Dissertations / Theses on the topic 'Blood disorder'

This study explored the narratives people living with sickle cell disorder construct to explain how the experience of employment influences their identity and subsequently their quality of life. Nine individuals with sickle cell disorder were interviewed and gave detailed autobiographical narratives which were transcribed and processed using narratives analysis. Participants were 4 men and 5 women aged between 22 and 60 years. Participants' passion for their job roles was illustrated by their positive work identities. Earlier experiences were felt to be influential on drive and resilience to work challenges. In the narratives, poor understanding of sickle cell disorder by organisations and reduced ability to manage their condition impacted physical health; psychological and emotional wellbeing; social and cultural experiences. This study has implications for the clinical practice and future research of adults living with sickle cell disorder, contributing to the broadening general understanding of sickle cell disorder.

Obstructive sleep apnea (OSA) is a disease characterized by nighttime airflow limitation, hypoxemia, arousal from sleep, and elevated sympathetic activity and blood pressure. With time, this nighttime dysfunction gives rise to daytime hypertension and a heightened risk for cardiovascular disease. Current treatment options for OSA are not always effective for all patients and the gold-standard intervention, continuous positive airway pressure, has discouraging compliance rates. The work set forth in this dissertation has as its focus a novel intervention for sleep apnea known as inspiratory muscle training (IMT). IMT improves respiratory function and cardiovascular health but has not been implemented previously as a treatment for OSA. As such, Study 1 implements IMT in individuals with mild and moderate OSA, with the objective of assessing the effects of training on the cardio- respiratory parameters of this disease. We randomly assigned 24 individuals with mild- moderate OSA into one of two groups: training vs. placebo, to assess the effects of 6 weeks of training on overnight polysomnography, subjective sleep quality, blood pressure, circulating inflammatory T cells, and plasma catecholamine content. Our results show IMT- related improvements in sleep quality, reduction in the number of arousals from sleep and in periodic limb movements following 6 weeks of training. Most important, IMT was associated with a significant reduction in systolic (~12 mmHg) and diastolic (~5 mmHg) blood pressure, relative to sleep apneics who undertook 6 weeks of placebo training. Additionally, individuals in the training group exhibited ~30% lower levels of sympathetic activity, as measured by plasma catecholamines, relative to placebo trained peers. The mechanism(s) that underlie the IMT-related reductions in blood pressure and sympathetic activity remain to be determined. However, in an effort to determine the precise respiratory stimulus that contributes to the results obtained in Study 1, we subsequently assessed the specific respiratory components of IMT to determine which component (large intrathoracic pressures and/or large lung volumes) likely contributes to the reduction in blood pressure in Study 1. The results of this study conducted in normotensive adults show that respiratory training that entails either large negative or positive intrathoracic pressures reduces systolic and diastolic blood pressure in healthy young adults. Importantly, neither the generation of large lung volumes alone nor performance of daily paced breathing is sufficient to lower blood pressure. Study 3 is a methodologic study that has as its focus upper airway electromyography (EMG) and the utility of assessing EMG activity across a range of conditions and breathing tasks in wakefulness. Because OSA traditionally has been viewed as the result of neuromuscular dysfunction of the upper airway that occurs during sleep, the aim of this work was to develop a "fingerprint" of healthy electromyographic activities during the day in healthy adults across a range of breathing tasks, body positions, and from two different muscle compartments of the upper airway. The findings from this study demonstrate regional differences in muscle activity that vary as a function of body position and task. These data from healthy subjects provide the basis of comparison for subsequent studies in individuals with obstructive sleep apnea.

Health-Related Quality of Life (HRQOL) is an important outcomes measure in cancer and there are specific issues depending on the site, stage, treatment and patient age. Although numerous instruments are available for cancer HRQOL, most are designed for adults, some for children, but none for adolescents and young adults (AYA) who have special age-specific concerns and poor improvement in survival compared with other age groups. An existing HRQOL instrument was modified to ensure its suitability for AYA, its validity, reliability and sensitivity were tested in Australians aged 16 to 25 years old diagnosed with cancer or a blood disorder. Varni’s PedsQLTM Measurement Model (13-18 year olds) was selected, modified then administered to families recruited from haematology/oncology clinics and wards at three Sydney Metropolitan Hospitals in person or by telephone. The Memorial Symptom Assessment Scale was used to categorise participants into groups reflecting sensitivity of symptom severity (slight, moderate and severe). The instruments demonstrated excellent internal consistency reliability, making them suitable for both group and individual comparisons. Clinical validity, construct validity, and discriminant validity were demonstrated by “known-groups” analysis, exploratory factor analysis and correlations, respectively. These new versions of the PedsQL Generic Core and Cancer Module are reliable, valid and sensitive measures of HRQOL in patients aged 16-25 years diagnosed with cancer or a blood disorder. The measures will soon be available for use as outcome measures in clinical trials and clinical practice with this age cohort in Australasia and internationally.

<p>This thesis explored the epidemiology of Posttraumatic Stress Disorder (PTSD) and different aspects of the disorder. Firstly, we investigated the lifetime prevalence of traumatic experiences and PTSD in the general adult population in Sweden and evaluated the impact of different trauma types, trauma frequency, and perceived distress. The results show that traumatic experiences are common and PTSD is not rare; roughly one out of ten traumatic events results in PTSD, with a 5.6% lifetime prevalence. The female/male ratio is 2:1. The risk for PTSD increases considerably with a high trauma-associated emotional impact. The distressing impact of a given trauma appears to be higher in women than in men, indicating an increased vulnerability in women. Secondly, we hypothesized that traffic road accidents (TRA’s) are one of the most prevalent types of traumatic events in Swedish society; therefore, we examined the impact of event and response characteristics associated with TRA’s on PTSD development. The data demonstrate that of those who had experienced a TRA (n=1074, 58.9%), 6.1% reported lifetime PTSD. TRA’s associated with fatal accidents and injury to oneself and related to high distress more than double the risk for PTSD. Thirdly, we compared the relative merits of the DSM-IV’s three-factor solution for PTSD symptoms to alternative models. We found that the symptomatology is equally well accounted for using all factor analytic models as yet presented in the literature; the DSM-IV, we found, provides as good a fit to data as other models. Fourthly, we examined the neurofunctional correlates of PTSD symptoms and whether a treatment-induced (serotonin reuptake inhibitor - SSRI) reduction of PTSD symptoms is associated with altered rCBF during symptom provocation. Our results indicate that PTSD symptoms correlates with areas involved in memory, emotion, attention, and motor control and that SSRI treatment normalizes provocation-induced rCBF in these areas.</p>

During these three-years PhD we analyzed the clinical, phenotypic and molecular aspects of a rare bleeding disorder: the Factor X (FX) deficiency. We contributed to:  create a new European database for collecting data on rare bleeding disorders (RBDs) based on a common data collection model and computer technology to obtain consistent statistic data that may be useful for the drafting of definitive guidelines  confirm that the prevalence of FX within RBDs is about 8%  demonstrate that FX deficiency is among the most severe disorders and patients suffering from it present severe bleedings such as haematoma and haemarthrosis as well as central nervous system, gastrointestinal tract and umbilical cord bleedings  describe heterozygous subjects presenting bleeding episodes, only sporadically reported  increase by 10% the number of causative genetic mutations and identify a rare case of FX deficiency associated to a chromosomal deletion  reports new structure/function protein relations through the characterization of some of the identified mutations by in vitro studies on recombinant wild type and mutant FXs such as: transient and stable transfections techniques, immunofluorescence analyses as well as pulse-chase experiments. In conclusion the obtained results allow to deepen the comprehension of genetic aspects and underlying mechanisms that might clarify the pathogenesis and, in future, improve the diagnosis and the treatment, for this rare but highly disabling disease

