Search

Introduction Childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) can be traced back to birth using leukemic clone-specific immunoglobulin heavy chain (IGH) rearrangements, implying prenatal origin of this disease. Methods We retrospectively analyzed neonatal blood spots (Guthrie cards) of 24 patients with childhood BCP-ALL aged 1–9.6 years (median 3.1 years) for the presence of clonotypic IGH rearrangements identified in diagnostic bone marrow samples. Based on the sequences of IGH rearrangements, 2 patient-specific primers were designed for each patient and used in semi-nested polymerase chain reaction for the detection of preleukemic clones at birth. Results Clonotypic IGH rearrangements were detected in neonatal blood spots of 54.2% of patients (13/24). In two cases with double IGH rearrangements detected at diagnosis, only one rearrangement was present at birth, while in the third case both leukemic rearrangements were detected in neonatal blood. Guthrie card-positive findings were significantly more frequent in children ≤5 years of age than in older children (p = 0.011). Regarding patients' characteristics at birth and at diagnosis, Guthrie card-positivity was not associated with sex, birth weight and mother's age, as well as with white blood cell count, percentage of bone marrow blasts, immunophenotype and the presence of ETV6/RUNX1 and TCF3/PBX1 fusion genes at diagnosis. Conclusion Our study confirms that a large proportion of childhood BCP-ALL originates in utero, regardless of the molecular subtype defined by chromosomal aberrations. The observed trend toward younger age at diagnosis in Guthrie card-positive versus Guthrie card-negative patients implies that the age at diagnosis depends on the presence of preleukemic clone at birth, as well as on the timing of postnatal transforming genetic events.

Background: The long non-coding RNA (lncRNA) MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) dysregulated expression and prognostic significance have been reported in a variety of cancers, including hematological malignancies, but have been poorly investigated in chronic lymphocytic leukemia (CLL). Acting through regulation of gene expression at transcriptional and post-transcriptional level, lncRNA MALAT1 is involved in many cellular processes such as proliferation, apoptosis, migration and drug resistance. However, its role as either an oncogene or a tumor-supressor is still controversial, and clearly tumor type-dependent. Aims: To analyze the expression pattern of lncRNA MALAT1 in CLL, and evaluate its prognostic relevance. Methods: This study enrolled 114 unselected CLL patients (pts) and 20 healthy controls (hcs). Clinical and laboratory characteristics of pts were determined at diagnosis, while genetic analyses were performed during the period prior to first treatment. The expression of MALAT1 was analyzed in peripheral blood mononuclear cells by RQ-PCR, using TaqMan chemistry and GAPDH as endogenous control; relative quantification was made by comparative ddCt method, using hcs as calibrator. Results: CLL cohort consisted of 81 males and 33 females (male/female=2.45), with median age at diagnosis of 59 years (range 33-80). Hcs group consisted of 15 males and 5 females (male/female=3), with median age at diagnosis of 71 years (range 65-85). Distribution of Binet stages (112/114 pts) was as follows: A-46.4%, B-39.3%, C-14.3%. Del13q, normal karyotype, trisomy12, del11q and del17p were detected by FISH in 33%, 35%, 9.3%, 10.3% and 12.4% of pts, respectively (97/114 pts). CD38 status (85/114 pts) was negative in 70.6% and positive in 29.4% of pts. Regarding IGHV mutational status (114 pts), 41.2% of pts were mutated, and 58.8% unmutated. Median follow-up was 72 months (range 1-360). LncRNA MALAT1 was overexpressed in CLL pts compared to hcs (p<0.0

Introduction: Childhood B-cell precursor acute lymphoblastic leukemic (BCP-ALL) can be traced back to birth using leukemic clone-specific immunoglobulin heavy chain (IGH) rearrangements, implying prenatal origin of this disease. The aim of this study was to analyze neonatal blood spots (Guthrie cards) of childhood BCP-ALL patients for the presence of clonotypic IGH rearrangements. Methods: The study enrolled 24 patients aged 1 to 9.6 years. Based on the sequences of IGH rearrangements identified in diagnostic lymphoblasts, 2 patient-specific primers were designed for each patient and used in semi-nested PCR for the detection of preleukemic clones at birth. Results: Clonotypic IGH rearrangements were detected in neonatal blood spots of 54.2% of patients. In two cases that had double IGH rearrangements at diagnosis, only one rearrangement was present at birth, while in the third case both leukemic rearrangements were detected in neonatal blood. Guthrie card-positive findings were significantly more frequent in children ≤5 years of age than in older children (p=0.011). Regarding patients’characteristics at birth and at diagnosis, Guthrie card-positivity was not associated with sex, birth weight and mother’s age, as well as with white blood cell count, percentage of bone marrow blasts, immunophenotype and the presence of ETV6/RUNX1 and TCF3/PBX1 fusion genes at diagnosis. Conclusion: Our study confirms that a large proportion of childhood BCP-ALL originates in utero, regardless of the molecularsubtype defined by chromosomal aberrations. The latency period to the overt leukemia depends on the presence of preleukemic clone at birth, as well as on the postnatal transforming genetic events.

