Programmed cell death

Cilj ovog seminara upoznavanje je s osnovnim mehanizmima apoptoze i glavnim ulogama programirane stanične smrti u višestaničnim organizmima. Apoptoza je programirani proces stanične smrti praćen aktivacijom kaspaza, cisteinskih aspartat – specifičnih proteaza. Postoje dva puta apoptoze – intrinzični i ekstrinzični. Intrinzični put započinje permeabilizacijom vanjske membrane mitohondrija, a ekstrinzični vezanjem signalnih molekula na receptore smrti u staničnoj membrani. Apoptoza ima veliku ulogu u pravilnom razvoju organizama. Osim održavanja ravnoteže između novonastalih i umirućih stanica, jedan je od glavnih pokretača morfogeneze i reorganizacije tkiva. Pojačana ili smanjena apoptoza nekih stanica jedan su od uzroka starenja. Programirana stanična smrt kompleksni je proces koji se može regulirati na mnogo razina, a greške u regulaciji dovode do pojave bolesti. Usmjereno ciljanje mehanizama apoptoze moglo bi biti ključ za liječenje raka, što čini istraživanja na ovu temu vrlo značajnima.The goal of this review is to introduce the reader with basic mechanisms of apoptosis and the main functions of programmed cell death in multicellular organisms. Apoptosis is a programmed process of cell death accompanied by activation of caspases, cysteinyl aspartate – specific proteases. There are two main pathways of apoptosis – intrinsic and extrinsic. Intrinsic pathway begins with permeabilization of outer mitochondrial membrane, while extrinsic pathway begins with binding of signal molecules to death receptors in cell membrane. Apoptosis plays a great role in correct development of organisms. Apart from its role in keeping the balance between newly-formed and dying cells, it is also one of the main initiators of morphogenesis and tissue reorganization. Upregulated or downregulated apoptosis of some cells is one of the main causes of ageing. Programmed cell death is a complex process that can be regulated on many levels, and misregulation of that process can often lead to an occurrence of illnesses. Direct targeting of apoptotic mechanisms could hold the key to curing cancer, which makes researches of this topic very significant

miR-7 AND miR-34a sequence cloning and expression in a1235 glioblastoma cell line

miRNAs are small non-coding RNAs which have an important role in signalling circuits regulating different cell processes. miR-7 and miR-34a are known as tumour suppressors, and both of them can interfere with cell proliferation, differentiation, apoptosis and migration. We constructed plasmids containing pri-miRNA sequences for these two miRNAs and introduced them into the A1235 glioblastoma cell line. Clones containing increased expression of processed miR-7 and miR-34a were obtained. The proliferation and sensitivity to alkylation agent of transfected cells were similar to those of control cells. Our results indicate that an increase in miR-7 and miR34 expression alone in A1235 glioblastoma cells is not sufficient to change their proliferation or sensitivity to the influence of alkylating agents

Pharmacogenetic distinction of the Croatian population from the European average

Aim To compare the Croatian and European population in terms of allele frequencies of clinically relevant polymorphisms in drug absorption, distribution, metabolism, and excretion (ADME) genes. Methods In 429 Croatian participants, we genotyped 27 loci in 20 ADME genes. The obtained frequencies were merged with the published frequencies for the Croatian population by sample size weighting. The study sample obtained in this way was compared with the average data for the European population from the gnomAD database. Results Variant allele frequencies in the Croatian population were higher in three and lower in two polymorphisms (Benjamini-Hochberg-corrected P values: 0.0027 for CYP2B6*4 rs2279343, CYP2C9*2 rs1799853, and VKORC1 rs9923231; 0.0297 for GSTP1 rs1695; 0.0455 for CYP2A6 rs1801272) compared with the European population. The most marked difference was observed for CYP2B6*4 (9.3% in Europe vs 24.3% in Croatia). The most clinically relevant findings were higher variant allele frequencies in two polymorphisms related to lower warfarin requirements: VKORC1*2 (34.9% in Europe vs 40.1% in Croatia) and CYP2C9*2 (12.3% in Europe vs 14.7% in Croatia). This indicates that three-quarters of Croatian people have at least one variant allele at these loci. Variants in genes GSTP1 and CYP2A6 were significantly less frequently observed in Croatia. Conclusions Croatian population has a higher bleeding and over-anticoagulation risk, which is why we recommend the prescription of lower doses of anticoagulation drugs such as warfarin and acenocoumarol. Lower phenytoin, and higher bupropion and efavirenz doses are also recommended in the Croatian population