Regional pulmonary blood flow and volume was measured in ten rabbits anesthetized with pentobarbital (30 mg/kg). Tracheostomy was performed and catheters were placed into the jugular vein and carotid artery. The cardiac ⁹⁹mtc output was measured using the indicator-dilution technique using Tc labelled RBC followed by an injection of radiolabelled macroaggregates (MAA) to mark regional blood flow. Measurements were made both before and after either exposure to cigarette smoke (3 cigarettes for ten minutes at 4 puffs/minute) or sham exposure to air. The animals were sacrificed and the lungs were removed with the vessels tied. The lungs were then inflated and rapidly frozen over liquid nitrogen. The lungs were sampled into slices by vertical height, each slice was further sampled and then gamma counted for the injected isotopes. Regional pulmonary blood flow was calculated by setting the total lung MAA counts for each MAA equal to the cardiac output so that the sample flow was calculated as the fraction of sample counts to total counts times the cardiac output. The blood volume was marked by the labelled RBC and RBC transit was calculated as blood volume (ml) divided by blood flow (ml/sec). In a second series of experiments (N=10) , ⁵¹Cr PMN were injected as a bolus along with ⁹⁹mtc RBC in an indicator-dilution run. Following the injection of the cells, the blood flow was marked with MAAs and then the same sham or smoke treatments were given as in the previous experiments. At the end of ten minutes, the animals were sacrificed and the lungs were processed the same as before. Regional PMN retention was calculated as the [formula omitted]. The data show that smoke exposure increased pulmonary blood volume (p<.01), pulmonary transit time (p<C.05) and the ratio of lung blood volume to central blood volume (p <C-05) without changing central blood volume or cardiac output. Smoke exposure also caused a redistribution of blood flow from upper to lower lung regions (p <C-05). This lengthened the regional RBC transit times in all regions but particularly in the upper zones. These changes in RBC transit had no effect on PMN retention. We conclude that acute smoke exposure lengthens the RBC transit through the pulmonary circulation by increasing blood volume and redistributing blood flow. This change in red cell behavior was not associated with a consistent change in PMN retention in the lungs.<br>Medicine, Faculty of<br>Pathology and Laboratory Medicine, Department of<br>Graduate

Potential risk factors for development of Post-Traumatic Stress Disorder (PTSD) are still unclear. One potential risk factor for the development of PTSD is an individual’s cardiovascular reactivity and recovery in response to stressor tasks. The current study was conducted with 763 Army National Guard and Army Reserve soldiers. Participants completed a stressful induction along with self-report measures prior to deployment. Post-deployment, self-report measures were completed to assess PTSD symptomatology and experiences related to deployment and combat. Multiple regression was used to determine the ability of blood pressure response to stress to predict PTSD symptoms immediately and one-year after return from deployment. Results indicated that soldiers who had a less reactive systolic blood pressure response to and recovery from stressor tasks reported more PTSD symptomatology immediately after and one year after return from deployment. These results suggest that soldiers who develop PTSD after deployment have less pre-deployment emotion regulation ability.

The negative energy balance experienced by dairy cows in early lactation caused by the lack of trade-off between energy intake (input) and requests for lactogenesis (output) is responsible for the occurrence of metabolic disorders. Blood metabolites are important indicators to monitor nutritional and energy status of the cows, and to detect the presence of metabolic disorders. In particular, the hyperketonemia (HYK) is one of the most frequent and costly metabolic diseases in early-lactation dairy cows, and it is commonly diagnosed through the determination of β-hydroxybutyrate (BHB) concentration in blood. With this background, the overall objectives of the present thesis were: i) to summarise literature results on phenotypic and genetic aspects of BHB concentration in blood and milk of dairy cows; ii) to develop mid-infrared (MIR) spectroscopy prediction models for routine determination of blood metabolites; iii) to describe phenotypic variation of MIR-predicted blood metabolites in Brown Swiss, Holstein-Friesian and Simmental cattle breeds; iv) to assess the genetic variation of blood BHB and nonesterified fatty acids (NEFA) predicted by MIR spectroscopy, and their correlations with milk production and composition traits in early-lactation Italian Holstein dairy cows. Hyperketonemia is an abnormal concentration of circulating ketone bodies in the blood; in particular, concentration of blood BHB ≥ 1.2 mmol/L is commonly recognized as indicator of HYK. In general, HYK impairs health of dairy cows by increasing the risk of the onset of other early-lactation diseases, and it negatively affects reproductive performance. Although the relationship with milk yield is still controversial, HYK has a detrimental effect on milk composition. Costs of HYK are mainly imputable to impaired fertility and milk loss. From a genetic point of view, results from the literature suggested the feasibility of selecting cows with low susceptibility to HYK. Milk is the most promising matrix to monitor HYK, taking advantage of using MIR spectroscopy during routine milk recording. The effectiveness of using routine milk MIR spectra to predict main blood metabolites in early-lactation dairy cows was evaluated. Blood BHB, urea and NEFA were the most predictable traits. Predicted blood BHB showed an improved performance in detecting cows with HYK, compared with commercial calibration equation for milk BHB. Factors associated with the phenotypic variation of MIR-predicted blood metabolites were investigated on a large spectral multi-breed database. Holstein-Friesian cows had the greatest concentration of blood BHB and NEFA, and the lowest blood urea content. Blood BHB and NEFA concentrations generally increased with parity. The greatest BHB concentration was observed in the first 10 days of lactation, except for Simmental cows. From 5 to 35 days in milk, NEFA concentration decreased, whereas urea content increased for all considered breeds. The maximum levels of blood BHB and NEFA concentrations were recorded in spring and early summer. Blood urea generally increased across the year, from spring to winter. Genetic parameters for MIR-predicted blood BHB and NEFA concentrations were estimated. The greatest heritability for both metabolites was assessed in the first 10 days after calving (0.32 for BHB and 0.23 for NEFA), and their genetic correlation varied from 0.50 to 0.60. Moreover, an unfavourable trend of estimated breeding values for both blood BHB and NEFA concentrations across year of birth of the bulls was detected. Genetic correlations of BHB and NEFA with milk yield, somatic cell score, protein, lactose and urea content were similar or at least in the same direction, whereas opposite correlations were observed with fat content and fat-to-protein ratio.

Against the background of a dilemma experienced by researchers during a genomics study at an established biomedical research centre in Kenya, the broad aims of this thesis are to develop appropriate responses to important ethical questions on sharing information on a common and serious genetic condition, sickle cell disorder, and assess the responsibilities of researchers in this regard. Using an empirical approach to normative reflection across two phases of qualitative research, I explore the nature of important moral concerns related to sharing sickle cell disease information from researchers’ and community members’ points of view; and develop a bottom-up normative analysis around the questions generated. This analysis interweaves community experiences, processes of community reasoning and ex situ normative reflection; placing community views and values centrally while referencing these to wider ethical debates, commentaries and guidelines in the literature. Two main outputs of this thesis are to provide recommendations for information sharing on SCD findings in the genomics study in Kilifi; and to propose a set of key issues to consider for this type of information in other studies and geographic settings. I conclude that researchers have a strong responsibility to share SCD information on affected children with families as a form of ancillary service (validating tests, counselling and care); but less responsibility to actively share carrier information. Concurrent responsibilities are working collaboratively with the Ministry of Health/District General Hospital to plan and implement services for SCD; ensuring counselling services support family stability as far as reasonably possible; and to build forms of community engagement and informed consent that counter risks of diagnostic interpretations of research.

Sickle cell disease (SCD) is a genetic disorder that affects millions of people worldwide. According to the Centers for Disease Control and Prevention, over 100,000 Americans have SCD, and more than 2 million Americans have a sickle cell trait (SCT). People with SCD are more likely than others to suffer premature mortality. Genetic screening is an important step in improving quality of life and increasing longevity for those with SCD. Early detection may lead to effective management of the disease and reduction of complicating factors. The purpose of this quasi-experimental study was to determine whether health education about SCD would impact college students' knowledge, attitudes, perceived risk, and intention to seek genetic screening and counseling in relation to the disease. The theoretical foundation for this study was the health belief model (HBM). This study involved 80 college students selected from a North Texas college. These students completed pre and post versions of an SCD questionnaire. Independent samples t tests were used to determine if there were significant differences in pre- and posttest scores of participants in both groups, and a MANOVA was used to determine differences among the scores of participants in the experimental group when grouped by age, gender, race, religiosity, and socioeconomic status. The results of this study showed that SCD health education improved the knowledge of and attitudes towards participants. Future research could explore barriers to seeking SCD screening and genetic counseling. Results of this study may further social change by encouraging the development of college-based health education efforts to increase awareness about SCD.

