A esquizofrenia é um transtorno neuropsiquiátrico crônico que afeta aproximadamente 1% da população mundial. As teorias neurobiológicas descrevem que a esquizofrenia é essencialmente causada por alterações bioquímicas e estruturais do cérebro, devido às disfunções nos sistemas glutamatérgico, dopaminérgico e serotoninérgico. Desta forma, a determinação das concentrações de aminoácidos e neurotransmissores em amostras de plasma de pacientes esquizofrênicos pode auxiliar na avaliação da eficácia da terapia. Além dos antipsicóticos, medicação de primeira linha no tratamento inicial da esquizofrenia, a maioria dos pacientes também faz uso concomitante de outras classes de fármacos, tais como antidepressivos, anticonvulsivantes e ansiolíticos para minimizar os sintomas associados a esta doença. Nesta tese, um método empregando a precipitação de proteínas (PPT) e a cromatografia líquida por interação hidrofílica acoplada à espectrometria de massas em tandem (HILIC-MS/MS) foi adequadamente desenvolvido e validado para a determinação de aminoácidos (aspartato, serina, glicina, alanina, metionina, leucina, tirosina e triptofano) e neurotransmissores (glutamato e ácido -aminobutírico) em amostras de plasma de 35 pacientes esquizofrênicos em tratamento com clozapina (27 pacientes) e olanzapina (8 pacientes) para avaliar a eficácia do tratamento, tendo como controle 38 voluntários sadios. O método HILIC-MS/MS apresentou linearidade do LIQ (9,7 pmol mL-1 - 13,3 nmol mL-1) ao LSQ (19,4 nmol mL-1 - 800 nmol mL-1), tempo de análise de 3,0 min, exatidão com EPR de -18 a 19% e precisão com CV de 0,1 a 16% (LIQ). A análise de variância (ANOVA), seguida por teste post-hoc de Duncan, revelou que os níveis médios plasmáticos (nmol mL-1) de metionina (F2,70 = 3,14, p = 0,049) de pacientes esquizofrênicos em tratamento com olanzapina foram significativamente mais elevados, quando comparados aos valores obtidos com o grupo controle (voluntários saudáveis), já o nível de glutamato em pacientes esquizofrênicos em tratamento com clozapina apresentaram tendência a valores mais altos (F2.70 = 2,50, p = 0,090). Já os métodos, PPT/LC-MS/MS e LC-MS/MS no modo column switching utilizando uma coluna monolítica de sílica híbrida com grupos cianopropil na primeira dimensão, foram desenvolvidos e validados para a determinação dos antipsicóticos (olanzapina, quetiapina, clozapina, haloperidol e clorpromazina), antidepressivos (mirtazapina, paroxetina, citalopram, sertralina, imipramina, clomipramina e fluoxetina), anticonvulsivantes (carbamazepina e lamotrigina), e ansiolíticos (diazepam e clonazepam) em amostras de plasma de pacientes esquizofrênicos para fins de monitorização terapêutica. O método PPT/LC-MS/MS apresentou linearidade do LIQ (0,2 ng mL-1 - 5,0 ng mL-1) ao LSQ (40,5 ng mL-1 - 10,5 g mL-1), exatidão com EPR de -9,7 a 8,0%, e precisão com CV de 0,1 a 12%. Já o método LC-MS/MS no modo column switching apresentou linearidade do LIQ (63,0 pg mL-1 - 1250,0 pg mL-1) ao LSQ (40,5 ng mL-1 - 10,5 g mL-1), exatidão com EPR de -14 a 12% e precisão com CV de 0,6 a 6,5%. A pré-concentração seletiva dos fármacos na coluna monolítica com grupos cianopropil incorporados e a remoção dos componentes endógenos da amostra biológica, antes da separação cromatográfica, favoreceram a seletividade e detectabilidade do método LC-MS/MS no modo column switching. Este método quando comparado ao de referência PPT/LC-MS/MS, através da análise de 10 amostras de pacientes esquizofrênicos, não apresentou diferença significativa (teste t) entre as concentrações plasmáticas, podendo ser aplicado na monitorização terapêutica. Além deste fato, este método automatizado favoreceu