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Road detection using LiDAR data acquired from aerial surveys of the Republic of Croatia
No full textCilj ovog rada je primjenom dostupnih ulaznih slojeva – digitalnog ortofoto (DOF) rastera, LiDAR (engl. Light Detection and Ranging) podataka te vektorskog sloja cesta preuzetog s OpenStreetMap-a detektirati i izdvojiti cestovni sloj u urbanom području grada Karlovca. Korišteni softver, eCognition Developer, nudi napredne funkcije za segmentiranje i klasificiranje slika. Za segmentaciju se koristi multirezolucijska segmentacija, pristup temeljen na analizi objekata (engl. Object-based Image Analysis - OBIA). Klasifikacija je nadzirana, što uključuje ručno odabiranje reprezentativnih uzoraka za svaku klasu. Proces klasifikacije koristi srednje vrijednosti ulaznih slojeva i algoritam klasifikatora najbližeg susjeda (engl. k-nearest neighbors - kNN) kako bi dodijelio preostale segmente jednoj od dvije definirane klase: cesta i ne-cesta. Analizom utjecaja pojedinih ulaznih slojeva na rezultate klasifikacije, cilj je dublje razumjeti izazove u izdvajanja cesta u urbanom okruženju te raspraviti moguća rješenja. Kombinacija spektralnih kanala, visinskih podataka i vektorskog sloja cesta pokazala se ključnom za poboljšanje točnosti klasifikacije. Međutim, najveći izazov ostaje pravilna klasifikacija ostalih asfaltiranih površina, poput parkirališta i pločnika, koje su spektralno slične cestama. Iako su neki segmenti ovih površina uspješno eliminirani, varijacije u rezultatima i dalje su prisutne. Ovaj rad pruža temelj za buduća istraživanja i unaprjeđenja u području segmentacije i klasifikacije urbanih cesta, ukazujući na važnost integracije različitih izvora podataka i naprednih algoritama za analizu slika.The aim of this study is to detect and extract the road surfaces in the urban area of Karlovac by applying available input layers – digital orthophoto (DOF) raster, LiDAR data, and a line vector layer of roads obtained from OpenStreetMap. eCognition Developer software was used. It offers advanced functions for image segmentation and classification. Multiresolution segmentation, an approach based on object-based image analysis (OBIA), is used for segmentation. The classification is supervised, which includes manually selecting representative samples for each class. The classification process utilizes the mean values of input layers and the k-nearest neighbor (kNN) classifier algorithm to assign the remaining segments to one of two defined classes: road and non-road. By analyzing the impact of individual input layers on classification results, the goal is to gain a deeper understanding of the challenges in road extraction in urban environments and to discuss possible solutions. The combination of spectral bands, elevation data, and the line vector road layer proved to be crucial for improving classification accuracy. However, the greatest challenge remains the accurate classification of other asphalt surfaces, such as parking lots and sidewalks, which are spectrally similar to roads. Although some segments of these surfaces were successfully eliminated, variations in results are still present. This study provides a base for future research and advancements in the field of urban road segmentation and classification, highlighting the importance of integrating different data sources and advanced image analysis algorithms
Assessment of the impact of erythromycin on bacteria Pseudomonas putida isolated from pharmaceutical wastewater
