Mutações na enzima Cu,Zn-superóxido dismutase (SOD1) estão associadas a casos familiares de Esclerose Lateral Amiotrófica (ELA), uma doença neurodegenerativa motora fatal. Entretanto, a toxicidade das SOD1s mutantes não está totalmente compreendida. Sabe-se que o desenvolvimento da doença está associado ao acúmulo de lesões oxidativas em biomoléculas, mas o papel da SOD1 neste processo não está claro. Estudos com sistemas modelo são, ainda, necessários para desvendar os mecanismos envolvidos. Para contribuir na compreensão dos mecanismos de danos em DNA promovidos pela SOD1, foram realizados estudos in vitro com SOD1/H2O2/HCO3-, e estudos com neuroblastomas em cultura transfectados com SOD1 mutante G93A, característica de ELA. Através da quantificação de quebras em DNA plasmidial e dos níveis de 8-oxo-7,8-dihidro-2´-desoxiguanosina (8-oxodGuo) e 1,N2-eteno-2\'-desoxiguanosina (1,N2-εdGuo) em DNA de timo de bezerro, concluiu-se que o cobre liberado da SOD1 tem um papel central na formação de lesões no DNA promovidas pela SOD1 na presença de H2O2, e que o bicarbonato pode modular a reatividade do cobre liberado. Níveis aumentados de quebras no DNA, 8-oxodGuo e 1,N2-εdGuo foram encontrados nos neuroblastomas transfectados com SOD1 G93A. Maior atividade de p53 foi também observada nestas células, indicando que o acúmulo de lesões no DNA pode desencadear o processo de apoptose neste modelo celular de ELA. Observou-se que a SOD1 pode estar associada à cromatina, e que a SOD1 G93A possui maior afinidade pelo DNA e maior atividade peroxidásica no núcleo. Estes resultados indicam que as lesões no DNA observadas no modelo celular de ELA podem ser diretamente promovidas pela SOD1 mutante. / Mutations in the gene encoding Cu,Zn-superoxide dismutase (SOD1) have been linked to familial Amyotrophic Lateral Sclerosis (ALS), a fatal motor neuron disease. However, the toxicity of mutant SOD1s was not completely understood. It is known that the development of the disease is associated with oxidative damage to biomolecules, but the role of SOD1 in this process is not clear. Model studies are still necessary to reveal the mechanisms involved. To understand the mechanism of DNA damage promoted by SOD1, in vitro studies with SOD1/H2O2/HCO3-, and studies with neuroblastoma cells transfected with the G93A ALS-mutant SOD1, were performed. Through the quantification of strand breaks in plasmid DNA and the quantification of 8-oxo-7,8-dihydro-2´-deoxyguanosine (8-oxodGuo) and 1,N2-etheno-2\'-deoxyguanosine (1,N2-εdGuo) levels in calf thymus DNA, it was concluded that copper liberated from SOD1 has a central role in DNA damage promoted by SOD1 in the presence of H2O2, and that bicarbonate can modulate the reactivity of released copper. Increased levels of DNA strand breaks, 8-oxodGuo and 1,N2-εdGuo were found in neuroblastoma cells transfected with G93A SOD1. Increased p53 activity was also observed in these cells, indicating that accumulation of DNA damage can lead to apoptosis in this ALS cellular model. Western blot analysis showed that G93A SOD1 is present in the nucleus, being associated to the DNA. Nuclear G93A SOD1 has identical superoxide dismutase-activity but displays increased peroxidase activity, when compared to wild-type. These results indicate that DNA damage observed in this ALS cellular model may be directly promoted by mutant SOD1.

Introduction: Amyotrophic lateral sclerosis (ALS) is a deadly, progressive neuromuscular disease that affects individuals all over the world. About 10% of the patients have a familial predisposition (FALS) while the remainder of cases are isolated or sporadic (SALS) and of unknown cause. To date, the principal recognized risk factors for ALS are higher age, male gender, slim figure (BMI<23) and a family history of ALS. In 1993, Rosen et al. observed that some FALS cases were associated with mutations in the gene encoding the CuZn superoxide dismutase enzyme (SOD1). Since then, several mutations in the SOD1 gene have been discovered, and mutations in more than 18 other genes have been associated with causing ALS. The aim of this thesis was to identify new mutations associated with ALS pathogenesis, and by comparing patients from different countries, were we also able to identify population-specific genetic variations. The studies are referred to as I–V. Methods: With written informed consent and adhering to the tenets of the Declaration of Helsinki, through a national network of ALS clinicians´, venous blood samples were collected from ALS patients and healthy subjects in Europe and the USA. The patients were diagnosed according to the El Escorial criteria, and as having FALS according to the criteria of Byrne et al. (2011). The DNA variations were amplified by various PCR techniques. (I, III and IV) The amplicons of ataxin 2 (ATXN2), profilin 1 (PFN1), and vesicle-associated membrane protein type B (VAPB) were characterised by direct sequencing. (II) After quantitative PCR, a genotype-phenotype correlation was performed to assess whether the survival motor neuron gene (SMN) modulates the phenotype of ALS. (V) The amplicons of the 50 base pair deletion in the SOD1 promotor (50 bp) were separated by electrophoresis on agarose. Results: (I) We observed a significant association between CAG expansions in the ATXN2 gene and ALS in a European cohort. (II) Abnormal copy number of the SMN1 gene was identified as a risk factor in France, but not in Sweden. Homozygosity of the SMN2 deletion prolonged survival among Swedish ALS patients, compared to French patients. (III) We identified two mutations in the PFN1 gene, the novel p.Thr109Met mutation and the p.Gln117Gly mutation, in two unrelated FALS patients. (IV) In our cohort, we identified five VAPB mutations