Introduction The B-cell lymphoma/leukaemia 11A (BCL11A) gene encodes a Kruppel-like transcription factor involved in lymphocyte development during normal haematopoiesis. Aberrant expression of BCL11A has been observed in several haematological malignancies, including chronic lymphocytic leukaemia (CLL). However, its functions in the regulatory networks of malignant B lymphocytes are poorly understood, as are the relations to clinical course and outcome of B-cell malignancies, particularly CLL. Methods The expression of BCL11A was analysed in peripheral blood mononuclear cells of 87 newly-diagnosed CLL patients by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), and association with clinical and molecular variables was assessed. Results BCL11A was significantly overexpressed in CLL samples compared to control samples (p lt 0.001). BCL11A expression level exhibited no association with age, sex, leukocyte, lymphocyte and platelet counts, haemoglobin level, serum beta 2-microglobulin, CD38 status and cytogenetic abnormalities. On the other hand, high BCL11A expression was associated with low serum lactate dehydrogenase (p = 0.031), Binet A stage (p = 0.047) and mutated IGHV (p = 0.028). In addition, a positive correlation with BCL2/BAX mRNA ratio was observed (r = 0.36; p lt 0.001). Regarding the association with the time to first treatment (TTFT), a trend towards longer median TTFT in BCL11A high- versus BCL11A low-expressing cases was detected (21 vs. 6 months; p = 0.164). Conclusion The results of this study show that BCL11A is upregulated in CLL patients, and that high BCL11A expression at diagnosis may be associated with better prognosis. These data are consistent with the role of BCL11A expression in CLL biology, and imply its potential prognostic relevance.

Uvod/Cilj Prognoza hronične limfocitne leukemije (HLL) značajno je unapređena u poslednje vreme. Među nekoliko prognostičkih modela čiji je cilj predviđanje vremena do prve terapije (eng. TTFT) izdvajaju se skor rizika od progresije (eng. PRS) i skor Centra za rak MD Anderson iz 2011. God. (eng. MDACC 2011), dok se internacionalni prognostički indeks za HLL (eng. CLL-IPI), iako primarno ustanovljen za predikciju ukupnog preživljavanja (eng. OS), dobro pokazao i u predikciji TTFT. Cilj ovog rada je da se ispita značaj pomenutih prognostičkih modela u pogledu predviđanja TTFT i OS. Metode Analizirana kohorta je obuhvatila 57 neselektovanih bolesnika sa HLL Univerzitetskog kliničkog centra Srbije sa prosečno agresivnijim profilom bolesti u odnosu na opštu populaciju de novo bolesnika sa HLL. Bolesnici su ocenjivani prema navedenim skorovima uz analizu TTFT i OS. Rezultati Bolesnici sa višim vrednostima CLL-IPI, PRS i MDACC 2011 primili su prvu terapiju značajno ranije u poređenju sa bolesnicima sa nižim vrednostima ovih skorova (p = 0,002, p = 0,019 i p lt 0,001, redom). U multivarijantnoj analizi, MDACC 2011 i CLL-IPI su zadržali prognostički značaj u predikciji TTFT (p = 0,001, odnosno p = 0,018), dok je PRS ovaj značaj izgubio. CLL-IPI je bio jedini značajan prediktor OS u univarijantnoj (p = 0,005) i u multivarijantnoj analizi (p = 0,013). Zaključak CLL-IPI, PRS i naročito MDACC 2011 su dobri prediktori TTFT čak i u kohortama bolesnika sa agresivnijom bolešću, dok je za predikciju OS od ova tri prognostička modela CLL-IPI jedini primenljiv. Ovi rezultati pokazuju da bi prognostičke modele trebalo ispitati na bolesnicima sa HLL u različitim fazama bolesti, kakvi se sreću u realnoj kliničkoj praksi., Introduction/Objective Prognostication of chronic lymphocytic leukemia (CLL) has been substantially improved in recent times. Among several prognostic models (PMs) focused on the prediction of time to first treatment (TTFT), progression-risk score (PRS), and MD Anderson Cancer Center score 2011 (MDACC 2011) are the most relevant, while CLL-International Prognostic Index (CLL-IPI), although originally developed to predict overall survival (OS), is also being used to estimate TTFT. The aim of this study was to investigate CLL-IPI, PRS, and MDACC 2011 prognostic values regarding TTFT and OS. Methods The analyzed cohort included 57 unselected Serbian CLL patients from a single institution, with the basic characteristics reflecting more aggressive disease than in the general de novo CLL population. The eligible patients were assigned investigated PMs, and TTFT and OS analyses were performed. Results Patients with higher risk scores according to CLL-IPI, PRS, and MDACC 2011 underwent treatment significantly earlier than patients with lower risk scores (p = 0.002, p = 0.019, and p lt 0.001, respectively). In multivariate analysis, MDACC 2011 and CLL-IPI retained their significance regarding TTFT (p = 0.001 and p = 0.018, respectively), while PRS did not. CLL-IPI was the only significant predictor of OS both at the univariate (p = 0.005) and multivariate (p = 0.013) levels. Conclusion CLL-IPI, PRS, and particularly MDACC 2011 are able to predict TTFT even in cohorts with more advanced-disease patients, while for prediction of OS, CLL-IPI is the only applicable among the three PMs. These results imply that PMs should be investigated in more diverse CLL populations, as it is in real-life setting.

