Production d'IgG sialylées en CHO et impact sur leurs fonctions effectrices

La sialylation des N-glycanes du fragment Fc des immunogobulines G (IgG) est une modification peu fréquente des IgG humaines. Pourtant, elle est l’objet de beaucoup d’attention depuis que deux articles fondateurs ont été publiés, qui montrent l’un que la sialylation des IgG diminue leur capacité à déclencher la cytotoxicité cellulaire dépendant de l’anticorps (ADCC), et l’autre que les IgG sialylées en α2,6 seraient la fraction efficace des IgG intraveineuses (IgIV) anti-inflammatoires. Les anticorps monoclonaux thérapeutiques, qui sont le plus souvent des IgG recombinantes produites en culture de cellules de mammifère, connaissent depuis la fin des années 90 un succès et une croissance phénoménaux sur le marché pharmaceutique. La maîtrise de la N-glycosylation du Fc des IgG est une clé de l’efficacité des anticorps monoclonaux. Si les IgG sialylées sont des molécules peu fréquentes in vivo, elles sont très rares en culture cellulaire. Dans cette étude, nous avons développé une méthode de production d’IgG avec une sialylation de type humain en cellules CHO. Nous avons travaillé principalement sur la mise au point d’une stratégie de production d’IgG sialylées par co-expression transitoire d’une IgG1 avec la β1,4-galactosyltransférase I (β4GTI) et la β-galactoside-α2,6-sialyltransférase I (ST6GalI). Nous avons montré que cette méthode permettait d’enrichir l’IgG1 en glycane fucosylé di-galactosylé mono-α2,6-sialylé G2FS(6)1, qui est le glycane sialylé présent sur les IgG humaines. Nous avons ensuite adapté cette méthode à la production d’IgG présentant des profils de glycosylation riches en acides sialiques, riches en galactose terminal, et/ou appauvris en fucosylation. L’analyse des profils de glycosylation obtenus par la co-expression de diverses combinaisons enzymatiques avec l’IgG1 native ou une version mutante de l’IgG1 (F243A), a permis de discuter des influences respectives de la sous-galactosylation des IgG1 en CHO et des contraintes structurales du Fc dans la limitation de la sialylation des IgG en CHO. Nous avons ensuite utilisé les IgG1 produites avec différents profils de glycosylation afin d’évaluer l’impact de la sialylation α2,6 sur l’interaction de l’IgG avec le récepteur FcγRIIIa, principal récepteur impliqué dans la réponse ADCC. Nous avons montré que la sialylation α2,6 augmentait la stabilité du complexe formé par l’IgG avec le FcγRIIIa, mais que ce bénéfice n’était pas directement traduit par une augmentation de l’efficacité ADCC de l’anticorps. Enfin, nous avons débuté le développement d’une plateforme d’expression stable d’IgG sialylées compatible avec une production à l’échelle industrielle. Nous avons obtenu une lignée capable de produire des IgG enrichies en G2FS(6)1 à hauteur de 400 mg/L. Cette étude a contribué à une meilleure compréhension de l’impact de la sialylation sur les fonctions effectrices des IgG, et a permis d’augmenter la maîtrise des techniques de modulation du profil de glycosylation des IgG en culture cellulaire.

A Reliable Downscaling of ECG Signals for the Detection of T wave Heterogeneity Features

