Doctor of Philosophy / Biochemistry and Molecular Biophysics / Timothy P. Durrett / The membrane bound O-acyltransferase (MBOAT) family contains multi-pass membrane proteins that add fatty acids to different compounds. Despite their importance in economic activity and human health, little is known about the localization of the active site and regions important for determining substrate specificity of MBOATs. Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) is the only known MBOAT enzyme that exhibits a high preference for acetyl-CoA, the shortest possible acyl-CoA. EaDAcT catalyzes the transfer of the acetate group from acetyl-CoA to the sn-3 position of diacylglycerol to form 3-acetyl-1,2-diacyl-sn-glycerol. Our goal was to investigate the structural features and the amino acid residues that define substrate specificity of EaDAcT to provide insights into the mechanism by which MBOAT family controls substrate selection. By mapping the membrane topology of EaDAcT we obtained the first experimentally determined topology model for a plant MBOAT. The EaDAcT model contains four transmembrane domains with both the N- and C- termini oriented toward the endoplasmic reticulum lumen. The MBOAT signature region including the putative active site His-257 of the protein is embedded in the third transmembrane domain close to the interface between the membrane and the cytoplasm. In order to identify amino acid residues important for acetyltransferase activity, we isolated and characterized orthologs of EaDAcT from other acetyl-TAG producing plants. Among them, the acetyltransferase from Euonymus fortunei possessed the highest activity in vivo and in vitro. Mutagenesis of conserved residues of DAcTs revealed that Ser-253, His-257 and Asp-258 are essential for enzyme activity of EaDAcT, suggesting their involvement in the enzyme catalysis. Alteration of residues unique to acetyltransferases did not alter the acyl donor specificity of EaDAcT, implying that multiple amino acids are important for substrate recognition. Together, this work identifies the structural features of EaDAcT and offers an initial view of the amino acids important for activity of the enzyme.

Doctor of Philosophy / Department of Agronomy / Johanna A. Dille / Kochia (Kochia scoparia L. Schrad.), one of the most problematic weeds in the Great Plains of United States, has evolved resistance to some herbicides including glyphosate (5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitor) which was first reported in western Kansas in 2007. The objectives of this research were to (1) characterize six kochia populations from western Kansas on the basis of glyphosate resistance or multiple herbicide resistance, (2) determine germination characteristics of the populations and evaluate their growth and fecundity in the field, and (3) determine if EPSPS gene amplification responsible for glyphosate resistance in kochia was associated with growth and fecundity cost in the plants. Six kochia populations were from Scott, Finney, Thomas, Phillips, Wallace, and Wichita counties. Based on shikimate assay for glyphosate and recommended field rates for four tested herbicides, three kochia populations (Scott (SC-R), Finney (FN-R), and Thomas (TH-R)) were grouped into glyphosate-resistant (GR) and three populations (Phillips (PH-S), Wallace (WA-S) and Wichita (WI-S)) were grouped into glyphosate-susceptible (GS). All populations were resistant to dicamba (synthetic auxin) and chlorsulfuron (acetolactate synthase inhibitor), however, atrazine (PS II inhibitor) resistance in FN-R was noted as exceptional among the GR populations. Across the three germination temperatures (5, 10 and 15 C), the GR populations consistently had less total cumulative germination and at 15 C, they consistently required more time to attain 50% of maximum cumulative germination than the GS populations. Both the field study and evaluation of relationship between EPSPS gene amplification and plant performance showed that differences in plant height, biomass accumulation and fecundity among populations were not in respect to glyphosate resistance but rather, differences in their inherent ability to grow and produce seeds in the presence or absence of neighbors. This research suggests that fitness differences between GR and GS kochia populations could be identified in germination characteristics but not in their growth or fecundity.

