Development of new methodologies in sample treatment for proteomics workflow based on enzymatic probe sonication technology and mass spectrometry

Thesis submitted to the Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia, for the degree of Doctor of Philosophy in Biochemistry

Descending pain modulation in a Kaolin-induced hydrocephalus rat model

Pain transmission at the spinal cord is modulated by descending actions that arise from supraspinal areas which collectively form the endogenous pain control system. Two key areas involved of the endogenous pain control system have a circunventricular location, namely the periaqueductal grey (PAG) and the locus coeruleus (LC). The PAG plays a crucial role in descending pain modulation as it conveys the input from higher brain centers to the spinal cord. As to the LC, it is involved in descending pain inhibition by direct noradrenergic projections to the spinal cord. In the context of neurological defects, several diseases may affect the structure and function of the brain. Hydrocephalus is a congenital or acquired disease characterized by an enlargement of the ventricles which leads to a distortion of the adjacent tissues, including the PAG and LC. Usually, patients suffering from hydrocephalus present dysfunctions in learning and memory and also motor deficits. It remains to be evaluated if lesions of the periventricular brain areas involved in pain control during hydrocephalus may affect descending pain control and, herein, affect pain responses. The studies included in the present thesis used an experimental model of hydrocephalus (the rat injected in the cisterna magna with kaolin) to study descending modulation of pain, focusing on the two circumventricular regions referred above (the PAG and the LC). In order to evaluate the effects of kaolin injection into the cisterna magna, we measured the degree of ventricular dilatation in sections encompassing the PAG by standard cytoarquitectonic stanings (thionin staining). For the LC, immunodetection of the noradrenaline-synthetizing enzyme tyrosine hydroxylase (TH) was performed, due to the noradrenergic nature of the LC neurons. In general, rats with kaolin-induced hydrocephalus presented a higher dilatation of the 4th ventricle, along with a tendency to a higher area of the PAG. Due to the validated role of detection the c-fos protooncogene as a marker of neuronal activation, we also studied neuronal activation in the several subnuclei which compose the PAG, namely the dorsomedial, dorsolateral, lateral and ventrolateral (VLPAG) parts. A decrease in the numbers of neurons immunoreactive for Fos protein (the product of activation of the c-fos protooncogene) was detected in rats injected with kaolin, whereas the remaining PAG subnuclei did not present changes in Fos-immunoreactive nuclei. Increases in the levels of TH in the LC, namely at the rostral parts of the nucleus, were detected in hydrocephalic animals. The following pain-related parameters were measured, namely 1) pain behavioural responses in a validated pain inflammatory test (the formalin test) and 2) the nociceptive activation of spinal cord neurons. A decrease in behavioral responses was detected in rats with kaolin-induced hydrocephalus was detected, namely in the second phase of the test (inflammatory phase). This is the phase of the formalin test in which the motor behaviour is less important, which is important since a semi-quantitative analysis of the motor performance of rats injected with kaolin indicates that these animals may present some motor impairments. Collectively, the results of the behavioral studies indicate that rats with kaolin-induced hydrocephalus exhibit hypoalgesia. A decrease in Fos expression was detected at the superficial dorsal layers of the spinal cord in rats with kaolin-induced hydrocephalus, further indicating that hydrocephalus decreases nociceptive responses. It remains to be ascertained if this is due to alterations in the PAG and LC in the rats with kaolin-induced hydrocephalus, which may affect descending pain modulation. It remains to be evaluated what are the mechanisms underlying the increased pain inhibition at the spinal dorsal horn in the hydrocephalus rats. Regarding the VLPAG, the decrease in neuronal activity may impair descending modulation. Since the LC has higher levels of TH in rats with kaolininduced hydrocephalus, which also appears to increase the noradrenergic innervation in the spinal dorsal horn, it is possible that an increase in the release of noradrenaline at the spinal cord accounts for pain inhibition. Our studies also determine the need to study in detail patients with hydrocephalus namely in what concerns their thresholds to pain and to perform imaging studies focused on the structure and function of pain control areas in the brain.

