Bullying research has grown tremendously throughout the years, and yet, there is a lack of research investigating the biological underpinnings of bullying victimization and perpetration. The single nucleotide polymorphism catechol-O-methyltransferase (COMT) Val158Met is an important candidate gene that has been demonstrated to interact with environmental factors and play an important role in emotion processing. However, it remains unknown whether COMT Val158Met influences youth and adolescents’ involvement as both targets and perpetrators of bullying, considering bully-victims are found to struggle with emotion regulation. To address this knowledge gap, the role of COMT Val158Met on the joint trajectories of bullying victimization and perpetration was investigated in a longitudinal community sample. A latent class growth analysis (LCGA) was used to identify distinct patterns of bullying victimization and perpetration across the ages 10 to 18 years (n = 648). A three-class solution was chosen for bullying victimization where most participants were reflected in a trajectory of low decreasing bullying victimization (74%), followed by moderate stable trajectory of bullying victimization (23%) and a final group following a high stable trajectory of bullying victimization (3%). A two-class solution was chosen for bullying perpetration. As predicted, most participants were reflected in the low stable bullying perpetration group (83%) and a small group followed a moderate increasing/decreasing trajectory of bullying perpetration (16.4%). Dual trajectory models revealed distinct subgroup of individuals involved in bullying either as targets, perpetrators, or bully-victims. Conditional probabilities results suggest that highly victimized youth would in time perpetrate against others while remaining targets of high levels of perpetration (i.e., target to bully-victim), whereas youth moderately victimized were more likely to be uninvolved in bullying perpetration. There was no significant difference in allelic variations (i.e., any Met allele vs Val/Val) of COMT Val158Met between bully-victims and children uninvolved in bullying. Implications of these findings are discussed from a differential susceptibility model. Gaining an understanding of the mechanisms behind the impact of bullying victimization and perpetration on children and adolescent will help provide insight and support for school and clinical prevention and intervention efforts.

Abstract The efforts to improve forest trees could be accelerated by means of genetic engineering. Thus, the performance and effects of genetically modified (GM) trees have been investigated in numerous studies, which have generally concluded that GM trees have similar effects on environment and/or other organisms as do conventionally bred trees. In the present study, GM silver birch (Betula pendula Roth) and hybrid aspen (Populus tremula L. × tremuloides Michx.) lines were utilized to study the influence of transgenes to the transcription of related endogenous genes and to the production of soluble phenolic compounds in relation to ectomycorrhizal symbiosis or herbivory. The GM silver birch lines had altered lignin composition, whereas the hybrid aspen lines produced the hemoglobin of Vitreoscilla sp. (VHb). The Pt4CL1a lines were generated using biolistic transformation and monitored under greenhouse conditions for three growing seasons. The Pt4CL1a and PtCOMT silver birch lines, with altered lignin syringyl/guaiacyl ratio, had also reduced transcript levels of endogenous genes, Bp4CL1 and BpCOMT, respectively. This indicates that these members of the 4CL and COMT multigene families are likely to contribute to the monolignol biosynthesis pathway of silver birch. No unintended effects were detected in the PtCOMT or Pt4CL1a lines in relation to ECM symbiosis or performance of insect larvae. Moreover, in soluble phenolic compounds, alterations were found mainly in cinnamic acid derivatives, a group of compounds involved in the biosynthesis of monolignols. In addition, the responses of the studied hybrid aspen lines that were exposed to herbivory for 24 hours were found to be comparable. Furthermore, the proportional weight gain of lepidopteran larvae was alike when fed with leaves of the VHb and non-transgenic hybrid aspen lines. Taken together, no unintended changes were found in the GM silver birch lines with altered lignin composition or in the VHb hybrid aspen lines. However, it is acknowledged that these short-term studies that were conducted under controlled conditions have certain limitations. / Tiivistelmä Puiden ominaisuuksia on mahdollista muuttaa geenitekniikkaa käyttämällä huomattavasti perinteistä jalostusta nopeammin. Geneettisen muuntamisen vaikutuksia puiden ominaisuuksiin ja vuorovaikutussuhteisiin on selvitetty useissa tutkimuksissa geenitekniikkaan liitettyjen riskien arvioimiseksi. Muunnettuja kohdeominaisuuksiaan lukuun ottamatta geneettisesti muunnettujen (GM) puiden ei ole yleisesti ottaen tutkimuksissa havaittu eroavan ympäristövaikutuksiltaan perinteisellä jalostuksella tuotetuista puista. Tässä työssä tutkittiin siirrettyjen geenien vaikutuksia GM-rauduskoivun (Betula pendula Roth) sekä hybridihaavan (Populus tremula L. × tremuloides Michx.) endogeenisten geenituotteiden ja liukoisten fenoliyhdisteiden määriin. Lisäksi työssä tarkasteltiin ligniinirakenteeltaan muunnettujen rauduskoivulinjojen ektomykorritsasymbioosia sekä ligniinimuunnettujen ja Vitreoscilla sp. -bakteerin hemoglobiinia (VHb) tuottavien hybridihaapalinjojen lehtien laatua perhostoukkien ravintona. Biolistisella geeninsiirrolla tuotetuista Amerikan haavan 4-kumaraattikoentsyymi A-ligaasi -geeniä (Pt4CL1) ilmentävistä rauduskoivulinjoista yhdessä havaittiin ligniinin syringyyli- ja guaiasyyliyksikköjen suhteessa muutos. Havaittu muutos aiheutui todennäköisesti koivun Bp4CL1-geenituotteiden määrän vähenemisestä. Myös kaffeaatti/5-hydroksylaatti O-metyylitransferaasi -geeniä (PtCOMT) ilmentävissä, ligniinirakenteeltaan muunnetuissa rauduskoivulinjoissa havaittiin endogeenisen BpCOMT-geenin tuotteiden määrän väheneminen. Tulokset viittaavat siihen, että Bp4CL1- ja BpCOMT-geenien tuottamat entsyymit toimivat rauduskoivun monolignolien biosynteesissä. Ligniiniominaisuuksiltaan muunnettujen rauduskoivujen liukoisista fenoliyhdisteistä todettiin muutoksia ensisijaisesti kanelihappojohdannaisissa, jotka liittyvät läheisesti monolignolien biosynteesireittiin. Ektomykorritsasymbioosissa tai perhostoukkien kasvunopeudessa ei havaittu kasvien geneettisestä muuntamisesta johtuvia eroja. Merkitseviä eroja ei todettu myöskään hybridihaapalinjojen herbivoria-vasteissa. On kuitenkin otettava huomioon, että kaikki tutkimuksen kokeet suoritettiin kasvihuoneissa käyttäen vasta juveniilivaiheessa olevia kasveja. Jotta abioottisten ja bioottisten ympäristötekijöiden sekä GM-puiden vuorovaikutusta olisi mahdollista arvioida kokonaisvaltaisesti, puita pitäisi tutkia pitkäaikaisissa kenttäkokeissa.