p.Asp130Glu, p.Ser160del, p.Asp162Glu, p.Met170Ile, and p.Arg184Trp, two of which are novel. (V) The 50 bp deletion upstream of the SOD1 gene was found in equal frequencies in both the patient and control cohorts. The 50 bp deletion did not affect SOD1 enzymatic activity. Furthermore, we found no differences in age of onset or disease duration in relation to the 50 bp deletion genotype.VI Conclusions: (I) Our findings indicate that ATXN2 plays an important role in the pathogenesis of ALS, and that CAG expansions in ATXN2 are a significant risk factor for the disease. (II) We suggest that abnormal SMN1 gene copynumber cannot be considered a universal genetic susceptibility factor for ALS. We also propose that the effect of abnormal SMN2 gene copy number on ALS phenotype may differ between populations. (III) This work provides evidence that PFN1 mutations can cause ALS as a Mendelian dominant trait. The novel p.Thr109Met mutation also shows that disturbance of actin dynamics can cause motor neuron degeneration. (IV) We find it unlikely that the VAPB mutations cause ALS in our cohorts. (V) We find it unlikely that the 50 bp region contains important regulatory elements for SOD1 expression. This thesis supports the theory that ALS is a multigenetic disease, but there appears to be great genetic variation among apparently identical populations. These studies emphasise the importance of continuous genetic screening, to identify further mutations and genes involved in ALS disease, but it also highlights the importance of cooperation and comparison between countries. / On the aetiology of ALS: A comprehensive genetic study

Protein misfolding and aggregation underlie several neurodegenerative proteinopathies including amyotrophic lateral sclerosis (ALS). Superoxide dismutase 1 (SOD1) was the first gene found to be associated with familial ALS. Overexpression of human mutant or wild type SOD1 in transgenic mouse models induces motor neuron (MN) degeneration and an ALS-like phenotype. SOD1 mutations, leading to the destabilization of the SOD1 protein is associated with ALS pathogenesis. However, how misfolded SOD1 toxicity specifically affects human MNs is not clear. The aim of this thesis was to develop patient-derived, cellular models of ALS to help understand the pathogenic mechanisms underlying SOD1. To understand which cellular pathways impact on the level of misfolded SOD1 in human cells, we established a model using patient-derived fibroblasts and quantified misfolded SOD1 in relation to disturbances in several ALS-related cellular pathways. Misfolded SOD1 levels did not change following reduction in autophagy, inhibition of the mitochondrial respiratory chain, or induction of endoplasmic reticulum (ER)-stress. However, inhibition of the ubiquitin-proteasome system (UPS) lead to a dramatic increase in misfolded SOD1 levels. Hence, an age-related decline in proteasome activity might underlie the late-life onset that is typically seen in SOD1 ALS. To address whether or not SOD1 misfolding is enhanced in human MNs, we used mixed MN/astrocyte cultures (MNCs) generated in vitro from patient-specific induced pluripotent stem cells (iPSCs). Levels of soluble misfolded SOD1 were increased in MNCs as well as in pure iPSC-derived astrocytes compared to other cell types, including sensory neuron cultures. Interestingly, this was the case for both mutant and wild type human SOD1, although the increase was enhanced in SOD1 FALS MNCs. Misfolded SOD1 was also found to exist in the same form as in mouse SOD1 overexpression models and was identified as a substrate for 20S proteasome degradation. Hence, the vulnerability of motor areas to ALS could be explained by increased SOD1 misfolding, specifically in MNs and astrocytes. To investigate factors that might promote SOD1 misfolding, we focussed on the stability of SOD1 mediated by a crucial, stabilizing C57-C146 disulphide bond and its redox status. Formation of disulphide bond is dependent on oxidation by O2 and catalysed by CCS. To investigate whether low O2 tension affects the stability of SOD1 in vitro we cultured fibroblasts and iPSC-derived MNCs under different oxygen tensions. Low oxygen tension promoted disulphide-reduction, SOD1 misfolding and aggregation. This response was much greater in MNCs compared to fibroblasts, suggesting that MNs may be especially sensitive to low oxygen tension and areas with low oxygen supply could serve as foci for ALS initiation. SOD1 truncation mutations often lack C146, and cannot adopt a native fold and are rapidly degraded. We characterized soluble misfolded and aggregated SOD1 in patient-derived cells carrying a novel SOD1 D96Mfs*8 mutation as well as in cells fom an unaffected mutation carrier. The truncated protein has a C-terminal fusion of seven non-native amino acids and was found to be extremely prone to aggregation in vitro. Since not all mutation carriers develop ALS, our results suggested this novel mutation is associated with reduced penetrance. In summary, patient derived cells are useful models to study factors affecting SOD1 misfolded and aggregation. We show for the first time that misfolding of a disordered and disease associated protein is enhanced in disease-related cell types. Showing that misfolded SOD1 exists in human cells in the same form as in transgenic mouse models strengthens the translatability of results obtained in the two species. Our results demonstrate disulphide-reduction and misfolding/aggregation of SOD1 and suggest that 20S proteasome could be an important therapeutic target for early stages of disease. This model provides a great opportunity to study pathogenic mechanisms of both familial and sporadic ALS in patient-derived models of ALS. / Varje år insjuknar omkring 5300 personer i världen i motorneuronsjukdomen Amyotrofisk lateralskleros (ALS). Sjukdomen kännetecknas av degeneration av motorneuron i hjärnan och ryggmärgen, de nervceller som styr kroppens muskler, vilket leder till musklerförtvining och gradvis förlamning. ALS-patienter avlider oftast till följd av andningssvikt när sjukdomen når andningsmuskulaturen. I de allra flesta fall uppkommer ALS sporadiskt (SALS), det vill säga utan känd genetisk orsak, medan ärftliga fall (FALS) drabbar omkring 10 % och beror på mutationer i ett antal kända gener. Upp till 6 % av alla ALS fall kan härledas till mutationer i genen superoxid dismutas 1 (SOD1). SOD1 är ett enzym som ansvarar för att omvandla och oskadliggöra fria syreradikaler som bildas vid normal ämnesomsättning. 