Cloning of the ligand JAGGED1 and its effect on the proliferation of lymphocytes

Signalni put Notch igra ključnu ulogu u određivanju stanične sudbine u brojnim tkivima. Ovisno o vrsti stanica, aktivacija puta Notch može rezultirati diferencijacijom, proliferacijom ili apoptozom. Receptori Notch transmembranski su proteini s velikom ekstracelularnom domenom koja veže ligande, transmembranske proteine na susjednoj stanici. Prilikom vezanja liganda i receptora intracelularna domena receptora se proteolitički cijepa i putuje u jezgru, gdje se veže s transkripcijskim faktorom CSL i tako regulira transkripciju gena. Ligandi receptora Notch dijele se u dvije skupine – ligande "Delta-like" (DLL) i ligande Jagged. Signalni put Notch ima važnu ulogu u diferencijaciji krvnih stanica, a pogreške u regulaciji tog procesa mogu dovesti do transformacije stanica i nastanka leukemija. Cilj ovog istraživanja bio je analizirati utjecaj liganda JAGGED1 na proliferaciju limfocita i aktivaciju puta Notch. Konstruiran je ekspresijski plazmid sa sekvencijom liganda JAGGED1. Leukemijski B-limfociti uzgajani su u kokulturi s adherentnim stanicama koje eksprimiraju ovaj ligand. Pratila se proliferacija limfocita u kokulturi u odnosu na kulturu samih limfocita te onih tretiranih inhibitorom puta Notch, kako bi se utvrdilo utječe li aktivacija puta Notch ligandom JAGGED1 na biologiju limfocita.Notch signaling pathway plays a crucial role in the determination of the cell fate in many tissues. Depending on the cell type, activation of Notch signaling can result in cell differentiation, proliferation or apoptosis. Notch receptors are transmembrane proteins with a large extracellular ligand-binding domain which binds to the ligand on the neighbouring cell. When a ligand binds to a receptor, the intracellular domain of the receptor is proteolytically cleaved and travels to the nucleus. There it binds CSL transcription factor and in that manner regulates gene transcription. Notch ligands belong to two families – "Delta-like" ligands (DLL) and Jagged. Notch signaling pathway plays an important role in the blood cell differentiation, and misregulations of that process could lead to malignant transformation and development of leukemia. The aim of this thesis was to analyse the effect of JAGGED1 ligand on the lymphocyte proliferation and activation of Notch signaling pathway. An expression plasmid with a coding sequence JAGGED1 was constructed, and leukemic B- lymphocytes were cocultured with adherent cell expressing JAGGED1 ligand. Proliferation of lymphocytes in coculture was analyzed in comparison with proliferation of lymphocytes grown alone, and lymphocytes treated with Notch inhibitor, to determine the effect of JAGGED1 ligand signaling on the lymphocyte biology

Cloning of the ligand JAGGED1 and its effect on the proliferation of lymphocytes

Signalni put Notch igra ključnu ulogu u određivanju stanične sudbine u brojnim tkivima. Ovisno o vrsti stanica, aktivacija puta Notch može rezultirati diferencijacijom, proliferacijom ili apoptozom. Receptori Notch transmembranski su proteini s velikom ekstracelularnom domenom koja veže ligande, transmembranske proteine na susjednoj stanici. Prilikom vezanja liganda i receptora intracelularna domena receptora se proteolitički cijepa i putuje u jezgru, gdje se veže s transkripcijskim faktorom CSL i tako regulira transkripciju gena. Ligandi receptora Notch dijele se u dvije skupine – ligande "Delta-like" (DLL) i ligande Jagged. Signalni put Notch ima važnu ulogu u diferencijaciji krvnih stanica, a pogreške u regulaciji tog procesa mogu dovesti do transformacije stanica i nastanka leukemija. Cilj ovog istraživanja bio je analizirati utjecaj liganda JAGGED1 na proliferaciju limfocita i aktivaciju puta Notch. Konstruiran je ekspresijski plazmid sa sekvencijom liganda JAGGED1. Leukemijski B-limfociti uzgajani su u kokulturi s adherentnim stanicama koje eksprimiraju ovaj ligand. Pratila se proliferacija limfocita u kokulturi u odnosu na kulturu samih limfocita te onih tretiranih inhibitorom puta Notch, kako bi se utvrdilo utječe li aktivacija puta Notch ligandom JAGGED1 na biologiju limfocita.Notch signaling pathway plays a crucial role in the determination of the cell fate in many tissues. Depending on the cell type, activation of Notch signaling can result in cell differentiation, proliferation or apoptosis. Notch receptors are transmembrane proteins with a large extracellular ligand-binding domain which binds to the ligand on the neighbouring cell. When a ligand binds to a receptor, the intracellular domain of the receptor is proteolytically cleaved and travels to the nucleus. There it binds CSL transcription factor and in that manner regulates gene transcription. Notch ligands belong to two families – "Delta-like" ligands (DLL) and Jagged. Notch signaling pathway plays an important role in the blood cell differentiation, and misregulations of that process could lead to malignant transformation and development of leukemia. The aim of this thesis was to analyse the effect of JAGGED1 ligand on the lymphocyte proliferation and activation of Notch signaling pathway. An expression plasmid with a coding sequence JAGGED1 was constructed, and leukemic B- lymphocytes were cocultured with adherent cell expressing JAGGED1 ligand. Proliferation of lymphocytes in coculture was analyzed in comparison with proliferation of lymphocytes grown alone, and lymphocytes treated with Notch inhibitor, to determine the effect of JAGGED1 ligand signaling on the lymphocyte biology