Wie bereits mehrfach in anderen Studien beschrieben (Falkenstein et al., 1991; Gehring et al., 1993; Holroyd & Coles, 2002) konnte ein signifikanter Unterschied im EEG nach richtigen Antworten im Vergleich zu Fehlern in einem Zeitfenster von -35 bis 108 ms und von 110 bis 450 ms gezeigt werden. Die als Differenz aus den negativsten beziehungsweise positivsten Peaks nach falschen und richtigen Antworten berechnete „error-related negativity“ (ERN) und „error positivity“ (Pe) als wichtigste Komponenten der Fehlerverarbeitung zeigten im Vergleich zu anderen Studien zwar geringere, aber trotzdem vergleichbare Werte. Um den Einfluss von genetischen Unterschieden auf die Fehlerverarbeitung deutlich zu machen, wurde in dieser Studie untersucht, inwieweit Polymorphismen des Dopamintransporters (DAT), der Catechol-O-Methyl-Transferase (COMT) und des Phosphoproteins Stathmin Einfluss auf die Ausprägung der ERN und der Pe nehmen. Bezüglich des DAT-Polymorphismus konnte ein signifikanter Einfluss weder auf die ERN noch auf die Pe nachgewiesen werden. Hier ist zu vermuten, dass der Polymorphismus den Dopaminhaushalt der Basalganglien nicht nennenswert beeinträchtigt und demnach keinen Einfluss auf die Fehlerverarbeitung hat. Im Hinblick auf den COMT – Polymorphismus zeigte sich zwar kein Effekt auf die ERN, bei der Pe konnte man allerdings signifikante Unterschiede zwischen den Gruppen feststellen. Hier zeigten die homozygoten Träger des „val“ – Allels signifikant höhere Pe – Amplituden als die homozygoten Träger des „met“ – Allels. Heterozygote Personen lagen hinsichtlich der Pe – Amplitude zwischen den beiden anderen Gruppen. Dieser Effekt entspricht zwar nicht den Ergebnissen von anderen Studien (Egan et al., 2005; Frank et al., 2007), sollte aber dennoch Gegenstand weiterführender Forschung sein. Der Polymorphismus im Gen des Phosphoproteins Stathmin beeinflusste zwar die Amplitude der Pe nicht, zeigte aber signifikante Auswirkungen auf die ERN, wobei hier Träger des T-Allels signifikant kleinere ERN-Amplituden aufwiesen als Probanden, die dieses Allel nicht trugen. Allerdings wurde deutlich, dass dieser Effekt nach genauerer Analyse nicht stabil gegenüber Veränderungen war. Trotzdem ist davon auszugehen, dass dieser Polymorphismus die Funktion des ACC und damit auch die Fehlerverarbeitung beeinflusst, wodurch die Notwendigkeit weiterer Untersuchungen auf dem Gebiet des Phosphoproteins Stathmin gegeben ist. / As already published in several studies (Falkenstein et al., 1991; Gehring et al., 1993; Holroyd & Coles, 2002) a significant difference after correct answers compared to errors could be shown in EEG in a time window of -35 to 108ms and 110 to 450ms. The error related negativity (ERN) and error positivity (Pe) showed comparable values to other studies. To demonstrate the influence of genetic difference to the error processing, this study researched the influence of dopamine transporter (DAT), catechol-o-methyl transferase (COMT) and Stathmine to ERN and Pe. The DAT polymorphism does not have any significant influence to ERN and Pe. The COMT polymorphism shows no significant influence to ERN, but there is a significant influence to Pe. The genetic polymorphism of Stathmine does not have any impact on the amplitude of Pe but shows significant influence to ERN.