206 olika SOD1 mutationer har identifierats, alla orsakar inte ALS men många leder till att den tredimensionella proteinstrukturen förändras, vilket ökar proteinets benägenhet att felveckas. Initialt trodde man att SOD1 mutationer förhindrade proteinets normalfunktion och följaktligen orsakade ALS. Studier har emellertid visat att den enzymatiska funktionen ofta bevaras, även hos muterade proteiner. Däremot kan små mängder felveckat SOD1 störa andra viktiga cellulära funktioner. Felveckat SOD1 har en benägenhet att klumpa ihop sig och bilda aggregat i det centrala nervsystemet (CNS). Dessa aggregat återfinns hos patienter med såväl FALS som SALS vilket tyder på att även vildtyps-SOD1 kan felveckas och vara involverat i sjukdomsutvecklingen. De flesta studier är baserade på transgena musmodeller som uttrycker extremt stora mängder av muterat humant SOD1. Det är dock oklart hur väl studier i möss överensstämmer med sjukdomsutvecklingen hos ALS-patienter, där mängden SOD1 är betydligt lägre. En central fråga som fortfarande står obesvarad är varför just motorneuron degenererar i ALS, trots att SOD1 uttrycks i alla kroppens celler. Det övergripande syftet med den här avhandlingen har varit att karakterisera felveckat SOD1 i patientceller för att studera dess roll i ALSrelaterade sjukdomsmekanismer med fysiologiskt relevanta nivåer av SOD1. Samtliga studier är gjorda in vitro med celler från friska donatorer med vildtyps-SOD1, celler från patienter med SOD1-FALS, FALS som bär andra ALS-associerade gener, samt SALS. I de allra flesta fallen har vi analyserat både lösligt felveckat SOD1 samt aggregerade former av SOD1 proteinet.

Mutações na enzima Cu,Zn-superóxido dismutase (SOD1) estão associadas a casos familiares de Esclerose Lateral Amiotrófica (ELA), uma doença neurodegenerativa motora fatal. Entretanto, a toxicidade das SOD1s mutantes não está totalmente compreendida. Sabe-se que o desenvolvimento da doença está associado ao acúmulo de lesões oxidativas em biomoléculas, mas o papel da SOD1 neste processo não está claro. Estudos com sistemas modelo são, ainda, necessários para desvendar os mecanismos envolvidos. Para contribuir na compreensão dos mecanismos de danos em DNA promovidos pela SOD1, foram realizados estudos in vitro com SOD1/H2O2/HCO3-, e estudos com neuroblastomas em cultura transfectados com SOD1 mutante G93A, característica de ELA. Através da quantificação de quebras em DNA plasmidial e dos níveis de 8-oxo-7,8-dihidro-2´-desoxiguanosina (8-oxodGuo) e 1,N2-eteno-2\'-desoxiguanosina (1,N2-εdGuo) em DNA de timo de bezerro, concluiu-se que o cobre liberado da SOD1 tem um papel central na formação de lesões no DNA promovidas pela SOD1 na presença de H2O2, e que o bicarbonato pode modular a reatividade do cobre liberado. Níveis aumentados de quebras no DNA, 8-oxodGuo e 1,N2-εdGuo foram encontrados nos neuroblastomas transfectados com SOD1 G93A. Maior atividade de p53 foi também observada nestas células, indicando que o acúmulo de lesões no DNA pode desencadear o processo de apoptose neste modelo celular de ELA. Observou-se que a SOD1 pode estar associada à cromatina, e que a SOD1 G93A possui maior afinidade pelo DNA e maior atividade peroxidásica no núcleo. Estes resultados indicam que as lesões no DNA observadas no modelo celular de ELA podem ser diretamente promovidas pela SOD1 mutante. / Mutations in the gene encoding Cu,Zn-superoxide dismutase (SOD1) have been linked to familial Amyotrophic Lateral Sclerosis (ALS), a fatal motor neuron disease. However, the toxicity of mutant SOD1s was not completely understood. It is known that the development of the disease is associated with oxidative damage to biomolecules, but the role of SOD1 in this process is not clear. Model studies are still necessary to reveal the mechanisms involved. To understand the mechanism of DNA damage promoted by SOD1, in vitro studies with SOD1/H2O2/HCO3-, and studies with neuroblastoma cells transfected with the G93A ALS-mutant SOD1, were performed. Through the quantification of strand breaks in plasmid DNA and the quantification of 8-oxo-7,8-dihydro-2´-deoxyguanosine (8-oxodGuo) and 1,N2-etheno-2\'-deoxyguanosine (1,N2-εdGuo) levels in calf thymus DNA, it was concluded that copper liberated from SOD1 has a central role in DNA damage promoted by SOD1 in the presence of H2O2, and that bicarbonate can modulate the reactivity of released copper. Increased levels of DNA strand breaks, 8-oxodGuo and 1,N2-εdGuo were found in neuroblastoma cells transfected with G93A SOD1. Increased p53 activity was also observed in these cells, indicating that accumulation of DNA damage can lead to apoptosis in this ALS cellular model. Western blot analysis showed that G93A SOD1 is present in the nucleus, being associated to the DNA. Nuclear G93A SOD1 has identical superoxide dismutase-activity but displays increased peroxidase activity, when compared to wild-type. These results indicate that DNA damage observed in this ALS cellular model may be directly promoted by mutant SOD1.

