Proefschrift Rijksuniversiteit Limburg, Maastricht. / Met lit. opg. - Met een samenvatting in het Nederlands.

Proefschrift Universiteit Maastricht. / Auteursnaam op omslag: Roel Schins. Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

Proefschrift Universiteit Maastricht. / Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Diss. (sammanfattning) SLU, Alnarp : Sveriges lantbruksuniversitet, 2008. / Härtill 4 uppsatser.

Thesis (MSc)--Stellenbosch University, 2012. / Triticale is an amphidiploid that consists of wheat (A and B) and rye (R) genomes. This cereal is fast becoming important on a commercial basis and warrants further assessment for the better management and breeding of the hybrid. The assessment of the genetic diversity among the wheat and rye genomes within triticale can be obtained by using molecular markers developed in both donor genomes. Simple sequence repeats markers (SSRs) and amplified fragment length markers (AFLPs) have been previously used to assess the genetic diversity among triticale lines. SSRs are highly polymorphic markers that are abundant and which have been shown to be highly transferable between species in previous studies while AFLP markers are known to generate plenty of data as they cover so many loci. Thus, the aim of this study was to develop a marker system suitable to assess the genetic diversity and relationships of advanced breeding material (and cultivars) of the Stellenbosch University’s Plant Breeding Laboratory (SU-PBL). Therefore, both AFLP and SSR markers were initially analysed using eight triticale cultivars (with known pedigrees) to facilitate cultivar identification. Fourty-two AFLP primer combinations and 86 SSR markers were used to assess the genetic diversity among the Elite triticale cultivars. The AFLP primer combinations generated under average polymorphism information content (PIC) values. Furthermore, these markers generated neighbour-joining (NJ) and unweighted pair group method with arithmetic average (UPGMA) dendograms that displayed relationships that did not correspond with the available pedigree information. Therefore, this marker system was found not to be suitable. A set of 86 SSRs previously identified in both wheat and rye, was used to test the genetic diversity among the eight cultivars. The markers developed in wheat achieved 84% transferability while those developed in rye achieved 79.3% transferability. A subset of SSR markers was able to distinguish the cultivars, and correctly identify them by generating NJ and UPGMA dendograms that exhibited relationships that corroborated the available pedigree data. This panel of markers was therefore chosen as the most suitable for the assessment of the advanced breeding material. The panel of seven SSR markers was optimised for semi-automated analysis and was used to screen and detect the genetic diversity among 306 triticale entries in the F6, Senior and Elite phases of the SU-PBL triticale breeding programme. An average PIC value of 0.65 was detected and moderate genetic variation was observed. NJ and UPGMA dendograms generated showed no clear groupings. However, the panel of markers managed to accurately identify all cultivars within the breeding program. The marker panel developed in this study is being used to routinely distinguish among the advanced breeding material within the SU-PBL triticale breeding programme and as a tool in molecular-assisted backcross.