As plantas medicinais são consideradas importantes fontes de compostos biologicamente ativos. Para muitas doenças crônicas, como as neurodegenerações, substâncias que apresentam atividades simultâneas em mais de um alvo relacionado à etiopatologia dessas desordens, constituem potenciais agentes terapêuticos. Nesse contexto, os objetivos deste trabalho foram a avaliação química e biológica de três espécies de samambaias de ocorrência no sul do Brasil: Blechnum binervatum, B. brasiliense e B. occidentale. O isolamento bioguiado foi utilizado para a identificação da(s) substância(s) com potencial atividade em modelos in vitro e in silico relacionados às desordens neurodegenerativas. A avaliação dos extratos e frações permitiu destacar a fração acetato de etila de B. brasiliense como a mais ativa na estabilização de radicais hidroxila (CI50: 12,5 μg/mL) e na inibição da lipoperoxidação (CI50: 10,4 μg/mL), sendo uma das mais ativas frente ao óxido nítrico (CI50: 55,6 μg/mL). Adicionalmente, na inibição da isoforma A da enzima monoamina oxidase (MAO), esta fração foi a que apresentou menor valor de CI50 (28,6 μg/mL). As frações diclorometano também apresentaram bons resultados na inibição da MAO-A, sendo algumas ativas igualmente como antioxidantes. Frente à MAO-B, os extratos e frações das três espécies vegetais demonstraram menores efeitos e foram inativos frente às enzimas acetil e butiril colinesterase, não demonstrando toxicidade em células polimorfonucleares (PMN) de ratos Wistar, na concentração de 1 mg/mL. Utilizando células-tronco cultivadas, os extratos e frações selecionadas, nas concentrações de 100 a 500 μg/mL, não afetaram a viabilidade celular e não apresentaram efeitos tóxicos. Análises químicas por cromatografia líquida de alta eficiência, acoplada a detector de arranjo de diodos e espectrometria de massas, permitiram a identificação de isômeros dos ácidos cafeoil quínico e cafeoil chiquímico nos extratos das três espécies estudadas. B. binervatum apresentou, ainda, ácido cafeico glicosilado, ácido isosalvianólico A e ácido rosmarínico sulfatado. Ácido salvianólico F foi identificado em B. binervatum e B. occidentale, bem como isômeros do ácido brainico. Ácido rosmarínico foi caracterizado em B. binervatum e B. brasiliense. Quercetina 3-O-glicosídeo e vicenina-2 também foram identificadas. As análises por cromatografia gasosa, acoplada à espectrometria de massas demonstraram que o diterpeno neofitadieno foi o composto majoritário nas frações diclorometano de Blechunm e nas frações hexano de B. occidentale e B. binervatum. Para a fração hexano de B. brasiliense, β-sitosterol foi o principal componente. A partir da fração acetato de etila de B. brasiliense foi isolado o ácido rosmarínico, o qual se mostrou ativo nos ensaios de atividade antioxidante, na inibição da catecol-O-metil transferase (CI50: 26,7 μM) e da MAO-A (CI50: 50,1 μM), sendo proposto mecanismo reversível de inibição desta enzima. Nos estudos de docking foram verificadas as interações moleculares entre o composto e as enzimas MAO-A e COMT, por ligações de hidrogênio e interações hidrofóbicas nos sítios ativos enzimáticos. O composto não apresentou efeitos tóxicos em células PMN de roedores, nas concentrações de 0,5 e 5 mM. Os ácidos rosmarínico e clorogênico isolados do extrato de B. binervatum não influenciaram na viabilidade celular e não induziram efeito tóxico sobre células-tronco (100 a 500 μM), sendo que o ácido rosmarínico foi capaz, ainda, de induzir proliferação celular. Capacidade protetora contra danos celulares causados por H2O2 (1400 μM) foi observada para ambas as substâncias, nas concentrações de 10-100 μM, sendo os resultados corroborados pelas imagens de microscopia celular. O ácido rosmarínico apresentou melhores respostas quando comparado ao ácido clorogênico, sendo mais efetivo na inibição dos danos por H2O2. O conjunto dos resultados obtidos ressalta a importância dos estudos associados químico-biológico de espécies vegetais e aponta para as potencialidades de samambaias como fontes de produtos bioativos, capazes de atuar sobre múltiplos alvos enzimáticos e não-enzimáticos relacionados a doenças neurodegenerativas. / Medicinal plants are considered important sources of biologically active compounds. For many chronic diseases, such as neurodegenerations, substances that present simultaneous activities in more than one target related to the etiopathology of these disorders are considered potential therapeutic agents. In this context, the aims of the study were the chemical and biological evaluation of three fern species, occurring in south Brazil: Blechnum binervatum, B. brasiliense and B. occidentale. The bioguided isolation was employed to identify compound(s) with potential activities at in vitro and in silico models associated to neurodegenerative disorders. The biological evaluation of extracts and fractions allowed to highlight the ethyl acetate fraction of B. brasiliense, which was the most active in the stabilization of hydroxyl radicals (IC50: 12.5 μg/mL) and on lipoperoxidation inhibition (IC50: 10.4 μg/mL), being one of the most active sample against nitric oxide (IC50: 55.6 μg/mL). Furthermore, on the inhibition of isoform A from monoamine oxidase (MAO), this fraction showed the lowest IC50 value (28.6 μg/mL). The dichloromethane fractions also presented good results in the MAO-A inhibition, being some of them active as antioxidants, too. Against MAO-B, extracts and fractions of the three species demonstrated reduced effects and all samples were inactive in the acetyl and butyryl cholinesterase inhibition, showing no toxic effects to polymorphonuclear cells (PMN) from Wistar rats, at 1 mg/mL. Using cultured stem cells, the selected extracts and fractions at 100 to 500 μg/mL did not affect cell viability and absence of cytotoxic effects was observed. The chemical analysis by high performance liquid chromatography, coupled to photodiode array detector and mass spectrometry, allowed the identification of caffeoyl quinic acid and caffeoyl shikimic acid isomers in the three studied fern species. B. binervatum presented also glycosilated caffeic acid, isosalvianolic acid A and sulphated rosmarinic acid. Salvianolic acid F was identified in B. binervatum and B. occidentale, as well as, brainic acid isomers. Rosmarinic acid was characterized in B. binervatum and B. brasiliense. Quercetin 3-O-glycoside and vicenin-2 were also found. Analysis by gas chromatography with mass spectrometry showed the diterpene neophytadiene was the major compound in dichloromethane fractions of Blechunm and in hexane fractions of B. occidentale and B. binervatum. For the hexane fraction from B. brasiliense, β-sitosterol was majority. From the ethyl acetate fraction of B. brasiliense was isolated rosmarinic acid, which was shown to be active in antioxidant assays, in the inhibition of catechol-O-methyltransferase (IC50: 26.7 μM) and MAO-A (IC50: 50.1 μM), being suggested a reversible inhibition mechanism against this enzyme. In docking studies were observed molecular interactions between the compound and MAO-A and COMT enzymes, via hydrogen bonds and hydrophobic interactions in enzyme active sites. The compound did not induce toxic effects to rodent PMN cells, at concentrations of 0.5 and 5 mM. The rosmarinic and chlorogenic acids, isolated from B. binervatum extract, did not influence cell viability and did not induce toxicity to stem cells (100 to 500 μM), the rosmarinic acid was also capable to induce cell proliferation. Protective ability against H2O2-induced cell damage (1400 μM) was observed for both substances at concentrations of 10-100 μM, and the results were supported by cellular microscopy images. Rosmarinic acid presented better responses compared with chlorogenic acid, being powerful inhibitor against H2O2-induced damage. The overall results highlights the importance of associated chemical-biological studies of plant species and points at the potential of ferns as sources of bioactive compounds, capable of modulating multiple enzymatic and non-enzymatic targets related to neurodegenerative diseases.