Sickle cell disease (SCD) is a genetic disorder that affects millions of people worldwide. According to the Centers for Disease Control and Prevention, over 100,000 Americans have SCD, and more than 2 million Americans have a sickle cell trait (SCT). People with SCD are more likely than others to suffer premature mortality. Genetic screening is an important step in improving quality of life and increasing longevity for those with SCD. Early detection may lead to effective management of the disease and reduction of complicating factors. The purpose of this quasi-experimental study was to determine whether health education about SCD would impact college students' knowledge, attitudes, perceived risk, and intention to seek genetic screening and counseling in relation to the disease. The theoretical foundation for this study was the health belief model (HBM). This study involved 80 college students selected from a North Texas college. These students completed pre and post versions of an SCD questionnaire. Independent samples t tests were used to determine if there were significant differences in pre- and posttest scores of participants in both groups, and a MANOVA was used to determine differences among the scores of participants in the experimental group when grouped by age, gender, race, religiosity, and socioeconomic status. The results of this study showed that SCD health education improved the knowledge of and attitudes towards participants. Future research could explore barriers to seeking SCD screening and genetic counseling. Results of this study may further social change by encouraging the development of college-based health education efforts to increase awareness about SCD.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2014.<br>Cataloged from PDF version of thesis.<br>Includes bibliographical references (pages 136-144).<br>Red blood cells (RBCs) play a crucial role in delivering oxygen to the body tissues. During the 120 days of their lifespan, average RBCs would circulate for approximately 500,000 times and undergo repeated deformations in small blood capillaries and splenic cords. Increased RBC clearance in the spleen is considered as one of the direct consequences of reduced RBC deformability. On the other hand, deformability is also indirectly impacting on RBC functionality through its complex connections with underlying molecular mechanisms. With the aid of microfabrication and microfluidic, we are able to perform high-throughput single cell deformability measurement. Overall, we established RBC deformability as an important biomarker for several blood related real world problems such as malaria and blood storage lesion. The ultimate goal is through our quantitative assessment of population-wide single cell deformability, we could aid in the decision-making of various clinical scenarios including drug screening and blood transfusion. Malaria is the most deadly parasitic disease affecting hundred millions of people worldwide. Infected RBCs are found to be less deformable and therefore more susceptible to splenic RBC clearance. In this thesis, we identified several clinically used anti-malarial drugs that are capable of altering RBC deformability and ultimately modifying RBC retention in spleen. We also employed a rodent malarial model, confirming the important connection of RBC deformability with splenic RBC retention and consequently malarial anemia in vivo. Blood storage lesion is another important application of our work. Taking the advantage of device high-throughput, we profiled hundreds of single RBC deformability from a large population and identified subpopulations that are less deformable. These subpopulations also exhibited higher osmotic fragility and were therefore predicted to pose higher transfusion risk according clinical standard. Furthermore, a deformability based sorting device was also developed to filter the less deformable blood subpopulations, improving overall blood quality.<br>by Sha Huang.<br>Ph. D.

INTRODUÇÃO: Entre os tratamentos existentes para o transtorno obsessivocompulsivo (TOC) os mais eficazes são o farmacológico, com inibidores de recaptura de serotonina (IRS; inibidores seletivos de recaptura de serotonina [ISRS] e clomipramina), e o comportamental, baseado na técnica de exposição com prevenção de resposta (EPR). Ambas apresentam eficácia semelhante para os sintomas obsessivo-compulsivos e estudos demonstram que a EPR produz modificações neurobiológicas semelhantes às provocadas pelo tratamento com ISRS. Essas evidências sugerem que a resposta clínica à EPR está diretamente relacionada a um aumento da biodisponibilidade de 5HT no cérebro. A concentração de 5HT no sangue periférico é uma medida representativa do sistema serotonérgico central, e é utilizada como um marcador biológico indireto. O objetivo deste estudo foi comparar a concentração serotonérgica (basal e variação em 8 semanas) e a resposta à terapia comportamental baseada em EPR. MÉTODOS: Foram incluídos nesse estudo 30 pacientes com diagnóstico operacional de TOC. Destes, 29 iniciaram o tratamento, 27 chegaram até a quarta semana e 24 completaram o protocolo padronizado com 16 sessões (8 semanas) de terapia. As dosagens de 5-HT foram feitas nas semanas 0 e 8 e as avaliações clínicas pelas escalas Y-BOCS e CGI, bem como medidas de sintomas secundários (depressão, ansiedade e incapacidade) nas semanas 0, 4 e 8. RESULTADOS: Encontrou-se correlação positiva entre a concentração basal de 5HT e a resposta clínica em quatro semanas de EPR (p<0,05).Observouse maior concentração basal e maior redução em 8 semanas nos níveis de 5HT em pacientes respondedores comparados aos não respondedores. Entretanto não houve significância estatística. CONCLUSÃO: Na amostra estudada os dados sugerem que a alta concentração basal de 5-HT é um marcador biológico preditor de boa resposta clínica a quatro semanas de EPR. Amostras maiores talvez mostrassem a concentração de 5-HT no plasma rico em plaquetas como um preditor de resposta a 8 semanas de EPR. Protocolos com amostras maiores e com grupos controle são necessários para confirmar esses achados<br>INTRODUCTION: Both gold standard treatments for obsessive-compulsive disorder (pharmacotherapy with serotonin reuptake inhibitors, and behavioral with exposure and response prevention [ERP]) are equally effective. Studies have demonstrated similar neurobiological changes elicited by these different treatments in OCD patients. These findings are suggestive that the clinical response to ERP is directly related to an increase of the 5-HT concentration in the brain. The platelet 5-HT concentration have been shown as a representative measure of central serotonergic system and used as a biological marker of the synaptic transmition. OBJECTIVE: To compare the platelet 5-HT concentration (basal and variation in 8 weeks) and the clinical response to ERP treatment. METHODS: 30 OCD patients were included and 29 started the treatment. 27 patients compleated at list 4 weeks and 24 completed all 8 weeks (16 sesions) ERP protocol. Patients had the basal and final (after 8 weeks) platelet 5-HT concentrations dosed and the clinical response measured by Y-BOCS, CGI and measures of secondary symptoms as well (depression, anxiety and disability). RESULTS: Data shows a positive correlation between the basal concentration of 5-HT and the 4 week ERP response (p<0,05). There were higher basal concentration and reduction in 8 weeks of platelet 5-HT concentration in the responders group compared with non-responders. Nevertheless, this differences was not significant (p>0,05). CONCLUSION: In the studied sample data suggest that high basal 5-HT platelet concentration is a biologic predictor of fast onset (4 weeks) of clinical response to ERP. Probably larger samples would show the basal 5-HT platelet concentration as a predictor of 8 weeks ERP outcome. Controlled trials are needed to confirm these findings

