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Elmira Davasaz Tabrizi. Metagenomic studies in chronic prostatitis samples. Dissertation (2023) Skip to content Tez Künye Durumu Metagenomic studies in chronic prostatitis samples / Kronik prostatit örneklerinde metagenomik çalışmalar Yazar:ELMIRA DAVASAZ TABRIZI Danışman: PROF. DR. ERCAN ARICAN Yer Bilgisi: İstanbul Üniversitesi / Fen Bilimleri Enstitüsü / Moleküler Biyoloji ve Genetik Ana Bilim Dalı / Moleküler Biyoloji ve Genetik Bilim Dalı Konu:Biyoloji = Biology ; Moleküler Tıp = Molecular Medicine ; Üroloji = Urology Dizin: Onaylandı Doktora İngilizce 2023 121 s. Kronik prostatit/kronik pelvik ağrı sendromu (CP/CPPS) olarak da bilinen, kategori III prostatit olarak sınıflandırılan, birçok insanı etkileyen ve yaşam kalitesini önemli ölçüde düşüren bir kronik hastalıktır. Kronik prostatit en yaygın prostatit türü olarak tanınmaktadır. Semptomlar iyileşebilir ve daha sonra herhangi bir uyarı vermeden geri gelebilir. Nedeni halen net olarak bilinmemektedir. Günümüzde etkileyici bir tedavisi açıklanmamak ile beraber, Karmaşık bir etiyolojiye sahip olan multimodal tedavi, klinik olarak bu hastalık için tanına en iyi tedavi gibi görünmektedir. Bakteriyel prostatit ve idrar yolu enfeksiyonları ortak semptomları paylaştığından, altta yatan nedenin geleneksel toplumlarda ortaya çıkmayan “gizli” bir hastalık olduğuna uzun zamandır inanılmaktadır. Bu yanlış algılamanın bir sonucu olarak hastalara yanlış ve gereksiz dozlarda antibiyotik kullanılmaktadır. Kronik bakteriyel prostatit hastalığı durumunda bile bu enfeksiyona prostat bezindeki bakteriler sebep olmaktadır. Antibiyotiklerin tüm bakterileri öldürecek kadar prostat dokusuna yeterince nüfuz edememesi nedeniyle prostat enfeksiyonu geri gelebilmektedir. Prostat büyümesi veya kronik prostatit tedavi edilmezse prostat bezinizin büyümesi idrar yolunun tıkanmasına neden olmaktadır. Belirtiler daha şiddetli hale gelip ve zamanla tıbbi müdahale gerektiren, üretranın tamamen tıkanması gibi ciddi durumlar geliştire bilmektedir. Metagenomik, numunenin doğasına ve mikrobiyal varlığın bolluğuna bakılmaksızın mikrobiyal genomu doğrudan çevresel örneklerden alma tekniğidir. Mikrobiyal kompozisyon ve çeşitliliğin tespiti, yeni genler, antibiyotik direnç genleri ve etkileşimlerin belirlenmesinde faydalı bir metottur. Bu tez çalışmasında, kronik prostatitli (CP/CPPS) ve hasta olmayan bireylerdeki mikrobiyatanın tespiti ve bu hastalıkta rol alabilecek organizmaların moleküler düzeyde tespiti amaçlanmıştır. Bu araştırmada, kronik prostatit belirtileri olmayan 25 (29 yaş ve üzeri) ve kronik prostatit hastası olan 68 erkek (29-39, 39-49 ve 50 yaş ve üzeri) bireyden orta akıntı idrar örnekleri alınmıştır. Orta akıntıdan idrar örnekleri clean-catch yöntemiyle toplanmıştır. İdrar örneklerinden DNA izolasyonu yapıldıktan sonra 16S rDNA’nın tamamı PCR ile çoğaltılmıştır. Çoğaltılan 16S rDNA bölgelerinin dizi analizleri Nanopore teknolojisi MinION cihazıyla gerçekleştirilmiştir. Öncül biyoinformatik analizleri için Oxford Nanopore Technology (ONT) guppy 5.0.11 aracı kullanılmıştır. Elde edilen consensus dizilerin taksonomik açıda değerlendirmeleri ise NCBI ve Silva (SLV) biyoinformatik data bankaları aracılığıyla tespit edilmiş ve hasta ve kontrol dizilerin benzerliği Shapiro-Wilk-W testi ve Unweighted Pair Group Method Arithmetic (UPGMA) ağacı kullanılarak değerlendirilmiştir. Bakteri 16S rDNA dizilerin taksonomik ve fonksiyonel özelliklerinin açığa çıkartılması için BV-BRC (Bakteriyel ve Viral Biyoinformatik Kaynak Merkezi) kullanılmıştır. Kronik prostatitit vakalarının yaklaşık % 20’si yaşamları boyunca prostat kanserine yakalanma potansiyeli taşımaktadırlar. Bu çalışmada ek olarak, prostat kanser dizileri, ERR3840084 (S1 plazma mikrobiyomu) ve SRR15244457 (S2 Gut mikrobiyomu) SRR numarasıyla NCBI’den alındı ve bu prostat kanser örneklerinin dizileri, kronik prostatit örneklerinden elde edilen diziler ile karşılaştırılarak kapsamlı genom analizleri gerçekleştirildi ve Antibiotic Resistance Genes (ARGs) taşıyıcısı açısından kromozomal ve plazmid DNA olarak karşılaştırıldı. Yapılan deneyler ve analizlerden elde edilen sonuçlara göre; mikrobiyom çeşitliliği hasta numunelerinde kontrol numunelerinden farklı olarak tespit edilmiştir. Ayrıca UPGMA ağacına göre 29-39 yaş aralığındaki hasta bireylerin diğer hasta gruplarından daha fazla mikrobiyom çeşitliliğine sahip olduğu bulunmuştur. Buna ek olarak, mikrobiyom çeşitliliği hasta gruplarında sağlam bireylere göre daha fazla olduğu tespit edilmiştir. Bu çalışmada elde edilen sonuçlar, mikrobiyom dengesizlikleri, bakteri popülasyonların proteaz aktiviteleri, kronik prostatit hastalığın muhtemel sebepleri ve olası tedavi süreçlerinin geliştirilmesinde ışık tutacak niteliktedir. Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), also known as category III prostatitis, is a chronic disease that affects many people and significantly reduces their quality of life. Chronic prostatitis is recognized as the most common type of prostatitis. Symptoms may get better and then return without warning. The reason is still not clearly known. Although no effective treatment has been announced till today, multimodal treatment seems to be the best clinically diagnosed treatment for this disease, which has a complex etiology. Because bacterial prostatitis and urinary tract infections share common symptoms, it has long been believed that the underlying cause is a “hidden” disease that does not manifest in traditional societies. As a result of this misperception, patients are given incorrect and unnecessary doses of antibiotics. Even in the case of chronic bacterial prostatitis, this infection is caused by bacteria in the prostate gland. The prostate infection may return because the antibiotics do not penetrate the prostate tissue sufficiently to kill all the bacteria. If prostate enlargement or chronic prostatitis is not treated, the enlargement of the prostate gland causes obstruction of the urinary tract. The prostate infection may return because the antibiotics couldn’t penetrate the prostate tissue sufficiently to kill all the bacteria. If prostate enlargement or chronic prostatitis aren’t treated, the enlargement of the prostate gland causes obstruction of the urinary tract. Symptoms may become more severe, and, over time, serious conditions may develop, such as complete obstruction of the urethra, requiring medical intervention. Metagenomics is the technique of obtaining the microbial genome directly from environmental samples, regardless of the nature of the sample or the abundance of microbial presence. The determination of microbial composition and diversity is a useful method for identifying new genes, antibiotic resistance genes, and interactions. In this study, it was aimed to detect the microbiota in individuals with and without chronic prostatitis (CP/CPPS) and to identify the organisms that may play a role in this disease at the molecular level. Midstream urine samples were collected from 25 men (29 years and older) without symptoms of chronic prostatitis and 68 men (29–39, 39–49, and 50 years and older) with chronic prostatitis. Urine samples from midstream were collected by the clean-catch method. After DNA isolation from urine samples, the entire 16S rDNA was amplified by PCR. Sequence analysis of the replicated 16S rDNA regions was performed with the Nanopore Technology MinION device. The Oxford Nanopore Technology (ONT) Guppy 5.0.11 tool was used for preliminary bioinformatics analyses. Taxonomic evaluations of the obtained consensus sequences were determined through NCBI and Silva (SLV) bioinformatics data banks, and the similarity of patient and control sequences was evaluated using the Shapiro-Wilk-W test and Unweighted Pair Group Method Arithmetic (UPGMA) tree. BV-BRC (Bacterial and Viral Bioinformatics Resource Center) was used to reveal the taxonomic and functional properties of bacterial 16S rDNA sequences. Approximately 20% of chronic prostatitis cases have the potential to develop prostate cancer during their lifetime. In addition, in this study, prostate cancer sequences were obtained from NCBI with SRR numbers ERR3840084 (S1 plasma microbiome) and SRR15244457 (S2 Gut microbiome), and comprehensive genome analyzes were performed by comparing the sequences of these prostate cancer samples with the sequences obtained from chronic prostatitis samples. Chromosomal and plasmid DNA were compared in terms of Antibiotic resistance genes (ARGs) carrier. According to the results obtained from the experiments and analysis; Microbiome diversity was detected differently in patient samples than control ones. Additionally, according to the UPGMA tree, sick individuals between the ages of 29 and 39 were found to have more microbiome diversity than other patient groups. In addition, microbiome diversity has been found to be greater in patient groups than in healthy individuals. The results obtained in this study will shed light on microbiome imbalances, protease activities of bacterial populations, possible causes of chronic prostatitis, and the development of possible treatment processes.
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