As plantas medicinais são consideradas importantes fontes de compostos biologicamente ativos. Para muitas doenças crônicas, como as neurodegenerações, substâncias que apresentam atividades simultâneas em mais de um alvo relacionado à etiopatologia dessas desordens, constituem potenciais agentes terapêuticos. Nesse contexto, os objetivos deste trabalho foram a avaliação química e biológica de três espécies de samambaias de ocorrência no sul do Brasil: Blechnum binervatum, B. brasiliense e B. occidentale. O isolamento bioguiado foi utilizado para a identificação da(s) substância(s) com potencial atividade em modelos in vitro e in silico relacionados às desordens neurodegenerativas. A avaliação dos extratos e frações permitiu destacar a fração acetato de etila de B. brasiliense como a mais ativa na estabilização de radicais hidroxila (CI50: 12,5 μg/mL) e na inibição da lipoperoxidação (CI50: 10,4 μg/mL), sendo uma das mais ativas frente ao óxido nítrico (CI50: 55,6 μg/mL). Adicionalmente, na inibição da isoforma A da enzima monoamina oxidase (MAO), esta fração foi a que apresentou menor valor de CI50 (28,6 μg/mL). As frações diclorometano também apresentaram bons resultados na inibição da MAO-A, sendo algumas ativas igualmente como antioxidantes. Frente à MAO-B, os extratos e frações das três espécies vegetais demonstraram menores efeitos e foram inativos frente às enzimas acetil e butiril colinesterase, não demonstrando toxicidade em células polimorfonucleares (PMN) de ratos Wistar, na concentração de 1 mg/mL. Utilizando células-tronco cultivadas, os extratos e frações selecionadas, nas concentrações de 100 a 500 μg/mL, não afetaram a viabilidade celular e não apresentaram efeitos tóxicos. Análises químicas por cromatografia líquida de alta eficiência, acoplada a detector de arranjo de diodos e espectrometria de massas, permitiram a identificação de isômeros dos ácidos cafeoil quínico e cafeoil chiquímico nos extratos das três espécies estudadas. B. binervatum apresentou, ainda, ácido cafeico glicosilado, ácido isosalvianólico A e ácido rosmarínico sulfatado. Ácido salvianólico F foi identificado em B. binervatum e B. occidentale, bem como isômeros do ácido brainico. Ácido rosmarínico foi caracterizado em B. binervatum e B. brasiliense. Quercetina 3-O-glicosídeo e vicenina-2 também foram identificadas. As análises por cromatografia gasosa, acoplada à espectrometria de massas demonstraram que o diterpeno neofitadieno foi o composto majoritário nas frações diclorometano de Blechunm e nas frações hexano de B. occidentale e B. binervatum. Para a fração hexano de B. brasiliense, β-sitosterol foi o principal componente. A partir da fração acetato de etila de B. brasiliense foi isolado o ácido rosmarínico, o qual se mostrou ativo nos ensaios de atividade antioxidante, na inibição da catecol-O-metil transferase (CI50: 26,7 μM) e da MAO-A (CI50: 50,1 μM), sendo proposto mecanismo reversível de inibição desta enzima. Nos estudos de docking foram verificadas as interações moleculares entre o composto e as enzimas MAO-A e COMT, por ligações de hidrogênio e interações hidrofóbicas nos sítios ativos enzimáticos. O composto não apresentou efeitos tóxicos em células PMN de roedores, nas concentrações de 0,5 e 5 mM. Os ácidos rosmarínico e clorogênico isolados do extrato de B. binervatum não influenciaram na viabilidade celular e não induziram efeito tóxico sobre células-tronco (100 a 500 μM), sendo que o ácido rosmarínico foi capaz, ainda, de induzir proliferação celular. Capacidade protetora contra danos celulares causados por H2O2 (1400 μM) foi observada para ambas as substâncias, nas concentrações de 10-100 μM, sendo os resultados corroborados pelas imagens de microscopia celular. O ácido rosmarínico apresentou melhores respostas quando comparado ao ácido clorogênico, sendo mais efetivo na inibição dos danos por H2O2. O conjunto dos resultados obtidos ressalta a importância dos estudos associados químico-biológico de espécies vegetais e aponta para as potencialidades de samambaias como fontes de produtos bioativos, capazes de atuar sobre múltiplos alvos enzimáticos e não-enzimáticos relacionados a doenças neurodegenerativas. / Medicinal plants are considered important sources of biologically active compounds. For many chronic diseases, such as neurodegenerations, substances that present simultaneous activities in more than one target related to the etiopathology of these disorders are considered potential therapeutic agents. In this context, the aims of the study were the chemical and biological evaluation of three fern species, occurring in south Brazil: Blechnum binervatum, B. brasiliense and B. occidentale. The bioguided isolation was employed to identify compound(s) with potential activities at in vitro and in silico models associated to neurodegenerative disorders. The biological evaluation of extracts and fractions allowed to highlight the ethyl acetate fraction of B. brasiliense, which was the most active in the stabilization of hydroxyl radicals (IC50: 12.5 μg/mL) and on lipoperoxidation inhibition (IC50: 10.4 μg/mL), being one of the most active sample against nitric oxide (IC50: 55.6 μg/mL). Furthermore, on the inhibition of isoform A from monoamine oxidase (MAO), this fraction showed the lowest IC50 value (28.6 μg/mL). The dichloromethane fractions also presented good results in the MAO-A inhibition, being some of them active as antioxidants, too. Against MAO-B, extracts and fractions of the three species demonstrated reduced effects and all samples were inactive in the acetyl and butyryl cholinesterase inhibition, showing no toxic effects to polymorphonuclear cells (PMN) from Wistar rats, at 1 mg/mL. Using cultured stem cells, the selected extracts and fractions at 100 to 500 μg/mL did not affect cell viability and absence of cytotoxic effects was observed. The chemical analysis by high performance liquid chromatography, coupled to photodiode array detector and mass spectrometry, allowed the identification of caffeoyl quinic acid and caffeoyl shikimic acid isomers in the three studied fern species. B. binervatum presented also glycosilated caffeic acid, isosalvianolic acid A and sulphated rosmarinic acid. Salvianolic acid F was identified in B. binervatum and B. occidentale, as well as, brainic acid isomers. Rosmarinic acid was characterized in B. binervatum and B. brasiliense. Quercetin 3-O-glycoside and vicenin-2 were also found. Analysis by gas chromatography with mass spectrometry showed the diterpene neophytadiene was the major compound in dichloromethane fractions of Blechunm and in hexane fractions of B. occidentale and B. binervatum. For the hexane fraction from B. brasiliense, β-sitosterol was majority. From the ethyl acetate fraction of B. brasiliense was isolated rosmarinic acid, which was shown to be active in antioxidant assays, in the inhibition of catechol-O-methyltransferase (IC50: 26.7 μM) and MAO-A (IC50: 50.1 μM), being suggested a reversible inhibition mechanism against this enzyme. In docking studies were observed molecular interactions between the compound and MAO-A and COMT enzymes, via hydrogen bonds and hydrophobic interactions in enzyme active sites. The compound did not induce toxic effects to rodent PMN cells, at concentrations of 0.5 and 5 mM. The rosmarinic and chlorogenic acids, isolated from B. binervatum extract, did not influence cell viability and did not induce toxicity to stem cells (100 to 500 μM), the rosmarinic acid was also capable to induce cell proliferation. Protective ability against H2O2-induced cell damage (1400 μM) was observed for both substances at concentrations of 10-100 μM, and the results were supported by cellular microscopy images. Rosmarinic acid presented better responses compared with chlorogenic acid, being powerful inhibitor against H2O2-induced damage. The overall results highlights the importance of associated chemical-biological studies of plant species and points at the potential of ferns as sources of bioactive compounds, capable of modulating multiple enzymatic and non-enzymatic targets related to neurodegenerative diseases.