Doutoramento em Bioquímica<br>The work presented in this thesis aimed to investigate the impact of healthy pregnancy and selected prenatal disorders on the metabolome and lipidome of maternal blood plasma, in order to define new potential biomarkers for non-invasive prediction and diagnosis. Chapter 1 describes the present status and challenges of the clinically relevant prenatal disorders, along with a presentation of the metabolomics strategy applied and the state of the art of metabolomics in prenatal research. All experimental details are described in Chapter 2, comprising sample metadata, sample collection and preparation, data acquisition protocols and data analysis procedures. The plasma metabolome and lipidome viewed by 1D and 2D NMR experiments are presented in Chapter 3. In this chapter, the use of Multiple Quantum NMR spectroscopy was explored, for the first time, for assignment of complex lipid mixtures. Chapter 4 contributes to filling in some existing gaps regarding human plasma degradability during handling and storage, as well as the importance of fasting conditions at collection. The use of heparin collection tubes resulted in no interference of the polysaccharide and full conservation of spectral information, while EDTA tubes produced a number of interfering signals from free and Ca2+/Mg2+ complexed EDTA, the impact of which on metabolomic analysis is discussed. Regarding temperature stability, large changes in lipoproteins and choline compounds were observed in plasma kept at room temperature for  2.5 hours, whereas short-term storage at -20ºC was found suitable up to 7 days, with storage at -80ºC being recommended, particularly for long-term periods (at least up to 2.5 years). Regarding freeze-thaw cycles, no more than 3 consecutive cycles were found advisable, while the use of non-fasting conditions (instead of fasting) was found acceptable. Chapter 5 presents the first NMR metabolomics study of maternal plasma throughout pregnancy, including correlation between plasma and urine metabolites. Some of the metabolic alterations observed confirmed known metabolic effects, while novel changes were observed, suggesting adjustments in energy and gut microflora metabolisms (citrate, lactate and dimethyl sulfone) and alterations in glomerular filtration rate (creatine and creatinine). Correlations studies unveiled specific lipoprotein/protein metabolic aspects of healthy pregnancy with impact on the excreted metabolome, providing further understanding of pregnancy metabolism. In Chapter 6, the impact of prenatal disorders on maternal plasma metabolome and lipidome is described for fetal chromosomal disorders (CD), including Trisomy 21 (T21). High classification rates were obtained for CD (88-89%) and T21 (85-92%) in 1st and 2nd trimesters, based on variable selection of NMR data. In addition, novel metabolic deviations were found through plasma/urine correlations, namely in low density and very low density lipoproteins (LDL+VLDL), sugar and gut microflora metabolisms. Changes in plasma phospholipid profile, namely in phosphatidylcholines, were further confirmed and characterised by hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-LC/MS). In Chapter 7, metabolic biomarkers of pre- and post-diagnosis GDM were sought by NMR metabolomics of whole maternal plasma and plasma lipid profile in the 2nd trimester. Metabolic alterations found to be predictive of GDM comprised increases in cholesterol, fatty acids, triglycerides and small metabolites changes in glucose, amino acids, betaine, urea, creatine and metabolites related with gut microflora. Post-diagnosis GDM was successfully classified using a 26-resonance plasma biomarker corresponding to 10 metabolites and lipids, advancing the possibility of using a multi-metabolite biomarker as a complementary tool in the clinical management of GDM. Chapter 8 describes the results obtained for prenatal disorders shown to have lower impact on maternal plasma metabolome, namely diagnosed fetal malformations and pre-diagnosis premature rupture of membranes, preterm delivery and preeclampsia. Finally, Chapter 9 describes the general conclusions and future perspectives in the context of this thesis, highlighting how this work contributes with new knowledge on prenatal disease mechanisms and possible biomarkers for prenatal diagnosis and prediction methods.<br>O trabalho apresentado nesta tese teve como principal objetivo investigar o impacto da gravidez saudável e algumas doenças pré-natais no metaboloma e lipidoma de plasma sanguíneo materno, com vista à definição de novos biomarcadores para a previsão e diagnóstico não invasivos daquelas doenças. O Capítulo 1 descreve a perspectiva atual e os desafios das doenças pré-natais mais relevantes, assim como a estratégia metabolómica e estado da arte na investigação pré-natal. Todos os detalhes experimentais do trabalho realizado estão descritos no Capítulo 2, incluindo as condições de amostragem, recolha e preparação das amostras, bem como os protocolos de aquisição e análise dos dados. No Capítulo 3 descreve-se o metaboloma e lipidoma de plasma detectados por RMN 1D e 2D. Neste capítulo, a utilização de espectroscopia de RMN de quantum-múltiplo foi explorada, pela primeira vez, para caracterização de misturas lipídicas complexas. O Capítulo 4 contribui para colmatar algumas falhas no conhecimento sobre a degradibilidade do plasma humano durante o manuseamento da amostra e armazenamento, e a importância de condições de colheita como o jejum. A utilização de tubos de colheita com heparina não mostrou interferência do polissacarídeo nos espectros conservando-se toda a informação espectral, enquanto que os tubos com EDTA deram origem a sinais interferentes provenientes do EDTA livre e complexado com Ca2+/Mg2+, cujo impacto na análise metabolómica é discutido. Relativamente à estabilidade do plasma à temperatura ambiente, foram observadas alterações nas lipoproteínas e compostos de colina a partir de 2.5 horas, enquanto que o armazenamento a -20ºC mostrou ser adequado até 7 dias, sendo o armazenamento a -80ºC aconselhado, particularmente para períodos de tempo longos (pelo menos até 2.5 anos). Relativamente aos ciclos de congelação-descongelação, não se aconselham mais de 3 ciclos consecutivos, enquanto que o efeito da colheita das amostras em não-jejum (em vez de jejum) foi considerado aceitável. O Capítulo 5 apresenta o primeiro estudo de metabolómica por RMN do plasma materno ao longo da gravidez, incluindo correlação entre plasma e urina. Algumas das alterações metabólicas observadas confirmaram efeitos metabólicos conhecidos, tendo outras sido observadas pela primeira vez sugerindo alterações no metabolismo energético, na microflora bacteriana (citrato, lactato e dimetil sulfona) e na taxa de filtração glomerular (creatina e creatinina). Os estudos de correlação revelaram aspetos metabólicos específicos das lipoproteínas/proteínas com impacto no metaboloma excretado. No Capítulo 6 descreve-se o impacto das doenças cromossómicas (CD), incluindo Trissomia 21 (T21) no metaboloma e lipidoma de plasma materno. Obtiveram-se elevadas taxas de classificação para CD (88-89%) e T21 (85-92%) no 1º e 2º trimestres baseadas na seleção de variáveis dos dados de RMN. A correlação de plasma e urina revelou novos desvios metabólicos, nomeadamente no metabolismo das lipoproteínas de baixa densidade e de muito baixa densidade (LDL+VLDL), dos açúcares e da microflora bacteriana. As alterações observadas no perfil de fosfolípidos do plasma, nomeadamente das fosfatidilcolinas, foram confirmadas e caracterizadas por cromatografia liquida hidrofílica acoplada a espetrometria de massa (HILIC-LC/MS). No Capítulo 7 apresentam-se os resultados obtidos na prospecção de biomarcadores metabólicos de diabetes mellitus gestacional (GDM) pré- e pós-diagnóstico por metabolómica de RMN de plasma materno do 2º trimestre. Observaram-se alterações metabólicas com poder de previsão de GDM, nomeadamente um aumento no colesterol, ácidos gordos, triglicerídeos e pequenas variações metabólicas na glucose, aminoácidos, betaína, ureia, creatina e metabolitos relacionados com a microflora bacteriana. O grupo de GDM pós-diagnóstico foi bem classificado utilizando como biomarcador um conjunto de 26 ressonâncias do espectro de plasma correspondendo a lípidos e 10 metabolitos de baixo peso molecular, sugerindo-se a possibilidade de usar este marcador conjunto na gestão clínica da GDM. O Capítulo 8 descreve os resultados obtidos para as doenças pré-natais que mostraram ter um menor impacto no metaboloma de plasma materno, nomeadamente as malformações fetais (FM), e os estados de pré-diagnóstico da rutura prematura das membranas (PROM), parto pré-termo (PTD) e pré-eclampsia. Finalmente, no Capítulo 9 são descritas as conclusões gerais e perspetivas futuras no contexto desta tese, realçando-se como este trabalho contribui para o novo conhecimento dos mecanismos das doenças pré-natais e possíveis biomarcadores para a sua previsão e diagnóstico.

Anxiety disorders are very common and the primary feature is abnormal or inappropriate anxiety. Fear and anxiety is often mediated by the amygdala, a brain structure rich in substance P (SP) and neurokinin 1 (NK1) receptors. To learn more about how the human amygdala is modulated by fear and anxiety in event-triggered anxiety disorders and to investigate if the SP/NK1 receptor system is affected, regional cerebral blood flow (rCBF) ([15O]-water; Study I and II) and the SP/NK1 receptor system ([11C]GR205171; Study III and IV) were studied with positron emission tomography (PET). In Study I we investigated the neural correlates of affective startle modulation in persons with specific phobia by measuring rCBF during exposure to fearful and non-fearful pictures, paired and unpaired with acoustic startle stimuli. Fear-potentiated startle was associated with activation of the affective part of the anterior cingulate cortex and the left amygdaloid–hippocampal area. In Study II short-term drug treatment effects on rCBF in patients diagnosed with social phobia was evaluated, comparing the NK1 receptor antagonist GR205171 to the selective serotonin reuptake inhibitor citalopram and placebo. Social anxiety and neural activity in the medial temporal lobe including the amygdala was significantly reduced by both drugs but not placebo. In Study III we investigated if activity in the SP/NK1 receptor system in the amygdala would be affected by fear provocation in individuals with specific snake or spider phobia. Fear provocation was associated with a decreased uptake of the NK1 antagonist [11C]GR205171 in the amygdala, possibly explained by an increase in endogenous SP release occupying the NK1 receptors. Study IV was conducted to explore the resting state NK1 receptor availability in PTSD patients as compared to healthy controls. Increased resting state binding of the tracer [11C]GR205171 in the amygdala of patients with PTSD suggested an increased amount of available receptors. In summary, fear and fear-potentiated startle modulates the human amygdala, possibly through the SP/NK1 receptor system.

<p>The present thesis explored the neurobiological basis of three aspects of defense behaviors in humans. Positron emission tomography methodology was used, and changes in regional cerebral blood flow (rCBF) were measured as an index of neural activity. Firstly, brain function was studied in a group of patients suffering from combat-related posttraumatic stress disorder, using a symptom provocation paradigm with combat sounds in order to elicit fear. Exposure to auditory trauma reminders relative to neutral sounds was associated with increased rCBF in sensorimotor areas, the cerebellar vermis, the periaqueductal gray matter, and the right amygdala, whereas decreased activity was observed in the retrosplenial area of the posterior cingulate cortex. Secondly, the neural circuitry mediating the acoustic startle response and its habituation was studied in a group of healthy subjects. During acoustic startle stimulation as compared to a resting condition, increased rCBF was found in a medial posterior area of the pons corresponding to the nucleus reticularis pontis caudalis. As a result of startle repetition, altered activity was found in the cerebellum, pointing to its involvement in startle habituation. Thirdly, neural activity associated with startle modulation by phobic fear was studied in a group of subjects with specific animal phobias during exposure to pictures of their feared and non-feared objects, paired and unpaired with acoustic startle stimuli. As a result of startle potentiation, increased rCBF was found in the left amygdaloid-hippocampal region, and medially in the affective division of the anterior cingulate cortex. In conclusion, these results provide evidence for the involvement of limbic and paralimbic brain areas during fear provocation and fear-potentiated startle and for a similar neurocircuitry underlying startle in humans and animals.</p>