Introduction Acute myeloid leukemia with normal karyotype (AML-NK) is the largest group of AML patients with very heterogeneous disease outcome. In order to ensure more precise risk stratification new molecular markers have been introduced, like expression level for BAALC (Brain and Acute Leukemia, Cytoplasmic) and MN1 (Meningioma 1) genes. Methods In this study, we investigated expression level of both genes in 111 adult AML-NK at diagnosis and examined their prognostic potential. Results BAALC and MN1 expression were detected in about one third of the patients, and positive correlation between these two genes was found. The BAALC(+)/or MN1(+) status was not associated with the presence of FLT3-ITD mutations, but exhibited strong correlation with NPM1(wt) status (P lt .001). Therefore, among BAALC(+)/or MN1(+) patients the most frequent ones were FLT3-ITD-/NPM1(-) double negative patients with intermediate prognosis. When BAALC(+)/or MN1(+) patients were divided into BAALC(high)/BAALC(low) (21/21) and MN1(high)/MN1(low) (21/22) groups, we detected that BAALC(high)/or MN1(high) patients had a tendency toward lower complete remission rate. Also, survival analysis showed that BAALC(high)/or MN1(high) patients had shorter disease-free survival and overall survival (OS). The most pronounced influence on prognosis was detected in FLT3-ITD-/NPM1(-) group of patients that are lacking reliable prognostic markers, where OS in BAALC(high)/or MN1(high) was only 5 months vs 25 months in BAALC(low)/or MN1(low). Conclusion These findings indicate that BAALC and MN1 expression level could be used for more precise risk stratification of AML-NK patients and especially FLT3-ITD-/NPM1(-) patients, transforming this intermediate-risk group, into a group with an adverse prognosis.

Fludarabine plus cyclophosphamide (FC) chemotherapy is the basis of treatment protocols used in management of chronic lymphocytic leukemia (CLL). In some patients, response to therapy may be affected by aberrant function of genes involved in pharmacokinetics and pharmacodynamics of the drugs. The aim of this research was to assess the impact of pharmacogenetic variability, namely expression of SLC28A3 gene and the presence of CYP2B6*6 variant allele, on the FC treatment efficacy. Forty-four CLL patients with functional TP53 gene at the time of FC initiation were enrolled in this study. CYP2B6 genotyping was performed by polymerase chain reaction and direct sequencing. SLC28A3 expression was measured by quantitative reverse-transcriptase polymerase chain reaction. Significantly higher pretreatment levels of SLC28A3 mRNA were detected in patients who failed to respond to FC in comparison to patients who achieved complete and partial response (p = 0.01). SLC28A3 high-expressing cases were almost ten times more likely not to respond to FC than low-expressing cases (OR = 9.8; p = 0.046). However, association of SLC28A3 expression with progression-free survival (PFS) and overall survival (OS) was not observed. CYP2B6*6 allele, detected in 24 patients (54.6%), exerted no association with the attainment of response to FC, as well as with PFS and OS. The results of this study demonstrate that SLC28A3 expression is a significant predictor of FC efficacy in CLL patients with intact TP53. Elevated SLC28A3 mRNA levels are associated with inferior short-term response to FC, suggesting that, if validated on larger cohorts, SLC28A3 expression may become a biomarker useful for pretreatment stratification of patients.

According to current criteria, patients with acute myeloid leukemia with normal karyotype (AML-NK) are classified as intermediate risk patients. There is a constant need for additional molecular markers that will help in substratification into more precise prognostic groups. One of the potential new markers is Ecotropic viral integration 1 site (EVI1) transcriptional factor, whose expression is dissregulated in abnormal hematopoietic process. The purpose of this study was to examine EVI1 gene expression in 104 adult AML-NK patients and on 10 healthy bone marrow donors using real-time polymerase chain reaction method, and to evaluate association between EVI1 expression level and other molecular and clinical features, and to examine its potential influence on the prognosis of the disease. Overexpression of EVI1 gene (EVI1(+) status) was present in 17% of patients. Increased EVI1 expression was predominantly found in patients with lower WBC count (P = 0.003) and lower bone marrow blast percentage (P = 0.005). EVI1(+) patients had lower WT1 expression level (P = 0.041), and were negative for FLT3-ITD and NPM1 mutations (P = 0.036 and P = 0.003). Patients with EVI1(+) status had higher complete remission rate (P = 0.047), but EVI1 expression didn't influence overall and disease free survival. EVI1 expression status alone, cannot be used as a new marker for more precise substratification of AML-NK patients. Further investigations conducted on larger number of patients may indicate how EVI1 expression could influence the prognosis and outcome of AML-NK patients, by itself, or in the context of other molecular and clinical parameters.