Cloning of the ligand JAGGED1 and its effect on the proliferation of lymphocytes

Signalni put Notch igra ključnu ulogu u određivanju stanične sudbine u brojnim tkivima. Ovisno o vrsti stanica, aktivacija puta Notch može rezultirati diferencijacijom, proliferacijom ili apoptozom. Receptori Notch transmembranski su proteini s velikom ekstracelularnom domenom koja veže ligande, transmembranske proteine na susjednoj stanici. Prilikom vezanja liganda i receptora intracelularna domena receptora se proteolitički cijepa i putuje u jezgru, gdje se veže s transkripcijskim faktorom CSL i tako regulira transkripciju gena. Ligandi receptora Notch dijele se u dvije skupine – ligande "Delta-like" (DLL) i ligande Jagged. Signalni put Notch ima važnu ulogu u diferencijaciji krvnih stanica, a pogreške u regulaciji tog procesa mogu dovesti do transformacije stanica i nastanka leukemija. Cilj ovog istraživanja bio je analizirati utjecaj liganda JAGGED1 na proliferaciju limfocita i aktivaciju puta Notch. Konstruiran je ekspresijski plazmid sa sekvencijom liganda JAGGED1. Leukemijski B-limfociti uzgajani su u kokulturi s adherentnim stanicama koje eksprimiraju ovaj ligand. Pratila se proliferacija limfocita u kokulturi u odnosu na kulturu samih limfocita te onih tretiranih inhibitorom puta Notch, kako bi se utvrdilo utječe li aktivacija puta Notch ligandom JAGGED1 na biologiju limfocita.Notch signaling pathway plays a crucial role in the determination of the cell fate in many tissues. Depending on the cell type, activation of Notch signaling can result in cell differentiation, proliferation or apoptosis. Notch receptors are transmembrane proteins with a large extracellular ligand-binding domain which binds to the ligand on the neighbouring cell. When a ligand binds to a receptor, the intracellular domain of the receptor is proteolytically cleaved and travels to the nucleus. There it binds CSL transcription factor and in that manner regulates gene transcription. Notch ligands belong to two families – "Delta-like" ligands (DLL) and Jagged. Notch signaling pathway plays an important role in the blood cell differentiation, and misregulations of that process could lead to malignant transformation and development of leukemia. The aim of this thesis was to analyse the effect of JAGGED1 ligand on the lymphocyte proliferation and activation of Notch signaling pathway. An expression plasmid with a coding sequence JAGGED1 was constructed, and leukemic B- lymphocytes were cocultured with adherent cell expressing JAGGED1 ligand. Proliferation of lymphocytes in coculture was analyzed in comparison with proliferation of lymphocytes grown alone, and lymphocytes treated with Notch inhibitor, to determine the effect of JAGGED1 ligand signaling on the lymphocyte biology

Relevance of <i>CYP2D6</i> Gene Variants in Population Genetic Differentiation

A significant portion of the variability in complex features, such as drug response, is likely caused by human genetic diversity. One of the highly polymorphic pharmacogenes is CYP2D6, encoding an enzyme involved in the metabolism of about 25% of commonly prescribed drugs. In a directed search of the 1000 Genomes Phase III variation data, 86 single nucleotide polymorphisms (SNPs) in the CYP2D6 gene were extracted from the genotypes of 2504 individuals from 26 populations, and then used to reconstruct haplotypes. Analyses were performed using Haploview, Phase, and Arlequin softwares. Haplotype and nucleotide diversity were high in all populations, but highest in populations of African ancestry. Pairwise FST showed significant results for eleven SNPs, six of which were characteristic of African populations, while four SNPs were most common in East Asian populations. A principal component analysis of CYP2D6 haplotypes showed that African populations form one cluster, Asian populations form another cluster with East and South Asian populations separated, while European populations form the third cluster. Linkage disequilibrium showed that all African populations have three or more haplotype blocks within the CYP2D6 gene, while other world populations have one, except for Chinese Dai and Punjabi in Pakistan populations, which have two