As plantas medicinais são consideradas importantes fontes de compostos biologicamente ativos. Para muitas doenças crônicas, como as neurodegenerações, substâncias que apresentam atividades simultâneas em mais de um alvo relacionado à etiopatologia dessas desordens, constituem potenciais agentes terapêuticos. Nesse contexto, os objetivos deste trabalho foram a avaliação química e biológica de três espécies de samambaias de ocorrência no sul do Brasil: Blechnum binervatum, B. brasiliense e B. occidentale. O isolamento bioguiado foi utilizado para a identificação da(s) substância(s) com potencial atividade em modelos in vitro e in silico relacionados às desordens neurodegenerativas. A avaliação dos extratos e frações permitiu destacar a fração acetato de etila de B. brasiliense como a mais ativa na estabilização de radicais hidroxila (CI50: 12,5 μg/mL) e na inibição da lipoperoxidação (CI50: 10,4 μg/mL), sendo uma das mais ativas frente ao óxido nítrico (CI50: 55,6 μg/mL). Adicionalmente, na inibição da isoforma A da enzima monoamina oxidase (MAO), esta fração foi a que apresentou menor valor de CI50 (28,6 μg/mL). As frações diclorometano também apresentaram bons resultados na inibição da MAO-A, sendo algumas ativas igualmente como antioxidantes. Frente à MAO-B, os extratos e frações das três espécies vegetais demonstraram menores efeitos e foram inativos frente às enzimas acetil e butiril colinesterase, não demonstrando toxicidade em células polimorfonucleares (PMN) de ratos Wistar, na concentração de 1 mg/mL. Utilizando células-tronco cultivadas, os extratos e frações selecionadas, nas concentrações de 100 a 500 μg/mL, não afetaram a viabilidade celular e não apresentaram efeitos tóxicos. Análises químicas por cromatografia líquida de alta eficiência, acoplada a detector de arranjo de diodos e espectrometria de massas, permitiram a identificação de isômeros dos ácidos cafeoil quínico e cafeoil chiquímico nos extratos das três espécies estudadas. B. binervatum apresentou, ainda, ácido cafeico glicosilado, ácido isosalvianólico A e ácido rosmarínico sulfatado. Ácido salvianólico F foi identificado em B. binervatum e B. occidentale, bem como isômeros do ácido brainico. Ácido rosmarínico foi caracterizado em B. binervatum e B. brasiliense. Quercetina 3-O-glicosídeo e vicenina-2 também foram identificadas. As análises por cromatografia gasosa, acoplada à espectrometria de massas demonstraram que o diterpeno neofitadieno foi o composto majoritário nas frações diclorometano de Blechunm e nas frações hexano de B. occidentale e B. binervatum. Para a fração hexano de B. brasiliense, β-sitosterol foi o principal componente. A partir da fração acetato de etila de B. brasiliense foi isolado o ácido rosmarínico, o qual se mostrou ativo nos ensaios de atividade antioxidante, na inibição da catecol-O-metil transferase (CI50: 26,7 μM) e da MAO-A (CI50: 50,1 μM), sendo proposto mecanismo reversível de inibição desta enzima. Nos estudos de docking foram verificadas as interações moleculares entre o composto e as enzimas MAO-A e COMT, por ligações de hidrogênio e interações hidrofóbicas nos sítios ativos enzimáticos. O composto não apresentou efeitos tóxicos em células PMN de roedores, nas concentrações de 0,5 e 5 mM. Os ácidos rosmarínico e clorogênico isolados do extrato de B. binervatum não influenciaram na viabilidade celular e não induziram efeito tóxico sobre células-tronco (100 a 500 μM), sendo que o ácido rosmarínico foi capaz, ainda, de induzir proliferação celular. Capacidade protetora contra danos celulares causados por H2O2 (1400 μM) foi observada para ambas as substâncias, nas concentrações de 10-100 μM, sendo os resultados corroborados pelas imagens de microscopia celular. O ácido rosmarínico apresentou melhores respostas quando comparado ao ácido clorogênico, sendo mais efetivo na inibição dos danos por H2O2. O conjunto dos resultados obtidos ressalta a importância dos estudos associados químico-biológico de espécies vegetais e aponta para as potencialidades de samambaias como fontes de produtos bioativos, capazes de atuar sobre múltiplos alvos enzimáticos e não-enzimáticos relacionados a doenças neurodegenerativas. / Medicinal plants are considered important sources of biologically active compounds. For many chronic diseases, such as neurodegenerations, substances that present simultaneous activities in more than one target related to the etiopathology of these disorders are considered potential therapeutic agents. In this context, the aims of the study were the chemical and biological evaluation of three fern species, occurring in south Brazil: Blechnum binervatum, B. brasiliense and B. occidentale. The bioguided isolation was employed to identify compound(s) with potential activities at in vitro and in silico models associated to neurodegenerative disorders. The biological evaluation of extracts and fractions allowed to highlight the ethyl acetate fraction of B. brasiliense, which was the most active in the stabilization of hydroxyl radicals (IC50: 12.5 μg/mL) and on lipoperoxidation inhibition (IC50: 10.4 μg/mL), being one of the most active sample against nitric oxide (IC50: 55.6 μg/mL). Furthermore, on the inhibition of isoform A from monoamine oxidase (MAO), this fraction showed the lowest IC50 value (28.6 μg/mL). The dichloromethane fractions also presented good results in the MAO-A inhibition, being some of them active as antioxidants, too. Against MAO-B, extracts and fractions of the three species demonstrated reduced effects and all samples were inactive in the acetyl and butyryl cholinesterase inhibition, showing no toxic effects to polymorphonuclear cells (PMN) from Wistar rats, at 1 