Die NADPHd-Färbung stellt bekanntermaßen Neurone dar, die die neuronale NOS exprimieren. Die Anfärbung der Neurone ist in ihrer Qualität dabei mit Golgi-basierten Versilberungstechniken vergleichbar. Aufgrund dieser Eigenschaften ermöglicht diese Methode morphologische und funktionelle Untersuchungen. Somit ist sie geradezu zur Bearbeitung neuropathologischer Fragestellungen prädestiniert. Im Putamen werden durch diese Technik vorwiegend Interneurone angefärbt. Anhand morphologischer Kriterien wurden die nitrinergen Neurone klassifiziert. Im menschlichen Putamen konnten dabei 12 Neuronentypen (NADPHd I bis XII) unterschieden werden, die nur zum Teil in bereits bestehende Klassifikationssysteme eingeordnet werden konnten. Ausgehend von dieser Klassifikation ist es möglich, in vergleichenden Studien Veränderungen NADPHd-positiver Neurone im Rahmen neurodegenerativer Erkrankungen festzustellen. Im Falle der vorliegenden Arbeit wurde dabei das Putamen schizophrener Patienten untersucht. Aufgrund der geringen Anzahl von drei untersuchten schizophrenen Gehirnen ließen sich nur vorläufige Aussagen in Bezug auf Unterschiede NADPHd-positiver Neurone im Putamen Gesunder und Schizophrener treffen. Solche Unterschiede wurden in der Morphologie dieser Neurone gefunden, aber auch in deren Dichte: Im Putamen Schizophrener lag die Dichte NADPHd-positiver Neurone signifikant unter der bei der gesunden Kontrollgruppe ermittelten Dichte. Neben diesem numerischen Unterschied konnten auch morphologisch auffällige Neurone gefunden werden, die in der gesunden Kontrollgruppe nicht vorhanden waren. Sowohl im Claustrum als auch in der das Claustrum umgebenden weißen Substanz der Capsulae externa et extrema konnten NADPHd-positive Neurone nachgewiesen werden. Die NADPHd-positiven Neurone des Claustrums ließen sich zum Teil nach bereits bestehenden Einteilungen klassifizieren. In den äußeren Kapseln lagen sie zumeist parallel zur Richtung der Fasermassen angeordnet und zählten zu den interstitiellen Zellen der weißen Substanz. / The NADPHd-staining is known to stain selectively neurons expressing the neuronal NOS. The staining results are comparable to Golgi impregnation techniques because not only the cell soma is stained but also the dendrites. Thus, morphological and functional aspects can be examined using that techniqe. This method was used to stain, characterize and classify nNOS-positive neurons of the human putamen. Predominantly, interneurons were stained. They displayed a homogenous staining of the cell soma and the dendrites showing clear morphological differences. The interneurons could be classified into 12 different types (NADPHd I to XII) which only partially corresponded to previously described neuron types. Based on this classification system of a healthy brain, it is possible to find abnormalities of NADPHd-positive interneurons in neurodegenerative diseases. In this study, the putamen of three schizophrenic subjects was examined. Differences could not only be found for the morphology of NADPHd-positive interneurons but also for their frequency: The number of NADPHd-positive interneurons was significantly reduced in the putamen of schizophrenics. However, since only three brains of schizophrenics were examined these results can only be judged preleminary. In the claustrum and in the white matter surrounding the claustrum NADPHd-positive neurons were found as well. Regarding the claustrum, the NADPHd-positive neurons fit partially in previous classification systems. The NADPHd-positive neurons of the external capsules were part of the interstitial cells of the white matter.

Der gasförmige Neurotransmitter Stickstoffmonoxid (NO) spielt eine Rolle bei verschiedenen physiologischen Vorgängen, aber auch psychiatrischen Erkrankungen wie Aggression, Ängstlichkeit, Depression und auch bei kognitiven Funktionen. Um mehr über die physiologische Rolle von NO herauszufinden untersuchten wir mittels Gen-Expressionsanalyse und Verhaltensversuchen Mäuse, bei denen die neuronale Isoform der Stickstoffmonoxidsynthase ausgeschaltet wurde. Die so genannte NOS-I ist die hauptsächliche Quelle von NO im zentralen Nervensystem. Knockout Tiere sind wertvolle Werkzeuge um sowohl den Einfluss eines Gens auf Verhalten als auch möglicherweise damit zusammenhängende Veränderungen des Transkriptoms zu identifizieren. Dies ist wichtig um herauszufinden, mit welchen molekularen Pfaden bestimmte Verhaltensweisen korreliert sind. In Bezug auf NOS-I gibt es zwei bisher beschriebene Knockout Mäuse Stämme. Es existieren KOex6 Knockout Mäuse, in welchen es überhaupt keine katalytisch aktive NOS-I gibt und es gibt einen Mausstamm, bei dem Exon 1 deletiert wurde, was aufgrund alternativer NOS-I Splicevarianten zu einer residualen Expression von bis zu 7% führt. Daher sind diese Mäsue besser zutreffend als Knockdown Mäuse zu bezeichnen. In der vorliegenden Arbeit untersuchten wir die Nos1 Knockdown Mäuse, da die hier vorliegende Situation wohl ähnlicher zu der bei menschlicher genetischer Varianten ist, da eine komplette Disruption bisher noch nicht beim Menschen beschrieben wurde. Es gibt diverse Studien, welche den behaviouralen Phänotyp der Nos1 Knockdown Mäuse untersuchen, aber diese widersprechen sich zum