Relationship of cryptosporidiosis to abdominal pain and diarrhea in Mayan Indians

Demonstration of cryptosporidiosis in Mayan Indians living around Lake Atitlan provided an opportunity to correlate infection with abdominal pain and/or diarrhea in different age groups of children. 94 subjects experiencing abdominal pain and/or diarrhea, between the ages of 2 and 13 were studied in towns around Lake Atitlan, Guatemala, over a two-year period. Cryptosporidium oocysts were found in the feces of 29% of children who presented with abdominal pain and 21% with diarrhea. Of the 60 infected subjects, 45% were experiencing abdominal pain and 33% diarrhea, 22% had abdominal pain and diarrhea. Both abdominal pain and diarrhea were significantly higher in children under 10 years of age and were most prevalent in the 6-9 year old age group but the correlation of symptoms to infection was not significantly different as the ages of the children increased. The high frequency of abdominal pain and/or diarrhea with infection in children was consistent with cryptosporidiosis, a disease considered as one of several common intestinal infections that produce these symptoms.

Wii-Workouts on Chronic Pain, Physical Capabilities and Mood of Older Women: A Randomized Controlled Double Blind Trial

Chronic Low Back Pain (CLBP) is a public health problem and older women have higher incidence of this symptom, which affect body balance, functional capacity and behavior. The purpose of this study was to verifying the effect of exercises with Nintendo Wii on CLBP, functional capacity and mood of elderly. Thirty older women (68 ± 4 years; 68 ± 12 kg; 154 ± 5 cm) with CLBP participated in this study. Elderly individuals were divided into a Control Exercise Group (n = 14) and an Experimental Wii Group (n = 16). Control Exercise Group did strength exercises and core training, while Experimental Wii Group did ones additionally to exercises with Wii. CLBP, balance, functional capacity and mood were assessed pre and post training by the numeric pain scale, Wii Balance Board, sit to stand test and Profile of Mood States, respectively. Training lasted eight weeks and sessions were performed three times weekly. MANOVA 2 x 2 showed no interaction on pain, siting, stand-up and mood (P = 0.53). However, there was significant difference within groups (P = 0.0001). ANOVA 2 x 2 showed no interaction for each variable (P > 0.05). However, there were significant differences within groups in these variables (P < 0.05). Tukey's post-hoc test showed significant difference in pain on both groups (P = 0.0001). Wilcoxon and Mann-Whitney tests identified no significant differences on balance (P > 0.01). Capacity to Sit improved only in Experimental Wii Group (P = 0.04). In conclusion, physical exercises with Nintendo Wii Fit Plus additional to strength and core training were effective only for sitting capacity, but effect size was small.

Postaerobic Exercise Blood Pressure Reduction in Very Old Persons With Hypertension.

BACKGROUND AND PURPOSE: A single bout of aerobic exercise acutely decreases blood pressure, even in older adults with hypertension. Nonetheless, blood pressure responses to aerobic exercise in very old adults with hypertension have not yet been documented. Therefore, this study aimed to assess the effect of a single session of aerobic exercise on postexercise blood pressure in very old adults with hypertension. METHODS: Eighteen older adults with essential hypertension were randomized into exercise (N = 9, age: 83.4 ± 3.2 years old) or control (N = 9, age: 82.7 ± 2.5 years old) groups. The exercise group performed a session of aerobic exercise constituting 2 periods of 10 minutes of walking at an intensity of 40% to 60% of the heart rate reserve. The control group rested for the same period of time. Anthropometric variables and medication status were evaluated at baseline. Heart rate and systolic and diastolic blood pressures were measured at baseline, after exercise, and at 20 and 40 minutes postexercise. RESULTS: Systolic blood pressure showed a significant interaction for group × time (F3,24 = 6.698; P = .002; ηp = 0.153). In the exercise group, the systolic blood pressure at 20 (127.3 ± 20.9 mm Hg) and 40 minutes (123.7 ± 21.0 mm Hg) postexercise was significantly lower in comparison with baseline (135.6 ± 20.6 mm Hg). Diastolic blood pressure did not change. Heart rate was significantly higher after the exercise session. In the control group, no significant differences were observed. CONCLUSIONS: A single session of aerobic exercise acutely reduces blood pressure in very old adults with hypertension and may be considered an important nonpharmacological strategy to control hypertension in this age group.