a precisão, a exatidão e a freqüência analítica. / Schizophrenia is a chronic neuropsychiatric disorder that affects approximately 1% of the world population. According to neurobiological theories, schizophrenia stems from biochemical and structural alterations in the brain due to dysfunction in the glutamatergic, dopaminergic, and serotonergic systems. Determining the concentrations of amino acids and neurotransmitters in plasma samples from schizophrenic patients may assist evaluation of therapy effectiveness. In addition to antipsychotics (the first-line drug in the initial treatment of schizophrenia), most patients concomitantly use other classes of drugs such as antidepressants, anticonvulsants, and anxiolytics to minimize the symptoms associated with this disease. To evaluate treatment efficacy, in this thesis a method based on protein precipitation (PPT) and hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry (HILIC-MS/MS) has been properly developed and validated to determine amino acids (aspartate, serine, glycine, alanine, methionine, leucine, tyrosine, and tryptophan) and neurotransmitters (glutamate and -aminobutyric acid) in plasma samples obtained from 35 schizophrenia patients treated with clozapine (27 patients) or olanzapine (8 patients); 38 healthy volunteers served as controls. The HILIC-MS/MS method was linear for concentrations ranging from the LLOQ (9.7 pmol mL-1 - 13.3 nmol mL-1) to the ULOQ (19.4 nmol mL-1 - 800 nmol mL-1). The analysis time was 3.0 min. In the case of accuracy, RSE ranged from -18 to 19%. As for precision, CV lay between 0.1 and 16% (LLOQ). Analysis of variance (ANOVA) followed by post-hoc Duncan showed that the average methionine serum levels (nmol mL-1) (F2.70 = 3.14, p = 0.049) in schizophrenic patients treated with olanzapine were significantly higher as compared with the control group (healthy volunteers). The glutamate level in schizophrenic patients treated with clozapine tended to higher values (F2.70 = 2.50, p = 0.090). Concerning the analytical methods, PPT/LC-MS/MS and LC-MS/MS operating in the column-switching mode were developed and validated to determine antipsychotic (olanzapine, quetiapine, clozapine, haloperidol, and chlorpromazine), antidepressants (mirtazapine, paroxetine, citalopram, sertraline, imipramine, clomipramine, and fluoxetine), anticonvulsants (carbamazepine and lamotrigine), and anxiolytics (diazepam and clonazepam) in plasma samples taken from schizophrenic patients for therapeutic drug monitoring. A monolithic hybrid column containing silica with cyanopropyl groups in the first dimension was employed. The PPT/LC-MS/MS method was linear from the LLOQ (0.2 ng mL-1 - 5.0 ng mL-1) to the ULOQ (40.5 ng mL-1 - 10.5 g mL-1). In the case of accuracy, RSE ranged from -9.7 to 8.0%; as for precision, CV lay between 0.1 and 12%. LC-MS/MS in the column-switching mode was linear from the LLOQ (63.0 pg mL-1 - 1250.0 pg mL-1) to the ULOQ (40.5 ng mL-1 - 10.5 g mL-1). RSE ranged from -14 to 12%; CV lay between 0.6 and 6.5%. The drugs were selectively pre-concentrated in the monolithic column containing silica incorporated with cyanopropyl groups. For the LC-MS/MS method operating in the column-switching mode, the endogenous components of the biological sample of the LC-MS/MS method were removed before analysis. Analysis of 10 plasma samples obtained from schizophrenic patients did not reveal any significant differences (t test) between the LC-MS/MS method and the reference PPT/LC-MS/MS method. Therefore, LC-MS/MS can be applied in therapeutic monitoring, with the advantage that this method offers improved precision, accuracy, and analytical frequency.