No full textDemografski rast stanovništva, širenje urbanih sredina, razvoj industrije te ubrzani način života, pokretačke su sile za povećanu upotrebu i proizvodnju farmaceutika. Različiti su izvori njihova dospijevanja u okoliš, a uslijed abiotičkih i biotičkih djelovanja mogu promijeniti svoju prvotnu strukturu. Potrebno je pratiti koncentracije farmaceutika u okolišu te standardnim metodama određivati njihov utjecaj na izložene organizme. Makrolidni se antibiotici navode kao štetni po okoliš uslijed velike potrošnje i duljeg zadržavanja u okolišu. U svrhu uklanjanja farmaceutika iz okoliša, proučavaju se razni postupci, no sve zastupljenije postaje iskorištavanje enzimatske sposobnosti mikroorganizama za njihovo uklanjanje. U ovome je radu provedena procjena utjecaja makrolidnog antibiotika eritromicina na bakterijsku kulturu Pseudomonas putidu izoliranu iz otpadne farmaceutske vode. Testovi toksičnosti provedeni pomoću bakterije Vibrio fischeri svrstavaju eritromicin kao slabo toksičnu tvar uz mogući rizik po okoliš. Istraživan je potencijal biorazgradnje eritromicina bakterijskom kulturom P. putidom s obzirom na rezultate testova osjetljivosti koji su pokazali otpornost kulture na raspon koncentracija eritromicina 0,2-2000 mg L^-1. Nakon uzgoja stanica baktrerijske kulture na Tryptic Soy Broth-u, praćena je aktivnost enzima esteraze u uvjetima bez i uz dodatak 0,2 mg L^-1 i 200 mg L^-1 eritromicina. Aktivnost enzima je rasla bez obzira na koncentraciju eritromicina, ali su pri 200 mg L^-1 uočene morfološke promjene stanica P. putide.Demographic growth of population, urbanization, industrialization and hectic lifestyle are driving forces for increasing utilization of pharmaceuticals and their production. There are different sources of their entry into the environment and due to abiotic and biotic activity their primary structures can be changed. It is necessary to control concentration of pharmaceuticals in the environment and determine their impact on exposed organisms by standard methods. Macrolide antibiotics are harmful for the environment due to their large consumption and longer environmental persistence. In order to remove pharmaceuticals from the environment, different processes are being researched with emphasis on enzymatic capability of microorganisms. In this study, assessment of the macrolide antibiotic erythromycin on bacterial culture Pseudomonas putida isolated from pharmaceutical wastewater was investigated. Bioassays conducted with bacteria Vibrio fischeri classified erythromycin as low toxic substance with possible risk for the environment. Biodegradation potential of erythromycin with bacterial culture Pseudomonas putida was investigated with susceptibility tests which indicated the resistance of bacteria at range of erythromycin concentration from 0.2 to 2000 mg L^-1. After cultivation of bacterial culture on Tryptic Soy Broth, activity of enzyme esterase was determined in conditions without and with addition of 0.2 mg L^-1 and 200 mg L^-1 of erythromycin. Activity of enzyme increased regardless of erythromycin concentration, while morphological changes of P. putida were noticed at 200 mg L^-1 of erythromycin
Primjena fotogrametrije u inženjerskoj geologiji
No full textU okviru ovog diplomskog rada izrađeni su 3D modeli terena (ogoline). Područje od interesa je sjeverna Istra gdje prevladaju flišne zaravni. U fokusu rada je mogućnost korištenja trodimenzionalnih modela za praćenje geodinamičkih procesa, prije svega ekscesivne erozije. Teorijski dio obuhvaća definicije fotogrametrije, s osvrtom na njezine metode, aerofotogrametriju i terestričku fotogrametriju. Nadalje, obrađene su osnove opće i inženjerske geologije, uključujući razvoj Zemljine kore, klasifikaciju stijena
te razlikovanje egzogenih procesa koji oblikuju reljef, kako bi se pružio temelj za razumijevanje pojave erozije. Obilježavanjem područja od interesa (ogoline u neposrednoj blizini mjesta Jakomići), pristupa se izradi 3D modela. U praktičnom dijelu je opisan sam postupak izrade modela koji uključuje informacije o vrstama mjerne opreme korištene za prikupljanje podataka, rad unutar softvera Agisoft Metashape-a te konačan prikaz podataka i njihovu analizu. Zaključno, napravljena je kritička procjena korištenih tehnologija, kroz isticanje njihovih prednosti i ograničenja u kontekstu inženjersko-geološke primjene
Validation of LC-MS/MS method for the determination of N-nitroso-hydrochlorothiazide in hydrochlorothiazide active substance