Intraocular Pressure Correlates with Optic Nerve Sheath Diameter in Patients with Normal Tension Glaucoma

PURPOSE: 1. Identify differences in optic nerve sheath diameter (ONSD) as an indirect measure of intracranial pressure (ICP) in glaucoma patients and a healthy population. 2. Identify variables that may correlate with ONSD in primary open-angle glaucoma (POAG) and normal tension glaucoma (NTG) patients. METHODS: Patients with NTG (n = 46) and POAG (n = 61), and healthy controls (n = 42) underwent B-scan ultrasound measurement of ONSD by an observer masked to the patient diagnosis. Intraocular pressure (IOP) was measured in all groups, with additional central corneal thickness (CCT) and visual field defect measurements in glaucomatous patients. Only one eye per patient was selected. Kruskal-Wallis or Mann-Whitney were used to compare the different variables between the diagnostic groups. Spearman correlations were used to explore relationships among these variables. RESULTS: ONSD was not significantly different between healthy, NTG and POAG patients (6.09 ± 0.78, 6.03 ± 0.69, and 5.71 ± 0.83 respectively; p = 0.08). Visual field damage and CCT were not correlated with ONSD in either of the glaucoma groups (POAG, p = 0.31 and 0.44; NTG, p = 0.48 and 0.90 respectively). However, ONSD did correlate with IOP in NTG patients (r = 0.53, p < 0.001), while it did not in POAG patients and healthy controls (p = 0.86, p = 0.46 respectively). Patient's age did not relate to ONSD in any of the groups (p > 0.25 in all groups). CONCLUSIONS: Indirect measurements of ICP by ultrasound assessment of the ONSD may provide further insights into the retrolaminar pressure component in glaucoma. The correlation of ONSD with IOP solely in NTG patients suggests that the translaminar pressure gradient may be of particular importance in this type of glaucoma.

Physical Activity, Obesity Status, and Blood Pressure in Preschool Children

Objective To examine the combined effects of physical activity and weight status on blood pressure (BP) in preschool-aged children. Study design The sample included 733 preschool-aged children (49% female). Physical activity was objectively assessed on 7 consecutive days by accelerometry. Children were categorized as sufficiently active if they met the recommendation of at least 60 minutes daily of moderate-to-vigorous physical activity (MVPA). Body mass index was used to categorize children as nonoverweight or overweight/obese, according to the International Obesity Task Force benchmarks. BP was measured using an automated BP monitor and categorized as elevated or normal using BP percentile-based cut-points for age, sex, and height. Results The prevalence of elevated systolic BP (SBP) and diastolic BP was 7.7% and 3.0%, respectively. The prevalence of overweight/obese was 32%, and about 15% of children did not accomplish the recommended 60 minutes of daily MVPA. After controlling for age and sex, overweight/obese children who did not meet the daily MVPA recommendation were 3 times more likely (OR 3.8; CI 1.6-8.6) to have elevated SBP than nonoverweight children who met the daily MVPA recommendation. Conclusions Overweight or obese preschool-aged children with insufficient levels of MVPA are at significantly greater risk for elevated SBP than their non overweight and sufficiently active counterparts. (J Pediatr 2015;167:98-102).

Exercise-Induced Intraventricular Obstruction in a Child with Near Syncope and Chest Pain During Exercise

We report the case of a 10-year-old girl with two episodes of light-headedness and chest pain during exercise. She had an unremarkable clinical record, physical examination, ECG, and echocardiogram. Noninvasive ischemia tests were positive, but coronary angiography was normal. Exercise stress echocardiogram revealed an exercise-induced intra-left-ventricular obstruction with a peak gradient of 78 mmHg and replicated her symptoms. After starting beta-blocker therapy her clinical status improved and no residual obstruction was detected. The authors review this unsuspected clinical condition, seldom reported in the adult population and, to our knowledge, never before in a child.