<p>Akrilamid&nbsp; je toksična hemijska supst anca koja je već dugi niz godina prisutna u životnoj sredini,&nbsp; jer se kao važan monomer koristi u različite industrijske i laboratorijske svrhe. U poslednjih petnaest godina, akrilamid je postao posebno zanimljiv za &scaron;ire naučne krugove jer&nbsp; se pokazalo da&nbsp; se&nbsp; nalazi&nbsp; i u&nbsp; hrani&nbsp; biljnog porekla, posebno hrani bogatoj skrobom, koja se priprema pečenjem ili prženjem na temperaturama vi&scaron;im od 120&deg;C.&nbsp; Do sada ustanovljeni negativni zdravstveni efekti akrilamida su veoma raznovrsni i mogu biti rezultat delovanja samog&nbsp; akrilamida ili delovanja njegovog metabolita glicidamida koji nastaje&nbsp; in vivo&nbsp; kada se jedan deo molekula akrilamida metaboli&scaron;e oksigenacijom dvostruke veze pomoću enzima citohrom P450 2E1 (CYP2E1). Akrilamid je supstanca koja ima dokazan negativan efekat&nbsp; na organske sisteme kod ljudi i životinja, i koja je svrstana u moguće humane karcinogene. Negativan efekat akrilamida na egzokrini pankreas je poznat, ali o mogućim efektima akrilamida na endokrini pankreas se i dalje veoma malo zna. Ima puno dokaza koji&nbsp; ukazuju na to da akrilamid ima citotoksični efekat koji se&nbsp; manifestuje kroz uticaj na redoks-status ćelija i dovodi do promena u vrednostima biomarkera oksidativnog i nitrozativnog stresa, kao i u aktivnosti antioksidativnih enzima. Pankreas&nbsp; je&nbsp; jedan od ciljnih&nbsp; organa za delovanje akrilamida te je&nbsp; glavni predmet istraživanja&nbsp; doktorske teze&nbsp; bio proučavanje potencijalnog efekta akrilamida na endokrini pankreas pacova.&nbsp; Ispitivanje je vr&scaron;eno na 3&nbsp; eksperimentalne grupe&nbsp; juvenilnih&nbsp; mužjaka pacova soja Wistar,&nbsp; od kojih je&nbsp; jedna grupa bila kontrolna, dok su dve bile tretirane&nbsp; sa akrilamidom u dozama od 25 mg/kg tm i 50 mg/kg tm,&nbsp; 5 dana nedeljno,&nbsp; tokom 3 nedelje. Po isteku tretmana,&nbsp; nakon dekapitacije, kompletno tkivo pankreasa&nbsp; je&nbsp; fiksirano u 10% rastvoru formalina&nbsp; tokom&nbsp; 24&nbsp; h i obrađeno prema&nbsp; standardnoj proceduri za kalupljenje u parafinu.&nbsp; Parafinski kalupi su sečeni na serijske preseke debljine 5 &micro;m, nakon čega su bojeni&nbsp; histohemijskom i imunohistohemijskim metodama.&nbsp; Kod eksperimentalnih grupa posmatrane&nbsp; su&nbsp; histolo&scaron;ke promene na endokrinom pankreasu, sa akcentom na &alpha;-&nbsp; i &beta;-ćelije.&nbsp; Takođe, posmatrana je&nbsp; i&nbsp; ekspresija&nbsp; hormona insulina i glukagona, enzima inducibilne azot -oksi d&nbsp; sintetaze (iNOS) i&nbsp; CYP2E1,&nbsp; kao&nbsp; i ekspresija&nbsp;&nbsp; antioksidativnih enzima&nbsp; katalaza&nbsp; (CAT) i superoksid dismut aza 1 i 2&nbsp; (SOD1 i SOD2)&nbsp; u ćelijama Langerhansovih ostrvaca. Potencijalna promena u funkcionalnosti &beta;-ćelija je ispitana i kroz analizu nivoa glukoze u serumu pacova tretiranih sa akrilamidom.<br />Budući da &beta;-ćelije čine 80% ćelija koje grade Langerhansova ostrvca pankreasa,&nbsp; pored in vivo&nbsp; eksperimenata, ispitana&nbsp; je&nbsp; i toksičnost akrilamida na&nbsp; Rin-5F ćelijsku liniju insulinoma &beta;-ćelija pacova u in vitro uslovima. Glavni cilj in vitro&nbsp; istraživanja je bio&nbsp; da se&nbsp; ispita&nbsp; uticaj&nbsp; rastućih&nbsp; koncentracija akrilamida na preživljavanje tretiranih&nbsp; Rin-5F&nbsp; ćelija, ali i efekat IC₅₀&nbsp; koncentracije ove supstance primenjene&nbsp; tokom&nbsp; različitih vremenskih intervala&nbsp; (0,5, 1, 3, 6, 12 i 24 h)&nbsp; na pojavu oksidativnog i nitrozativnog stresa. Redoks-status Rin-5F ćelija tretiranih&nbsp; sa akrilamidom je ispitan preko analize prisustva biomarkera oksidativnog i nitrozativnog stresa, akrivnosti CAT i ukupne SOD, kao i promene u ekspresiji gena za CAT, SOD1, SOD2&nbsp;&nbsp; i iNOS.&nbsp; Pored toga, analiziran je i efekat istog tretmana na&nbsp; ekspresiju gena za insulin, CYP2E1, Bax i Bcl-2. U okviru teze je pokazano da akrilamid ne dovodi do&nbsp; značajnih promena u histolo&scaron;koj građi, dijametru i broju Langerhansovih ostrvaca&nbsp; kod&nbsp; tretiranih životinja.&nbsp; Primena stereolo&scaron;kih metoda&nbsp; je&nbsp; ukazala&nbsp; na mikrostrukturne promene na&nbsp; endokrinom pankreasu na nivou &alpha;-&nbsp; i &beta;-ćelija. U ovoj tezi je po prvi put pokazano da tretman akrilamidom negativno utiče na broj i povr&scaron;inu &beta;-ćelija pankreasa.&nbsp; U tezi je, takođe,&nbsp; pokazan&nbsp; značajan dozno-zavisni pad u prisustvu insulina u &beta;-ćelijama&nbsp;&nbsp; pankreasa. Uprkos&nbsp; tome, kod&nbsp; akrilamidom tretiranih&nbsp; životinja&nbsp; nije konstatovana&nbsp; promena&nbsp; u&nbsp; koncentraciji serumske glukoze.&nbsp; U&nbsp; ovoj tezi je pokazano da tretman akrilamidom dovodi do&nbsp;&nbsp; statistički značajnog porasta&nbsp; u broju &alpha;-ćelija&nbsp; kod životinja koje su primale nižu dozu tretmana, dok se&nbsp; broj &alpha;-ćelija&nbsp; kod životinja koje su primale vi&scaron;u dozu tretmana&nbsp; ne razlikuje značajno od kontrole.