No full textValidacija je važan korak u stvaranju analitičke metode nakon njenog razvitka, te se validacija definira kao postupak dokazivanja je li metoda prikladna za namjeravanu upotrebu. Validacija analitičke metode izvodi se u laboratoriju provođenjem ispitivanja izvedbenih karakteristika metode kao što su selektivnost/specifičnost, linearnost, preciznost, točnost, granica detekcije i kvantifikacije, radno područje te robusnost. Ovisno o namjeni analitičke metode ne moraju se nužno ispitivati sve izvedbene karakteristike već samo odabrane. Glavni cilj ovog rada bio je potvrditi prikladnost LC-MS/MS metode za određivanje N-nitrozo-hidroklorotiazida u hidroklorotiazid aktivnoj supstanci. Eksperimentalni dio rada izveden je u laboratoriju Odjela Kontrole kvalitete, Pliva Hrvatske d.o.o. ispitivanjem svih potrebnih izvedbenih karakteristika. Postignuta je dobra linearnost (R^2 = 1,000) te su također zadovoljeni kriteriji za preciznost (RSD ≤ 20 %) i točnost (RSD ≤ 25 %). Definirana je granica kvantifikacije (LOQ = 0,60 ng/ml) te radno područje metode (0,60 ng/ml – 50 ng/ml). Dodatno je ispitana stabilnost otopina standarda N-nitrozo-hidroklorotiazida i hidroklorotiazid aktivne supstance i te robusnost metode. Rezultatima validacije potvrđena je prihvatljivost metode za određivanje N-nitrozo-hidroklorotiazida u hidroklorotiazid aktivnoj supstanci što omogućuje njenu primjenu u svakodnevnom rutinskom radu laboratorija.Validation is a key step in the creation of an analytical method after its development, and it is defined as the process of proving whether the method is suitable for the intended purpose. Analytical method validation is conducted in the laboratory by testing performance characteristics of the method, such as selectivity, specificity, linearity, working range, precision, trueness, limit of detection and quantification, and robustness. Depending on the purpose of the analytical method, it is not necessary to examine all performance features, but only selected ones. The main goal of this work is to validate the LC-MS/MS method for the determination of N-nitroso-hydrochlorothiazide in hydrochlorothiazide active substance. The experimental part of the work covered determination of all performance characteristics and was conducted in the Quality Control Department, Pliva Croatia d.o.o. The method achieved good linearity (R^2=1,000) and met the criteria for precision (RSD ≤ 20%) and accuracy (RSD ≤ 25%). The limit of quantification (LOQ = 0,60 ng/ml) as well as the working range of the method (0,60 ng/ml – 50 ng/ml) are defined. In addition, the stability of the solutions of N-nitroso-hydrochlorothiazide standard and hydrochlorothiazide active substance and the robustness of the method were evaluated. The validation results confirmed the acceptability of the method for the determination of N-nitroso-hydrochlorothiazide in hydrochlorothiazide active substance was confirmed, given that the obtained values of performance characteristics are within the given limits of acceptability. This enables the application of the method in the daily routine work of the laboratory
Validacija novog staničnog modela za mapiranje interaktoma Q398X mutacije optineurina iz ALS bolesnika
No full textAmyotrophic lateral sclerosis (ALS) is a progressive adult-onset neurodegenerative disease of upper and lower motor neurons that leads to a complete paralysis and death usually in 2-5 years from the disease onset. Although decades of research have been invested in understanding the ALS pathogenesis, the extremely heterogenous genetic background, with more than 20 genes being implicated, makes it difficult to pinpoint the exact mechanisms that lead to development of this disease. Optineurin protein, encoded by the OPTN gene, has been linked to ALS in a small subset of patients. It was suggested that OPTN acts solely through a loss-of-function mechanism in contrast to a toxic gain-of-function mechanism that was suggested for more common genes mutated in ALS, such as C9ORF72, SOD1 and TARDBP. We decided to apply the proximity-dependent labeling method to study the interactome of Q398X optineurin, a mutation that has been found in ALS patients. For that purpose, we generated new HEK293 Flp-In cell models that upon doxycycline treatment express constructs that carry BioID2 enzyme linked to