In cardiovascular disease the definition and the detection of the ECG parameters related to repolarization dynamics in post MI patients is still a crucial unmet need. In addition, the use of a 3D sensor in the implantable medical devices would be a crucial mean in the assessment or prediction of Heart Failure status, but the inclusion of such feature is limited by hardware and firmware constraints. The aim of this thesis is the definition of a reliable surrogate of the 500 Hz ECG signal to reach the aforementioned objective. To evaluate the worsening of reliability due to sampling frequency reduction on delineation performance, the signals have been consecutively down sampled by a factor 2, 4, 8 thus obtaining the ECG signals sampled at 250, 125 and 62.5 Hz, respectively. The final goal is the feasibility assessment of the detection of the fiducial points in order to translate those parameters into meaningful clinical parameter for Heart Failure prediction, such as T waves intervals heterogeneity and variability of areas under T waves. An experimental setting for data collection on healthy volunteers has been set up at the Bakken Research Center in Maastricht. A 16 – channel ambulatory system, provided by TMSI, has recorded the standard 12 – Leads ECG, two 3D accelerometers and a respiration sensor. The collection platform has been set up by the TMSI property software Polybench, the data analysis of such signals has been performed with Matlab. The main results of this study show that the 125 Hz sampling rate has demonstrated to be a good candidate for a reliable detection of fiducial points. T wave intervals proved to be consistently stable, even at 62.5 Hz. Further studies would be needed to provide a better comparison between sampling at 250 Hz and 125 Hz for areas under the T waves.

Hospital surfaces contamination with antineoplastic drugs: influence of cleaning procedures

Introduction: The raising frequency of cancer diseases is leading to a widespread application of antineoplastic drugs, thus increasing the probability of workplace surfaces contamination. Most of these drugs are classified by the International Agency for Research on Cancer as known or suspected human carcinogens. Skin absorption is the main route for antineoplastic drugs exposure in occupational settings, therefore cleaning protocols have paramount influence in surfaces contamination and, consequently, in exposure. The aim of this study was to assess surfaces contamination in a Portuguese chemotherapy unit before and during drug administration, in both preparation and administration facilities. Methods: Samples were collected by wipe-sampling from potentially contaminated surfaces selected by previous protocol observation. Samples were analyzed by HPLCDAD. Cyclophosphamide (CP), 5-fluorouracil (5FU), and paclitaxel (PTX) were used as surrogate markers for surfaces contamination for all cytotoxic drugs. Results: From the 34 samples collected before any preparation and administration activities, 41.2% were contaminated with 5-FU (4.0-84.7 ng/cm2) and 23.5% of the samples were contaminated with CP (19.8-139.6 μg/cm2). Only 2 samples presented contamination by PTX (5.9%) with a maximum value of 3.7 ng/cm2. Of the 37 samples collected during preparation and administration of antineoplastic drugs, 48.7% were contaminated with 5-FU (1.9-88.7 ng/cm2) and 24.3% with CP (12.0-93.9 μg/cm2). None of the samples showed contamination with PTX. Discussion: Data showed differences in contamination levels before and after the handling of antineoplastic drugs in preparation and in administration settings. These results point out the importance of cleaning procedures. This is well in accordance to previous studies that showed how the type of cleaning procedures and products used can be determinant for surfaces decontamination.

Is quality of life measurement likely to be a proxy for health needs assessment in patients with coronary artery disease?

BACKGROUND: The identification of patients' health needs is pivotal in optimising the quality of health care, increasing patient satisfaction and directing resource allocation. Health needs are complex and not so easily evaluated as health-related quality of life (HRQL), which is becoming increasingly accepted as a means of providing a more global, patient-orientated assessment of the outcome of health care interventions than the simple medical model. The potential of HRQL as a surrogate measure of healthcare needs has not been evaluated. OBJECTIVES AND METHOD: A generic (Short Form-12; SF-12) and a disease-specific questionnaire (Seattle Angina Questionnaire; SAQ) were tested for their potential to predict health needs in patients with acute coronary disease. A wide range of healthcare needs were determined using a questionnaire specifically developed for this purpose. RESULTS: With the exception of information needs, healthcare needs were highly correlated with health-related quality of life. Patients with limited enjoyment of personal interests, weak financial situation, greater dependency on others to access health services, and dissatisfaction with accommodation reported poorer HRQL (SF-12: p < 0.001; SAQ: p < 0.01). Difficulties with mobility, aids to daily living and activities requiring assistance from someone else were strongly associated with both generic and disease-specific questionnaires (SF-12: r = 0.46-0.55, p < 0.01; SAQ: r = 0.53-0.65, p < 0.001). Variables relating to quality of care and health services were more highly correlated with SAQ components (r = 0.33-0.59) than with SF-12 (r = 0.07-0.33). Overall, the disease-specific Seattle Angina Questionnaire was superior to the generic Short Form-12 in detecting healthcare needs in patients with coronary disease. Receiver-operator curves supported the sensitivity of HRQL tools in detecting health needs. CONCLUSION: Healthcare needs are complex and developing suitable questionnaires to measure these is difficult and time-consuming. Without a satisfactory means of measuring these needs, the extent to which disease impacts on health will continue to be underestimated. Further investigation on larger populations is warranted but HRQL tools appear to be a reasonable proxy for healthcare needs, as they identify the majority of needs in patients with coronary disease, an observation not previously reported in this patient group