Orientador: Jorge Fernando Brandão Pereira / Coorientador: Valéria de Carvalho dos Santos Ebinuma / Banca: João Vitor Dutra Molino / Banca: Marcel Otavio Cerri / Resumo: A proteína verde fluorescente (GFP, do inglês Green Fluorescent Protein) é um biomarcador utilizado na produção de proteínas de fusão, amplamente empregado in vivo e in vitro. Este trabalho avaliou a produção da GFP expressa em Escherichia coli BL21 (DE3) [pLysS; pET28(a)] em mesa incubadora rotativa e biorreator, e sua posterior extração utilizando Sistemas Micelares de Duas Fases Aquosas (SMDFA). Na etapa de produção em mesa incubadora rotativa, estudou-se a influência da taxa de agitação, tempo de indução e concentração do composto indutor, isopropil-β-1-D-tiogalactopiranosídeo (IPTG), sobre a produção da GFP, e apenas a variação da taxa de agitação e o tempo de indução alteraram a produção de GFP. A melhor produção de GFP (314,59 mg/L) foi obtida a 30°C após 22 h de cultivo a uma agitação de 150 rpm, induzindo com 0,5 mM de IPTG realizada 10 h após o início do cultivo (meio da fase exponencial de crescimento). Posteriormente, avaliou-se menores concentrações de IPTG (0,25; 0,125 mM) na produção de GFP e observou-se que os níveis de produção foram mantidos. Estes estudos iniciais, foram a base para as condições empregadas em biorreator tanque agitado, que operou no modo batelada a uma agitação de 200 rpm, aeração de 2 vvm (volume de ar/ volume de meio) e indução com 0,125 mM de IPTG após 6 h de cultivo. Nas condições citadas, obteve-se apenas uma produção de 128,13 mg/L de GFP, desse modo, apesar da potencialidade da ampliação de escala na produção desta biomolécula, estud... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Green Fluorescent Protein (GFP) is a in vivo and in vitro biomarker widely used in the production of fusion proteins. This work evaluated the production of GFP expressed in Escherichia coli BL21 (DE3) [pLysS; pET28 (a)] in shaker and bioreactor, and its subsequent extraction using Aqueous Micellar Two-Phase Systems (AMTPS). The influence of the agitation rate, induction time and concentration of the inductor, isopropyl-β-1-D-thiogalactopyranoside (IPTG), on GFP production, was studied using a shaker. The results shown that only the agitation rate and the induction time have affect the GFP production. The best GFP production (314.59 mg/L) was obtained at 30°C after 22 h of culture at 150 rpm, induced with 0.5 mM IPTG after 10 h after of the start of cultivation (at half of exponential growth). Subsequently, lower IPTG concentrations (0.25, 0.125 mM) were evaluated for the GFP production, being maintained the production levels. From these conditions, a cultivation using agitated tank bioreactor was then carried out. The bioreactor operated in batch mode at 200 rpm with a 2 vvm aeration (air volume / volume of medium), being the GFP production induced with 0.125 mM IPTG after 6 h of microorganism' growth. Under that conditions, a production of 128.13 mg/L of GFP was obtained. Thus, despite the potential of scale-up in the production of this biomolecule, further studies are still required. After the GFP production studies, the bacterial cells were disrupted using continuous freezing / thawing cycles. GFP recovered from the cell lysate was then used to perform stability studies at different pHs (3 - 12) and temperatures (25°, 37° and 50°C). The stability studies shown that GFP was stable between pHs 7 to 11 and at temperatures of 25°C and 37°C, but not stable at a temperature of 50°C. Afterwards, liquid-liquid extraction... (Complete abstract click electronic access below) / Mestre