lt b gt Uvod: lt /b gt Akutna limfoblastna leukemija (ALL) je najčešće maligno oboljenje kod dece, dok je kod odraslih njena učestalost mnogo niža. U današnjoj kliničkoj praksi kao najvažnije metode stratifikacije pacijenata u određene grupe rizika koriste se metode identifikacije citogenetičkih aberacija i malog broja molekulanih markera. Tehnologija sekvenciranja nove generacije (SNG) obezbeđuje veliku količinu podataka koji doprinose razjašnjavanju mutacionog profila dečje (dALL) i adultne ALL (aALL). lt b gt Metode: lt /b gt Uzorci DNK iz 34 dALL i aALL pacijenata analizirani su primenom SNG ciljanog sekvenciranja ("TruSeq Amplicon Cancer Panel - TSACP") kojim se sekvenciraju "hotspot" mutacije u 48 gena povezanih sa kancerom. lt b gt Rezultati: lt /b gt Identifikovano je ukupno 330 varijanti u kodirajućim regionima, od kojih je samo 95 njih za posledicu imalo potencijalnu promenu u proteinu. Posmatrano kod pojedinačnih pacijenata, detektovane mutacije su pretežno remetile Ras/RTK signalni put (STK11, KIT, MET, NRAS, KRAS, PTEN). Pored toga, identifikovano je 5 pacijenata sa istom mutacijom u HNF1A genu, koja je uzrokovala poremećaje u Wnt i Notch signalnom putu. Kod dva pa cijenta otkrivene su varijante u NOTCH1 genu. Nije detektovano istovremeno prisustvo varijanti u HNF1A i NOTCH1 genu, dok su geni uključeni u Ras/RTK signalni put pokazali tendenciju ka akumuliranju mutacija. lt b gt Zaključak: lt /b gt Naši rezultati pokazuju da ALL sadrži Mali broj mutacija, bez značajnih razlika između dALL i aALL (medijana po pacijentu 2 odnosno 3). Detektovane mutacije izazivaju poremećaje u nekoliko ključnih signalnih puteva, prvenstveno Ras/RTK kaskade. Ova studija doprinosi ukupnom znanju o mutacionom profilu ALL, što vodi ka boljem razumijevanju molekularne osnove ovog oboljenja., lt b gt Background: lt /b gt Acute lymphoblastic leukemia (ALL) is the most common cancer in children, whereas it is less common in adults. Identification of cytogenetic aberrations and a small number of molecular abnormalities are still the most important risk and therapy stratification methods in clinical practice today. Next generation sequencing (NGS) technology provides a large amount of data contributing to elucidation of mutational landscape of childhood (cALL) and adult ALL (aALL). lt b gt Methods: lt /b gt We analyzed DNA samples from 34 cALL and aALL patients, using NGS targeted sequencing TruSeq Amplicon - Cancer Panel (TSACP) which targets mutational hotspots in 48 cancer related genes. lt b gt Results: lt /b gt We identified a total of 330 variants in the coding regions, out of which only 95 were potentially protein-changing. Observed in individual patients, detected mutations predominantly disrupted Ras/RTK pathway (STK11, KIT, MET, NRAS, KRAS, PTEN). Additionally, we identified 5 patients with the same mutation in HNF1A gene, disrupting both Wnt and Notch signaling pathway. In two patients we detected variants in NOTCH1 gene. HNF1A and NOTCH1 variants were mutually exclusive, while genes involved in Ras/RTK pathway exhibit a tendency of mutation accumulation. lt b gt Conclusions: lt /b gt Our results showed that ALL contains low number of mutations, without significant differences between cALL and aALL (median per patient 2 and 3, respectively). Detected mutations affect few key signaling pathways, primarily Ras/RTK cascade. This study contributes to knowledge of ALL mutational landscape, leading to better understanding of molecular basis of this disease.