mg/mL. Using cultured stem cells, the selected extracts and fractions at 100 to 500 μg/mL did not affect cell viability and absence of cytotoxic effects was observed. The chemical analysis by high performance liquid chromatography, coupled to photodiode array detector and mass spectrometry, allowed the identification of caffeoyl quinic acid and caffeoyl shikimic acid isomers in the three studied fern species. B. binervatum presented also glycosilated caffeic acid, isosalvianolic acid A and sulphated rosmarinic acid. Salvianolic acid F was identified in B. binervatum and B. occidentale, as well as, brainic acid isomers. Rosmarinic acid was characterized in B. binervatum and B. brasiliense. Quercetin 3-O-glycoside and vicenin-2 were also found. Analysis by gas chromatography with mass spectrometry showed the diterpene neophytadiene was the major compound in dichloromethane fractions of Blechunm and in hexane fractions of B. occidentale and B. binervatum. For the hexane fraction from B. brasiliense, β-sitosterol was majority. From the ethyl acetate fraction of B. brasiliense was isolated rosmarinic acid, which was shown to be active in antioxidant assays, in the inhibition of catechol-O-methyltransferase (IC50: 26.7 μM) and MAO-A (IC50: 50.1 μM), being suggested a reversible inhibition mechanism against this enzyme. In docking studies were observed molecular interactions between the compound and MAO-A and COMT enzymes, via hydrogen bonds and hydrophobic interactions in enzyme active sites. The compound did not induce toxic effects to rodent PMN cells, at concentrations of 0.5 and 5 mM. The rosmarinic and chlorogenic acids, isolated from B. binervatum extract, did not influence cell viability and did not induce toxicity to stem cells (100 to 500 μM), the rosmarinic acid was also capable to induce cell proliferation. Protective ability against H2O2-induced cell damage (1400 μM) was observed for both substances at concentrations of 10-100 μM, and the results were supported by cellular microscopy images. Rosmarinic acid presented better responses compared with chlorogenic acid, being powerful inhibitor against H2O2-induced damage. The overall results highlights the importance of associated chemical-biological studies of plant species and points at the potential of ferns as sources of bioactive compounds, capable of modulating multiple enzymatic and non-enzymatic targets related to neurodegenerative diseases.

>Magister Scientiae - MSc / Anxiety, resilience and childhood trauma can be categorized as functional behavioural categories, with a wealth of research behind each. The research approach adopted for each, in most cases, is either from a genetic or neuropsychological standpoint, with few studies combining both to better understand all three functional behavioural categories as a multidimensional construct A number of candidate genes have been identified as markers for anxiety, resilience and childhood trauma, of which Catechol-methyl-transferase (COMT) and several respective single nucleotide polymorphisms (SNPs) are included. Although COMT SNPs have been linked to at least one of the functional categories, with a handful of haplotypes identified, to our knowledge no study has investigated the combination of SNPs selected for this study (rs6269, rs4818, rs4680, rs4633, rs737865, rs2075507) as a possible haplotype, specifically in a South African population. The use of SNaPshot for the genotyping of genes is an efficient and reliable means of identifying genotype frequencies and haplotypes in large sample groups, yet when selecting more than two SNPs of interest, the development of a multiplex assay is ideal. The first aim of the study was to design and optimize a multiplex assay to genotype several COMT SNPs. The primer design, multiplex optimization and SNaPshot conditions used showed good working parameters that can be utilized and further improved by optimization. Self-report measures are widely used to measure psychiatric disorders, such as anxiety, and has also been used for the measurement of resilience and childhood trauma. With each functional behavioural category well investigated in its respective domain, there is a need to investigate all three as a collective in a South African population due to the high rate of anxiety and childhood trauma exposure in communities. The second aim of the study was to investigate the prevalence of anxiety, resilience and childhood as functional behavioural categories in the full South African sample group; and the role of sex, through established self-report measures and respective normative data. Additionally, this carried over into investigating the correlation between anxiety, resilience and childhood trauma as a multidimensional construct in both the full South African sample and between sexes. There is a clear relationship which exists between all three functional behavioural categories, as they show a correlation in various dimensions independent of one another. Higher anxiety levels amongst females were reported, with no difference between sexes for resiliency. The empirical data collected from both COMT SNP and self-report measures for male and female where explored and reviewed against current literature for better understanding and insight into the association of COMT SNPs with anxiety, resilience and childhood trauma in a South African population. The results of this study to understand the complexity and association of all three functional behavioural categories as a multidimensional construct will be invaluable and may assist in the identification of possible risk factors which are essential for the promotion of better mental health in society.