the N-terminus of the wild-type or Q398X optineurin. Our results demonstrated that doxycycline successfully induced the expression of inserted constructs and that BioID2 enzyme was able to conjugate biotin to a wide range of proteins. Additionally, we detected interactions between both wild-type or Q398X optineurin and TBK1 demonstrating that linkage of BioID2 and Myc tag to optineurin N-terminus did not disturb its physiological interactions. Unexpectedly, we found that wild-type optineurin interacted with p-p65, but the Q398X optineurin did not. Although this was likely an indirect interaction of wild-type optineurin, it highlighted the possibility that Q398X mutation loses some relevant interactions in the NF-κB pathway, which contributes to the ALS development. On the other hand, we found no interaction between wild-type or Q398X optineurin with RIPK1, which we attributed to a small labeling radius of BioID2 that was likely insufficient for labeling interaction partners binding to the C-terminus of optineurin. This signified the importance of generating additional cell lines with BioID2 linked to optineurin C-terminus. Combined with our existing cell lines, they should enable the mapping of complete interactomes of wild-type and Q398X optineurin using mass spectrometry and hopefully contribute to the understanding of mechanisms by which Q398X optineurin causes ALS.Amiotrofična lateralna skleroza (ALS) je progresivna neurodegenerativna bolest odrasle dobi koja zahvaća gornje i donje motoričke neurone. Vodi do potpune paralize i smrti uglavnom u roku 2-5 godine nakon početka bolesti. Iako su već desetljeća istraživanja posvećena razumijevanju patogeneze ALS-a, izuzetna heterogenost genetske pozadine, s više od 20 gena vezanih s njezinim nastankom, stvara teškoće u razumijevanju točnih mehanizama koji vode ka razvoju ove bolesti. Protein optineurin, kojeg kodira gen OPTN, povezan je s ALS-om u maloj skupini bolesnika. Pretpostavlja se da OPTN djeluje isključivo mehanizmom gubitka funkcije (eng. loss-of-function) za razliku od djelovanja putem dobitka novih toksičnih funkcija (eng. gain-of-function) koji je bio predložen za učestalije mutirane gene u ALS-u poput C9ORF72, SOD1 i TARDBP. Odlučili smo koristiti metodu obilježavanja na temelju bliskosti (eng. proximity-dependent labeling) za istraživanje interaktoma Q398X optineurina, mutacije pronađene u pacijentima oboljelih od ALS-a. U tu smo svrhu napravili nove HEK293 Flp-In stanične modele koji po tretmanu s doksiciklinom eksprimiraju proteinske konstrukte koji sadrže optineurin divljeg soja ili Q398X povezan s BioID2 enzimom na N-kraju. Naši su rezultati pokazali da doksiciklin uspješno izaziva ekspresiju umetnutih konstrukata te da BioID2 enzim biotinilira širok raspon proteina. Nadalje, uočili smo interakcije optineurina divljeg soja i Q398X s TBK1, što je pokazalo da vezanje BioID2 enzima i Myc oznake za N-kraj optineurina nije omelo njegove fiziološke interakcije. Neočekivano, pronašli smo da optineurina divljeg soja ulazi u interakciju s p-p65, no ta interakcija nije pronađena u Q398X mutanta. Iako je posrijedi vjerojatno bila indirektna interakcija s optineurinom divljeg soja, ovo saznanje je istaknulo mogućnost da Q398X mutacija gubi neke značajne interakcije u NF-κB signalnom putu i time doprinosi razvoju ALS-a. S druge strane, interakcije s RIPK1 nisu pronađene u optineurina divljeg soja ni u Q398X mutacije, što smo pripisali malenom radijusu obilježavanja BioID2 koji je vjerojatno bio nedovoljan da obuhvati interakcije s partnerima koji se vežu za C-terminalni dio optineurina. To je istaknulo potrebu za stvaranjem dodatnih staničnih linija sa BioID2 enzimom vezanim za C-kraj optineurina. Te bi stanične linije, u kombinaciji s već postojećima, omogućile mapiranje cjelovitog interaktoma optineurina divljeg soja i Q398X mutacije pomoću masene spektrometrije te, nadajmo se, doprinijele razumijevanju mehanizama kojima Q398X optineurin uzrokuje ALS
The role of NK and ILC1 cells in the control of murine cytomegalovirus during ontogeny