Introduction Patients with acute promyelocytic leukemia (APL) are characterized by the highest expression of Wilms' tumor 1 (WT1) gene compared with other subtypes of acute myeloid leukemia, and yet this molecular marker is almost never used for risk stratification and in therapy response monitoring. Methods Quantitative assessment of Wilms' tumor 1 (WT1) gene transcripts was performed using real-time PCR method. The bone marrow samples were collected at the time of diagnosis for 47 APL patients, and for 31/47 patients during follow-up/relapse of the disease (129 samples in total). We examined how this molecular marker can be used for prognosis and minimal residual disease (MRD) monitoring. Results Increased WT1 expression was found in 34% of patients. WT1(high) status was an independent unfavorable factor for early death occurrence and was associated with shorter overall survival (OS). Assessment of log reduction value of WT1 expression in paired diagnosis/complete remission samples did not reveal its impact on relapse rate, disease-free survival, and OS. Also, measurement of WT1 expression level at different time points during therapy was not a reliable method for MRD monitoring. Conclusion Increased expression of WT1 gene detected in high proportion of APL patients could be considered as a marker for more precise risk stratification models in an attempt to further improve treatment and outcome of APL patients.

Leydig-ove ćelije testisa su primarno mesto sinteze muških polnih hormona. Ovi hormoni su neophodani za reproduktivno, ali i za opšte zdravlje budući da su ozbiljni zdravstveni problemi često povezani sa njihovom smanjenom produkcijom. Insulin i insulinu sličan faktor rasta 1, IGF1 (engl. insulin like growth factor 1), i signalizacija koju pokreću preko svojih receptora (INSR i IGF1R), su jedan od ključnih faktora koji regulišu specifični razvoj tkiva, pa i samih gonada. Ipak, uloga i mehanizmi delovanja ovih receptora u steroidogenim tkivima nisu u potpunosti poznati. Stoga je istraživanje uokviru ove doktorske disertacije koncipirano sa ciljem da se, na modelu prepubertalnih (P21) i adultnih (P80) mužjaka miševa sa kondicionalnom delecijom Insr i Igf1r gena u steroidogenim ćelijama (Insr/Igf1r-DKO), definiše uloga INSR i IGF1R u regulisanju diferencijacije i steroidogene funkcije Leydig-ovih ćelija. Pored toga, mužjaci i ženke P21 miševa sa istom delecijom su korišćeni za praćenje ekspresije glavnih markera mitohondrijalne biogeneze i fuzije/arhitekture u Leydigovim ćelijama, ovarijumima i nadbubrežnim žlezdama. Rezultati su potvrdili da delecija Insr i Igf1r u steroidogenim tkivima utiče na diferencijaciju i funkcionalne karakteristike Leydig-ovih ćelija P21 i P80 miševa, upućujući na pojavu tzv. „feminizacije“. Broj Leydig-ovih ćelija izolovanih iz P21 i P80 Insr/Igf1rDKO miševa bio je smanjen, a morfologija i ultrastruktura ovih ćelija izmenjene kod P21 Insr/Igf1rDKO miševa. Steroidogeni kapacitet i aktivnost, kao i ekspresija glavnih elemenata steroidogene mašinerije (Lhcgr, Star, Cyp11a1, Cyp17a1, Hsd3b1 i 6, Hsd17b3, Sf1) bili su smanjeni u Leydig-ovim ćelijama P21 i P80 Insr/Igf1r-DKO miševa, dok je ekspresija transkripcionih represora steroidogeneze (Arr19 i Dax1) bila povećana specifično u istim ćelijama, Leydig cells of testes are the primary site of the male sex hormones synthesis. These hormones are indispensable for both reproductive and general health since serious health problems are often associated with their reduced production. Insulin and insulin-like growth factor 1, IGF1 (insulin like growth factor 1), and signaling triggered through their receptors (INSR and IGF1R), are one of the key factors that regulate specific development of tissue including gonads. However, the role and mechanisms of these receptors action in steroidogenic tissues are not known enough. This study was designed to observe the role of INSR and IGF1R in regulating the differentiation and steroidogenic function of Leydig cells by using the model of prepubertal (P21) and adult (P80) male mice with the conditional deletion of the Insr and Igf1r genes in steroidogenic cells (Insr/Igf1r-DKO). In addition, male and female P21 mice with the samedeletion were used to monitor the expression of the main markers of mitochondrial biogenesis and fusion/architecture in Leydig cells, ovaries and adrenal glands. The results confirmed that deletion of Insr and Igf1r in steroidogenic tissues influences differentiation and functional characteristics of Leydig cells isolated from P21 and P80 mice, suggesting an appearance of "feminization". The number of Leydig cells isolated from both P21 and P80 Insr/Igf1r-DKO mice was reduced. Morphology and ultrastructure of Leydig cells were disturbed in P21 Insr/Igf1r-DKO mice. Steroidogenic capacity and activity, as well as expression of the main elements of steroidogenic machinery (Lhcgr, Star, Cyp11a1, Cyp17a1, Hsd3b1 and 6, Hsd17b3, Sf1) were decreased in Leydig cells from P21 and P80 Insr/Igf1r-DKO mice, while the expression of transcriptional repressors of steroidogenesis (Arr19 and Dax1) was increased in the sa