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Succédant à une théorisation centrée sur le rôle des déficits des fonctions exécutives, les modèles contemporains du trouble déficit de l'attention / hyperactivité (TDAH) mettent en avant l’hétérogénéité d’une catégorie diagnostique impliquant des déficits neuropsychologiques, voies cérébrales et mécanismes étiopathogéniques multiples. En dépit de cette évolution, la majorité des études d'imagerie cérébrale des corrélats structurels du trouble menées à ce jour ont été conduites au niveau de la catégorie diagnostique, sans spécification supplémentaire. Cette approche comparant en moyenne un groupe de patients avec TDAH à un groupe de sujets sains a donné des résultats très variables d'une étude à l'autre, la comparaison inter-étude étant toutefois rendue difficile par la présence de facteurs confondants, tels que des différences en terme de régions d’intérêt examinées, de comorbidités acceptées chez les patients, de pourcentages de sujets masculins et féminins, de fenêtre d’âge sélectionnée, de méthodologie d'analyse ou encore de pourcentage de patients traités par méthylphénidate. Dans ce doctorat, nous nous sommes appuyés sur la morphométrie voxel-à-voxel pour isoler l’influence sur les volumes de matière grise de deux facteurs d’hétérogénéité intra-catégorielle dans le TDAH : le genre d’une part, et un polymorphisme génétique (Val158Met du gène Catéchol-O-méthyltransferase (COMT)) d’autre part ; ces deux facteurs présentant l’intérêt de moduler le risque associé de développer un trouble de type externalisé. Nous avons également comparé les volumes de matière grise d’enfants avec TDAH ayant reçu un traitement par méthylphénidate, de patients n'ayant jamais été exposé à la médication, et de sujet sains. Ces recherches expérimentales ont été inscrites dans une discussion plus générale de l’hétérogénéité des résultats de la littérature structurelle consacrée au TDAH et des sources neuropsychologiques de cette hétérogénéité. Dans notre étude des effets du genre sur les volumes de matière grise dans le TDAH, nous reportons pour la première fois une interaction entre genre et diagnostic, avec des corrélats structurels du trouble différents chez les garçons et les filles avec TDAH dans des régions de la ligne médiane du cerveau, impliquées à la fois dans la régulation émotionnelle et dans le fonctionnement du mode de réseau par défaut. Nous suggérons que ces différences structurelles pourraient contribuer aux différences de risque associé pour les troubles internalisés et externalisés présentées par les garçons et filles avec TDAH. Dans notre étude explorant l'influence du polymorphisme Val158Met sur les volumes de matière grise, nous mettons en évidence une modulation génétique des corrélats structurels du trouble : les sujets homozygotes pour l'allèle Val158, identifiés dans la littérature comme à risque pour le développement d'un trouble des conduites, présentent des volumes de matière grise supérieurs dans le noyau caudé comparativement aux sujets sains, tandis que les patients avec TDAH porteurs d'un allèle Met158 présentent des volumes de matière grise plus faibles dans le cortex préfrontal inférieur droit, une région cruciale pour les processus de contrôle attentionnel. Enfin, dans notre étude des corrélats structurels de l'exposition au méthylphénidate, nous reportons un effet potentiellement normalisateur du traitement sur les volumes de matière grise de l'insula et du pole temporal, des volumes de matière grise plus faibles chez les patients traités comparativement aux sujets sains dans le gyrus frontal moyen et dans le gyrus précentral, et une association entre volume de matière grise dans le nucleus accumbens gauche et durée d'exposition au méthylphénidate chez les sujets traités. (...) / Previous models of Attention Deficit / Hyperactivity Disorder (ADHD) such as Barkley’s or Brown’s conceptualized ADHD as essentially a developmental impairment of executive function. Against this view, it is now recognized that ADHD is a heterogeneous disorder, involving multiple deficits and multiple neuronal pathways. Despite this current theoretical framework, most structural brain imaging studies in ADHD have compared groups of children with ADHD with typically developing children, without trying to identify subgroups within the diagnostic category. This approach has yielded heterogeneous findings, possibly due to inter-studies variations in the type and number of comorbidities, the percentage of medicated participants included, the number of girls included, and/or methodological and statistical differences. Patients participating in these studies were also often exposed to methylphenidate, and potential medication effects on grey matter volumes are still unclear in certain brain regions such as the frontal lobe, despite a therapeutic action involving the preferential activation of catecholamine neurotransmission within the prefrontal cortex. In this thesis, we used voxel-based morphometry to study the influence of two important risk factors for the development of comorbid conditions in ADHD. The first of these two factors was gender, and the second a genetic polymorphism of the Catechol-O-methyltransferase gene known to put children with ADHD at risk for developing a conduct disorder (Val158Met). We also compared grey matter volumes in children with ADHD exposed to methylphenidate, never-medicated children with ADHD and typically developing children. These experimental studies were part of a more general discussion of ADHD neuropsychological and neurobiological heterogeneity. In our study exploring the influence of gender on the structural correlates of ADHD, we report for the first time a gender-by-diagnosis interaction, with grey matter volume differences in boys and girls with ADHD in midline cortical structures, involved in emotional regulation and part of the default mode network. We propose that these differences may contribute to explain why girls with ADHD more often develop inattentive and internalizing symptoms, whereas externalizing symptoms are predominant in boys with ADHD. In our study investigating the effects of Val158Met in ADHD, we report the first evidence of a COMT-related genetic modulation of ADHD-related grey matter volume alterations. Indeed, children with ADHD at higher risk for developing a conduct disorder (children homozygotes for the Val158 allele) presented increased grey matter volumes in the caudate nucleus when compared with typically developing children, whereas children carrying a Met158 allele presented with decreased grey matter volumes in the right inferior frontal cortex, a region known for its key role in attention. Finally, we measured grey matter volumes in medicated children with ADHD, never-medicated children with ADHD and typically developing children using both whole-brain voxel-based morphometry and automated tracing procedures in chosen regions of interest. We document potential methylphenidate-related grey matter volume normalization and deviation in previously unexplored frontal and temporal regions, and report a positive association between treatment history and grey matter volume in the nucleus accumbens, a key region for reward processing. Our first two experimental studies therefore contribute to a better understanding of the influence of important sources of within-category heterogeneity, while the third helps clarifying the potential confounding effect of medication exposure in previous structural brain imaging studies in ADHD.