No full textLjudski citomegalovirus (HCMV) je jedan od najrasprostranjenijih virusa diljem svijeta. Njegova raširenost predstavlja veliki problem u zdravstvenom sektoru zbog toga što može nakon infekcije ostaviti ozbiljne posljedice, pogotovo ukoliko dođe do prijenosa virusa s trudnice na plod, čime uzrokuje prirođenu infekciju HCMV-om koja može imati i letalan ishod. Imunosni sustav u prenatalnom i ranom postnatalnom razdoblju nije razvijen kao u odraslih te je nedostatak zrelosti razlog tome što infekcije u tom periodu mogu uzrokovati ozbiljne posljedice. Mehanizmi kako nerazvijeni imunosni sustav, odnosno prirođena imunost, štiti plod i novorođenčad od infekcija različitim patogenima su slabo istraženi. Za potrebe istraživanja HCMV-a koristi se mišjicitomegalovirus (MCMV) s kojim dijeli mnoge sličnosti u temeljnoj biologiji i patogenezi, te zbog toga infekcija miševa MCMV-om predstavlja najčešće korišteni model infekcije citomegalovirusom. U ovom istraživanju koristili smo transgenične miševe, pristup uklanjanja imunoloških stanica monoklonskim protutijelima, te rekombinantni MCMV kako bi istražili ulogu stanica NK i ILC1 u nadzoru infekcije MCMV-om u novookoćenih miševa. Transgenične miševe smo genotipizirali PCR-om, prisustvo stanica NK i ILC1 odredili smo metodom protočne citometrije, dok smo testom virusnih čistina odredili količinu virusa u ispitivanim organima. Rezultati ovog rada pokazali su kako stanice urođene imunosti, stanice NK i ILC1, imaju važnu ulogu u nadzoru infekcije MCMV-om u jetri novookoćenih miševa. Daljnja istraživanja će pružiti bolje razumijevanje neonatalnog imunosnog sustava.Human cytomegalovirus (HCMV) is one of the most widespread viruses worldwide. It is a major problem in healthcare because the infecion can leaveserious consequences, especially if the virus is transmitted from the pregnant woman to the fetus, i.e., if it causes congenital HCMV infection that can havea fatal outcome. The immune system in the prenatal and early postnatal period is immature as compared to adults, and the immaturity is the reason why infections in that period can result in serious consequences. The mechanisms by which an underdeveloped immune system, i.e. innate immunity, protects the fetus and newborns from infections by various pathogens have been poorly investigated. To study HCMV, murine cytomegalovirus (MCMV) is often used, which shares many biological similarities and pathogenesis; therefore, infection of mice with MCMV is the most commonly used model of cytomegalovirus infection. In this study, we used transgenic mice, an immune cell depletion approach with monoclonal antibodies, and recombinant MCMV to investigate the role of NK cells and ILC1 cells in the control of MCMV infection in newborn mice. The transgenic mice were genotyped by PCR, the presence of NK and ILC1 cells was determinedby flow cytometry method, while the levels of virus in the examined organs was determined by the plaque assay. The results of this work showed that innate immune cells, NK cells and ILC1, play an important role in controlling MCMV infection in the liver of newborn mice. Further research will provide a better understanding of the neonatal immune system
Removal of the cyanide and the rhodanide from waters
No full textCijanidi i rodanidi su toksični spojevi sa štetnim utjecajem na okoliš i ljudsko zdravlje. Cijanidi su poznati kao bitna sirovina kod proizvodnje farmaceutika, kozmetike, medicinskih i poljoprivrednih proizvoda te kod procesa obrade u metalurškoj industriji. Reakcijama cijanida sa sumporom nastaju rodanidi ili tiocijanati koji su karakterizirani kao spojevi manje toksičnosti, ali veće stabilnosti od cijanida. Razvojem industrije i povećavanjem proizvodnih kapaciteta raste i količina otpadnog cijanida točnije otpadnim vodama dospjevaju u okoliš. Stoga je potrebno provoditi obradu otpadnih voda prije ispuštanja iz postrojenja kako bi se smanjila njihova prisutnost u okolišu. Napredni oksidacijski procesi su vrsta procesa koji se koriste pri obradi takvih onečišćenih voda. Temelje se na korištenju oksidacijskih sredstava pomoću kojih dolazi do uspješne razgradnje i uklanjanja neželjenih organskih spojeva.