A depressão é um transtorno psiquiátrico altamente prevalente e oneroso, sendo uma das principais causas mundiais de morbimortalidade. Contudo, seu status nosológico é tema de profundas controvérsias, uma vez que apresenta uma definição pouco precisa, gerando uma enorme heterogeneidade clínica. Esta tese é composta por quatro artigos, e visa estudar a heterogeneidade da depressão investigando suas interações com o construto de personalidade e com marcadores biológicos. Os dois primeiros artigos visam viabilizar o uso de um instrumento de avaliação de estilos de personalidade, o Temperament and Personality Questionnaire (T&P). O primeiro artigo descreve o processo de tradução e adaptação transcultural do T&P para o português brasileiro, utilizando uma metodologia estandardizada. O segundo artigo examina as propriedades psicométricas do T&P em uma amostra de pacientes deprimidos. Nesse artigo, análises fatoriais confirmatórias foram realizadas para testar os sete níveis do instrumento, sendo encontrados índices de ajuste adequados para modelos com dois até quatro fatores, com alta consistência interna para todos os níveis. Foram encontradas as associações esperadas entre os estilos de personalidade e dois validadores externos, com estilos de personalidade resilientes apresentando menor qualidade de vida e maiores níveis de depressão, enquanto estilos de personalidade desordenados apresentaram o inverso. Além disso, a performance dos itens foi explorada utilizando a Teoria de Resposta ao Item, com a maioria dos itens apresentando melhor padrão de respostas dicotômico. Por fim, correlações altas entre os estilos de personalidade de níveis de baixa ordem com seus correspondentes construtos molares, confirmaram a organização hierárquica do instrumento. O terceiro artigo estudou a heterogeneidade da depressão em relação a personalidade. Nele, foi avaliada a associação entre dimensões depressivas (definidas por análise fatorial exploratória) e quatro classes de personalidade (criadas a partir de uma análise de classes latentes) em uma amostra de pacientes deprimidos. As classes de personalidade foram denominadas resilientes e desordenadas de acordo com seu nível de funcionamento e qualidade de vida. A classes desordenadas apresentaram maiores escores de sintomatologia depressiva, principalmente na dimensão cognitiva, com maior comorbidade com transtornos ansiosos. As dimensões depressivas melancólicas foram pouco ou nada discriminativas em relação às classes de personalidade, reforçando sua maior relevância para as depressões não-melancólicas. O quarto artigo analisou subgrupos depressivos classificados por um instrumento de avaliação de alterações da psicomotricidade (o CORE). Os grupos melancólico e não-melancólico foram comparados em relação a três dimensões de marcadores biológicos (estresse oxidativo, neurotrofina e marcadores imunológicos). Os resultados replicaram e estenderam achados sobre diferenças biológicas de subtipos depressivos, mostrando a importância da avaliação dos sinais de melancolia. Assim, essa tese contribui no estudo da heterogeneidade da depressão viabilizando o uso de um instrumento de avaliação da personalidade e estendendo achados que podem possibilitar que subtipos depressivos possam ser avaliados em relação a estilos de personalidade e marcadores biológicos. / Depression is a highly prevalent and burdensome psychiatric disorder, and one of the main causes of morbidity and mortality. However, its nosological status remains the subject of deep controversies, since it presents an imprecise definition, generating an enormous clinical heterogeneity. This thesis comprises four papers, and aims to study the heterogeneity of depression investigating its interactions with the construct of personality and with biologic markers. The first two papers aim to enable the use of an instrument for assessing personality styles, the Temperament and Personality Questionnaire (T&P). The first paper describes the process of translation and cross-cultural adaptation of the T&P into Brazilian Portuguese using standardized methodology. The second paper examines the psychometric properties of the T&P in a sample of depressed patients. In this paper, confirmatory factor analyzes were conducted in order to test the seven tiers of the instrument, with proper goodness fit for models from two to four factors, and high internal consistency for all levels. The expected associations between personality styles and two external validators (quality of life and depression) were found, with resilient personality styles presenting lower quality of life and higher levels of depression, while disordered personality styles showed the reverse. Moreover, the performance of the items was explored using the Item Response Theory, and most items showed a better dichotomous response pattern. Finally, high correlations between lower-order personality styles and their corresponding molar personality constructs were found, confirming the hierarchical organization of the instrument. The third paper studied the heterogeneity of depression in relation to personality. In it, the association between depressive dimensions (defined by exploratory factor analysis) and four personality classes (created by latent class analysis) was evaluated in a sample of depressed patients. Classes were named resilient and disordered according to their level of functioning and quality of life. The disordered classes had higher scores of depressive symptoms, especially in the cognitive dimension, and higher comorbidity with anxiety disorders. The melancholic depressive dimensions were little or no discriminative with respect to the personality classes, reinforcing its greater relevance to non-melancholic depression. The fourth paper examined depressive subgroups classified by an assessment instrument of psychomotor changes (the CORE). The melancholic and non-melancholic groups were compared with respect to three dimensions of biomarkers (oxidative stress, neurotrophin and immunological markers). Results replicated and have extended findings on biological differences in depressive subtypes, showing the importance of evaluating signs of melancholia. Thus, this thesis contributes to the study of depression heterogeneity enabling the use of an instrument for assessing personality and extending findings that may allow that depressive subtypes can be assessed in relation to personality styles and biological markers.