Teil. Bei unserer Untersuchung legten wir den Schwerpunkt auf Verhaltenstests, welche spezifische Symptome des Aufmerksamkeitsdefizit-/Hyperaktivitätssyndrom (ADHS) aufdecken sollten. Wir führten den Elevated Plus Maze Test (EPM) und ein modifiziertes Lochbrett-Paradigma, die COGITAT-Box, durch. Um die den gefundenen Verhaltensänderungen zugrunde liegenden molekularen Mechanismen herauszufinden, suchten wir nach Unterschieden der Expression des Serotonin- (5HTT) und des Dopamintransporters (DAT) zwischen den Knockdown und den Wildtyp Mäusen. Wir hatten spekuliert, dass die Disruption der NOS-I zu einer modifizierten Expression des DAT oder des 5HTT geführt habe könnte wegen den bekannten engen Interaktionen zwischen dem nitrinergen und den monoaminergen Systemen. Wir fanden einen diskret anxiolytischen Phänotyp, da die Knockdown Mäuse eine längere Zeit auf dem offenen Arm des EPM verbrachten bzw. häufiger den offenen Arm betraten im Vergleich zu dem Wildtypen. Dies war nicht durch eine höhere lokomotorische Aktivität zu erklären. Auch beobachteten wir ein geschlechterunabhängiges kognitives Defizit im Arbeits- und Referenzgedächtnis in der COGITAT-Box. Überraschenderweise fanden wir keine signifikante Dysregulation der Monoamin-Transporter in der Expressionsanalyse mittels der quantitativen Real Time PCR. Dies war eher unerwartet, da vorherige Studien verschiedene Veränderungen im serotonergen und dopaminergen System bei den Nos1 Knockdown Mäusen gefunden hatten, wie z.B. einen verminderten Serotonin-Umsatz in frontalen Cortex und hypofunktionale 5 HT1A and 5HT1B Rezeptoren. Auch ist bekannt, dass NO direkt Monoamin-Transporter nitrosyliert. Zusammenfassend zeigen die Nos1 Knockdown Mäuse ein charakteristisches behaviourales Profil mit reduzierter Ängstlichkeit und Defiziten im Gedächtnis. Weitere Studien sollten folgen um zu klären, ob diese Mäuse als Tiermodell für z.B. die Alzheimer-Erkrankung oder das Aufmerksamkeitsdefizit-/Hyperaktivitätssyndrom dienen könnten und die weitere pathophysiologische Rolle des NO bei neuropsychiatrischen Erkrankungen herauszufinden. / The gaseous messenger nitric oxide (NO) has been implicated in a wide range of behaviours, including aggression, anxiety, depression and cognitive functioning. To further elucidate the physiological role of NO and its down-stream mechanisms, we conducted behavioral and expressional phenotyping of mice lacking the neuronal isoform of nitric oxide synthase (NOS1), the major source of NO in the central nervous system. Knockout animals are valuable tools to identify both the behavioural impact of a given gene, as well as subsequent changes of the transcriptome to correlate behaviour to molecular pathways. With respect to NOS-I, two genetically modified mouse strains have been described in the literature. There are the KOex6 knockout mice in which you find a complete absence of catalytically active NOS-I and previously generated animals with a targeted deletion of exon 1 which show a residual NOS-I expression up to 7% rendering those mice actually Nos1 knockdown animals. In this study we used the knockdown animals because this situation seems to be more closely to human genetic variation, since a complete disruption of the gene has not yet been described in man. There a several studies on the behavioural phenotype of these animals but they are in part contradictory. In our investigations we had a special emphasis on ADHD-relevant tests, we performed the Elevated Plus Maze and a modified holeboard paradigm, the COGITAT Box. To further examine the underlying molecular mechanisms, we searched for differences in the expression of the serotonine and the dopamine transporter between the knockdown and the wildtype mice because we had speculated that disruption of the NOS-I might lead to a modified expression of the DAT or the 5HTT because of the tight interactions of NO and both the serotonergic as well as the dopaminergic system. A subtle anxiolytic phenotype was observed, with knockdown mice displaying a higher open arm time in the Elevated Plus Maze (student's t-test p>0.05) as compared to their respective wildtypes which was not caused by a higher locomotor acivity. Also there was gender-independent cognitive impairment in spatial learning and memory, as assessed by an automatized holeboard paradigm, the COGITAT Box. Here the working memory error and the reference memory error were in parts significantly different between knockdown animals and their respective wildtypes. Surprisingly no significant dysregulation of monoamine transporters was evidenced by qRT PCR. This we did not expect because of previous findings in Nos1 knockout mice that showed for example a reduced 5 HT turnover in the frontal cortex along with a concomitant increase in frontal 5-HT as well as 5 HT1A and 5HT1B receptor hypofunctioning and the fact that NO directly nitrosylates monoamine transporters. Taken together, Nos1 knockdown mice display a characteristic behavioural profile consisting of reduced anxiety and impaired learning and memory. Further research has to assess the value of these mice as animal models e.g. for Alzheimer’s disease or attention deficit disorder, in order to clarify a possible pathophysiological role of NO therein.