Caractérisation d'IGFBP-2 comme biomarqueur intégrateur de la santé cardiométabolique

La protéine de liaison aux facteurs de croissance analogues à l’insuline (IGFBP)-2 est une protéine circulante fortement associée à la résistance à l’insuline qui module les effets métaboliques d’IGF-I et IGF-II en s’y associant directement, et qui exerce aussi des actions IGF-indépendantes via sa liaison à la matrice extracellulaire et aux intégrines. Chez l’homme, de faibles niveaux d’IGFBP-2 sont associés à un profil lipidique délétère, ainsi qu’à une augmentation de la masse grasse et de la résistance à l’insuline. Les travaux décrits dans cette thèse montrent chez l’humain et la souris que les niveaux d’IGFBP-2 sont associés de manière indépendante aux composantes du risque cardiométabolique. Chez l’homme, de faibles niveaux d’IGFBP-2 sont associés à la dyslipidémie athérogène. Une valeur seuil d’IGFBP-2 de 221.5 ng/mL a permis de discriminer entre les sujets métaboliquement sains et ceux répondant aux critères du syndrome métabolique. En plus de son association avec la résistance à l’insuline et les composantes du profil lipidique, de faibles niveaux d’IGFBP-2 sont associés à une fonction cardiaque diminuée chez les patients atteints de sténose aortique, tel qu’évaluée par le volume d’éjection indexé, un indice de fonction global du ventricule gauche qui intègre la fonction pompe et le remodelage du tissu. Chez l’homme, des niveaux d’IGFBP-2 élevés sont associés à un tissu adipeux brun plus volumineux ainsi qu’à une activité métabolique plus importante de ce dernier. Ces observations, telles qu’évaluées par PET/CT, sont aussi validées chez les souris surexprimant la forme humaine d’IGFBP-2. Nos travaux démontrent que les niveaux d’IGFBP-2 sont fortement associés au métabolisme des lipoprotéines et des lipides, à la fonction cardiaque ainsi qu’à l’activité du tissu adipeux brun. L’influence des niveaux d’IGFBP-2 par différentes altérations métaboliques menant à l’augmentation du risque cardiométabolique pourrait faire de ce dernier un biomarqueur précoce et intégrateur. Les travaux exposés dans la présente thèse soulignent aussi un rôle mécanistique potentiel pour IGFBP-2 dans la protection contre certaines altérations du métabolisme.

Ultra high pressure homogenization (UHPH) inactivation of Bacillus amyloliquefaciens spores in phosphate buffered saline (PBS) and milk

Ultra high pressure homogenization (UHPH) opens up new areas for dynamic high pressure assisted thermal sterilization of liquids. Bacillus amyloliquefaciens spores are resistant to high isostatic pressure and temperature and were suggested as potential surrogate for high pressure thermal sterilization validation. B. amyloliquefaciens spores suspended in PBS buffer (0.01 M, pH 7.0), low fat milk (1.5%, pH 6.7), and whole milk (3.5%, pH 6.7) at initial concentration of similar to 10(6) CFU/mL were subjected to UHPH treatments at 200, 300, and 350 MPa with an inlet temperature at similar to 80 degrees C. Thermal inactivation kinetics of B. amyloliquefaciens spores in PBS and milk were assessed with thin wall glass capillaries and modeled using first-order and Weibull models. The residence time during UHPH treatments was estimated to determine the contribution of temperature to spore inactivation by UHPH. No sublethal injury was detected after UHPH treatments using sodium chloride as selective component in the nutrient agar medium. The inactivation profiles of spores in PBS buffer and milk were compared and fat provided no clear protective effect for spores against treatments. Treatment at 200 MPa with valve temperatures lower than 125 degrees C caused no reduction of spores. A reduction of 3.5 log(10)CFU/mL of B. amyloliquefaciens spores was achieved by treatment at 350 MPa with a valve temperature higher than 150 degrees C. The modeled thermal inactivation and observed inactivation during UHPH treatments suggest that temperature could be the main lethal effect driving inactivation.