&nbsp; Tretman akrilamidom je doveo do značajnog&nbsp; porasta u količini&nbsp;&nbsp; prisutnog glukagona&nbsp; u &alpha;-ćelijama pankreasa.<br />Tretman akrilamidom nije doveo do značajne promene u ekspresiji CAT, SOD1 i SOD2 u ćelijama Langerhansovih ostrvaca.&nbsp; Kod&nbsp; tretiranih životinja&nbsp; do&scaron;lo do značajnog dozno-zavisnog porasta&nbsp; u ekspresiji&nbsp; enzima iNOS,&nbsp; dok je ekspresija&nbsp; CYP2E1 značajno dozno-zavisno opala&nbsp; nakon tretmana. U&nbsp; tezi je pokazano da tretman akrilamidom negativno utiče na vijabilnost Rin-5F ćelija, i utvrđeno je da IC50&nbsp; koncentracija akrilamida za Rin-5F ćelije iznosi 10 mM.&nbsp; Rezultati teze pokazuju da tretman akrilamidom u IC₅₀&nbsp; koncentraciji u Rin-5F ćelijskoj liniji značajno povećava nivo malondialdehida (MDA) nakon tretmana u trajanju od 1, 12 i 24 h.&nbsp; Isti tretman&nbsp; značajno smanjuje nivo redukovanog GSH nakon tretmana od 1, 3, 6, 12 i<br />24 h, kao i nivo slobodnih&nbsp; &ndash;SH grupa nakon tretmana od 3 i 6 h. Tretman akrilamidom u IC_50&nbsp; koncentraciji signifikantno pojačava aktivnost CAT nakon tretmana od 1 h, dok tretman u trajanju od 12 h značajno smanjuje aktivnost ovog enzima. Ovaj tretman smanjuje aktivnost SOD nakon 1, 12 i 24 h, dok&nbsp; tretman u trajanju od 6 h značajno pojačava aktivnost enzima SOD.&nbsp; U tezi je, takođe, pokazan i veoma značajan porast&nbsp; u nivou prisutnih nitrita,&nbsp; koji&nbsp; je direktno proporcionalan&nbsp; sa nivoom azot-oksida i nivoom akivnosti enzima iNOS.&nbsp; Ovaj&nbsp; nalaz ukazuje na potencijalnu pojavu nitrozati vnog stresa u akrilamidom-tretiranim Rin-5F ćelijama.&nbsp; U&nbsp; tezi je po prvi put pokazano da tretman&nbsp; akrilamidom dovodi do&nbsp; značajnih&nbsp; varijacija&nbsp; u transkripciji gena za iNOS, SOD1, SOD2,&nbsp; CAT,&nbsp; CYP2E1,&nbsp; Bax i Bcl-2 u tretiranim Rin-5F ćelijama, dok isti tretman ne dovodi do&nbsp; promene nivoa&nbsp; transkripcije gena za insulin.&nbsp; Tretman akrilamidom u koncentraciji od 10<br />mM tokom rastućih vremenskih perioda dovodi do porasta u relativnoj količini iRNK<br />gena za iNOS u svim tačkama tretmana, do porasta&nbsp; nivoa&nbsp; iRNK za SOD1 i SOD2 nakon tretmana od 12 i 24 h, kao i do porasta&nbsp; količine&nbsp; iRNK za CAT nakon tretmana od 3 h.&nbsp; U&nbsp; tezi je pokazano&nbsp; i&nbsp; da akrilamid&nbsp; izaziva&nbsp; promene&nbsp; u sintezi&nbsp; iRNK&nbsp; za enzim&nbsp; CYP2E1&nbsp; koji je&nbsp; posebno značajan u kontekstu detoksikacije ove toksične supstance.&nbsp; Porast u transkripciji gena za&nbsp; CYP2E1&nbsp; je uočen&nbsp; nakon tretmana u trajanju od 0,5 i 1 h, dok je&nbsp; do smanjenja transkripcije&nbsp; do&scaron;lo&nbsp; nakon tretmana od 12&nbsp; i 24&nbsp; h.&nbsp; Tretman akrilamidom u koncentraciji od&nbsp; 10 mM tokom rastućih vremenskih perioda dovodi do porasta u relativnoj količini iRNK&nbsp; gena za Bax u svim tačkama tretmana, i do porasta u transkripciji gena za Bcl-2 nakon tretmana od 0,5, 1 i 3 h.<br />Sumirajući&nbsp; sve&nbsp; rezultate&nbsp; ove teze,&nbsp; moze se zaključiti&nbsp; da je endokrini pankreas&nbsp; jedno od&nbsp; ciljnih tkiva, na koje akrilamid ostvaruje vi&scaron;estruki negativni uticaj.</p> / <p>Acrylamide is a toxic chemical used as an important monomer for various industrial and laboratory purposes, which makes it highly present in the environment. In the last fifteen years, acrylamide has become especially interesting for wider scientific circles when it was found in staple foodstuff rich in starch, prepared at temperatures higher than 120&deg;C. The established negative health effects of acrylamide are very diverse and can be the result of the acrylamide action itself or the action of its metabolite glycidamide that occurs in vivo, when acrylamide molecule is metabolized via oxygenation of the double bond by the cytochrome P450 2E1 (CYP2E1). Acrylamide is a substance with a proven adverse effect on humans and animals, and it is classified as a possible human carcinogen. The negative effect of acrylamide on the exocrine pancreas has already been recognized, but the possible effects of acrylamide&nbsp; on endocrine pancreas are still mostly undetermined. There is a significant amount of evidence to suggest that acrylamide exerts a cytotoxic effect which manifests through the changes in level of oxidative and nitrosative stress biomarkers, as well as in the activity of antioxidant enzymes. Since, pancreas is one of the target organs for acrylamide, the main subject of doctoral thesis was to investigate the potential effect of acrylamide on the rat endocrine pancreas. The investigation was conducted on 3 experimental groups of juvenile male Wistar rats, of which one group was the control group, while two groups were treated with acrylamide at doses of 25 mg/kg bw and 50 mg/kg bw, 5 days a week, during 3 weeks. After termination of the treatment, decapitation was performed, and the complete pancreatic tissue was fixed in a 10% formalin solution for 24 h and treated according to the standard paraffin embedding