Doctor of Philosophy

dissertationAn intact lung capillary glycocalyx is vital to normal vascular barrier function and subsequently normal pulmonary function. Evidence suggests that the glycocalyx provides active regulatory functions, which are fundamental to normal lung fluid balance and that endothelial surface glycoproteins participate in agonist-mediated signaling. Heparan sulfates and hyaluronan glycosaminoglycans are of particular interest in mechanostimulation and subsequent mechanotransduction because of direct and indirect attachment to intracellular components involved with barrier maintenance. Also important to glycocalyx structure are associated blood serum proteins. The component contribution to the overall glycocalyx mechanical environment is integral to its transfer of extracellular mechanical signals to intracellular signals. These components have not been characterized in terms of their mechanical contribution to the glycocalyx stiffness, which allows for endothelial mechanotransduction. Understanding these components will assist in developing a strategy to treat acute inflammation of the lungs. In this dissertation, the mechanical contributions of glycosaminoglycans (heparan sulfate and hyaluronan) and associated macromolecules (albumin and hydroxyethyl starch) to lung glycocalyx mechanical structure are measured with novel applications of two optical micromechanical techniques: atomic force microscopy and reflectance interference contrast microscopy. This information is combined into an inclusive mechanical model. Specifically, the biomechanical properties of the microvascular glycocalyx were acquired and analyzed by probing with physiologically relevant normal forces. The techniques and experiments described in this dissertation provided means to measure and potentially other soft biologic materials, including the local glycocalyx microenvironment

Analyzing the Catalytic Role of Asp97 in the Methionine Aminopeptidase from \u3cem\u3eEscherichia coli\u3c/em\u3e

An active site aspartate residue, Asp97, in the methionine aminopeptidase (MetAPs) from Escherichia coli (EcMetAP-I) was mutated to alanine, glutamate, and asparagine. Asp97 is the lone carboxylate residue bound to the crystallographically determined second metal-binding site in EcMetAP-I. These mutant EcMetAP-I enzymes have been kinetically and spectroscopically characterized. Inductively coupled plasma–atomic emission spectroscopy analysis revealed that 1.0 ± 0.1 equivalents of cobalt were associated with each of the Asp97-mutated EcMetAP-Is. The effect on activity after altering Asp97 to alanine, glutamate or asparagine is, in general, due to a ∼ 9000-fold decrease in kca towards Met-Gly-Met-Met as compared to the wild-type enzyme. The Co(II) d–d spectra for wild-type, D97E and D97A EcMetAP-I exhibited very little difference in form, in each case, between the monocobalt(II) and dicobalt(II) EcMetAP-I, and only a doubling of intensity was observed upon addition of a second Co(II) ion. In contrast, the electronic absorption spectra of [Co_(D97N EcMetAP-I)] and [CoCo(D97N EcMetAP-I)] were distinct, as were the EPR spectra. On the basis of the observed molar absorptivities, the Co(II) ions binding to the D97E, D97A and D97N EcMetAP-I active sites are pentacoordinate. Combination of these data suggests that mutating the only nonbridging ligand in the second divalent metal-binding site in MetAPs to an alanine, which effectively removes the ability of the enzyme to form a dinuclear site, provides a MetAP enzyme that retains catalytic activity, albeit at extremely low levels. Although mononuclear MetAPs are active, the physiologically relevant form of the enzyme is probably dinuclear, given that the majority of the data reported to date are consistent with weak cooperative binding