Epidemiology and genetic architecture of blood pressure: a family based study of Generation Scotland

Hypertension is a major risk factor for cardiovascular disease and mortality, and a growing global public health concern, with up to one-third of the world’s population affected. Despite the vast amount of evidence for the benefits of blood pressure (BP) lowering accumulated to date, elevated BP is still the leading risk factor for disease and disability worldwide. It is well established that hypertension and BP are common complex traits, where multiple genetic and environmental factors contribute to BP variation. Furthermore, family and twin studies confirmed the genetic component of BP, with a heritability estimate in the range of 30-50%. Contemporary genomic tools enabling the genotyping of millions of genetic variants across the human genome in an efficient, reliable, and cost-effective manner, has transformed hypertension genetics research. This is accompanied by the presence of international consortia that have offered unprecedentedly large sample sizes for genome-wide association studies (GWASs). While GWAS for hypertension and BP have identified more than 60 loci, variants in these loci are associated with modest effects on BP and in aggregate can explain less than 3% of the variance in BP. The aims of this thesis are to study the genetic and environmental factors that influence BP and hypertension traits in the Scottish population, by performing several genetic epidemiological analyses. In the first part of this thesis, it aims to study the burden of hypertension in the Scottish population, along with assessing the familial aggregation and heritialbity of BP and hypertension traits. In the second part, it aims to validate the association of common SNPs reported in the large GWAS and to estimate the variance explained by these variants. In this thesis, comprehensive genetic epidemiology analyses were performed on Generation Scotland: Scottish Family Health Study (GS:SFHS), one of the largest population-based family design studies. The availability of clinical, biological samples, self-reported information, and medical records for study participants has allowed several assessments to be performed to evaluate factors that influence BP variation in the Scottish population. Of the 20,753 subjects genotyped in the study, a total of 18,470 individuals (grouped into 7,025 extended families) passed the stringent quality control (QC) criteria and were available for all subsequent analysis. Based on the BP-lowering treatment exposure sources, subjects were further classified into two groups. First, subjects with both a self-reported medications (SRMs) history and electronic-prescription records (EPRs; n =12,347); second, all the subjects with at least one medication history source (n =18,470). In the first group, the analysis showed a good concordance between SRMs and EPRs (kappa =71%), indicating that SRMs can be used as a surrogate to assess the exposure to BP-lowering medication in GS:SFHS participants. Although both sources suffer from some limitations, SRMs can be considered the best available source to estimate the drug exposure history in those without EPRs. The prevalence of hypertension was 40.8% with higher prevalence in men (46.3%) compared to women (35.8%). The prevalence of awareness, treatment and controlled hypertension as defined by the study definition were 25.3%, 31.2%, and 54.3%, respectively. These findings are lower than similar reported studies in other populations, with the exception of controlled hypertension prevalence, which can be considered better than other populations. Odds of hypertension were higher in men, obese or overweight individuals, people with a parental history of hypertension, and those living in the most deprived area of Scotland. On the other hand, deprivation was associated with higher odds of treatment, awareness and controlled hypertension, suggesting that people living in the most deprived area may have been receiving better quality of care, or have higher comorbidity levels requiring greater engagement with doctors. These findings highlight the need for further work to improve hypertension management in Scotland. The family design of GS:SFHS has allowed family-based analysis to be performed to assess the familial aggregation and heritability of BP and hypertension traits. The familial correlation of BP traits ranged from 0.07 to 0.20, and from 0.18 to 0.34 for parent-offspring pairs and sibling pairs, respectively. A higher correlation of BP traits was observed among first-degree relatives than other types of relative pairs. A variance-component model that was adjusted for sex, body mass index (BMI), age, and age-squared was used to estimate heritability of BP traits, which ranged from 24% to 32% with pulse pressure (PP) having the lowest estimates. The genetic correlation between BP traits showed a high correlation between systolic (SBP), diastolic (DBP) and mean arterial pressure (MAP) (G: 81% to 94%), but lower correlations with PP (G: 22% to 78%). The sibling recurrence risk ratio (λS) for hypertension and treatment were calculated as 1.60 and 2.04 respectively. These findings confirm the genetic components of BP traits in GS:SFHS, and justify further work to investigate genetic determinants of BP. Genetic variants reported in the recent large GWAS of BP traits were selected for genotyping in GS:SFHS using a custom designed TaqMan® OpenArray®. The genotyping plate included 44 single nucleotide polymorphisms (SNPs) that have been previously reported to be associated with BP or hypertension at genome-wide significance level. A linear mixed model that is adjusted for age, age-squared, sex, and BMI was used to test for the association between the genetic variants and BP traits. Of the 43 variants that passed the QC, 11 variants showed statistically significant association with at least one BP trait. The phenotypic variance explained by these variant for the four BP traits were 1.4%, 1.5%, 1.6%, and 0.8% for SBP, DBP, MAP, and PP, respectively. The association of genetic risk score (GRS) that were constructed from selected variants has showed a positive association with BP level and hypertension prevalence, with an average effect of one mmHg increase with each 0.80 unit increases in the GRS across the different BP traits. The impact of BP-lowering medication on the genetic association study for BP traits has been established, with typical practice of adding a fixed value (i.e. 15/10 mmHg) to the measured BP values to adjust for BP treatment. Using the subset of participants with the two treatment exposure sources (i.e. SRMs and EPRs), the influence of using either source to justify the addition of fixed values in SNP association signal was analysed. BP phenotypes derived from EPRs were considered the true phenotypes, and those derived from SRMs were considered less accurate, with some phenotypic noise. Comparing SNPs association signals between the four BP traits in the two model derived from the different adjustments showed that MAP was the least impacted by the phenotypic noise. This was suggested by identifying the same overlapped significant SNPs for the two models in the case of MAP, while other BP traits had some discrepancy between the two sources