procedure. Paraffin molds were cut into 5 &mu;m thick serial sections, after which they were stained with histochemical and immunohistochemical methods. Histological changes ofthe endocrine pancreas, with the emphasis on &alpha;- and &beta;-cells, were examined in three experimental groups of rats. In addition, the expression of insulin and glucagon hormone, the inducible nitric oxide synthase (iNOS) and CYP2E1 enzymes, and the expression of antioxidative enzymes catalase (CAT) and superoxide dismutases 1 and 2&nbsp; (SOD1 and SOD2) in the islets of Langerhans were also investigated. A potential change in the functionality of &beta;-cells was also examined by analyzing glucose level in the serum of rats treated with acrylamide. In pancreatic islets of Langerhans the majority of cells (&gt;80%) are &beta;-cells. Therefore, in addition to in vivo experiments, the toxicity of acrylamide was examined in vitro on rat insulinoma Rin-5F cell line.The main goal of in vitro research was to investigate the impact of increasing acrylamide concentrations on the viability of treated Rin-5F cells, and also to examine whether IC50 concentration of this substance, applied at different intervals of time (0.5, 1, 3, 6, 12 and 24 h), induce oxidative and nitrosative stress. Redox-status of Rin-5F cells treated with acrylamide was examined by analyzing oxidative and nitrosative stress biomarkers, CAT and total SOD activity, as well as changes in the expression of the CAT, SOD1, SOD2 and iNOS. In addition, the effect of the same treatment on the transcription of the insulin, CYP2E1, Bax and Bcl-2 gene was analyzed.The results of the thesis showed that acrylamide treatment does not lead to significant changes in the histological structure, diameter and number of islets of Langerhans of treated animals. Application of stereological methods indicated microstructural changes of &alpha;- and &beta;-cells ofendocrine pancreas. It has been shown for the first time that treatment with acrylamide negatively affects the number and surface area of pancreatic &beta;-cells. In addition, a significant dose-dependent decline in the amount of insulin in pancreatic &beta;-cells was also demonstrated. However, no change in serum glucose level was observed in treated animals. Acrylamide treatment led to a statistically significant increase in the number of &alpha;-cells in animals receiving a lower dose of treatment, while the number of &alpha;-cells in animals receiving a higher dose of treatment did not differ significantly from the control. Treatment with acrylamide led to a significant increase in the amount of the glucagon in &alpha;-cells. Treatment with acrylamide did not cause a significant change in the expression of CAT, SOD1 and SOD2 in islets of Langerhans. However, there was a significant dosedependent increase in the&nbsp; expression of iNOS enzyme, whereas expression of CYP2E1 significantly decreased in dose-dependent manner in treated animals. Results of the thesis showed that acrylamide exerts a negative effect on the viability of Rin-5F cell line. It has been established that the IC50 concentration of acrylamide for the Rin-5F cell line is 10 mM. The results of the thesis indicate that treatment of Rin-5F cell line with IC50 concentration of acrylamide for 1, 12, and 24 h significantly increased the level of malondialdehyde (MDA). Exposure to acrylamide for 1, 3, 6, 12 and 24 h significantly decreased the level of reduced GSH, while the level of free -SH groups was reduced after 3 and 6 h of acrylamide treatments. Treatment with IC50 concentration of acrylamide significantly enhanced CAT activity after 1 h of acrylamide exposure, while 12 h exposure significantly reduced the activity of this enzyme. Application of acrylamide reduced SOD activity after 1, 12, and 24 h exposure, while 6 h exposure significantly increased the activity of SOD enzymes. Results of the thesis also showed a very significant increase of the nitrite level, which is directly proportional to the level of nitrogen oxide (NO) and the level of the iNOS activity. This finding points to the potential occurrence of nitrosative stress in acrylamide-treated Rin-5F cells. It has been shown for the first time that acrylamide treatment leads to significant variations in transcription of iNOS, SOD1, SOD2, CAT, CYP2E1, Bax and Bcl-2 genes in treated Rin-5F cells, while the same treatment does not affect transcription of the insulin gene. Treatment with acrylamide at a concentration of 10 mM for increasing periods of time leads to an increase in the relative amount of the iNOS gene iRNA at all treatment points. Twelve and and 24 h of acrylamide exposure increased the transcription of the SOD1 and SOD2 genes. Transcription of CAT gene was increased after 3 h&nbsp; ofacrylamide exposure. Furthermore, it has been shown that acrylamide treatment leads to variations in the mRNA synthesis of CYP2E1 gene, which is particularly significant in the context of detoxification of this toxic substance. An increase in the transcription ofthe CYP2E1&nbsp; gene was observed after 0.5 and 1 h of acrylamide exposure, while the reduction of&nbsp; transcription occurred after 12 and 24 h of acrylamide exposure. The treatment with 10 mM acrylamide has led to an increase of the transcription of the Bax gene at all treatment points, and also to an increase of transcription of the Bcl-2 gene after of 0.5, 1, and 3 h of acrylamide exposure. Summarizing all the results of this thesis, it can be concluded that the endocrine pancreas&nbsp; is one of the target tissues of acrylamide, to which this substance exerts a multiple adverse effects.</p>