Intaktes Endothel und die ausreichende Funktion der endothelialen NO-Synthase (eNOS) sind Voraussetzungen für gesunde Gefäße. Eine endotheliale Dysfunktion besteht bei Patienten mit kardiovaskulären Risikofaktoren bzw. manifester koronarer Herzerkrankung (KHK). Endotheliale Vorläuferzellen (EPC) sind ein wichtiger Faktor für die Aufrechterhaltung der Homöostase des Endothels. Im klinischen Teil der vorliegenden Arbeit konnte gezeigt werden, dass diese Vorläuferzellen mit zunehmender Schwere der KHK in geringerem Maß im Blut zirkulieren. Die Anzahl der EPC war bei Patienten mit einer koronaren 3-Gefäßerkrankung um 77 % geringer, die Anzahl der kolonie-bildenden Einheiten (CFUs) um 50,3%, jeweils verglichen mit Patienten ohne KHK. Bei diesen Patienten konnte ebenfalls gezeigt werden, dass sich die Konzentration des endogenen eNOS-Inhibitors asymmetrisches Dimethylarginin (ADMA) im Plasma mit zunehmender Schwere der KHK erhöhte (0,47 ± 0,02 μmol/l bei fehlender KHK gegenüber 0,58 ± 0,02 μmol/l bei koronarer 3-Gefäßerkrankung). ADMA ist über eine Hemmung der eNOS an der Entstehung und Aufrechterhaltung einer endothelialen Dysfunktion beteiligt. Über diesen Weg wird vermutlich auch die Funktion der EPC erheblich eingeschränkt. Dies konnten wir anhand der In­vitro­Versuche mit EPC gesunder Spender zeigen. Dabei reduzierte sich unter ADMA-Einfluß die Anzahl der EPC in Kultur, die Anzahl und Größe der CFUs und ihre Funktion bzw. ihre Fähigkeit, sich in gefäßähnliche Strukturen zu integrieren. Eine gleichzeitige Gabe des HMG-CoA-Reduktase-Inhibitors Rosuvastatin wirkte in all diesen In-vitro-Versuchen der hemmenden Wirkung von ADMA entgegen. Die vorliegende Arbeit zeigt erstmals eine inverse Korrelation zwischen ADMA-Spiegeln und der Anzahl und Funktion der EPC. Der negative Einfluss auf EPCs ist vermutlich ein wichtiger Mechanismus, über den ADMA der Entstehung und dem Fortschreiten kardiovaskulärer Erkrankungen Vorschub leistet. / Endothelial progenitor cells play a pivotal role in regeneration of injured endothelium, thereby limiting the formation of atherosclerotic lesions. Reduced numbers of EPCs may affect progression of coronary artery disease. Regulation of EPC mobilization and function is mediated in part by nitric oxide (NO). Endogenous inhibitors of NO synthases, such as ADMA, contribute to endothelial dysfunction and injury. We tested the hypothesis that asymmetric dimethylarginine (ADMA) may be an endogenous inhibitor of endothelial progenitor cells (EPCs). We used flow cytometry and in vitro assays to investigate the relationship between EPC number and function with ADMA plasma levels in patients with stable angina. The plasma concentration of ADMA was related to the severity of coronary artery disease and correlated inversely with the number of circulating CD34+/CD133+ progenitor cells (r = -0.69; p < 0.0001) and endothelial colony forming units (CFUs) (r = -0.75; p < 0.0001). Adjusting for all patient characteristics, we confirmed these findings in multivariate regression analyses. In vitro differentiation of EPCs was repressed by ADMA in a concentration-dependent manner. Compared with untreated cells, ADMA reduced EPC incorporation into endothelial tube-like structures to 27 +/- 11% (p < 0.001). Asymmetric dimethylarginine repressed the formation of CFUs from cultured peripheral blood mononuclear cells to 35 +/- 7% (p < 0.001). Asymmetric dimethylarginine decreased endothelial nitric oxide synthase activity in EPCs to 64 +/- 6% (p < 0.05) when compared with controls. Co-incubation with the hydroxymethyl glutaryl coenzyme A reductase inhibitor rosuvastatin abolished the detrimental effects of ADMA. CONCLUSIONS: Asymmetric dimethylarginine is an endogenous inhibitor of mobilization, differentiation, and function of EPCs. This contributes to the cardiovascular risk in patients with high ADMA levels and may explain low numbers and function of EPCs in patients with coronary artery disease.