Optimizing Western Blots for the Detection of Endogenous α-Synuclein in the Enteric Nervous System

International audience

Soft computing based parameter identification in pavements and geomechanical systems

Accurate estimation of road pavement geometry and layer material properties through the use of proper nondestructive testing and sensor technologies is essential for evaluating pavement’s structural condition and determining options for maintenance and rehabilitation. For these purposes, pavement deflection basins produced by the nondestructive Falling Weight Deflectometer (FWD) test data are commonly used. The nondestructive FWD test drops weights on the pavement to simulate traffic loads and measures the created pavement deflection basins. Backcalculation of pavement geometry and layer properties using FWD deflections is a difficult inverse problem, and the solution with conventional mathematical methods is often challenging due to the ill-posed nature of the problem. In this dissertation, a hybrid algorithm was developed to seek robust and fast solutions to this inverse problem. The algorithm is based on soft computing techniques, mainly Artificial Neural Networks (ANNs) and Genetic Algorithms (GAs) as well as the use of numerical analysis techniques to properly simulate the geomechanical system. A widely used pavement layered analysis program ILLI-PAVE was employed in the analyses of flexible pavements of various pavement types; including full-depth asphalt and conventional flexible pavements, were built on either lime stabilized soils or untreated subgrade. Nonlinear properties of the subgrade soil and the base course aggregate as transportation geomaterials were also considered. A computer program, Soft Computing Based System Identifier or SOFTSYS, was developed. In SOFTSYS, ANNs were used as surrogate models to provide faster solutions of the nonlinear finite element program ILLI-PAVE. The deflections obtained from FWD tests in the field were matched with the predictions obtained from the numerical simulations to develop SOFTSYS models. The solution to the inverse problem for multi-layered pavements is computationally hard to achieve and is often not feasible due to field variability and quality of the collected data. The primary difficulty in the analysis arises from the substantial increase in the degree of non-uniqueness of the mapping from the pavement layer parameters to the FWD deflections. The insensitivity of some layer properties lowered SOFTSYS model performances. Still, SOFTSYS models were shown to work effectively with the synthetic data obtained from ILLI-PAVE finite element solutions. In general, SOFTSYS solutions very closely matched the ILLI-PAVE mechanistic pavement analysis results. For SOFTSYS validation, field collected FWD data were successfully used to predict pavement layer thicknesses and layer moduli of in-service flexible pavements. Some of the very promising SOFTSYS results indicated average absolute errors on the order of 2%, 7%, and 4% for the Hot Mix Asphalt (HMA) thickness estimation of full-depth asphalt pavements, full-depth pavements on lime stabilized soils and conventional flexible pavements, respectively. The field validations of SOFTSYS data also produced meaningful results. The thickness data obtained from Ground Penetrating Radar testing matched reasonably well with predictions from SOFTSYS models. The differences observed in the HMA and lime stabilized soil layer thicknesses observed were attributed to deflection data variability from FWD tests. The backcalculated asphalt concrete layer thickness results matched better in the case of full-depth asphalt flexible pavements built on lime stabilized soils compared to conventional flexible pavements. Overall, SOFTSYS was capable of producing reliable thickness estimates despite the variability of field constructed asphalt layer thicknesses.