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting the upper and lower motor neurons causing muscle atrophy and paralysis followed by death. Aggregates containing superoxide dismutase (SOD1) are found as pathological hallmark in diseased ALS patients. Consequently ALS is regarded as a protein misfolding disorder like Alzheimer’s disease and Parkinson’s disease. So far, little is known about the cause and mechanism behind SOD1 aggregation but the inherent property of all polypeptide chains to form stable aggregated structures indicates that the protein misfolding diseases share a common mechanism. Our results show that SOD1 aggregation starts from the globally unfolded state, since fibrillation is fastest at full occupancy of denatured protein induced either by chemical denaturation or mutation. Even so, the fibrillation rate shows a surprisingly weak dependence on the concentration of globally unfolded SOD1 indicating fibril fragmentation as the dominant mechanism for aggregate formation. This is further supported by the observation that the SOD1 sample has to be mechanically agitated for fibrillation to occur.  Interestingly, we observe a similar SOD1 aggregation behaviour in vivo, where the survival times of ALS transgenic mice correlates with mutant stability, and aggregate growth depends weekly on the concentration of unfolded monomer. Additionally, in-cell NMR measurements reveal that in live cells the thermodynamic equilibrium is shifted towards the unfolded state of SOD1, which is also more fully extended than in vitro. This suggests that the globally unfolded aggregation competent protein is more abundant in the crowded environment in vivo than dilute in vitro conditions. Finally, antibody analysis of aggregates from ALS transgenic mice reveals the existence of aggregate strains involving different parts of the protein depending on mutation, which may offer an explanation for the various disease phenotypes observed in ALS. Altogether these findings provide important clues for understanding SOD1 aggregation with implications for ALS, as well as other protein misfolding diseases.

Many mutations in superoxide dismutase 1 (SOD1) cause destabilization and misfolding of the protein and are implicated in amyotrophic lateral sclerosis. Likewise, a few post-translational modifications (PTMs) on SOD1 have been shown to cause the same phenotype. However, relatively few PTMs on SOD1 have been studied in depth and, in particular, very few studies have demonstrated how these PTMs affect SOD1's various biological roles. SOD1 is traditionally known for its role in reactive oxygen species (ROS)-scavenging but has also been found to have a few other biological roles, including transcription factor activity to promote genomic stability, preservation of cytoskeletal activity, maintaining zinc and copper homeostasis, and suppressing respiration. We have used the computational analysis tool, SAPH-ire, to find PTM 'hotspots' on SOD1 that have a high likelihood of affecting its biological functions. Interestingly, the top seven ranked PTM 'hotspots' were found in a small region of SOD1, between S98-K128. We focused our studies on one of the PTM 'hotspots' found in this region, lysine-122 (K122). K122 is found in the electrostatic loop of SOD1, a loop that is important for shuttling in superoxide radicals to be neutralized. According to our data, and other studies, this lysine is both succinylated and acetylated. We found that acetyl and succinyl-mimetics (K122Q and K122E, respectively) of this site do not affect its ROS scavenging activity but do prevent SOD1 from suppressing respiration and decrease its localization to the mitochondria. Further, when cells are depleted of SIRT5 (the desuccinylase for K122), SOD1 can no longer suppress respiration. Additionally, we found that SOD1 appears to suppress respiration at complex I, whether directly or through an indirect pathway is unknown. When HCT116 colon cancer cells were depleted of endogenous SOD1, the overexpressed succinyl K122-mimetic (K122E) could not recover growth as well as overexpressed WT SOD1. The K122E SOD1 expressing cells also exhibited increased mitochondrial ROS and unhealthier mitochondria. We propose a mechanism whereby SOD1 suppression of respiration acts as an additional regulator of oxidative stress: SOD1 suppresses the electron transport chain to decrease reactive oxygen species leakage and to promote healthier mitochondria and growth.