Lajdigove ćelije intersticijuma testisa su primarno mesto sinteze androgenih hormona, a dominantno testosterona (T). Ovaj hormon je zajedno sa svojim metabolitom dehidrotestosteronom (DHT) neophodan, kako za produženje vrste obezbeđivanjem pravilnog razvoja i funkcionisanja muškog reproduktivnog sistema, tako i za opšte zdravlje individue. U cilju tretiranja brojnih kliničkih poremećaja, kao i u svrhu kontracepcije, sintestisani su derivati androgena čija se primena u klinici zasniva na dejstvu koje ostvaruju posredstvom androgenih i/ili anaboličkih efekata, te su zajedničkim imenom nazvani anabolički androgeni steroidi (AAS). Nažalost, AAS se često zloupotrebljavaju, ne samo od strane profesionalnih i rekreativnih sportista, nego i velike populacije adolescenata, iako je dobro poznato da njihova upotreba u neterapeutske svrhe može izazvati niz neželjenih zdravstvenih posledica. Stoga su AAS svrstani u grupu farmakoloških preparata čija je upotreba strogo regulisana i nisu dostupni bez lekarskog recepta. Uprkos široko rasprostranjenoj kliničkoj upotrebi i zloupotrebi AAS, kao i velikom interesovanju naučne zajednice za ovu problematiku, nisu u potpunosti razjašnjeni molekulski događaji koji su posledica njihove kratkoročne i dugoročne primene. S obzirom na značaj T za reprodukciju i produženje vrste, ali i zdravlje i kvalitet života indivudue, kao i široku primenu i zloupotrebu T i njegovih derivata, neophodno je okarakterisati precizne molekulske događaje nastale kao posledica poremećene homeostaze T. Ovo je važno zbog toga što, prema trenutno dostupnoj literaturi, ne postoji dovoljan broj podataka o funkcionalnosti i obrascima signalnih puteva Lajdigovih ćelija, čija je osnovna uloga sinteza i sekrecija T. Stoga je glavni cilj ovog istraživanja bio da ispita funkcionalnost i obrasce signalnih puteva važnih za održavanje steroidogene funkcije Lajdigovih ćelija, narušene primenom egzogenih agonista i/ili antagonista T u in vivo ili in vitro uslovima. U tu svrhu prim, Leydig cells of testis interstitium represent the major site for synthesis of androgenic hormone, primarily testosterone (T). This hormone, together with its metabolite dihydrotestosterone (DHT), is required, not only for the continuation of the species by ensuring proper development and functioning of male reproductive system, but for overall health of an individual as well. For the purpose of treatment of multiple clinical disorders, as well as for contraception purposes, androgen derivatives have been synthesized, the clinical application of which is based on the influence they have through anabolic and/or androgenic effects, thus having a common name anabolic androgenic steroids (AASs). Unfortunately, AASs are often abused, not only by professional and recreational athletes, but by a large population of adolescents as well, although it is well known that non-therapeutic use thereof may cause a series of adverse health effects. Therefore, AASs are classified into a group of pharmacological preparations, the use of which is strictly regulated and which are not available without a medical prescription. Despite widespread clinical use and abuse of AASs, as well as the great interest shown by the scientific community in this field, molecular events resulting from their short-term and long-term use have not been fully clarified. Given the importance of T for reproduction and continuation of the species, but also for health and quality of life of an individual, and the widespread use and abuse of T and its derivatives, it is necessary to characterize precise molecular events resulting from disturbed T homeostasis. This is important because, according to the currently available literature, there is insufficient data on the functionality and patterns of signaling pathways of Leydig cells, the basic role of which is the synthesis and secretion of T. Therefore, the main aim of this study is to examine the functionality and patterns of signaling pathways relevant for mainta