Introduction: Haemostasis is the arrest of bleeding. In recent years, in-vitro studies have suggested that secondary haemostasis (blood coagulation) is subject to activation by reactive oxygen species (ROS). It is known that patients who suffer from vascular disease are typically hypoxaemic and in the case of peripheral occlusive artery disease (POAD), physical exercise is used to improve symptom free mobility in the affected limbs. Hypoxia and physical exercise are two potent independent and synergistic initiators of ROS. We identified a clear need for in-vivo analysis of this novel area of research. Aims: There were two main aims of this research. 1. To explore the in-vivo influences of inspiratory hypoxia and physical exercise on biomarkers of haemostasis; and in doing so and subsequently carry out a randomised double blind placebo control trial to explore the interaction between oxidative stress (ROS accumulation) and haemostasis. Hypothesis: It was hypothesised that hypoxia and exercise would be independently and synergistically associated with an increase in oxidative stress, resulting in coagulation activation. We hypothesised that intervention with free radical reaction-chain breaking antioxidant vitamins would attenuate oxidative stress and thus attenuate the activation of coagulation. Methods: study 1 - Healthy males were subjected to six hours of normobaric hypoxia (12% inspired oxygen) and then a physical exercise challenge to exhaustion (cycling ramp-test). Citrated plasma was collected pre hypoxic exposure, post six hours of exposure, then immediately post exercise and analysed for routine clinical markers of coagulation (aPTT, PT, TT and fibrinogen) and analysed with and without correction for plasma volume shift. Data were analysed using a one-factor repeated measures ANOVA incorporating one within (condition: time point) subjects factor. Following a significant main effect and interaction, paired samples t-tests were employed to make post hoc comparisons at each level of the within-subjects factor. Study! - Healthy males were subjected to a double blind, randomised, placebo controlled intervention with vitamin C (a water soluble) and vitamin E (a lipid soluble), two ROS-scavenging, chain breaking antioxidants. The intervention lasted eight weeks to insure membrane enrichment with antioxidants. The methods of study one were repeated but with a pre-intervention time point added and the addition of two extra markers of thrombin generation (PF1+2 and T-AT). Data were analysed using a two-factor mixed ANOVA incorporating one between (group: antioxidant intervention vs. placebo control) and one within (condition: time point) subjects factor. Following a significant main effect and interaction, a paired samples t-test was used to make post hoc comparisons at each level of the within-subjects factor, with the alpha level Bonferroni corrected for multiple comparisons Between-group comparisons were assessed using independent samples t-tcsts applied to each level of the between-subjects factor. Results: Study 1 - Hypoxia was not associated with activation of coagulation. Physical exercise increased the activity of contact factor coagulation pathway activation. Study 2 - The intervention increased thrombin generation in the antioxidant group. This was met with an antagonistic antithrombin activation. Hypoxia did not impact the placebo group, but normalised the thrombin generation of the antioxidant group. Physical exercise increased contact factor pathway (CFP) as per study 1, but thrombin generation was unaltered. Hypoxia suppressed fibrinolysis post exercise, which is known to be activated in normoxic exercise. Discussion: hi study one, hypoxia alone did not activate coagulation. We hypothesised that this could be tentative evidence of a ROS concentration threshold for activation since once exercise was superimposed the accumulation of ROS activated the CFP. Correction for changes in plasma volume nullified the increased activity of the CFP. Corrections for shifts in plasma volume are routinely ignored in the literature and this was a novel finding. Study 2 was the first intervention of its kind. The increase in thrombin generation pre-post intervention with antioxidants suggests compelling evidence of in-vivo regulation of coagulation by ROS. But the direction of change was completely contrary to the original hypothesis. The confirmation that hypoxia does not activate coagulation is important, especially given the controversy surrounding long-haul flight deep vein thrombosis. Interestingly, exercise did not increase thrombin generation, despite the increase in the CFP. These findings suggest haemostasis is indeed subject to control by the body's redox state invivo via an as of yet, unknown mechanism.

Correction of blood genetic disorders requires permanent gene transfer into self renewing, hematopoietic stem cells (HSC), and regulation of transgene expression in specific cell lineages. HIV-derived lentiviral vectors are very effective in transducing rare, non-dividing stem cell populations without altering their long-term repopulation and differentiation capacity. We developed a strategy for transcriptional targeting' of lentiviral vectors based on replacing the viral LTR control elements with cell lineage specific, genomic control elements. An upstream enhancer (HS2) of the erythroidspecific GATA-l gene was cloned in a second-generation lentiviral vector to replace most of the U3 region of the LTR, immediately upstream of the HIV-l promoter. The modified LTR was used to drive the expression of a reporter gene (GFP), while a second gene (~LNGFR) was placed under the control of an internal, constitutive promoter to monitor cell transduction, or immunoselect transduced cells, independently from the expression of the targeted promoter. The vector was used to transduce cell lines, human CD34⁺ hematopoietic stem/progenitor cells, and murine bone marrow HSCs. Gene expression was analyzed in the differentiated progeny of transduced stem cells in vitro and in vivo, after transplantation into lethally irradiated co-isogenic (for murine cells) or NOD/SCID (for human cells) mice. The transcriptionally targeted HIV LTR allowed very high level of transgene expression specifically in mature erythroblasts, in a <i>tat</i>-independent fashion and with no alteration in titer, infectivity, and genomic stability of the lentiviral vector. Expression from the targeted LTR was higher, better restricted, and showed significantly less position effect variegation than that obtained by the same combination of enhancer/promoter elements placed in the conventional, internal position. Cloning of the woodchuck hepatitis virus post transcriptional regulatory element (WPRE) at a defined position in the targeted vector, allowed selective accumulation of the genomic with respect to the internal RNA transcript, with no loss of cell-type restriction. A critical advantage of this targeting strategy is the use of the spliced, major viral transcript to express a therapeutic gene, and that of an internal, independently regulated promoter to express an additional gene for either cell marking or <i>in vivo</i> selection purposes.

Mestrado em Métodos biomoléculares<br>A aplicação da metabonómica na pesquisa de novos biomarcadores de doenças tem ganho um interesse crescente na investigação e desenvolvimento, tanto ao nível do processamento analítico como do tratamento de dados. Nomeadamente, a análise metabonómica usando espectroscopia de Ressonância Magnética Nuclear (NMR) fornece uma grande quantidade de dados de uma forma rápida e não invasiva sobre a composição de amostras complexas como o plasma sanguíneo. Uma vez que as doenças pré-natais têm um elevado impacto no metabolismo materno e fetal, sendo responsáveis por várias complicações durante e depois da gravidez, esta estratégia foi aplicada ao estudo destas doenças através da análise de sangue de senhoras grávidas (colhido entre 15-24 semanas de gestação), com o objectivo de investigar possíveis metabolitos marcadores ou com poder de previsão para a diabetes gestacional e malformações fetais. Num primeiro passo, foram estudados os perfis metabólicos em RMN dos controlos (n=20) e gravidezes com diagnóstico ou suspeita de malformações fetais (n=11) e pré-diabetes gestacional (com posterior diagnóstico clínico entre 22-34 semanas de gestação). A análise multivariada (análise de componentes principais, PCA; análise discriminante pelo método de mínimos quadrados parcias, (PLS-DA) e duas versões deste último, interval PLS-DA e ortogonal PLS-DA (OPLS-DA)) foram aplicados com o objectivo de pesquisar por correlações de solidez estatística entre a composição do plasma e a ocorrência das doenças em estudo. Os resultados mostraram que as amostras controlo e doença podem ser diferenciadas com base no seu perfil metabólico, nomeadamente mostrando níveis mais elevados de compostos que contêm colina em mulheres que desenvolveram diabetes gestacional mais tarde na gravidez. Adicionalmente, níveis mais elevados de piruvato, manose e compostos que contêm colina, e níveis mais baixos de vários aminoácidos e acetato foram encontrados nas gravidezes afectadas por malformações fetais. Numa segunda etapa do trabalho, as mesmas amostras foram analisadas por espectroscopia de Infravermelho com Transformadas de Fourier (FTIR), um método mais barato e acessível para eventual uso clínico. O perfil dos espectros de FTIR também revelou algumas diferenças entre controlos e doenças, no entanto a sua interpretação específica torna-se difícil devido à grande sobreposição de bandas característica de espectros de infravermelho. Estes resultados mostraram que a análise metabonómica de plasma de mulheres grávidas por RMN e FTIR pode ser uma ferramenta poderosa para obter informação bioquímica sobre a saúde pré-natal e encontrar possíveis novos marcadores com potencial para prever doenças, particularmente no caso do diabetes gestacional. ABSTRACT: The use of Metabonomics to search for new disease biomarkers has gained increasing interest in the research community and continuous developments, both at the analytical and data processing levels have boosted this area into new quests in biomarker research. Namely, Nuclear Magnetic Resonance (NMR)-metabonomics provides a large amount of compositional data on complex samples such as blood plasma, in a rapid and non-invasive manner. Since prenatal diseases have a high impact on both maternal and fetal metabolisms, being responsible for a range of complications both during and after pregnancy, this strategy was hereby applied to the study of prenatal diseases, through the analysis of blood (collected at 15-24 gestational weeks), in order to probe for possible marker/predictor metabolites for gestational diabetes and fetal malformations. In the first stage of this work, the plasma metabolic profiles of controls (n=20) and pregnancies affected by diagnosed or suspected fetal malformations (n=11) and pre-gestational diabetes (with posterior clinical diagnosis at 22-34 gestational weeks) were evaluated by NMR spectroscopy. Multivariate analysis (principal component analysis, PCA; partial least squares discriminant analysis, PLS-DA and two extended versions of the latter, interval PLS-DA (iPLS-DA) and orthogonal PLS-DA (OPLS-DA) were applied in order to search for consistent statistical correlations between plasma composition and the occurrence of the diseases. It was found that controls and diseased subjects could be differentiated with basis on their plasma profile, namely showing higher levels of choline-containing compounds in pregestational diabetic women. In addition, higher contents of pyruvate, mannose and choline-containing compounds and lower contents of several amino acids and acetate were found in pregnancies affected by fetal malformations. In a second stage of the work, the same samples were analysed by Fourier Transform Infrared (FTIR) spectroscopy, a cheaper and more-accessible method, more suited to straightforward clinical use. The FTIR spectral profiles also revealed some differences between controls and diseased subjects, the interpretation of which posing a harder challenge than that of NMR. These results have shown that NMR and FTIR metabonomics of pregnant women blood plasma may be a powerful tool to gain insight into prenatal diseases and find possible new markers with potential predictive value, particularly in the case of gestational diabetes.