The purpose of this study was the investigation of several transport systems expressed at the BBB. The identification and functional characterization of such transport systems is essential to provide a basis for strategies to regulate drug disposition into the brain. Immortalized rat brain endothelial cells (RBE4 cells) have been used in this study as an in vitro model of the BBB. The present study has shown that the RBE4 cells are a suitable model of the BBB for transporter studies. These cells do express the amino acid transport systems L and y+, which are known to be present at the BBB. The uptake of L-tryptophan, a neutral amino acid transported by system L, exhibited a half saturation constant (Kt) of 31 µM and a maximal velocity rate (Vmax) of about 1 nmol/mg/min in RBE4 cells. The kinetic constants of the L-arginine uptake, representing system y+ transport activity, into RBE4 cells were determined with a Kt value of about 55 µM and a Vmax of 0.56 nmol/mg/min. Furthermore the expression of two sodium dependent transporters, the 5-HT transporter (SERT) and the organic cation/carnitine transporter OCTN2, was shown at the RBE4 cells. Uptake studies with radiolabeled 5-HT exhibited a saturable, sodium dependent transport at RBE4 cells with a Kt value of about 0.40 µM and a Vmax of about 52 fmol/mg/min. L-carnitine and TEA (tetraethylammonium) are known to be transported by the OCTN2 transporter. The uptake of L-carnitine into RBE4 cells was shown to be sodium dependent and saturable with a Kt value of 54 µM and a maximal velocity of about 3.6 pmol/mg/min. In contrast, the organic cation TEA follows a sodium independent uptake mechanism at RBE4 cells. Also a sodium independent choline uptake into the cells was discovered but the molecular identity remained unknown. This saturable choline transport exhibited a Kt value of about 22 µM and a maximal velocity of about 52 pmol/mg/min.

Every individual is born with different natural competencies that can be honed by both voluntary and involuntary environmental stimuli. The response our genotype decides to make, if any, towards those stimuli, determines how well our competencies develop. Each person’s coding and variations of genes will result in unique qualities in their phenotype, or physical structure. As a result, a person has various traits that are displayed through their behavior. DNA is genetically shown to express itself through traits by up to 75%. This leaves a sort of buffer of around 25%. This region is available for us to adapt to our environmental stimuli. Your innate qualities will not reach their full potential without stimulation from the environment, in a leadership case, with education and training and therefore it can be argued that environmental exposure is necessary to fully expose the potentials and capabilities of an individual, rather than instill a new skill or develop a talent that was not existent before. Innate leadership is not a permanent state, on the contrary, it is a continuously adaptive situation demanding contextual evolutionary changes or resignation from the subject occupying the role. When the needs and demands of a society or era outweigh the relevance of the innate leaders' traits and competencies, an evolution of leadership is needed to maintain a positive relationship between all parties involved. As a result, the innate leader will begin to lose their innateness in their role and unless they evolve and adapt (because the two actions are not the same) to new contextual needs, their tenure as leader will begin to be detrimental and counter-functional. What we want to put forward is a real, universal and constructive understanding of what makes a human happy, motivated and productive and how an innate person in context is a much better solution in the short and long run, for those around them when put to a task.