Production d'IgG sialylées en CHO et impact sur leurs fonctions effectrices

La sialylation des N-glycanes du fragment Fc des immunogobulines G (IgG) est une modification peu fréquente des IgG humaines. Pourtant, elle est l’objet de beaucoup d’attention depuis que deux articles fondateurs ont été publiés, qui montrent l’un que la sialylation des IgG diminue leur capacité à déclencher la cytotoxicité cellulaire dépendant de l’anticorps (ADCC), et l’autre que les IgG sialylées en α2,6 seraient la fraction efficace des IgG intraveineuses (IgIV) anti-inflammatoires. Les anticorps monoclonaux thérapeutiques, qui sont le plus souvent des IgG recombinantes produites en culture de cellules de mammifère, connaissent depuis la fin des années 90 un succès et une croissance phénoménaux sur le marché pharmaceutique. La maîtrise de la N-glycosylation du Fc des IgG est une clé de l’efficacité des anticorps monoclonaux. Si les IgG sialylées sont des molécules peu fréquentes in vivo, elles sont très rares en culture cellulaire. Dans cette étude, nous avons développé une méthode de production d’IgG avec une sialylation de type humain en cellules CHO. Nous avons travaillé principalement sur la mise au point d’une stratégie de production d’IgG sialylées par co-expression transitoire d’une IgG1 avec la β1,4-galactosyltransférase I (β4GTI) et la β-galactoside-α2,6-sialyltransférase I (ST6GalI). Nous avons montré que cette méthode permettait d’enrichir l’IgG1 en glycane fucosylé di-galactosylé mono-α2,6-sialylé G2FS(6)1, qui est le glycane sialylé présent sur les IgG humaines. Nous avons ensuite adapté cette méthode à la production d’IgG présentant des profils de glycosylation riches en acides sialiques, riches en galactose terminal, et/ou appauvris en fucosylation. L’analyse des profils de glycosylation obtenus par la co-expression de diverses combinaisons enzymatiques avec l’IgG1 native ou une version mutante de l’IgG1 (F243A), a permis de discuter des influences respectives de la sous-galactosylation des IgG1 en CHO et des contraintes structurales du Fc dans la limitation de la sialylation des IgG en CHO. Nous avons ensuite utilisé les IgG1 produites avec différents profils de glycosylation afin d’évaluer l’impact de la sialylation α2,6 sur l’interaction de l’IgG avec le récepteur FcγRIIIa, principal récepteur impliqué dans la réponse ADCC. Nous avons montré que la sialylation α2,6 augmentait la stabilité du complexe formé par l’IgG avec le FcγRIIIa, mais que ce bénéfice n’était pas directement traduit par une augmentation de l’efficacité ADCC de l’anticorps. Enfin, nous avons débuté le développement d’une plateforme d’expression stable d’IgG sialylées compatible avec une production à l’échelle industrielle. Nous avons obtenu une lignée capable de produire des IgG enrichies en G2FS(6)1 à hauteur de 400 mg/L. Cette étude a contribué à une meilleure compréhension de l’impact de la sialylation sur les fonctions effectrices des IgG, et a permis d’augmenter la maîtrise des techniques de modulation du profil de glycosylation des IgG en culture cellulaire.