Uvod: Nekoliko studija u literaturi navode dokaze o povećanoj incidenci hematoloških komplikacija u bolesnika sa Gošeovom bolešću, uključujući monoklonsku i poliklonsku gamapatiju i hematološke malignitete, a posebno multipli mijelom. Metode: Određivana je serumska koncentracija imunoglobulina kao i rearanžman gena za teški lanac imunoglobulina - IGH, PCR analizom. Klonalni PCR produkti su direktno sekvencirani i analizirani koristeći adekvatne alate i baze podataka. Monoklonski proteini seruma su detektovani i identifikovani metodom elektroforeze. Rezultati: Među 27 bolesnika, klonalni IGH rearanžman je otkriven kod osmoro, od kojih je petoro imalo i monot klonski protein u serumu. Hipergamaglobulinemija je otkrivena u 9/27 bolesnika. Podaci o praćenju za 17 bolesnika su pokazali da je klonalni rearanžman ostao isti u četiri bolesnika, dok je u jednog bolesnika iščezao tokom perioda praćenja. Preostalih 12/17 bolesnika nisu imali klonalni IGH rearanžman niti su ga stekli nakon perioda praćenja. Zaključak: Iako klonska ekspanzija može da nastane relativno rano u toku Gošeove bolesti, barem sudeći prema rearanžmanu IGH gena, detektovani klonovi mogu biti tranzitorni. Pažljivo kliničko praćenje ovih bolesnika je obavezno, uključujući i nadzor nad limfoidnim neoplazmama, posebno multiplim mijelomom., Background: Several studies support the evidence of increased incidence of hematological complications in Gaucher disease including monoclonal and polyclonal gammopathies and blood malignancies, especially multiple myeloma. Methods: Serum concentrations of immunoglobulins and PCR analysis of the IGH gene rearrangements were performed. The clonal PCR products were directly sequenced and analyzed with the appropriate database and tools. Serum monoclonal proteins were detected and identified by electrophoresis. Results: Among 27 Gaucher patients, clonal IGH rearrangement was discovered in eight, with 5/8 having also serum monoclonal protein. Elevated immunoglobulins were detected in 9/27 patients. Follow-up data for 17 patients showed that the clonal rearrangement remained the same in four of them, however, in one patient it disappeared after the follow-up period. The remaining 12/17 patients were without previous IGH clonal rearrangement and remained so after the follow-up. Conclusions: Although clonal expansion may occur relatively early in the disease course, at least judging by the IGH gene rearrangements in Gaucher patients, the detected clones may be transient. A careful clinical follow-up in these patients is mandatory, including monitoring for lymphoid neoplasms, especially multiple myeloma.

Reproduktivno starenje muškaraca razvija se uporedo sa starenjem organizma, a manifestuje se značajnim promena u funkciji endokrinog sistema i smanjenim fekunditetom. U kontekstu izmenjene funkcionalnosti endokrinog sistema ističe se smanjen nivo testosterona koji zajedno sa brojnim psiho-fizičkim promenama smanjuje kvalitet života jedinke. Konstantno produženje životnog veka i potencijalno neželjena dejstva zamenske terapije androgenima usmeravaju buduća istraživanja ka boljem razumevanju molekularnih osnova muškog hipogonadizma i pospešivanju endogene produkcije androgena u starijem životnom dobu muškaraca. U skladu sa iznetim činjenicama i usmerenjma postavljeni su sledeći ciljevi istraživanja: 1. Karakterizacija staračkog hipogonadizma kod pacova soja Wistar kao modela za istraživanje reproduktivnog starenja; 2. Ispitivanje udela promena cAMP- i cGMP-signalizacije u formiranju starog fenotipa Lć. Najvažnijim rezultatima ustanovljeno je da starenje Wistar pacova prati pojava primarnog hipogonadizma koji je iskazan smanjenom produkcijom testosterona i smanjenim eksprimiranjem steroidogenih gena i proteina od dvanaestog meseca. Zajedno sa smanjenim androgenim kapacitetom poremećena je i cAMP, MAPK i NO-cGMP signalizacija kao i energetska homeostaza u Lć starog fenotipa. Tokom istraživanja ustanovljeno je da smanjen nivo cAMP nije jedini uzročnik smanjenog androgenog kapaciteta, a akutna i hronična inhibicija PDE5 povećava steroidogeni kapacitet i normalizuje energetsku homeostazu u Lć starog fenotipa. Takođe je ustanovljeno da cAMP i cGMP različito regulišu energetsku homeostazu Lć, pri čemu cGMP normalizuje parametre funkcionalnosti mitohondrija i eksprimiranje regulatora energetske homeostaze., Reproductive aging of males develops along with the aging of the organism, and it is manifested by a significant change in the endocrine system and decreased fecundity. The decreased level of testosterone stands out in the context of the changed functionality of the endocrine system which together with numerous psycho-physical changes, reduces the quality of life of the individual. In respect with extended human life and undesirable effects of androgens replacement therapy, further investigations has been dedicated to better understanding of male hypogonadism by the molecular approach as well as endogen testosterone production during the aging. According to the presented facts we defined the two aims of the study: 1. Characterization of the age-related hypogonadism in the male Wistar rats as a model for reproductive aging, and 2. Investigation of the impact of a cAMP and a cGMP disturbances in development of aged Leydig cells. Our results showed appearance of primary hypogonadism during the aging of male Wistar rats, including impair testosterone production and impaired expression of steroidogenic genes and proteins from the 12th month of age. Beside that cAMP, MAPK and NO-cGMP signaling has been disturbed in aged Lc as well as energy homeostasis. We also showed that less production of cAMP is not the only cause of aged Lc sub-functionality and that acute and chronic inhibition of the PDE5 showed positive effect at steroidogenesis and energy homeostasis in aged Lc. Further, cAMP and cGMP differently regulated energy homeostasis of aged LC, with positive impact of the cGMP treatment on mitochondrial functionality and energy homeostasis regulators.