<p>Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with an incidence of 1.5-2.7/100000 people/year. Today there is no cure for the disease and only symptomatic treatments are available. ALS progresses rapidly and only 50% of the patients are alive three years after the symptom debut. In ALS, the upper and lower motor neurons undergo degeneration in a process resembling apoptosis. This leads to muscle atrophy and paralysis. The causes of neuronal death are however unknown. In this thesis we have studied transgenic mice carrying human mutant superoxide dismutase, as a model for familial ALS. These mice develop ALS-like symptoms after four months of age with degeneration of the motor neurons. Our results show an involvement of endoplasmic reticulum stress, caspase-12, -9, -3 and procaspase-7 in the ALS mice spinal cord. Overexpression of the antiapoptotic protein XIAP in spinal cord neurons inhibited the activation of caspase-12 and reduced caspase-3 and calpain activity. Calpastatin, the regulator of calpain activity, was kept intact in the ALS-XIAP mice. These mice showed a 12% increase in the mean survival suggesting a beneficial effect of XIAP in ALS. The reason for the ultimate cell death of motor neurons in the ALS-XIAP mice may be due to the activation of additional cell death pathways. Thus, we observed that lysosomal proteases particularly, cathepsinB, -D, and -L were activated in the ALS mice spinal cord together with a less marked upregulation of the inhibitors, cystatinB and -C. We also found activation of astrocytes and microglial cells in the spinal cord of ALS mice indicating their involvement in the disease. The results show that both caspase-dependent and -independent pathways are activated during neuronal degeneration in the ALS spinal cord. The results obtained may help to identify novel drug targets for future treatments of ALS.</p>

Light and oxygen generate harmful reactive oxygen species (ROS) in the lens, causing biochemical changes that gradually disarrange the lens fibres resulting in light scattering and loss of transparency. In the healthy eye, this chronic exposure to oxidative stress may lead to age-related cataract. However, there are also some conditions that accelerate cataract formation, such as diabetes mellitus, in which increased glucose levels may contribute to increased generation of ROS. The superoxide dismutases (SOD) participate in the defence against ROS by catalysing the dismutation of superoxide radicals. The main SOD isoenzyme in the lens is copper-zinc superoxide dismutase (SOD1). The aim of this thesis was to explore if this antioxidant enzyme is important for the protection against age-related and diabetes-induced cataract development. Lenses from wild-type mice and mice lacking SOD1 were incubated in high levels of glucose in vitro and their transparency and damage evaluated daily. Also, the impact of nitric oxide was studied by adding a nitric oxide synthase inhibitor. Furthermore, in vivo cataract formation in relation to the oxidative status of the lens was evaluated in streptozotocin-induced diabetic mice as well as in non-diabetic mice of both genotypes. Finally, the spontaneous age-related cataract development was studied in both genotypes. In vitro, the SOD1 null lenses showed increased levels of superoxide radicals and developed dense nuclear lens opacities upon exposure to high levels of glucose. They also showed increased lens leakage of lactate dehydrogenase, reduced transport function across cell membranes, and increased water contents. However, the lens damage and cataract formation were eliminated when the synthesis of nitric oxide was inhibited. This indicates that both superoxide and nitric oxide have important roles in glucose-induced cataract development possibly through their reaction with each other which generates the highly reactive peroxynitrite. In vivo, both the SOD1 null and the wild-type mice showed cortical cataract changes after 8 weeks of diabetes, although the SOD1 null mice showed a more pronounced cataract formation than the wild-type mice in relation to the level of hyperglycaemia. As cataract formation was accentuated the lenses showed diminishing levels of glutathione but increasing amounts of protein carbonyls, suggesting a reduced lens antioxidant capacity as well as increased lens protein oxidation. Non-diabetic young (18 weeks of age) SOD1 null mice did not show any signs of cataract. At 1 year of age they had developed some cortical lens obscurity as compared to the wild-type mice which did not show equivalent changes until 2 years of age. The results presented in this thesis show that SOD1 null mice are more prone to develop diabetes-induced and age-related cataract than wild-type mice. The findings thus further endorse the importance of oxidative stress as a contributor to cataract development and indicate that both superoxide and nitric oxide may be damaging to the lens. I therefore conclude that the antioxidant enzyme SOD1 is important for the protection against cataract.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with an incidence of 1.5-2.7/100000 people/year. Today there is no cure for the disease and only symptomatic treatments are available. ALS progresses rapidly and only 50% of the patients are alive three years after the symptom debut. In ALS, the upper and lower motor neurons undergo degeneration in a process resembling apoptosis. This leads to muscle atrophy and paralysis. The causes of neuronal death are however unknown. In this thesis we have studied transgenic mice carrying human mutant superoxide dismutase, as a model for familial ALS. These mice develop ALS-like symptoms after four months of age with degeneration of the motor neurons. Our results show an involvement of endoplasmic reticulum stress, caspase-12, -9, -3 and procaspase-7 in the ALS mice spinal cord. Overexpression of the antiapoptotic protein XIAP in spinal cord neurons inhibited the activation of caspase-12 and reduced caspase-3 and calpain activity. Calpastatin, the regulator of calpain activity, was kept intact in the ALS-XIAP mice. These mice showed a 12% increase in the mean survival suggesting a beneficial effect of XIAP in ALS. The reason for the ultimate cell death of motor neurons in the ALS-XIAP mice may be due to the activation of additional cell death pathways. Thus, we observed that lysosomal proteases particularly, cathepsinB, -D, and -L were activated in the ALS mice spinal cord together with a less marked upregulation of the inhibitors, cystatinB and -C. We also found activation of astrocytes and microglial cells in the spinal cord of ALS mice indicating their involvement in the disease. The results show that both caspase-dependent and -independent pathways are activated during neuronal degeneration in the ALS spinal cord. The results obtained may help to identify novel drug targets for future treatments of ALS.