O gene TrCit do fungo filamentoso Trichoderma reesei codifica a proteína citrato sintase, uma enzima chave do ciclo de Krebs. Análise da região 5´ upstream de TrCit mostra que o gene está adjacente ao gene TrIcl (que codifica a proteína isocitrato liase, uma enzima do ciclo de glioxalato), em uma orientação cabeça-cabeça. A região promotora intergênica de 647 pb rica em G + C, apresenta uma ilha CpG, seqüência INR, caixas GC, caixas CAAT, sítios de ligação para diversos fatores de transcrição e é isenta de caixa TATA. O gene TrCit de 1573 pb contém 3 éxons e 2 íntrons. Sua seqüência codificadora de 1422 pb produz uma proteína de 474 aminoácidos, com um peso molecular estimado de 52,3 kD. O gene TrIcl de 1880 pb contém 3 éxons e 2 íntrons. Sua seqüência codificadora de 1788 pb produz uma proteína de 596 aminoácidos, com um peso molecular estimado de 65,4 kD. A atividade transcricional da região promotora foi analisada utilizando como repórter o gene de higromicina B fosfotransferase (hph). Uma região funcional necessária à transcrição de ambos os genes foi identificada na região central do promotor e contém uma caixa GC que liga o putativo fator de transcrição Sp1 de T. reesei (TrZnFSp1). O gene do putativo fator de transcrição “zinc-finger" TrZnFSp1 de 1500 pb contém 3 éxons e 2 íntrons. Sua seqüência codificadora de 1344 pb produz uma proteína de 448 aminoácidos, com um peso molecular estimado de 48,4 kD. Os resultados mostram que ambos os genes são transcritos de forma divergente a partir de um promotor bidirecional que compartilha na região central uma caixa GC, necessária para a transcrição de ambos os genes. / The TrCit gene from the filamentous fungus Trichoderma reesei codes for the citrate synthase protein, a key enzyme in the Krebs cycle. Analysis of TrCit 5’ upstream region showed that it is adjacent to the TrIcl gene that codes for isocitrate lyase protein, an enzyme involved in the glyoxylate cycle. Both genes, on a head-to-head orientation, are separated by an intergenic GC-rich and TATA-less promoter region of 647 base pairs. This bidirectional promoter has diverse cis regulatory elements: a CpG island, two INR sequences, GC boxes, CAAT boxes and several putative interaction sites for different transcription factors. The TrCit gene, 1,573-base pair-long, has an open reading frame of 1,422 base pairs interrupted by two introns. The gene codes for a protein with an estimated molecular weight of 52.3 kD. The TrIcl gene, 1,880-base pair-long, contains 3 exons and 2 introns and a putative coding sequence of 1,788 base pairs. The estimated molecular weight of TrICL is 65.4 kD. he transcriptional activity of the intergenic promoter region was analyzed using hygromicin B phosphotransferase (hph) as a reporter gene. A functional region required for the transcription of both genes was identified in the centre of this promoter. It has a GC box that interacts with a putative transcription factor Sp1 from T. reesei (TrZnFSp1). The results presented in this work show that both genes are divergently transcribed from a bidirectional promoter that shares an essential central GC box.

Apesar da elevada prevalência dos transtornos depressivos em idosos, os mecanismos fisiopatológicos subjacentes a estes quadros são pouco conhecidos. Atualmente, o principal foco dos estudos sobre a fisiopatologia da depressão geriátrica são as alterações cerebrovasculares associadas a estes quadros. Outros mecanismos fisiopatológicos têm sido estudados, como as alterações em cascatas neurotróficas e inflamatórias. A enzima glicogênio sintase quinase 3 beta (GSK-3B) tem sido implicada na patogênese de diversos transtornos mentais, em especial os transtornos afetivos (i.e. depressão maior e o transtorno afetivo bipolar) e doenças neurodegenerativas (i.e. doença de Alzheimer). Entretanto, não há estudos que avaliam o papel desta enzima nos pacientes idosos com depressão maior. Desta maneira, o objetivo principal deste trabalho é avaliar a atividade da GSK-3B em pacientes idosos com depressão maior. A hipótese deste estudo é que a atividade enzimática está aumentada nos pacientes idosos deprimidos em relação a idosos saudáveis. Para este estudo, recrutamos 40 idosos com depressão maior (de acordo com os critérios diagnósticos do DSM-IV) e que não estavam em uso de antidepressivos. O grupo comparativo foi constituído por 13 idosos saudáveis, sem evidências de transtornos cognitivos ou do humor. A gravidade da sintomatologia depressiva foi avaliada pela escala de depressão de Hamilton de 21 itens (HAM-D); o desempenho cognitivo dos pacientes e controles foi avaliado pelo teste cognitivo de Cambridge (CAMCOG) e pelo mini-exame do estado mental (MEEM). A expressão da GSK-3B foi determinada em plaquetas através de ensaio imunoenzimático (EIA), sendo estabelecido os níveis totais da GSK-3B (T-GSK-3B) e de sua forma fosforilada (P-GSK-3B), inativa. A atividade enzimática foi inferida indiretamente pela razão P-GSK- 3B / T-GSK-3B. Nos pacientes idosos com depressão maior, observou-se uma redução significante dos níveis P-GSK-3B (p=0,03) e da razão da GSK- 3B (p=0,03). Os pacientes com sintomatologia depressiva mais grave (HAMD > 21) e déficits cognitivos