This thesis is about the experience of Muslim British Pakistani families coping with thalassaemia (a chronic, inherited blood disorder) and the implications for service delivery. Its central concern is to illustrate that simplistic and culturally-biased assumptions are an unsatisfactory base on which to devise health service delivery for minority populations, and that with careful study it is possible to deliver culturally sensitive and appropriate services. The thesis is written in four parts. The first part contains the research methods and the clinical aspects of thalassaemia. It also provides an introduction to the families in the study. The British Pakistani population is considered in the context of migration to Britain, which has created a plural society requiring adaptations to services to meet the diverse health needs of the different ethnic minorities. The second part deals with the socio-economic and cultural background of British Pakistanis in Pakistan: this (is) crucial to an understanding of their present situation. Family dynamics, marriage patterns and decision-making processes are explored, as is the central role of religion and kinship networks in the lives of British Pakistanis. It also examines their settlement process and present living conditions and illustrates how the social structures prevalent in Pakistan have been re-established in England, albeit in a modified form. The third part documents, using case studies, the experiences of British Pakistani families with thalassaemic children. These are analysed to highlight deficiencies in health service delivery and areas where cultural misconceptions exist. These areas require attention to provide an effective genetic counselling service for this population. The final part examines the social and clinical implications of consanguineous marriage. It gives the results of a study showing increased frequency of consanguineous marriage among British Pakistanis than among Pakistanis in Pakistan. It then illustrates how kinship networks within communities practising this marriage pattern provide an opportunity to offer a genetic counselling service in a unique way, by making positive use of the practice. This proposed approach applies not only to thalassaemia but also to other inherited diseases.

Actually, there is a strong incidence of psychiatric diseases, representing a 13% of total burden diseases and 450 million of people affected. The etiology of psychiatric diseases remains unknown. However, scientific evidences suggest a maldevelopment of nervous system (NS). The diagnosis is inaccurate, and international manuals (ICD-10 and DSM-IV) identify pathologies according to a list of symptoms but no underlying biological cause of disease. The aim of the thesis is to identify potential biomarkers -related to the development of NS- in peripheral blood of psychiatric patients diagnosed as different mental diseases, such as autism spectrum disorders (ASD), schizophrenia and bipolar disorders. It is intended to contribute with the improvement of diagnostic, prognostic and treatment of subjects. The thesis is divided into 4 chapters: 1) study of neurotrophins in ASD, where the results show the relationship of this family of molecules with the disease, 2) study of Latrophilin-3 (LPHN3) in the TEA, which was obtained in association with lower cognitive level of ASD, 3) study of the Eph-receptor A4 in the pathology of schizophrenia and bipolar disorder, results of which show no association, and finally 4) study of Ankyrin-3 (ANK3) in schizophrenia and bipolar disorder, which shown a relationship with bipolar disorder but not with schizophrenia.<br>Actualment, hi ha una forta incidència de les patologies psiquiàtriques, representant un 13% del total de les malalties i 450 milions de persones afectades. L’etiologia de les patologies psiquiàtriques és desconeguda. Tot i així, evidències científiques suggereixen un mal desenvolupament del sistema nerviós (SN). El diagnòstic és poc precís, i els manuals internacionals (ICD-10 i DSM-IV) identifiquen les patologies d’acord a un llistat de símptomes, però sense cap causa biològica subjacent de la patologia. L’objectiu de la tesi és la identificació de biomarcadors potencials –relacionats en el desenvolupament del SN- en sang perifèrica de pacients diagnosticats amb diferents patologies mentals, com ara els trastorns de l’espectre autista (TEA), esquizofrènia i desordres bipolars. És pretén contribuir amb la millora del diagnòstic, el pronòstic i el tractament de les persones que les pateixen. La tesi s’estructura en 4 capítols: 1) estudi de les neurotrofines en els TEA, on els resultats evidencien la relació d’aquesta família de molècules amb la patologia, 2) estudi de la Latrofilina-3 (LPHN3) en els TEA, on s’ha obtingut associació amb el nivell cognitiu més baix dels TEA, 3) estudi del receptor EPH A4 en les patologies d’esquizofrènia i desordres bipolars, resultats del qual no mostren associació i, per últim 4) estudi de la Ankirina-3 (ANK3) en l’esquizofrènia i els desordres bipolars, en el qual si que es troba una relació amb els desordres bipolars, però no amb l’esquizofrènia.<br>Actualmente, hay una fuerte incidencia de las patologías psiquiátricas, representando un 13% del total de las enfermedades y 450 millones de personas afectadas. La etiología de las patologías psiquiátricas es desconocida. Aún así, evidencias científicas sugieren un mal desarrollo del sistema nervioso (NS). El diagnóstico es poco preciso, y los manuales internacionales (ICD-10 y DSM-IV) identifican las patologías de acuerdo a un listado de síntomas, pero sin ninguna causa biológica subyacente de la patología. El objetivo de la tesis es la identificación de biomarcadores potenciales –relacionados con el desarrollo del SN- en sangre periférica de pacientes diagnosticados con diferentes patologías mentales, como son los trastornos del espectro autista (TEA), esquizofrenia y desordenes bipolares. Se pretende contribuir en la mejora del diagnóstico, el pronóstico i el tratamiento de las personas que las padecen. La tesis se estructura en 4 capítulos: 1) estudio de las neurotrofinas en los trastornos del espectro autista (TEA), en el cual los resultados evidencian la relación de esta familia de moléculas con la patología, 2) estudio de la Latrofilina-3 (LPHN3) en los TEA, donde se ha obtenido una asociación con el nivel cognitivo más bajo de los TEA, 3) estudio del receptor EPH A4 en las patologías de la esquizofrenia y los desordenes bipolares, resultados del cual no muestran asociación y, por último 4) estudio de la Ankirina-3 (ANK3) en la esquizofrenia y los desordenes bipolares, en el cual si que se ha encontrado una relación con los desordenes bipolares, pero no con la esquizofrenia.