Succédant à une théorisation centrée sur le rôle des déficits des fonctions exécutives, les modèles contemporains du trouble déficit de l'attention / hyperactivité (TDAH) mettent en avant l’hétérogénéité d’une catégorie diagnostique impliquant des déficits neuropsychologiques, voies cérébrales et mécanismes étiopathogéniques multiples. En dépit de cette évolution, la majorité des études d'imagerie cérébrale des corrélats structurels du trouble menées à ce jour ont été conduites au niveau de la catégorie diagnostique, sans spécification supplémentaire. Cette approche comparant en moyenne un groupe de patients avec TDAH à un groupe de sujets sains a donné des résultats très variables d'une étude à l'autre, la comparaison inter-étude étant toutefois rendue difficile par la présence de facteurs confondants, tels que des différences en terme de régions d’intérêt examinées, de comorbidités acceptées chez les patients, de pourcentages de sujets masculins et féminins, de fenêtre d’âge sélectionnée, de méthodologie d'analyse ou encore de pourcentage de patients traités par méthylphénidate. Dans ce doctorat, nous nous sommes appuyés sur la morphométrie voxel-à-voxel pour isoler l’influence sur les volumes de matière grise de deux facteurs d’hétérogénéité intra-catégorielle dans le TDAH : le genre d’une part, et un polymorphisme génétique (Val158Met du gène Catéchol-O-méthyltransferase (COMT)) d’autre part ; ces deux facteurs présentant l’intérêt de moduler le risque associé de développer un trouble de type externalisé. Nous avons également comparé les volumes de matière grise d’enfants avec TDAH ayant reçu un traitement par méthylphénidate, de patients n'ayant jamais été exposé à la médication, et de sujet sains. Ces recherches expérimentales ont été inscrites dans une discussion plus générale de l’hétérogénéité des résultats de la littérature structurelle consacrée au TDAH et des sources neuropsychologiques de cette hétérogénéité. Dans notre étude des effets du genre sur les volumes de matière grise dans le TDAH, nous reportons pour la première fois une interaction entre genre et diagnostic, avec des corrélats structurels du trouble différents chez les garçons et les filles avec TDAH dans des régions de la ligne médiane du cerveau, impliquées à la fois dans la régulation émotionnelle et dans le fonctionnement du mode de réseau par défaut. Nous suggérons que ces différences structurelles pourraient contribuer aux différences de risque associé pour les troubles internalisés et externalisés présentées par les garçons et filles avec TDAH. Dans notre étude explorant l'influence du polymorphisme Val158Met sur les volumes de matière grise, nous mettons en évidence une modulation génétique des corrélats structurels du trouble : les sujets homozygotes pour l'allèle Val158, identifiés dans la littérature comme à risque pour le développement d'un trouble des conduites, présentent des volumes de matière grise supérieurs dans le noyau caudé comparativement aux sujets sains, tandis que les patients avec TDAH porteurs d'un allèle Met158 présentent des volumes de matière grise plus faibles dans le cortex préfrontal inférieur droit, une région cruciale pour les processus de contrôle attentionnel. Enfin, dans notre étude des corrélats structurels de l'exposition au méthylphénidate, nous reportons un effet potentiellement normalisateur du traitement sur les volumes de matière grise de l'insula et du pole temporal, des volumes de matière grise plus faibles chez les patients traités comparativement aux sujets sains dans le gyrus frontal moyen et dans le gyrus précentral, et une association entre volume de matière grise dans le nucleus accumbens gauche et durée d'exposition au méthylphénidate chez les sujets traités. (...) / Previous models of Attention Deficit / Hyperactivity Disorder (ADHD) such as Barkley’s or Brown’s conceptualized ADHD as essentially a developmental impairment of executive function. Against this view, it is now recognized that ADHD is a heterogeneous disorder, involving multiple deficits and multiple neuronal pathways. Despite this current theoretical framework, most structural brain imaging studies in ADHD have compared groups of children with ADHD with typically developing children, without trying to identify subgroups within the diagnostic category. This approach has yielded heterogeneous findings, possibly due to inter-studies variations in the type and number of comorbidities, the percentage of medicated participants included, the number of girls included, and/or methodological and statistical differences. Patients participating in these studies were also often exposed to methylphenidate, and potential medication effects on grey matter volumes are still unclear in certain brain regions such as the frontal lobe, despite a therapeutic action involving the preferential activation of catecholamine neurotransmission within the prefrontal cortex. In this thesis, we used voxel-based morphometry to study the influence of two important risk factors for the development of comorbid conditions in ADHD. The first of these two factors was gender, and the second a genetic polymorphism of the Catechol-O-methyltransferase gene known to put children with ADHD at risk for developing a conduct disorder (Val158Met). We also compared grey matter volumes in children with ADHD exposed to methylphenidate, never-medicated children with ADHD and typically developing children. These experimental studies were part of a more general discussion of ADHD neuropsychological and neurobiological heterogeneity. In our study exploring the influence of gender on the structural correlates of ADHD, we report for the first time a gender-by-diagnosis interaction, with grey matter volume differences in boys and girls with ADHD in midline cortical structures, involved in emotional regulation and part of the default mode network. We propose that these differences may contribute to explain why girls with ADHD more often develop inattentive and internalizing symptoms, whereas externalizing symptoms are predominant in boys with ADHD. In our study investigating the effects of Val158Met in ADHD, we report the first evidence of a COMT-related genetic modulation of ADHD-related grey matter volume alterations. Indeed, children with ADHD at higher risk for developing a conduct disorder (children homozygotes for the Val158 allele) presented increased grey matter volumes in the caudate nucleus when compared with typically developing children, whereas children carrying a Met158 allele presented with decreased grey matter volumes in the right inferior frontal cortex, a region known for its key role in attention. Finally, we measured grey matter volumes in medicated children with ADHD, never-medicated children with ADHD and typically developing children using both whole-brain voxel-based morphometry and automated tracing procedures in chosen regions of interest. We document potential methylphenidate-related grey matter volume normalization and deviation in previously unexplored frontal and temporal regions, and report a positive association between treatment history and grey matter volume in the nucleus accumbens, a key region for reward processing. Our first two experimental studies therefore contribute to a better understanding of the influence of important sources of within-category heterogeneity, while the third helps clarifying the potential confounding effect of medication exposure in previous structural brain imaging studies in ADHD.