Cloninger’s Psychobiological Theory of Personality proposes four temperament dimensions, each underpinned by a different neurotransmitter system. The serotonergic system is purportedly linked to Harm Avoidance (HA). The aim of this study was to explore the relationship between HA and serotonin in migraine without aura (MO). A second aim was to explore the personality profile of MO patients. Sixty-six participants completed an online questionnaire and donated blood samples. Results indicated no significant association between HA and serotonin and a significant relationship between MO and HA. This study indicates that both Cloninger’s Psychobiological Theory of Personality and the Tridimensional Personality Questionnaire used for its assessment have value in South African personality research. In addition, the findings of the study reveal support for personality influences on the processes involved in migraine. This not only produces worthwhile avenues of research but also an alternative perspective for clinical practice. / Psychology / M.A. (Psychology (Research Consultation))

A esquizofrenia é um transtorno neuropsiquiátrico crônico que afeta aproximadamente 1% da população mundial. As teorias neurobiológicas descrevem que a esquizofrenia é essencialmente causada por alterações bioquímicas e estruturais do cérebro, devido às disfunções nos sistemas glutamatérgico, dopaminérgico e serotoninérgico. Desta forma, a determinação das concentrações de aminoácidos e neurotransmissores em amostras de plasma de pacientes esquizofrênicos pode auxiliar na avaliação da eficácia da terapia. Além dos antipsicóticos, medicação de primeira linha no tratamento inicial da esquizofrenia, a maioria dos pacientes também faz uso concomitante de outras classes de fármacos, tais como antidepressivos, anticonvulsivantes e ansiolíticos para minimizar os sintomas associados a esta doença. Nesta tese, um método empregando a precipitação de proteínas (PPT) e a cromatografia líquida por interação hidrofílica acoplada à espectrometria de massas em tandem (HILIC-MS/MS) foi adequadamente desenvolvido e validado para a determinação de aminoácidos (aspartato, serina, glicina, alanina, metionina, leucina, tirosina e triptofano) e neurotransmissores (glutamato e ácido -aminobutírico) em amostras de plasma de 35 pacientes esquizofrênicos em tratamento com clozapina (27 pacientes) e olanzapina (8 pacientes) para avaliar a eficácia do tratamento, tendo como controle 38 voluntários sadios. O método HILIC-MS/MS apresentou linearidade do LIQ (9,7 pmol mL-1 - 13,3 nmol mL-1) ao LSQ (19,4 nmol mL-1 - 800 nmol mL-1), tempo de análise de 3,0 min, exatidão com EPR de -18 a 19% e precisão com CV de 0,1 a 16% (LIQ). A análise de variância (ANOVA), seguida por teste post-hoc de Duncan, revelou que os níveis médios plasmáticos (nmol mL-1) de metionina (F2,70 = 3,14, p = 0,049) de pacientes esquizofrênicos em tratamento com olanzapina foram significativamente mais elevados, quando comparados aos valores obtidos com o grupo controle (voluntários saudáveis), já o nível de glutamato em pacientes esquizofrênicos em tratamento com clozapina apresentaram tendência a valores mais altos (F2.70 = 2,50, p = 0,090). Já os métodos, PPT/LC-MS/MS e LC-MS/MS no modo column switching utilizando uma coluna monolítica de sílica híbrida com grupos cianopropil na primeira dimensão, foram desenvolvidos e validados para a determinação dos antipsicóticos (olanzapina, quetiapina, clozapina, haloperidol e clorpromazina), antidepressivos (mirtazapina, paroxetina, citalopram, sertralina, imipramina, clomipramina e fluoxetina), anticonvulsivantes (carbamazepina e lamotrigina), e ansiolíticos (diazepam e clonazepam) em amostras de plasma de pacientes esquizofrênicos para fins de monitorização terapêutica. O método PPT/LC-MS/MS apresentou linearidade do LIQ (0,2 ng mL-1 - 5,0 ng mL-1) ao LSQ (40,5 ng mL-1 - 10,5 g mL-1), exatidão com EPR de -9,7 a 8,0%, e precisão com CV de 0,1 a 12%. Já o método LC-MS/MS no modo column switching apresentou linearidade do LIQ (63,0 pg mL-1 - 1250,0 pg mL-1) ao LSQ (40,5 ng mL-1 - 10,5 g mL-1), exatidão com EPR de -14 a 12% e precisão com CV de 0,6 a 6,5%. A pré-concentração seletiva dos fármacos na