Hereditary Neuropathy with Liability to Pressure Palsy Presenting with Hand Drop in a Young Child

Hereditary neuropathy with liability to pressure palsy (HNPP) results from the deletion of the PMP22 gene in chromosome 17p11.2. Clinically, it presents with painless pressure palsies, typically in the 2nd and 3rd decades of life, being a rare entity in childhood. We present the case study of a six-year-old male child who presented with left hand drop that he kept for over four weeks. Electrophysiological studies suggested HNPP and genetic studies confirmed it. With this paper, we pretend to create awareness to this entity as a diagnosis to be considered in a child with painless monoparesis and to emphasize the importance of electrophysiological studies in the diagnosis.

Women's Attachment as a Predictor of Pain During Labour and Post-Delivery: a Prospective Observational Study

INTRODUCTION: Labour is considered to be one of the most painful and significant experiences in a woman's life. The aim of this study was to examine whether women's attachment style is a predictor of the pain experienced throughout labour and post-delivery. MATERIAL AND METHODS:Thirty-two pregnant women were assessed during the third trimester of pregnancy and during labour. Adult attachment was assessed with the Adult Attachment Scale ' Revised. The perceived intensity of labour pain was measured using a visual analogue scale for pain in the early stage of labour, throughout labour and post-delivery. RESULTS:Women with an insecure attachment style reported more pain at 3 cm of cervical dilatation (p < 0.05), before the administration of analgesia (p < 0.01) and post-delivery (p < 0.05) than those securely attached. In multivariate models, attachment style was a significant predictor of labour pain at 3 cm of cervical dilatation and before the first administration of analgesia but not of the perceived pain post-delivery. DISCUSSION: These findings confirm that labour pain is influenced by relevant psychological factors and suggest that a woman's attachment style may be a risk factor for greater pain during labour. CONCLUSION:Future studies in the context of obstetric pain may consider the attachment style as an indicator of individual differences in the pain response during labour. This may have important implications in anaesthesiology and to promote a relevant shift in institutional practices and therapeutic procedures.

Intraocular Pressure in Eyes Receiving Intravitreal Antivascular Endothelial Growth Factor Injections

PURPOSE: The aim of the this study was to determine the effect of intravitreal antivascular endothelial growth factor injections on intraocular pressure (IOP) and identify possible risk factors for the development of increased IOP. MATERIALS AND METHODS: This prospective study included a total of 106 eyes receiving intravitreal injection of bevacizumab as treatment for macular edema or active choroidal neovascularization. IOP was measured by Goldmann applanation tonometry immediately before the intravitreal injection and 5 min, 1 h and 15 days after the procedure. The records of the study patients were reviewed for age, gender, history of glaucoma, diabetes mellitus, phakic status, systemic and topical medication and number of previous injections. Subconjunctival reflux was registered. IOP elevation was defined as IOP ≥21 mm Hg and/or a change from baseline of ≥5 mm Hg recorded at least on two or more measurements on the same visit. RESULTS: Mean preoperative IOP was 15.31 ± 3.90 mm Hg and postoperative IOP values were 27.27 ± 11.87 mm Hg (after 5 min), 17.59 ± 6.24 mm Hg (after 1 h) and 16.86 ± 3.62 mm Hg (after 15 days). The IOP variation was statistically significant between pre- and postoperative measurements (p < 0.05). Subconjunctival reflux was recorded in 11.3%, and in this subgroup the IOP at 5 min and at 1 h was lower than preoperative IOP (p < 0.05). CONCLUSIONS: More than one third of the eyes achieved IOPs >30 mm Hg 5 min after injection. Subconjunctival reflux contributed to a lower mean postoperative IOP (p < 0.05). Considerations for the management include prophylactic IOP lowering with medical therapy and/or preinjection ocular decompression for patients with a history of glaucoma or ocular hypertension and switching to an as-needed injection protocol in patients suffering a marked IOP rise in previous injections. © 2015 S. Karger AG, Basel.