Construção e produção de fragmentos de anticorpos anti-ssDNA em um contexto de desenvolvimento de linfócitos B

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Pós-Graduação em Biologia Molecular, 2016.

The use of subclinical vascular markers of atherosclerosis in youth

The foundations for cardiovascular disease (CVD) in adults are laid in childhood and accelerated by the presence of comorbid conditions. Early detection of manifestations of cardiovascular pathology is an important clinical objective to identify those at risk for subsequent cardiovascular morbidity and events, and to initiate behavioral and medical interventions to reduce risk. Children were once considered to be at low risk, but with the growing health concerns related to lifestyle, cardiovascular screening may be needed earlier. Several noninvasive procedures are available to assess the cumulative effect of these exposures. These include carotid ultrasound, flow-mediated dilation, pulse wave velocity and measures left ventricular mass. This dissertation analyzes the comorbid conditions that increase cardiovascular risk in youth, namely obesity and low physical fitness, using carotid intima-media thickness to objectively detect early manifestations of cardiovascular pathology. Until recently researchers have not used surrogate markers of subclinical atherosclerosis to examine the role of a single bout of exercise. Utilizing the acute exercise model can be advantageous as it allows for an efficient manipulation of exercise variables and permits greater experimental control of confounding variables. It is possible that the effects of a bout of exercise can predict the effects of chronic exercise. We analyze the physiological factors pertinent to arterial stiffness using arterial distensibility and pulse wave velocity in the context of acute exercise in children and adults. In some instances, those who amend their trajectory by not maintaining risk factors into adulthood experience reductions in subclinical markers to levels associated with never having had the risk factor. Though avoidance of risk factors in youth is ideal, there is still a window for intervention where long-lasting cardiovascular effects might be avoided. In this dissertation we present preliminary findings linking modifiable youth risk factors to subclinical markers of CVD in adulthood.

Kinematics and Local Motion Planning for Quasi-static Whole-body Mobile Manipulation

This thesis studies mobile robotic manipulators, where one or more robot manipulator arms are integrated with a mobile robotic base. The base could be a wheeled or tracked vehicle, or it might be a multi-limbed locomotor. As robots are increasingly deployed in complex and unstructured environments, the need for mobile manipulation increases. Mobile robotic assistants have the potential to revolutionize human lives in a large variety of settings including home, industrial and outdoor environments.

Mobile Manipulation is the use or study of such mobile robots as they interact with physical objects in their environment. As compared to fixed base manipulators, mobile manipulators can take advantage of the base mechanism’s added degrees of freedom in the task planning and execution process. But their use also poses new problems in the analysis and control of base system stability, and the planning of coordinated base and arm motions. For mobile manipulators to be successfully and efficiently used, a thorough understanding of their kinematics, stability, and capabilities is required. Moreover, because mobile manipulators typically possess a large number of actuators, new and efficient methods to coordinate their large numbers of degrees of freedom are needed to make them practically deployable. This thesis develops new kinematic and stability analyses of mobile manipulation, and new algorithms to efficiently plan their motions.