coluna monolítica com grupos cianopropil incorporados e a remoção dos componentes endógenos da amostra biológica, antes da separação cromatográfica, favoreceram a seletividade e detectabilidade do método LC-MS/MS no modo column switching. Este método quando comparado ao de referência PPT/LC-MS/MS, através da análise de 10 amostras de pacientes esquizofrênicos, não apresentou diferença significativa (teste t) entre as concentrações plasmáticas, podendo ser aplicado na monitorização terapêutica. Além deste fato, este método automatizado favoreceu a precisão, a exatidão e a freqüência analítica. / Schizophrenia is a chronic neuropsychiatric disorder that affects approximately 1% of the world population. According to neurobiological theories, schizophrenia stems from biochemical and structural alterations in the brain due to dysfunction in the glutamatergic, dopaminergic, and serotonergic systems. Determining the concentrations of amino acids and neurotransmitters in plasma samples from schizophrenic patients may assist evaluation of therapy effectiveness. In addition to antipsychotics (the first-line drug in the initial treatment of schizophrenia), most patients concomitantly use other classes of drugs such as antidepressants, anticonvulsants, and anxiolytics to minimize the symptoms associated with this disease. To evaluate treatment efficacy, in this thesis a method based on protein precipitation (PPT) and hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry (HILIC-MS/MS) has been properly developed and validated to determine amino acids (aspartate, serine, glycine, alanine, methionine, leucine, tyrosine, and tryptophan) and neurotransmitters (glutamate and -aminobutyric acid) in plasma samples obtained from 35 schizophrenia patients treated with clozapine (27 patients) or olanzapine (8 patients); 38 healthy volunteers served as controls. The HILIC-MS/MS method was linear for concentrations ranging from the LLOQ (9.7 pmol mL-1 - 13.3 nmol mL-1) to the ULOQ (19.4 nmol mL-1 - 800 nmol mL-1). The analysis time was 3.0 min. In the case of accuracy, RSE ranged from -18 to 19%. As for precision, CV lay between 0.1 and 16% (LLOQ). Analysis of variance (ANOVA) followed by post-hoc Duncan showed that the average methionine serum levels (nmol mL-1) (F2.70 = 3.14, p = 0.049) in schizophrenic patients treated with olanzapine were significantly higher as compared with the control group (healthy volunteers). The glutamate level in schizophrenic patients treated with clozapine tended to higher values (F2.70 = 2.50, p = 0.090). Concerning the analytical methods, PPT/LC-MS/MS and LC-MS/MS operating in the column-switching mode were developed and validated to determine antipsychotic (olanzapine, quetiapine, clozapine, haloperidol, and chlorpromazine), antidepressants (mirtazapine, paroxetine, citalopram, sertraline, imipramine, clomipramine, and fluoxetine), anticonvulsants (carbamazepine and lamotrigine), and anxiolytics (diazepam and clonazepam) in plasma samples taken from schizophrenic patients for therapeutic drug monitoring. A monolithic hybrid column containing silica with cyanopropyl groups in the first dimension was employed. The PPT/LC-MS/MS method was linear from the LLOQ (0.2 ng mL-1 - 5.0 ng mL-1) to the ULOQ (40.5 ng mL-1 - 10.5 g mL-1). In the case of accuracy, RSE ranged from -9.7 to 8.0%; as for precision, CV lay between 0.1 and 12%. LC-MS/MS in the column-switching mode was linear from the LLOQ (63.0 pg mL-1 - 1250.0 pg mL-1) to the ULOQ (40.5 ng mL-1 - 10.5 g mL-1). RSE ranged from -14 to 12%; CV lay between 0.6 and 6.5%. The drugs were selectively pre-concentrated in the monolithic column containing silica incorporated with cyanopropyl groups. For the LC-MS/MS method operating in the column-switching mode, the endogenous components of the biological sample of the LC-MS/MS method were removed before analysis. Analysis of 10 plasma samples obtained from schizophrenic patients did not reveal any significant differences (t test) between the LC-MS/MS method and the reference PPT/LC-MS/MS method. Therefore, LC-MS/MS can be applied in therapeutic monitoring, with the advantage that this method offers improved precision, accuracy, and analytical frequency.