mais intensos (CAMCOG < 86) apresentaram maior atividade enzimática (p=0,03 e teste, p=0,01, respectivamente). Os resultados deste trabalho sugerem que a atividade da GSK-3B está significantemente aumentada em pacientes idosos com depressão maior e que está alteração é mais pronunciada nos pacientes com sintomatologia depressiva e déficits cognitivos mais graves. Neste contexto, a atividade da GSK-3B pode ser considerada um marcador de estado em pacientes idosos com episódios depressivos mais graves e ser um importante alvo para o desenvolvimento de estratégias terapêuticas para estes quadros / Despite the high prevalence of depressive disorders in the elderly, its main physiopathological mechanisms are largely unknown. In the recent years, most of the research efforts focused on the association between cerebrovascular changes and geriatric depression. Nonetheless, other mechanisms have been studied, such as changes in neurotrophic and inflammatory cascades. The enzyme glycogen synthase kinase 3 beta (GSK- 3B) has been implicated in many mental disorders, in particular affective disorders (i.e. major depression and bipolar disorder) and neurodegenerative disorders (i.e. Alzheimers disease). However, there is no study so far that addressed the role of this enzyme in elderly patients with major depression. Therefore, the main objective of this study was to evaluate if GSK-3B activity is changed in elderly patients with major. The working hypothesis is that enzyme activity is significantly increased in elderly patients with major depression as compared to elderly controls. We recruited 40 elderly patients with current major depressive episode (according to the DSM-IV criteria) that was not under antidepressant treatment. The comparison group included 13 healthy elderly subjects with no evidence of cognitive impairment or major psychiatric disorder. The severity of depressive symptoms was assessed by the Hamilton Depression Scale 21 items; cognitive performance was assessed by the Cambridge Cognitive test (CAMCOG) and the Mini-mental State Examination (MMSE). The levels of total and phosphorylated GSK-3B (T-GSK-3B and P-GSK-3B, respectively) levels were determined in platelets by immunoenzymatic assay (EIA). Enzyme activity was indirectly inferred by the ratio P-GSK-3B / T-GSK-3B. Elderly patients with major depression had a significant reduction in the P-GSK-3B levels (p = 0.03) and GSK-3B ratio (p= 0.03). The patients with severe depressive episode (HAM-D scores above 21 points) and cognitive impairment (CAMCOG scores below 86 points) presented the more significant reduction of GSK-3B ratio (p = 0.03 and p = 0.01, respectively). These data altogether suggest that GSK-3B activity is significantly increased in elderly patients with major depression, in particular in those with more severe depressive episode and worse cognitive performance. In this context, the increased enzyme activity may be regarded as a state marker of severe depressive episodes and may an important target to the development of therapeutic strategies to this disorder

Epicoccum nigrum é um fungo ubíquo conhecido por sua capacidade de produzir vários metabólitos secundários bioativos e pelo seu uso potencial como agente de biocontrole contra vários fitopatógenos. Entre os compostos produzidos por E. nigrum, epicolactona é um policetídeo com uma estrutura bastante complexa. O objetivo desta tese foi identificar e caracterizar genes relacionados à biossíntese da epicolactona em E. nigrum. Três mutantes defectivos para a produção de epicolactona anteriormente gerados por mutagênese aleatória foram analisados. Entretanto, os resultados mostraram que o T-DNA provavelmente estava inserido em regiões regulatórias. Usando ferramentas de bioinformática, seis genes de PKSs foram selecionados para deleção. A deleção do gene PKSi12 mostrou que os seus produtos estão relacionados à atividade antagonista. A análise química permitiu a identificação putativa dos preditos precursores da epicolactona, os quais tiveram sua produção afetada no mutante &#916;PKSi12. Uma possível via de biossíntese de epicoccona B e epicoccina por E. nigrum foi proposta. / Epicoccum nigrum is a ubiquitous fungus mainly known for its ability to produce many bioactive secondary metabolites and for its potential use as a biocontrol agent against many phytopathogens. Among the compounds produced by E. nigrum, epicolactone is a polyketide with a very complex structure. The aim of this thesis was to identify and characterize genes related to epicolactone biosynthesis in E. nigrum. Three defective epicolactone mutants previously generated by random mutagenesis were analyzed. However, the results showed that the T-DNA was probably inserted in regulatory regions. Using a genome mining approach, six PKS genes were selected for deletion. The deletion of PKSi12 gene showed that its products are related to E. nigrum antagonistic activity against fungal phytopathogens. The chemical analysis allowed a putative identification of the previously proposed epicolactone precursors, which production was affected in the &#916;PKSi12 mutant. A proposed biosynthesis of epicoccone B and epicoccine by E. nigrum was suggested.