U ovom radu provedena su istraživanja uklanjanja cijanida (CN-) i tiocijanata (SCN-) iz modelnih otopina naprednim oksidacijskim procesima fotolizom, UV/H2O2 i UV/S2O82- pri pH vrijednostima 4, 7 i 10. Omjer onečišćivala (CN- i SCN-) prema oksidacijskom sredstvu (H2O2 i S2O82-) iznosio je 1:10 dok je valna duljina UV zračenja iznosila 254 nm. Rezultati su pokazali kako je najučinkovitija primjena UV/S2O82- za oba istraživana onečišćivala pri nižim pH. Metoda UV/S2O82- je najučinkovitija s 99,26% uklonjenog CN- i 80,26% uklonjenog SCN- pri pH = 4. Minimalne učinkovitosti pri uklanjanju oba onečišćivala zabilježene su kod čiste fotolize gdje je uklonjeno samo 40,90% CN- (pH = 7) i 21,49% SCN- (pH = 10). Najbrža je kinetika razgradnje CN- metodom UV/S2O82- (k = 0,0806 s^-1) pri pH=4 dok je najsporija tijekom čiste fotolize (k = 0,0076 s^-1) pri pH=7. Najbrža razgradnja SCN- je metodom UV/S2O82- (k =0,027 s^-1) pri pH=4 dok je najsporija tijekom čiste fotolize (k = 0,0041 s^-1) pri pH = 10. Navedene brzine uklanjanja CN- i SCN- su prvog reda odnosno kinetika se ipak smatra pseudo-prvog reda zbog velikog broj radikalskih reakcija.Cyanides and rhodanides are toxic compounds with a negative influence on the environment and the human health. Cyanides are known as a important raw material in pharmaceutical, cosmetics, medical and agricultural product manufacture and in processing processes of the metallurgical industry. Reactions between cyanides and sulphur result with the formation of rhodanides or thiocyanites which are charaterized as compunds with lower toxicity but higher stability than cyanides. With the development of the industry and the increase in production capacity, the amount of cyanide and rhodanides that ends up in the environment within wastewaters increases. Because of that, it is necessary to carry out the wastewater treatment before the discharging to decrease their presence in the environment. Advanced oxidation processes are a type of processes which are used for treating that kind of polluted water. They are based on the usage of oxidizing agents which successfully result with the decomposition of unwanted organic compounds. This work deals with the removal od cyanides (CN-) and rhodanides (SCN-) from model solutions by the advanced oxidation processes using photolysis, UV/H2O2 and UV/S2O82- at pH values 4, 7 and 10. The ratio of pollutants (CN- and SCN-) to oxidizing agents (H2O2 and S2O82-) was 1:10, while the wavelength of the UV radiation was 254 nm The results showed that the usage of the UV/S2O82- method under lower pH values was the most effient for both pollutants. The UV/S2O82- method is the most efficient with 99.26% of CN- and 80.26% of SCN- removed at pH=4. Minimal efficiencies in the removal of both pollutants were recorded with pure photolysis, where only 40.90% of CN- (pH=7) and 21.49% of SCN- (pH=10) were removed. The fastest kinetics of the CN- degradation is by the UV/S2O82- method (k = 0.0806 s^-1) at pH=4, while the slowest is during pure photolysis (k = 0.0076 s^-1) at pH=7. The fastest degradation of SCN- is by the UV/S2O82- method (k = 0.027 s^-1) at pH=4, while the slowest is during pure photolysis (k = 0.0041 s^-1) at pH=10. The stated removal processes of CN- and SCN- contain first order reactions, that are actually considered to be pseudo-first order due to the large number of radical reactions
L'ischemie myocardique : analyse terminologique et traduction d'un article scientifique
No full textOvaj diplomski rad se bavi terminološkom analizom i prijevodom znanstvenog članka o ishemiji miokarda. Podijeljen je na dva glavna dijela : teorijski i praktični dio. U teorijskom dijelu definirana je terminologija i njeni ključni pojmovi, opisan je razvoj kardiologije, obrađeni su članci koji govore o problematici medicinske terminologije, te su navedena referentna djela koja su nam pomogla u prijevodu i stvaranju konceptualnih prikaza. Praktični dio obuhvaća prijevod stručnog teksta, komentar na prijevod, 10 terminoloških kartica, te glosar s francuskim i hrvatskim terminima.Ce mémoire de master porte sur l'analyse terminologique et la traduction d'un article scientifique sur l'ischémie myocardique. Il est divisé en deux parties principales : une partie théorique et une partie pratique. Dans la partie théorique, nous définissons la terminologie et ses concepts clés, nous décrivons l’évolution de la cardiologie, nous examinons des articles qui traitent des problèmes de la terminologie médicale et nous citons les ouvrages de référence qui nous ont aidés dans la traduction et la création de représentations conceptuelles. La partie pratique comprend la traduction du texte spécialisé, un commentaire sur la traduction, 10 fiches terminologiques, et un glossaire avec des termes français et leurs équivalents croates
Uloga optineurina u fagocitozi fibrilarnog amiloida b u makrofagima i dendritičkim stanicama proizvedenim iz koštane srži
No full textOptineurin is a multifunctional ubiquitin-binding adaptor protein involved in
regulation of cellular processes, for instance vesicle trafficking, autophagy
and inflammatory signalling. The mutations of the OPTN gene that encodes
for optineurin were linked to several neurodegenerative diseases, the most
important being amyotrophic lateral sclerosis (ALS), which leads to
destruction of motor neurons. Research has shown that phagocytosis, a
process of internalizing and degrading particles larger than 0.5 µm in
diameter, is disrupted in various neurodegenerative diseases, including ALS
and Alzheimer’s disease (AD). Previous research in our lab showed no effect
of optineurin during phagocytosis of dead neurons and pHrodo™ Green E.