The purpose of this study was the investigation of several transport systems expressed at the BBB. The identification and functional characterization of such transport systems is essential to provide a basis for strategies to regulate drug disposition into the brain. Immortalized rat brain endothelial cells (RBE4 cells) have been used in this study as an in vitro model of the BBB. The present study has shown that the RBE4 cells are a suitable model of the BBB for transporter studies. These cells do express the amino acid transport systems L and y+, which are known to be present at the BBB. The uptake of L-tryptophan, a neutral amino acid transported by system L, exhibited a half saturation constant (Kt) of 31 µM and a maximal velocity rate (Vmax) of about 1 nmol/mg/min in RBE4 cells. The kinetic constants of the L-arginine uptake, representing system y+ transport activity, into RBE4 cells were determined with a Kt value of about 55 µM and a Vmax of 0.56 nmol/mg/min. Furthermore the expression of two sodium dependent transporters, the 5-HT transporter (SERT) and the organic cation/carnitine transporter OCTN2, was shown at the RBE4 cells. Uptake studies with radiolabeled 5-HT exhibited a saturable, sodium dependent transport at RBE4 cells with a Kt value of about 0.40 µM and a Vmax of about 52 fmol/mg/min. L-carnitine and TEA (tetraethylammonium) are known to be transported by the OCTN2 transporter. The uptake of L-carnitine into RBE4 cells was shown to be sodium dependent and saturable with a Kt value of 54 µM and a maximal velocity of about 3.6 pmol/mg/min. In contrast, the organic cation TEA follows a sodium independent uptake mechanism at RBE4 cells. Also a sodium independent choline uptake into the cells was discovered but the molecular identity remained unknown. This saturable choline transport exhibited a Kt value of about 22 µM and a maximal velocity of about 52 pmol/mg/min.

Anxiety disorders are the most prevalent psychiatric conditions within primary care, affecting up to 29% of people across their lifetime. Generalized Anxiety disorder (GAD) is frequently comorbid with Major Depressive Disorder (MDD), resulting in greater functional impairment. Gut microbiota have been shown to modulate brain chemistry and function, possibly also playing a role in the genesis of anxiety. Bacteria are also able to produce, or interact with the host metabolism of neuroactive substances, including classical neurotransmitters and trace amines, like octopamine, which although found in trace concentrations in the mammalian brain, can affect CNS function. Specifically, trace amines can affect catecholamine release, reuptake and biosynthesis, and modulate dopamine and serotonin metabolism. We investigated whether microbiota from patients with GAD with no signs of immune activation can alter behaviour in gnotobiotic mice and whether this is accompanied by changes in metabolites within the gastrointestinal tract. Germ-free NIH Swiss mice (n=35) were colonized with microbiota from either a GAD patient (n=18) with severe anxiety, comorbid depression, and low serum and fecal octopamine, or an age and sex-matched healthy control (HC) (n=17). Three weeks post- colonization, mouse behaviour was assessed by standard psychometric tests. Emotionality z-scores were calculated to provide a robust integrated behavioural assessment. Microbiota profiles were assessed by 16S rRNA based Illumina, fecal β-defensin-3 level was measured by ELISA. After sacrifice, mouse brain BDNF and GDNF expression was assessed by immunofluorescence, and gene expression in the hippocampus, amygdala, and olfactory bulbs was assessed by Nanostring. Stool and cecum metabolites were measured in all colonized mice by multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). There were no differences in fecal β-defensin levels between mice colonized with GAD microbiota as compared to mice colonized with HC microbiota. However, GAD mice exhibited greater anxiety and depressive-like behavior compared to HC mice in the digging and tail suspensions tests. Behavioural z-scoring across all six standard psychometric tests showed a significant increase in group emotionality score means of GAD-colonized mice compared to HC-colonized mice. Mice colonized with microbiota from a GAD patient had distinct bacterial profiles from mice colonized with HC microbiota. Compared to HC mice, GAD mice had lower levels of dopamine, octopamine and acetylcholine in cecum contents. Furthermore, GAD mice had higher expression of BDNF in the amygdala, lower expression of BDNF in the hippocampus, and lower expression of GDNF in the midbrain. GAD mice also had lower expression of CCR2 in the hippocampus, higher Cnlp/CAMP in the amygdala and olfactory bulb, and higher Nfkb1 in the olfactory bulb compared to HC mice. Our results suggest that microbiota from a selected patient with GAD has the ability to induce anxiety and depressive-like behavior, by mechanisms independent of immune system, likely by altered production of biogenic amines and neurotransmitters. / Thesis / Master of Science (MSc)

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