A maior parte dos insetos possui uma estrutura anatômica em forma de filme (membrana peritrófica, MP) composta de quitina e proteínas (peritrofinas), que separa o alimento do epitélio do intestino médio. A MP protege o epitélio de microorganismos e da abrasão, e possui outras funções baseadas no fato de que a MP promove a compartimentalização de enzimas, que incluem: aumento da eficiência digestiva através da diminuição da taxa de excreção das enzimas e de outros mecanismos postulados que são testados nesta tese. A síntese das peritrofinas é mais conhecida do que a da quitina componente da MP, tornando desejável um esforço no detalhamento dessa última. Foram realizadas a caracterização e expressão de genes de S. frugiperda que codificam uma peritrofina e enzimas responsáveis pela síntese e degradação de quitina (quitina sintases 1 (SfCHS1) e 2 (SfCHS2), e quitinase (SfCHI), respectivamente). As sequências dos cDNAs correspondentes foram determinadas através da amplificação de fragmentos de PCR que se sobrepõem. Os padrões de expressão dos genes envolvidos no metabolismo da quitina da MP foram analisados durante o desenvolvimento do inseto por RT-PCR. SfCHS2 é expresso no intestino médio durante os estágios de alimentação da larva, enquanto que SfCHI é expresso durante as fases de pós-alimentação, pré-pupa, e pupa. Ambos os genes são predominantemente expressos na região anterior no intestino médio com um gradiente decrescente de expressão ao longo do tubo digestivo. A citolocalização da quitina revelou que o polissacarídeo está presente somente quando SfCHS2 é expresso e não há quitina no intestino médio quando SfCHI é expresso. Esses resultados levaram a formulação da hipótese de que SfCHS2 é responsável pela síntese da quitina da MP durante o estágio larval e SfCHI degrada a quitina da MP durante a muda larva-pupa, sugerindo padrões inversos de expressão desses genes. Em Spodoptera frugiperda, Tenebrio molitor e Musca domestica é possível prever o sítio de secreção das enzimas digestivas (ventrículo anterior, médio ou posterior) a partir da distribuição antero-posterior das enzimas no espaço endoperitrófico. Também foi possível mostrar, usando vários modelos experimentais, que a separação de compartimentos luminais pela MP: a) impede a inibição de despolimerazes por remover oligômeros do espaço endoperitrófico; b) evita a inibição de oligômero hidrolases restringindo-as ao espaço ectoperitrófico e impedindo que entrem em contato com o alimento e c) anula a inibição de enzimas envolvidas na digestão terminal presentes na superfície do epitélio, impedindo que o alimento entre em contato com elas. / Most insects have a film-like anatomical structure (peritrophic membrane, PM) composed of chitin and proteins (peritrophins), which separates food from midgut tissue. It protects the epithelium against food abrasion and microrganisms and has other functions based on compartmentalization of enzymes, which include: increasing digestive efficiency by decreasing enzyme excretion and by other mechanisms that were tested in this thesis. The peritrophin synthesis is less known than PM chitin synthesis, which needs to be better understood. The characterization and expression of S. frugiperda genes encoding a peritrophin and enzymes responsible for the synthesis and degradation of chitin, chitin synthases 1(SfCHS1) and 2 (SfCHS2), and chitinase (SfCHI), respectively, were analysed. Sequences of corresponding cDNAs were determined by amplification of overlapping PCR fragments and the expression patterns of chitin metabolism genes were analyzed during insect development by RT-PCR. SfCHS2 is expressed in the midgut during the feeding stages, whereas SfCHI is expressed during the wandering and pupal stages. Both genes are predominantly expressed in the anterior portion of the midgut with a decreasing gradient of transcript levels in the medial and posterior portions. Chitin staining revealed that the polysaccharide is present in the PM only when SfCHS2 is expressed. There is little or no chitin in the midgut when SfCHI is expressed. These results support the hypothesis that SfCHS2 is responsible for PM chitin synthesis during the larval stage and SfCHI for PM chitin degradation during larval-pupal molting, suggesting inverse patterns of expression of these genes. The secretion site (anterior, middle or posterior midgut) of digestive enzymes can be predicted in Spodoptera frugiperda, Tenebrio molitor and Musca domestica based on enzyme activity distribution along the endoperitrophic space. We also have shown, using several experimental models, that the luminal compartment separation by PM: a) avoid the polimer hidrolases inhibition by removing oligomer from endoperitrophic space; b) decrease the oligomer hidrolases inhibition by restricting them to the ectoperitrophic space (by avoiding their contact with food); and c) block the inhibition of enzymes located at the cell surface involved in terminal digestion by avoiding their contact with food.

