mutant p97/VCP differs from other IBMPFD mutants by having a unique solubility profile and subcellular localization. Intriguingly, although almost all mutants exhibit enhanced p47 and Ufd1-Npl4 binding, the P137L mutation completely abolishes p97/VCP interactions with Ufd1, Npl4 and p47, while retaining its gp78 binding. While recombinant R155C mutant protein consistently interacts with both Ufd1 and VIM of gp78, P137L mutant protein lost binding ability to Ufd1 but not to VIM in vitro. The differential impairments in p97/VCP interactions with its functional partners and function should help our understanding of the molecular pathogenesis of IBMPFD

The effects of agomelatine in cisplatin-induced toxicity on the kidney and liver tissues: In vivo study

Nephrotoxicity and hepatotoxicity are frequently seen adverse effects during cisplatin chemotherapy. In this study, we investigated the effects of agomelatine on cisplatin-induced toxicity in the kidney and liver. Animals were administered with a single dose of cisplatin (7 mg/kg, i.p.) and treated with agomelatine (20 and 40 mg/kg, p.o) for seven days. Renal and hepatic functions were evaluated by measuring concentrations of creatinine, BUN, AST and ALT in the serum. Oxidative stress and protein peroxidation were assessed by measuring SOD, CAT, GSH and AOPP levels in both tissues. Serum PON-1 levels were also evaluated. Histopathological analysis was performed to determined structural changes in the kidney and liver. Agomelatine (20 mg/kg) treatment approximately halved cisplatin-related increase in serum creatinine, BUN, AST and ALT levels. Agomelatine (20 mg/kg) significantly prevented the cisplatin-induced excessive decrease in SOD, CAT, GSH in both tissues and serum PON-1 levels. Agomelatine (20 and 40 mg/kg) protected the structural integrity of the kidney against cisplatin-insult. Although agomelatine (40 mg/kg) protected the kidney and showed parallel results with 20 mg/kg biochemically, it failed to show the same liver tissue effects in both analyses. Although agomelatine protected against cisplatin-induced toxicity in the kidney and liver, care should be taken with higher doses for possible hepatotoxicity