I first develop detailed and novel descriptions of the kinematics governing the operation of multi- limbed legged robots working in the presence of gravity, and whose limbs may also be simultaneously used for manipulation. The fundamental stance constraint that arises from simple assumptions about friction and the ground contact and feasible motions is derived. Thereafter, a local relationship between joint motions and motions of the robot abdomen and reaching limbs is developed. Baseeon these relationships, one can define and analyze local kinematic qualities including limberness, wrench resistance and local dexterity. While previous researchers have noted the similarity between multi- fingered grasping and quasi-static manipulation, this thesis makes explicit connections between these two problems.

The kinematic expressions form the basis for a local motion planning problem that that determines the joint motions to achieve several simultaneous objectives while maintaining stance stability in the presence of gravity. This problem is translated into a convex quadratic program entitled the balanced priority solution, whose existence and uniqueness properties are developed. This problem is related in spirit to the classical redundancy resoxlution and task-priority approaches. With some simple modifications, this local planning and optimization problem can be extended to handle a large variety of goals and constraints that arise in mobile-manipulation. This local planning problem applies readily to other mobile bases including wheeled and articulated bases. This thesis describes the use of the local planning techniques to generate global plans, as well as for use within a feedback loop. The work in this thesis is motivated in part by many practical tasks involving the Surrogate and RoboSimian robots at NASA/JPL, and a large number of examples involving the two robots, both real and simulated, are provided.

Finally, this thesis provides an analysis of simultaneous force and motion control for multi- limbed legged robots. Starting with a classical linear stiffness relationship, an analysis of this problem for multiple point contacts is described. The local velocity planning problem is extended to include generation of forces, as well as to maintain stability using force-feedback. This thesis also provides a concise, novel definition of static stability, and proves some conditions under which it is satisfied.

Phase asymmetry of heart rate variability signal

Heart rate asymmetry (HRA) is considered as a physiological phenomenon in healthy subjects. In this article, we propose a novel HRA index, Slope Index (SI), to quantify phase asymmetry of heart rate variability (HRV) system. We assessed the performance of proposed index in comparison with conventional (Guzik's Index (GI) and Porta's Index (PI)) HRA indices. As illustrative examples, we used two case studies: (i) differentiate physiologic RR series from synthetic RR series; and (ii) discriminate arrhythmia subjects from Healthy using beat-to-beat heart rate time series. The results showed that SI is a superior parameter than GI and PI for both case studies with maximum ROC area of 0.84 and 0.82 respectively. In contrast, GI and PI had ROC areas {0.78, 0.61} and {0.50, 0.56} in two case studies respectively. We also performed surrogate analysis to show that phase asymmetry is caused by a physiologic phenomena rather than a random nature of the signal. In conclusion, quantification of phase asymmetry of HRV provides additional information on HRA, which might have a potential clinical use to discriminate pathological HRV in future.

Changes in intravenous fluid use patterns in Australia and New Zealand: evidence of research translating into practice

OBJECTIVES: To describe changes in the use of intravenous (IV) fluid by quantity and type in different regions of Australia and New Zealand. DESIGN, SETTING AND PARTICIPANTS: We conducted a retrospective ecological study examining regional and temporal trends in IV fluid consumption across Australia and New Zealand over the periods 2012-2013 and 2013- 2014, using national proprietary sales data as a surrogate for consumption, and demographic data from the public domain. RESULTS: More than 13.3 million litres of IV fluid were consumed in Australia and New Zealand in 2012-2013, and more than 13.9 million litres in 2013-2014, with colloid solutions accounting for < 2%. There was marked regional variation in consumption of fluids, by volumes and proportions used, when standardised to overall Australian and New Zealand values. There was no significant change in the overall volume of crystalloid solutions consumed but there was a significant decrease (9%; P = 0.02) in the ratio of unbalanced to balanced crystalloid solutions consumed. Consumption of all forms of colloid solutions decreased, with a 12% reduction overall (P = 0.02), primarily driven by a 67% reduction in the consumption of hydroxyethyl starch (HES) solutions. CONCLUSIONS: The amount and type of IV fluid use, as determined by fluid sales, is highly variable across Australia and New Zealand. However, overall use of balanced crystalloid solutions is increasing and the use of HES has decreased dramatically.