Thesis (Ph.D. in Clinical Science) -- University of Colorado Denver, 2008.<br>Typescript. Includes bibliographical references (leaves 136-146). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Includes bibliographical references<br>Background: South Africa has the fourth highest prevalence of Human Immunodeficiency Virus (HIV) in the world, with over 340 000 new infections in 2013 alone. Implementation of Highly Active Antiretroviral Treatment (HAART) regimens, has improved the lives of many HIV-infected individuals, and has led to a decrease in the incidence of extreme forms of HIV-associated dementia. However, the extended lifespan afforded by HAART has also resulted in an increase in the prevalence of milder forms of HIV-associated Neurocognitive Disorders (HAND). Clinical diagnosis of such cognitive decline currently relies on neurocognitive assessments, as no definitive biochemical test exists. A point-of-care test designed for use with easily accessible samples (e.g. blood) would be of great utility in both HAND research and clinical diagnosis, prognostication, and provision of timeous therapy. A blood-based biomarker test would also be less subjective than neurocognitive testing, which can be adversely affected by the patient's level of education as well as operator competency. Recently, Professor Simon Lovestone (University of Oxford) developed a panel of blood-based biomarkers for the diagnosis of Alzheimer's disease. Due to pathological similarities between HAND and Alzheimer's disease, it is hypothesised that there may be applicability of this panel to diagnosis of HAND. The differential protein expression profile of patients with and without HAND is thus compared. Aims and Objectives: The aim of this study was to assess the applicability of a set of Alzheimer's disease biomarkers to the diagnosis of HAND. In order to achieve this, targeted proteomic assays were developed to measure the markers in human serum. This assay was then applied to defined patient cohorts. Methods: Targeted proteomic assays were developed in order to quantify candidate markers in patient blood serum. Parallel Reaction Monitoring assays were developed on a Thermo Q Exactive Quadrupole-Orbitrap mass spectrometer, for application to defined patient samples collected from Groote Schuur Hospital (Cape Town, South Africa). Patient samples were divided into groups according to HAND severity (based on the HIV Dementia Scale), namely: Normal (HIV+), Minor Neurocognitive Disorders and HIV-associated Dementia. Discovery proteomic analysis was performed on pooled patient samples in order to determine the optimal peptides for identification and quantitation of candidate proteins. Targeted methods were then developed and refined using the same pooled samples. Finally, patient samples (N=81) from the three classes were analysed in a statistically rigorous manner. Data were normalised to correct for possible loading inconsistencies by two independent methods. Quantitative differences were investigated using t-tests and non-parametric statistical tests where appropriate. Results: Statistical assessment of final data indicated no significant differences in proteins between patient classes. Discussion and Conclusions: It is likely that the inflammatory nature of HIV itself influences the levels of these markers in HIV-positive patient samples to a greater degree than neurocognitive effects. Alternatively, possible co-infection by TB may have confounded the results. Regardless, it was concluded that the candidate Alzheimer's biomarkers were not applicable to diagnosis of HAND. Further analysis on a larger sample cohort is recommended, utilising the assays optimised herein. Prospective studies to obtain viable biomarkers for definitive diagnosis may lie in proteomic analysis of model infection systems and cerebrospinal fluid.

We live in an age of 'multimorbidity', yet have a rather poor understanding of the impacts of this phenomenon from cellular to organ and system levels. Bi-directional relationships are observed between cardiometabolic (diabetes mellitus, coronary heart disease) and mood disorders (major depressive disorder), each increasing disease risk and frequently co-existing. This cardiometabolic-mood interaction may reflect an under-appreciated commonality of underlying mechanisms across these pathologies. Importantly, comorbid development of these disorders exacerbates the already profound burdens of these individual chronic disorders. Further, psychosocial stress and ageing – increasingly prevalent features of modern societies - may promote these conditions and worsen outcomes. In assessing the intersections of these inter-related disorders, chronic low-grade inflammation is often highlighted and implicated as a common early substrate in cardiometabolic and mood disorders, linking these dominant diseases. Nonetheless, how immunoinflammatory dysregulation drives multimorbidity, and influences 'multifinality' (diverse disease outcomes from common risks/causes), are unclear. Whether immunoinflammatory changes in metabolic vs mood disorders are unique or involve common motifs, and how these interact in dictating cardiometabolic and mental health, remains to be established. Dysregulation of toll-like receptor (TLR) signalling has been specifically implicated in these diseases, potentially presenting a mechanistic nexus in comorbid development of cardiometabolic and mood disorders. The doctoral work presented in this thesis was designed to address the broad concept of mechanistic intersections contributing to rising multimorbidity. In order to practicably achieve this at a systems level, four major tissues impacted by and contributing to aspects of metabolic and mood disorders were studied: the brain, heart, liver and blood (circulating serum). The brain is an overarching and critical organ, regulating both mood and metabolic homeostasis, and influencing all other organ systems. Cardiac tissue is assessed as it is particularly impacted by type 2 diabetes (T2D); the risk of cardiovascular disease (CVD) is markedly elevated in T2D patients and is the lead cause of death in these patients. Further, CVD is an independent risk factor for both depression and T2D. The liver is fundamental to metabolic homeostasis (the central organ maintaining homeostasis across fed and fasted states via oxidisable substrate storage and release), links the gut to the periphery (gut-liver axis) and contains specialised macrophages which are front-line responders to gut-derived lipopolysaccharides (LPS) (arising with gut permeability changes). Finally, serum was assessed as it is the primary medium for transport of macronutrients, hormones and immunoinflammatory molecules throughout the body. To avoid the pitfalls of reliance upon gene-modified animal models, non-genetic murine models of type 1 and type 2 diabetes development in early adulthood were studied in Chapter 2 to explore potential changes in TLR-related signalling at the gene level. Somewhat unexpectedly, there was little evidence of shifts in TLR signalling in central nervous system (CNS), hepatic or cardiac tissues in these two conditions. Moreover, while modest elevations in circulating interleukin-6 (IL-6) are observed in T2D animals, tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) levels remained unchanged across models. Together with further analysis of gene expression, these data indicate that the T2D model does not develop an overtly pro-inflammatory phenotype, despite clear evidence of metabolic dysregulation. Given that a well-developed diabetic phenotype was observed (increased body weight, moderate hyperglycaemia, insulin resistance), this work called into question the essential role of TLR4 in the pathogenesis of T2D. Data does reveal marked changes in circulating adipokines and catecholamines, suggesting these endocrine systems may be key to disease progression, independent of inflammation. Surprisingly, assessment of circulating LPS revealed serum levels were decreased (although not significantly) in T2D animals, which casts doubt on whether 12 weeks of feeding with a ‘Western diet’ (30% of total calories from fat) is sufficient to disturb gut physiology and trigger the systemic innate immune response. The majority of animal models employ much higher fat percentages (45-60% calories from fat) in their diabetogenic diets, despite such high fat levels vastly exceeding those in a Western diet (~30%) and raising questions of pathophysiological relevance. Given that little evidence of TLR dysregulation was detected at a physiologically relevant fat percentage, a more chronic model (21-week dietary modification) with an intermediate fat content (43% calories from fat) was developed for subsequent experimentation. Studies in Chapter 3 demonstrate that a more profound weight gain is achieved with a higher fat content, which still remains below the extreme 60% levels often studied in animal models: this new feeding regime resulted in significantly greater body weight gain although fasting insulin and glucose levels were not altered when compared with the T2D model assessed in Chapter 2. Nonetheless, despite this more pronounced phenotype, limited TLR4-related gene changes were observed in the model. Selective changes to total cellular nuclear factor-kappa B (NF-κB) protein expression were observed in hepatic tissue, although analysis of the nuclear compartment revealed no changes in response to T2D. Thus, while a greater pool of this critical pro-inflammatory factor was evident, this was not necessarily associated with increased nuclear interaction and thus transcriptional control. These data suggest dysregulation of transcriptional control by NF-κB (particularly TLR4 pathway elements) may not be essential in the evolution of T2D in these animals. Examining a dietary intervention with an omega-3 polyunsaturated fatty acid (n-3 PUFA), α-linolenic acid (ALA), which has been linked to anti-inflammatory outcomes in some chronic disorders, revealed no improvements in the systemic sequalae of T2D. This is consistent with evidence PUFA supplementation may (somewhat paradoxically) have greater influences on metabolic homeostasis in healthy rather than diseased subjects. Unexpected elevations in key inflammatory transcripts were noted with ALA supplementation, potentially reflecting the highly pleiotropic actions of PUFAs. Proteomic profiling of cardiac tissue revealed that a number of inflammatory and related factors beyond TLR-related signalling were impacted by T2D (Serpin-1/PAI-1, leptin, resistin) and/or are sensitive to ALA (IL-10, CD40, VEGF). Data also suggest animal age may be a complicating factor in the protracted disease model, producing apparent independent effects on leptin/insulin expression levels, in turn complicating interpretation of these data. Finally, specific investigation of CNS changes in the model - via analysis of frontal cortex (FC) leptin receptor expression - indicates a sensitivity of central leptin signalling to T2D, which may not only participate in metabolic dysregulation but behavioural outcomes (as suggested in the pathogenesis of depression, for example). This highlights the likely importance of central control and behavioural determinants of disease outcomes. Investigating central control mechanisms and behaviour in more detail, studies presented in Chapter 4 revealed that the T2D phenotype involves induction of anxiety-related behaviours without impacting on hedonic behaviour. Further, despite evidence from other studies of the benefits of PUFA supplementation in mood disorders, ALA supplementation did not reverse anxiety-related behaviours, though increased locomotion in both healthy and T2D animals. Interestingly, analysis suggests ALA supplementation may confer benefits to locomotive activity independently of disease state, although outcomes are better in healthy controls. Examining elements of central reward pathways, neurotransmission, endocrine and inflammatory control in FC and hippocampus revealed select changes with T2D, including elevated Drd2 (dopamine D2 receptor) and reduced Htr1a (serotonin receptor 1A) in the FC, together with shifts in leptin receptor expression. Surprisingly, hippocampal Il1b gene expression remained similar between groups, though this is consistent with no change in hedonic behaviour. Circulating dopamine and leptin were also sensitive to T2D, with hippocampal dopamine levels selectively elevated in T2D animals supplemented with ALA (although the relevance of this finding is not clear). Overall, these data point to dysregulation of central dopamine and leptin signalling, which may contribute to behavioural disruption in T2D, in turn influencing disease development and outcomes. In the final studies, CNS responses were explored in greater detail via RNA-sequencing (Chapter 5), more broadly testing whether metabolic and mood disorders share common nervous system changes. Analyses of transcriptome profiles in the FC of the T2D model assessed in Chapter 5 together with a model of chronic social stress (SS) known to induce anxiety/depressive behaviours again provided limited support for an overarching immunoinflammatory dysregulation as a key driver of behavioural changes. These analyses reveal both distinct and common processes (beyond TLR-signalling) that are dysregulated in disease. Comparison between T2D and SS models reveal commonalities in CNS leptin and insulin receptor changes, congruent with evidence of insulin and leptin resistance in the T2D model and their implication in both human major depressive disorder (MDD) and T2D. A majority of over-represented genes were related to cell/tissue development, cell migration and proliferation, suggesting a dominant role for CNS 'remodelling' with both metabolic and mood disorders. Interestingly, down-regulation of ATP metabolic processes and mitochondrial genes was evident in the SS model but not T2D, supporting a more dominant effect of stress on energy production. Taken together, the findings presented in this doctoral project raise questions regarding the role of dysregulated TLR-signalling in T2D (and stress-related disorders). Despite a clear diabetic phenotype and shifts in behavioural profiles of T2D animals, findings challenge whether TLR-related dysregulation (reported by others) may be a consequence rather than causative factor in disease pathogenesis. Importantly, evidence is presented that insulin, leptin and dopamine signalling (together with other metabolic mediators) may be important linkages between metabolic and mood disorders, and underlie behavioural detriment in T2D. In terms of limiting the development or impacts of disease, n-3 PUFA supplementation resulted in selective benefits in T2D, though these appear unrelated to the metabolic profile of these animals. Finally, shared and unique CNS changes were identified in models of T2D and chronic social stress, supporting structural sensitivity and plasticity with inter-related metabolic and mood disorders.<br>Thesis (PhD Doctorate)<br>Doctor of Philosophy (PhD)<br>School of Medical Science<br>Griffith Health<br>Full Text