Optimal Dosing of Enoxaparin in Overweight and Obese Children

Aim:Current enoxaparin dosing guidelines in children are based on total bodyweight. This is potentially inappropriate in obese children as it may overestimate thedrug clearance. Current evidence suggests that obese children may require lower ini-tial doses of enoxaparin, therefore the aim of this work was to characterise the phar-macokinetics of enoxaparin in obese children and to propose a more appropriatedosing regimen.Methods:Data from 196 unique encounters of 160 children who received enoxa-parin treatment doses were analysed. Enoxaparin concentration was quantified usingthe chromogenic anti factor Xa (anti-Xa) assay. Patients provided a total of 552 anti-Xa samples. Existing published pharmacokinetic (PK) models were fitted and evalu-ated against our dataset using prediction-corrected visual predictive check plots(pcVPCs). A PK model was fitted using a nonlinear mixed-effects modelling approach.The fitted model was used to evaluate the current standard dosing and identify anoptimal dosing regimen for obese children.Results:Published models of enoxaparin pharmacokinetics in children did not capturethe pharmacokinetics of enoxaparin in obese children as shown by pcVPCs. A one-compartment model with linear elimination best described the pharmacokinetics ofenoxaparin. Allometrically scaled fat-free mass with an estimated exponent of 0.712(CI 0.66-0.76) was the most influential covariate on clearance while linear fat-freemass was selected as the covariate on volume. Simulations from the model showedthat fat-free mass-based dosing could achieve the target anti-Xa activity at steadystate in 77.5% and 78.2% of obese and normal-weight children, respectively, com-pared to 65.2% and 75.5% for standard total body weight-based dosing.Conclusions:A population PK model that describes the time course of anti-Xa activ-ity of enoxaparin was developed in a paediatric population. Based on this model, aunified dosing regimen was proposed that will potentially improve the success rate oftarget attainment in overweight/obese patients without the need for patient bodysize categorisation. Therefore, prospective validation of the proposed approach iswarranted

Taking after a parent: phenotypic resemblance and the professional familialisation of genomics

This article draws on 2 years’ worth of ethnographic observation of team meetings to explore decision-making in an NHS clinical genomics service. The focus of discussions was on ambiguous genomic results known as VUS or Variants of Uncertain Significance, which may be pathogenic but which also may turn out to be benign. In examining decision-making around such results, we note how, in contrast to much policy and promotional material in this area, clinicians in these meetings (clinical geneticists and genetic counsellors) place great emphasis on parental phenotypes and whether the parents of a patient share the symptoms and signs of the suspected condition. This information is then combined with the result of genomic tests to decide whether the variant a patient has is responsible for their condition. This article explores the way in which clinicians attempt to flexibly enrol parents into genomic explanations through informal diagnosis of their possible phenotypes and the way in which actually meeting parents allows some clinicians to trump explanations based on documentary or photographic data. The paper sheds light on the way that earlier scholarly understandings of such decisions (around, say dysmorphology) remain relevant and explores claims that laboratory tests overrule clinical decision-making

Reflexive standardization and the resolution of uncertainty in the genomics clinic

In genomics, the clinical application of Next Generation Sequencing technologies (such as Whole Genome or Exome Sequencing) has attracted considerable attention from UK policymakers, interested in the benefits such technologies could bring the National Health Service. However, this boosterism plays little attention to the challenges raised by a kind of result known as a Variant of Uncertain Significance, or VUS, which require clinical geneticists and related colleagues to classify ambiguous genomic variants as ‘benign’ or ‘pathogenic’. With a rigorous analysis based on data gathered at 290 clinical meetings over a two-year period, this paper presents the first ethnographic account of decision-making around NGS technology in a NHS clinical genomics service, broadening our understanding of the role formal criteria play in the classification of VUS. Drawing on Stefan Timmermans’ concept of ‘reflexive standardisation’ to explore the way in which clinical genetics staff classify such variants this paper explores the application of a set of criteria drafted by the American College of Medical Genetics and Genomics, highlighting the flexible way in which various resources – variant databases, computer programmes, the research literature – are drawn on to reach a decision. A crucial insight is how professionals’ perception of, and trust in, the clinical practice at other genomics centres in the NHS, shapes their own application of criteria and the classification of a VUS as either benign or pathogenic

Therapeutic Potential of Citrulline as an Arginine Supplement: A Clinical Pharmacology Review