coli BioParticles™. The main goal of this thesis was to investigate the role
optineurin has in phagocytosis of fibrillar amyloid beta (A-beta) . To investigate this,
we performed a flow cytometry analysis of the phagocytosis assay of
fluorescent A-beta in wild type (WT) and optineurin truncation Optn470T-carrying
bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs).
Optn470T mice mimic the C-terminal truncations found in a small percentage
of ALS patients. Our results showed no difference between BMDM genotypes
in the number of phagocytic cells, but there was a decrease of ~20% in the
phagocytic capacity of Optn470T cells compared to WT BMDMs. There was
also a very significant difference between WT and Optn470T BMDC in both
the number of phagocytosing cells and their phagocytic ability. Optn470T
BMDCs had a ~15-20% lower number of phagocytosing cells than WT
BMDCs. Their phagocytic ability was ~30-40% lower than that of WT cells.
The differences between the genotypes did not significantly increase if the
cells were pretreated with inflammatory stimuli. In conclusion, this study
demonstrates that optineurin has a role in phagocytosis of fibrillar fA-beta in
BMDCs, opening a possibility that this is the mechanism by which optineurin
contributes to disease pathogenesis.Optineurin je multifunkcionalni adapterski protein koji veže ubikvitin.
Uključen je u regulaciju staničnih procesa kao što je upalna signalizacija,
promet vezikulama i autofagija. Mutacije u OPTN genu koji kodira za
optineurin povezane su s nekoliko neurodegenerativnih bolesti, od kojih je
najvažnija amiotrofična lateralna skleroza (ALS), koja dovodi do odumiranja
motornih neurona. Istraživanja su pokazala da je fagocitoza, proces
internalizacije i razgradnje čestica većih od 0,5 µm u promjeru, poremećena
u raznim neurodegenerativnim bolestima uključujući ALS i Alzheimerovu
bolest (AD). Prethodna istraživanja u našem laboratoriju nisu pokazala
nikakav učinak optineurina tijekom fagocitoze mrtvih neurona i pHrodo™
Green E. coli BioParticles™. Cilj ovog diplomskog rada bio je istražiti ulogu
optineurina u fagocitozi fibrilarnog amiloida beta (A-beta). U tu smo svrhu proveli
analizu fagocitoze fluorescentnog A-beta na stanicama divljeg tipa (WT) i
stanicama s trunkacijom optineurina (Optn470T) na temelju protočne
citometrije. Korišteni su makrofagi (BMDM) i dendritičke stanicame (BMDC)
porijeklom iz koštane srži. Optn470T miševi oponašaju skraćenja C-terminala
pronađena u maloj podskupini pacijenata s ALS-om. Naši rezultati nisu
pokazali razliku između genotipova BMDM u broju fagocitnih stanica, ali
došlo je do smanjenja od ~20% u fagocitnom kapacitetu Optn470T stanica u
usporedbi s WT BMDM-ovima. Nadalje, postojala je značajna razlika između
WT i Optn470T BMDC-a u broju fagocitoznih stanica i njihovoj fagocitnoj
sposobnosti. Optn470T BMDC-i imali su ~15-20% niži broj stanica koje
fagocitiraju od WT BMDC. Njihova fagocitna sposobnost bila je ~30-40%
niža nego kod WT stanica. Razlike između genotipova nisu se značajno
povećale ako su stanice prethodno tretirane upalnim podražajima.