O gênero Hypostomus (Siluriformes: Loricariidae) com cerca de 130 espécies nominais, se destaca como um dos mais diversos e amplamente distribuídos gêneros de peixes de água doce neotropical. Devido a sua ampla distribuição e alta diversidade, os conhecimentos taxonômicos, filogenéticos e biogeográficos para as espécies do gênero são ainda consideravelmente incompletos. Consequentemente, pouco se sabe sobre processos naturais envolvidos em diversificação e variação morfológica para o gênero. Hypostomus ancistroides é uma espécie descrita para a bacia do Alto Paraná, uma eco-região hidrográfica tradicionalmente reconhecida por seu endemismo ictiofaunístico, ocorrendo também na bacia costeira do rio Ribeira de Iguape. Esta espécie apresenta considerável variação morfológica, cariotípica e isoenzimática em suas diferentes populações, sugerindo a existência de um complexo de espécies. Sua ampla área de distribuição, somada aos novos conhecimentos sobre padrões biogeográficos para diversas espécies de peixes do Alto Paraná, reforça essa possibilidade. Entretanto, a variação encontrada na morfologia das populações de H. ancistroides é ampla e contínua, impedindo que se defina diferentes espécies através das abordagens taxonômicas clássicas em ictiologia. Assim, este trabalho se propõe a utilizar ferramentas da sistemática molecular, genética de populações e filogeografia para responder questões fundamentais sobre a evolução desse potencial complexo de espécies. Sequências nucleotídicas completas do marcador mitocondrial ATP sintase (subunidades 6 e 8; 842 pb) foram obtidas para diversas espécies de Hypostomus, incluindo 162 exemplares de H. ancistroides provenientes de doze localidades abrangendo toda a sua área de ocorrência, além de outros gêneros da família Loricariidae, utilizados como grupos externos. Análises filogenéticas de Máxima Verossimilhança, Máxima Parcimônia e Neighbor Joining resultaram em topologias essencialmente semelhantes, sustentando a monofiletismo da espécie, e apontando como seus parentes mais próximos espécies de bacias hidrográficas adjacentes ao Alto Paraná. Esses resultados mostram ainda a existência de quatro filogrupos distintos para a espécie, com áreas de distribuição parcialmente sobrepostas. Análises populacionais e filogeográficas incluiram comparação de distância genética P, estruturação populacional baseada em distribuição de haplótipos e índices de diversidade, testes de neutralidade, índice de fixação FST, análise de variância molecular (AMOVA), análise espacial de variância molecular (SAMOVA), construção de rede haplotípica de parcimônia, e análise de clados hierarquizados (NCPA). Os resultados mostram 48 haplótipos repartidos em doze populações bem estruturadas, com baixo ou nenhum fluxo gênico entre si. Eventos de expansão geográfica podem ser identificados ao longo da história demográfica, sugerindo que a estruturação encontrada atualmente reflete não só as características ecológicas da espécie, como também uma história de mudanças nas condições ambientais, eventualmente favoráveis a migração e dispersão. Contatos entre populações de diferentes bacias podem ser mais frequentes através de capturas de cabeceiras do que ao longo do corpo dos rios principais. A hipótese mais plausível para a presença da espécie na bacia do Ribeira é a de uma captura de cabeceira do alto rio Tietê. Apesar de ser formado por quatro filogrupos distintos, algumas linhagens derivadas de H. ancistroides apresentam sobreposição de suas áreas de distribuição. Esse contato secundário revelado apenas por um marcador de herança matrilineal impossibilita a delimitação de diferentes espécies correspondentes aos filogrupos, sob os paradigmas clássicos de especiação em peixes neotropicais. / The genus Hypostomus (Siluriformes: Loricariidae), comprising ca. 130 nominal species, is one of the most species-rich and widely distributed genera of neotropical freshwater fish. Because of its wide distribution and vast diversity, knowledge on the taxonomy, phylogenetic relationships and biogeography of Hypostomus is still severely incomplete. Consequently, little is known about the processes involved in the diversification and morphological variation for the genus. Hypostomus ancistroides is a species described from the upper Rio Paraná drainage, a freshwater ecoregion known for its ichthyological endemism, also occurring in the coastal basin of Rio Ribeira de Iguape. This species shows considerable morphological, karyotypic and isoenzimatic variation among different populations, suggesting the existence of a species complex. Its wide distribution area, coupled with recent understanding on biogeographic patterns of several fish species from the Upper Parana, reinforces that possibility. However, morphological variation in populations of H. ancistroides is wide and continuous, and does not allow recognition of potential different species by means of traditional taxonomic approaches. Thus, this paper uses tools from molecular systematics, population genetics, and phylogeography in order to answer major questions about the evolution of this potential species complex. Complete sequences of the mitochondrial marker ATP synthase (subunits 6 and 8; 842 bp) were obtained for several species of Hypostomus, including 162 specimens of H. ancistroides from twelve localities covering its entire area of distribution, plus other loricariid genera as outgroups. Phylogenetic analysis using methods of Maximum Likelihood, Maximum Parsimony, Neighbor Joining and Bayesian Inference resulted in mostly similar topologies, supporting the monophyly of the species, and showing as its closest relatives other species from river basins bordering the Upper Parana. Results also reveal four distinct phylogroups for the species, with partially overlapping distribution areas. Population and phylogeographic analysis included comparisons of genetic distance P, population structure based on the haplotype distribution and diversity indices, neutrality tests, fixation index FST, analysis of molecular variance (AMOVA), spatial analysis of molecular variance (SAMOVA), construction of a parsimony haplotype network, and nested clade phylogeographical analysis (NCPA). Results show 48 haplotypes distributed into twelve well-structured populations with little or no gene flow. Geographic range expansion events can be identified along the demographic history of H. ancistroides, suggesting that the structure found today reflects not only the ecology of the species, but also a history of changing environmental conditions that on occasion weree favorable for migration and dispersal. Contact between populations from differente basins may be more intense through headwater stream capture than through the river channel. The most supported hypothesis for the presence of the species in the Rio Ribeira basin is a headwater capture from the upper Rio Tiete. Although H. ancistroides is split into four distinct phylogroups, some derived lineages of the species have overlapping distribution ranges. Such secondary contact is revealed only by a matrilineal inheritance marker and does not allow the recognition of separate species for the different phylogroups under the current paradigm of speciation and species limits in Neotropical fishes.