Thesis (M.S.)--Georgia State University, 2005.<br>Title from title screen. Roberta Attanasio, committee chair; P.C. Tai, W.C. Hooper, committee members. Electronic text (57 p. : col. ill.) : digital, PDF file. Description based on contents viewed Aug. 15, 2007. Includes bibliographical references (p. 55-57).

An individual who is diagnosed with an inherited bleeding disorder is expected to manage his or her condition on a daily basis. This chronic situation can totally disrupt psychosocial functioning and make it more difficult to adjust to the illness. Other researchers have studied this phenomenon in various other chronic illnesses; however, not in individuals with inherited bleeding disorders (Akkasilpa, et al, 2000, Pollack, 1989a, 1989b). Psychosocial problems are not restricted to individuals with one chronic illness and clinically, it is noted that some individuals adjust to chronic diseases better than others. Individuals living with inherited bleeding disorders may also have other chronic illnesses such as hypertension, asthma, diabetes mellitus (DM), congestive heart failure (CHF), arthritis, and hepatitis A, B, C and/or HN. The aims of this study are to describe health stressors, health related hardiness, perception of illness impact, self perception of health status and psychosocial adjustment to illness in individuals living with an inherited bleeding disorder; to determine relationships between demographic and illness variables, health stressors, health related hardiness, perception of illness impact, self­ perception of health status and psychosocial adjustment to illness; and to determine if perception of illness impact has a direct and/or mediating effect on the relationship between health stressors, health related hardiness, and self-perception ofhealth status and psychosocial adjustment to illness. A cross sectional survey design was used in this study. Sixty individuals of predominantly Asian Pacific Islander ethnicity diagnosed with hemophilia, von Willebrand's Disease, Factor V or as hemophilia carriers comprised the sample which was drawn from the Hemophilia Treatment Center of Hawaii. All participants were asked to complete five questionnaires: Demographic form and illness information, health related hardiness scale (Pollock, 1990), perception of illness impact scale, self-perception of health status and psychosocial adjustment to illness scale (Derogatis, 1990). Higher health stressors were associated with higher perception of illness impact, lower perception of health status and poorer psychosocial adjustment to illness. Individuals with higher hardiness were better adjusted to their illness. Higher perception of illness impact was associated with lower self-perception of health status and poorer psychosocial adjustment to illness. Higher self-perception of health status was associated with better psychosocial adjustment to illness. Perception of illness impact did mediate the relationship between health related hardiness and psychosocial adjustment to illness. Perception of illness impact did not mediate the relationship between health stressors and psychosocial adjustment to illness, between health stressors and self-perception of health status, and between health related hardiness and self-perception of health status. The knowledge generated from this study has the potential to impact the existing practices in improving evidence-based nursing practice in caring for individuals with inherited bleeding disorders. Future research is indicated with a large sample to determine differences between diagnosed individuals and carriers, between various Asian Pacific Islander cultural groups, and to determine replicability of the findings from this smaller study sample.

Polymorphisms of platelet glycoproteins (GPs) are frequently targets for anti-platelet antibodies. At least 19 antigenic polymorphisms have been identified on platelet GPs. Antibodies against the HPA-lb polymorphism (a Leu to Pro switch at amino acid residue 33 of the IIIa sub-unit of GP IIb/Illa) have been attributed to as much as 90% of all cases of neonatal alloimmune thrombocytopenic purpura and posttransfusion purpura in caucasians. The HPA-lb polymorphism has also been equivocally associated with coronary artery disease, particularly early onset (<60 years of age) myocardial infarction. Current technology for identifying individuals with the HPA-lb phenotype is limited to the labor-intensive, highly technical and expensive process of DNA amplification by polymerase chain reaction and restriction fragment length polymorphism (PCR/RFLP) analysis.This study proposes an alternative method for phenotyping individuals for the HPA-1 polymorphism using the Biocytex Platelet HPA-1 kit. The kit identifies the HPA-1 polymorphism utilizing two monoclonal CD61 (platelet glycoprotein IIb/IIIa) antibodies, one of which has a lowered affinity for GP llb/Illa possessing the HPA-lb polymorphism. Fluorescent labeling of bound antibody allows for flow cytometric quantitation of antibody binding capacity (ABC) for both monoclonal antibodies, and ratios derived from the ABC can be used to phenotype previously unknown samples.The Biocytex HPA-1 kit identified 73 of 74 (98.6%) individuals possessing the HPA1 a/HPA-1 a phenotype, 22 of 22 (100%) HPA-1 a/HPA-1 b individuals and 4 of 4 (100%) HPAIb/HPA-Ib individuals. All HPA-lb phenotypes were confirmed by PCR/RFLP. Total accuracy of the test system was 99%.<br>Department of Biology

Pre-eclampsia is a common disorder, specific to human pregnancy and a major cause of maternal and neonatal morbidity and mortality. The cause is unknown but the syndrome is thought to be the result of generalised endothelial dysfunction. There is increasing evidence that the maternal innate immune system may be involved in this systemic response. Peripheral blood monocytes, exquisitely sensitive cells pivotal to the immunosurveillance role of the innate immune system, interact with genetically different fetal derived cells, cellular organelles and secreted products throughout pregnancy. Monocytes secrete cytokines that could underlie endothelial activation in a process that resembles a systemic inflammatory response. The work in this thesis was designed to look for evidence of activation in monocytes in pre-eclampsia. Monocyte derived proinflammatory cytokines ILla, IL1[3, 1L6 and TNFOL and the anti-inflammatory cytokine ILlRa were used to characterise the behaviour of these cells in the menstrual cycle, normal pregnancy and in pre-eclampsia. There was evidence of activation of monocytes in the post-ovulatory phase of the menstrual cycle confirming their role in normal reproductive processes. In normal pregnancy there was evidence of both immunosuppression and immune activation of monocytes suggesting a tight and complex regulatory mechanism in vivo. Evidence of activation of monocytes in preeclampsia is consistent with the suggestion that that these cells contribute to a systemic inflammatory response, supporting the proposal that there is a continuum of activation in the innate immune system throughout normal pregnancy and that this is exaggerated in pre-eclampsia. Several polymorphisms in the 1L1 gene were investigated for an association with pre-eclampsia. Although no simple association was found, the gene family may contribute to the severity of this complex condition and therefore may be useful as part of a genetic susceptibility profile for pre-eclampsia.