L’autisme et la déficience mentale sont deux syndromes neuro-développementaux impliquant des facteurs génétiques. Notre travail a consisté à rechercher de nouveaux gènes candidats ou facteurs de susceptibilité chez 106 patients atteints d’autisme et 68 de déficience mentale non syndromique sporadique.Nous avons observé une association entre l’allèle 4 d’un marqueur microsatellite GXAlu localisé en 17q11.2 dans l’intron 27b du gène NF1 et des patients atteints de déficience mentale non-syndromique.Nous avons contribué à la mise en évidence d’une augmentation d’expression du transcrit NLGN4X, chez un patient autiste avec un retard mental non-syndromique présentant une mutation dans le promoteur du gène NLGN4X.L’étude de la région 22q13 par MLPA, nous a permis de mettre en évidence une délétion de novo d’au moins 1Mb chez un patient autiste.Les variations de nombre de copies (CNV) ont été étudiées chez des autistes par QPCR. Nous avons identifié 27 variations réparties sur 17 gènes parmi les 36 explorés. Les CNV observés dans les gènes ITGA6, TAGLN3, HOXA1, DLG4 et UBE2C sont intéressants en raison de l’implication de ces gènes dans le développement cérébral ou la fonction neuronale.L’ensemble de ces résultats nécessite des expériences complémentaires de validation. / Autism and mental retardation are two neurodevelopmental syndromes involving genetic factors. Our work consists in finding new candidate genes or susceptibility factors. 106 autistic patients and 68 sporadic non-syndromic mentally retardated patients were studied.We have shown an association between allele 4 of a microsatellite marker GXAlu locasized in 17q11.2, in intron 27b of the NF1 gene and patients with non-syndromic mental retardation.We contributed to the study on the NLGN4X gene. We demonstrated an increase of expression of NLGN4X transcript, in an autistic patient with non-syndromic mental retardation linked to a mutation in the NLGN4X gene promoter.We study the 22q13 region with MLPA method, we have demonstrated a deletion de novo of at least 1Mb in an autistic patient.The copy number variations (CNV) have been investigated in an autistic population by QPCR. We identified 27 variations on 17 genes among the 36 investigated. The CNV observed in ITGA6, TAGLN3, HOXA1, DLG4 and UBE2C genes are interesting because of the involvement of these genes in brain development or neuronal function.These results require further experiments for validation.

L’objectif de ce travail de thèse était de vérifier l’hypothèse selon laquelle les troubles olfactifs peuvent constituer des marqueurs de la dépression. Tout d’abord, deux études longitudinales ont évalué l’effet du traitement antidépresseur sur les atteintes olfactives et émotionnelles. Deux autres études ont étudié quelles atteintes olfactives peuvent aider à différencier la dépression d’une maladie d’Alzheimer débutante. Les résultats de ce travail ont confirmé la présence de marqueurs olfactifs d’état (pour les aspects hédonique et émotionnel) et de trait (pour les atteintes en rapport avec des capacités cognitives) dans la dépression. Par ailleurs, nos données ont mis en évidence des profils d’altérations différents dans la dépression et la maladie d’Alzheimer débutante, concernanat la mémoire de reconnaissance et l’identification des odeurs, ce qui pourrait aider à différencier ces deux maladies. D’autres travaux devront confirmer l’implication de ces marqueurs dans l’efficacité de la thérapie, le suivi et le diagnostic précoce des patients. / The aim of this study was to test the hypothesis of olfactory deficits as potential markers of depression. Firstly, two longitudinal studies have evaluated the effect of antidepressant treatment on olfactory and emotional alterations. In two other studies, we wanted to investigate which olfactory deficits could differentiate depression and early stage of Alzheimer’s disease. The results have confirmed the presence of state (for hedonic and emotional aspects) and trait (for alterations involving cognitive capacities) olfactory markers of depression. Besides, we have demonstrated two different profiles of alterations in depression and early stage of Alzheimer’s disease, concerning odors’ memory recognition and identification, which could help to discriminate these two diseases. Other investigations are necessary to confirm the implication of these markers in the efficiency of the therapy, the diagnostic and the take care of patients.