Supplemental arginine has shown promise as a safe therapeutic option to improve endogenous nitric oxide (NO) regulation in cardiovascular diseases associated with endothelial dysfunction. L-arginine, an endogenous amino acid, was reported in clinical studies in adults to improve cardiovascular function in hypertension, pulmonary hypertension, pre-eclampsia, angina, and mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome. L-citrulline, a natural precursor of L-arginine, is more bioavailable than L-arginine because of hepatic first-pass metabolism avoidance and longer circulation time. Although not yet well studied, arginine/citrulline has immense therapeutic potential in some life-threatening diseases of children. However, optimal clinical development of arginine or citrulline in children is dependent on more information about pharmacokinetics and exposure-response relationships at appropriate ages and under relevant disease states. This article summarizes the pre-clinical and clinical studies of arginine/citrulline in both adults and children, including currently available pharmacokinetic information. The pharmacology of arginine/citrulline is confounded by several patient-specific factors such as baseline variation of arginine/citrulline due to developmental ages and disease states. Currently available pharmacokinetic studies are not enough to inform the optimal design of clinical studies, especially those in children. Successful bench to bedside clinical translation of arginine supplementation awaits information from well-designed pharmacokinetic-pharmacodynamic studies, along with pharmacometric approaches

MCAK associates with the tips of polymerizing microtubules.

International audienceMCAK is a member of the kinesin-13 family of microtubule (MT)-depolymerizing kinesins. We show that the potent MT depolymerizer MCAK tracks (treadmills) with the tips of polymerizing MTs in living cells. Tip tracking of MCAK is inhibited by phosphorylation and is dependent on the extreme COOH-terminal tail of MCAK. Tip tracking is not essential for MCAK's MT-depolymerizing activity. We propose that tip tracking is a mechanism by which MCAK is preferentially localized to regions of the cell that modulate the plus ends of MTs

Current use of complementary and conventional medicine for treatment of pediatric patients with gastrointestinal disorders

Infants, children, and adolescents are at risk of experiencing a multitude of gastrointestinal disorders (GID). These disorders can adversely affect the quality of life or be life-threatening. Various interventions that span the conventional and complementary therapeutic categories have been developed. Nowadays, parents increasingly seek complementary options for their children to use concurrently with conventional therapies. Due to the high prevalence and morbidity of diarrhea, constipation, and irritable bowel syndrome (IBS) in children, in this review, we decided to focus on the current state of the evidence for conventional and complementary therapies used for the treatment of these diseases in children. Diarrhea treatment focuses on the identification of the cause and fluid management. Oral rehydration with supplementation of deficient micronutrients, especially zinc, is well established and recommended. Some probiotic strains have shown promise in reducing the duration of diarrhea. For the management of constipation, available clinical trials are insufficient for conclusive recommendations of dietary modifications, including increased use of fruit juice, fiber, and fluid. However, the role of laxatives as conventional treatment is becoming more established. Polyethylene glycol is the most studied, with lactulose, milk of magnesia, mineral oil, bisacodyl, and senna presenting as viable alternatives. Conventional treatments of the abdominal pain associated with IBS are poorly studied in children. Available studies investigating the effectiveness of antidepressants on abdominal pain in children with IBS were inconclusive. At the same time, probiotics and peppermint oil have a fair record of benefits and safety. The overall body of evidence indicates that a careful balance of conventional and complementary treatment strategies may be required to manage gastrointestinal conditions in children

Project #91: Optimizing Vascular Access to Reduce CLABSI

Henry Ford Macomb Hospital experienced an increase in Central Line Associated Bloodstream Infections (CLABSI) in 2021. A significant portion were occurring in the MICU and were associated with Candida sp. Bloodstream infections negatively impact patient outcomes, provider workload, and are costly, with a median cost of $48,108 based on a meta-analysis conducted by AHRQ in 2017. By end of 2022, HFM aimed to reduce CLABSI incidence by 50%.https://scholarlycommons.henryford.com/qualityexpo2023/1004/thumbnail.jp