Zaključno, ova studija pokazuje da optineurin ima ulogu u fagocitozi
fibrilarnog fA-beta u BMDC, otvarajući mogućnost da je to mehanizam kojim
optineurin doprinosi patogenezi bolesti
Doprinos mišićno-intrinzične toksičnosti ALS-mutiranog FUS-a motornoj neurodegeneraciji u ALS-u
No full textAmyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by loss of motor neurons. Most commonly exhibited symptoms include muscular weakness and atrophy. Clinical manifestations begin in adult age and the disease progresses until eventually, death occurs due to respiratory system failure, on average around 3-5 years after clinical onset. The majority of ALS cases are sporadic, with no previously recorded family history of the disease, while a minor proportion of cases are familial. Genes most commonly implicated in ALS are C9orf72, SOD1, FUS and TDP-43. FUS is an RNA/DNA binding protein involved in multiple cellular processes, including transcription regulation, splicing, RNA transport and degradation. Most ALS mutations in FUS are found in the nuclear localization signal, a domain responsible for import of FUS into the nucleus. Mutations in the NLS disrupt FUS import into the nucleus and cause FUS to aggregate within the cytoplasm. ALS is considered to be a distal axonopathy, and the dying-back hypothesis proposes that it originates in the neuromuscular junctions and progresses retrogradely, implicating the involvement of skeletal muscle in disease pathogenesis. The FusΔNLS mutant mice are an adequate model for FUS-ALS as their mutation mimicks those in ALS patients by preventing the transcription of the NLS. The mutation can be fully reversed to wild-type, and its reversal in just the skeletal muscle cells allows the study of non-cell-autonomous contribution of muscle tissue to ALS. FusΔNLS mice exhibit mild but progressive motor deficits, motor axon loss as well as muscular atrophy. Number and area of muscle fibers are slightly reduced, and signs of denervation in motor neurons are also present, as indicated by the reduction in axon numbers. Reversal of the mutation in skeletal muscle partially rescues some phenotypes. The findings suggest that ALS does indeed arise at least in part due to non-cell-autonomous mechanisms of skeletal muscle.Amiotrofična lateralna skleroza (ALS) je progresivna neurodegenerativna bolest karakterizirana gubitkom motornih neurona. Najčešći simptomi uključuju slabost i atrofiju mišića. Kliničke manifestacije započinju u odrasloj dobi i bolest napreduje dok naposljetku ne nastupi smrt uslijed zatajenja respiratornog sustava, u prosjeku 3-5 godina nakon nastupanja kliničkih znakova. Većina slučajeva ALS-a jest sporadično, bez prethodno zabilježene obiteljske povijesti bolesti, s manjim udjelom familijarnih slučajeva. Najčešći geni koji imaju ulogu u ALS-u su C9orf72, SOD1, FUS i TDP-43. FUS je protein koji veže RNA i DNA, i koji je uključen u mnoge stanične procese, poput regulacije transkripcije, „splicinga“, te transporta i razgradnje RNA. Većina ALS mutacija u FUS genu događa se u nuklearnom lokalizacijskom signalu, domeni odgovornoj za unos FUS-a u jezgru. Mutacije u NLS-u ometaju unos FUS-a u jezgru i uzrokuju agregaciju FUS-a unutar citoplazme. ALS se smatra distalnom aksonopatijom, i „dying-back“ hipoteza postulira da bolest započinje u neuromuskularnim spojevima te da napreduje retrogradno, sugerirajući na upletenost skeletnih mišića u patogenezi bolesti. FusΔNLS miševi su prikladan model za FUS-ALS budući da njihova mutacija oponaša mutacije zapažene kod ALS pacijenata time što sprječava transkripciju NLS-a. Mutacija se može u potpunosti poništiti preobraćanjem na divlji genotip, a preobraćanje mutacije samo u stanicama skeletnih mišića omogućava proučavanje ne-stanično-autonomnog doprinosa mišićnog tkiva ALS-u. FusΔNLS miševi iskazuju blage ali progresivne motorne defekte, gubitak motornih aksona, kao i atrofiju mišića. Broj i povšina mišićnih vlakana su blago umanjeni, i znakovi denervacije u motornim neuronima su također prisutni, što je dokazano smanjenjem broja aksona. Preobraćanje mutacije u skeletnim mišićima djelomično spašava neke fenotipe. Ova otkrića sugeriraju da se ALS razvija barem djelomično zbog ne-stanično-autonomnih mehanizama skeletnih mišića