Biološki časovnik organizuje metabolizam i fiziološke procese u cirkadijalne ritmove. Na nivou ćelije, on se sastoji od grupe gena koji preko negativnih povratnih sprega održavaju ritam sopstvene transkripcije, ali regulišu i ritmičnost transkripcije mnogih drugih gena. Iako je poznato da su određeni geni časovnika neophodni za sintezu testosterona i fertilnost mužjaka, još uvek nema preciznih podataka o cirkadijalnoj fiziologiji testosteron produkujućih Lajdigovih ćelija. Ova studija je dizajnirana da definiše (1) cirkadijalni obrazac endokrine funkcije Lajdigovih ćelija uključujući i eksprimiranje gena perifernog časovnika i (2) upletenost LH-cAMP signalizacije u sinhronizaciju ritma Lajdigovih ćelija korišćenjem in vivo modela poremećene cAMP homeostaze (hipogonadotropni hipogonadizam, starački hipogonadizam, pinealektomija) i in vitro stimulacije Lajdigovih ćellija. Rezultati su pokazali cirkadijaln ritam funkcije Lajdigovih ćelija koji se ogleda u vremenski koordinisanoj oscilatornoj produkciji testosterona i intracelularnog cAMP, cirkadijalnom eksprimiranju regulatora (Nur77 i Arr19), steroidogenih elementa (Star/StAR, Cyp11a1 i Cyp17a1), kao i elementa časovnika (Bmal1/BMAL1, Per1/2/3, Cry1/2, Rev-erba/b/REV-ERBA, Rorb, Dec1/2, Dbp i E4bp4). Ritam transkripcije osnovnih gena časovnika kao i ključnog elementa steroidogeneze (Star) se održava i u primarnoj kulturi ovih ćelija. Redukcija cAMP detektovana u Lajdigovim ćelijama pacova sa hipogonadotropnim hipogonadizmom stimuliše transkripciju većeg broja gena časovnika: Per2, Rorb, Rev-erbb, Dec1/2, E4bp4, Ck1e/d, i inhibiše Npas2. Sa druge strane, in vitro stimulacija cAMP-signalizacije povećava transkripciju Per1, Dec1/2, Rorb, Npas2 i E4bp4, i smanjuje transkripciju Rev-erba. Starenje, dovodi do opadanja robusnosti cirkadijalne funkcije Lajdigovih ćelija koja se ogleda u smanjenju oscilacija intracelularnog cAMP, smanjenja amplitude eksprimiranja najvažnijih gena časovnika (Bmal1/BMAL1, Per1/2, Rev-erba/REV-ER, Biological clock organizes metabolic and physiological processes in circadian rhythms. At cell level, it consists of group of genes that regulate its own transcription by negative feedback loop, also regulating transcription rhythmicity of other genes. Although, it is known that some clock genes are necessary for testosterone synthesis and male fertility, there is no precise data about circadian physiology of testosterone-producing Leydig cells. This thesis was design to define (1) circadian pattern of endocrine function of Leydig cells, including expression of clock genes, and (2) involvement of LH-cAMP signaling in synchronization of Leydig cells rhythm using in vivo model of disturbed cAMP homeostasis (hypogonadotropic hypogonadism, hypogonadism in aging, pinealectomy) and in vitro Leydig cell stimulation. Results confirmed circadian rhythmicity of Leydig cell function represented by temporal coordination of cyclic testosterone production and intracellular cAMP, circadian expression of regulators (Nur77, Arr19), steroidogenic (Star/StAR, Cyp11a1 i Cyp17a1) and clock elements (Bmal1/BMAL1, Per1/2/3, Cry1/2, Rev-erba/b/REV-ERBA, Rorb, Dec1/2, Dbp, E4bp4). Rhythm in transcription of core clock genes as well as key steroidogenic element (Star) was preserved in primary Leydig cell culture. Reduction in cAMP, detected in Leydig cells from hypogonadotropic hypogonadal rats, stimulated transcription of some clock genes: Per2, Rorb, Rev-erbb, Dec1/2, E4bp4, Ck1e/d, but inhibited Npas2. On the other hand, in vitro stimulation of cAMP signaling increased transcription of Per1, Dec1/2, Rorb, Npas2 and E4bp4, and reduced transcription of Rev-erba. Aging dulled robustness of circadian function of Leydig cells, represented by decline in intracellular cAMP oscillations and amplitude of expression of core clock genes (Bmal1/BMAL1, Per1/2, Rev-erba/REV-ERBA), genes involved in cholesterol metabolism (Lipe, Soat2, Scarb1) and steroidogenic genes (Star/StAR, Cyp11a1, Cyp17a1, Hsd3b1