Contributo para a validação da versão portuguesa da Quebec Back Pain Disability Scale : fiabilidade e poder de resposta em utentes com dor lombar crónica

RESUMO: Um dos principais resultados das intervenções de Fisioterapia dirigidas a utentes com Dor Lombar Crónica (DLC) é reduzir a incapacidade funcional. A Quebec Back Pain Disability Scale (QBPDS) é um instrumento amplamente aceite a nível internacional na medição do nível de incapacidade funcional reportada pelos indivíduos com DLC. O objetivo deste estudo é dar um contributo para a adaptação cultural da versão portuguesa da QBPDS (QBPDS-VP) e investigar o poder de resposta e interpretabilidade da escala. Metodologia: Realizou-se um estudo metodológico, multicentro, baseado num coorte prospetivo de 132 utentes com DLC. Os utentes foram recrutados a partir da lista de espera de 16 serviços de Medicina Física e de Reabilitação/Fisioterapia de várias áreas geográficas de Portugal. A QBPDS- VP foi administrada 3 vezes, em 3 momentos de recolha de dados distintos: T0 - momento inicial (utentes em lista de espera); T1 - 1 semana de intervalo (início dos tratamentos de Fisioterapia); e T2 - 6 semanas de intervalo (pós-intervenção de Fisioterapia). Os dados recolhidos em T0 foram utilizados para a análise fatorial e para o estudo da consistência interna (n=132); os dados da amostra emparelhada de T0 e T1 (n=132) para a fiabilidade teste-reteste; e os dados da amostra emparelhada de T0 e T2 (n=120) para a análise do poder de resposta e interpretabilidade. A âncora externa utilizada foi a perceção global de mudança, neste caso a PGIC- VP, que foi respondida em T1 e T2. O nível de significância para o qual os valores se consideraram satisfatórios foi de p≤ 0,05. O tratamento dos dados foi realizado no software IBM SPSS Statistics (versão 20). Resultados: A QBPDS- VP é uma escala unidimensional, que revela uma excelente consistência interna (α de Cronbach= 0,95) e uma fiabilidade teste-reteste satisfatória (CCI= 0,696; IC 95%: 0,581–0,783). Esta escala demonstrou um poder de resposta moderado, quando aplicada em utentes com DLC ( = 0,426 e AAC= 0,741; IC 95%: 0,645 – 0,837). A Diferença Mínima Detetável (DMD) estimada foi de 19 pontos e as estimativas da Diferença Mínima Clinicamente Importante (DMCI) variaram entre 7 (pelo método curva ROC) e 8 pontos (pelo método “diferença média de pontuação”). A estimativa pela curva ROC deriva do ponto ótimo de corte de 6,5 pontos, com Área Abaixo da Curva (AAC)= 0,741, sensibilidade de 72%, e especificidade de 71%. Uma análise complementar da curva ROC baseada nas diferenças de pontuações da QBPDS, expressa em percentagem, revelou um ponto ótimo de corte de - 24% (AAC= 0,737, sensibilidade de 71%, e especificidade de 71%). Para pontuações iniciais da QBPDS- VP mais altas (≥34 pontos), foi encontrado um ponto ótimo de corte de 10,5 pontos (AAC= 0,738, sensibilidade de 73%, e especificidade de 67%). Conclusão: A QBPDS-VP demonstrou bons níveis de fiabilidade e poder de resposta, recomendando-se o seu uso na medição e avaliação da incapacidade funcional de utentes com DLC. A DMD estimada, de 19 pontos, determinou uma amplitude válida da QBPDS-VP de 19 a 81 pontos. Este estudo propõe estimativas de DMCI da QBPDS- VP numa aplicação específica da escala (em utentes com DLC que são referidos para a intervenção de Fisioterapia). A pontuação inicial da QBPDS- VP deve ser considerada na interpretação de mudanças de pontuação, após a intervenção de Fisioterapia.------------ ABSTRACT: One of the main results of physiotherapy interventions for patients with Chronic Low Back Pain (CLBP) is decrease the functional disability. The Quebec Back Pain Disability Scale (QBPDS) is an instrument widely accepted internationally, in measuring the level of disability reported by individuals with CLBP. The purpose of this study is to contribute to the cultural adaptation of the Portuguese version of QBPDS (QBPDS - PV) and investigate the Responsiveness and Interpretability of QBPDS-PV. Methodology: This was a methodological and multicenter study, based on a sample of 132 subjects with CLBP. The patients were recruited from the waiting lists of 16 medicine rehabilitation service, in many Portugal districts. The Quebec Back Pain Disability Scale was administered in three different moments: T0 – baseline (patients in the waiting list); T1- one week after T0 (the beginning of treatment); and T2 – six weeks after T1 (the posttreatment). The data collected at T0 were used for factor analysis and to study the internal consistency (n = 132); paired sample data of T0 and T1 (n=132) were used for test-retest reliability, and sample data paired for T0 and T2 (n=120) used for responsiveness and interpretability analysis. The external anchor was the global perception of change, measured by the Portuguese version of Patient’s Global Impression of Change (PGIC) Scale. The minimal level of significance established was p ≤ 0,05. Data analysis was performed using the IBM SPSS Statistics software (version 20). Results: The QBPDS-PV is a unidimensional scale, demonstrates an excellent internal consistency (Cronbach's α=0.95) and satisfactory test-retest reliability (ICC= 0.696, 95% CI: 0.581–0.783). The scale revealed moderate responsiveness when applied to patients with CLBP ( = 0.426 and AUC= 0.741, 95% CI: 0.645 - 0.837). The Smallest Detectable Change (SDC) was 19 points, whereas the Minimal Clinically Important Change (MCIC) ranged between 7 (ROC curve method) and 8 points (by the "mean difference score"). The estimate was derived from the ROC curve by an optimal cutoff point of 6.5 points, with Area Under the Curve (AUC)= 0.741, sensitivity 72%, and specificity of 71%. A complementary analysis of the ROC curve based on differences in QBPDS scores from baseline, expressed in percentage, revealed an optimal cutoff point of -24% (AUC= 0.737, sensitivity of 71%, and specificity of 71%). For the highest initial scores of QBPDS-PV (≥ 34 points) was found an optimal cutoff of 10.5 points (AUC= 0.738, sensitivity of 73%, and specificity 67%). Conclusion: The QBPDS-PV demonstrated good levels of reliability and responsiveness, being recommended its use in the measurement and evaluation of disability of patients with CLBP. The SDC of 19 points determined the QBPDS‟ scale width of 19 to 81. This study proposes MCIC values for QBPDS –PV for this specific setting (in CLBP patients who are referred for physiotherapy intervention). The QBPDS –PV baseline score have to be taken into account while interpreting the score change after physiotherapy intervention.

Disturbed Correlation Between Arterial Resistance and Pulsatility in Glaucoma Patients

PURPOSE: (i) To investigate whether pulsatility index (PI) and mean flow velocities (MFV) are altered in glaucoma patients. (ii) To evaluate the significance of PI in retrobulbar autoregulation capacity. METHODS: Patients with primary open-angle glaucoma (POAG; n = 49), normal tension glaucoma (NTG; n = 62) and healthy controls (n = 48) underwent colour Doppler imaging measurements of the retrobulbar vasculature. Kruskal-Wallis test was used to compare variables between the three diagnostic groups. Restricted cubic splines were used to determine nonlinearities between the resistive index (RI) and PI correlations. RESULTS: Mean flow velocities (MFV) were lower in both short posterior ciliary arteries (SCPA) and central retinal arteries (CRA) from the two glaucoma groups (p < 0.04 versus healthy controls). No differences were detected in RI or PI in any arteries of the three diagnostic groups (p > 0.08). In healthy individuals, correlations between RI and PI were linear in all arteries. In both POAG and NTG patients, CRA presented a nonlinear curve with a cutpoint at RI 0.77 (p < 0.001) and 0.61 (p = 0.03), respectively, above which the slope increased nearly five- and tenfold (POAG: 1.96 to 10.06; NTG: -0.46-4.06), respectively. A nonlinear correlation in the ophthalmic artery was only observed in NTG patients, with a cutpoint at RI 0.82 (p < 0.001), above which the slope increased from 3.47 to 14.03. CONCLUSIONS: Glaucoma patients do not present the linear relationships between RI and PI observed in healthy individuals. Their nonlinear relations may be indicative of an altered autoregulation and suggest a possible threshold RI could be determined above which autoregulatory disturbances become more relevant.

Do hospitals react to random demand pressure by early discharges?

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Economics from the NOVA – School of Business and Economics