La plupart des conditions détectées par le dépistage néonatal sont reliées à l'une des enzymes qui dégradent les acyls-CoA mitochondriaux. Le rôle physiopathologique des acyls-CoA dans ces maladies est peu connue, en partie parce que les esters liés au CoA sont intracellulaires et les échantillons tissulaires de patients humains ne sont généralement pas disponibles. Nous avons créé une modèle animal murin de l'une de ces maladies, la déficience en 3-hydroxy-3-methylglutaryl-CoA lyase (HL), dans le foie (souris HLLKO). HL est la dernière enzyme de la cétogenèse et de la dégradation de la leucine. Une déficience chronique en HL et les crises métaboliques aigües, produisent chacune un portrait anormal et distinct d'acyls-CoA hépatiques. Ces profils ne sont pas prévisibles à partir des niveaux d'acides organiques urinaires et d'acylcarnitines plasmatiques. La cétogenèse est indétectable dans les hépatocytes HLLKO. Dans les mitochondries HLLKO isolées, le dégagement de 14CO2 à partir du [2-14C]pyruvate a diminué en présence de 2-ketoisocaproate (KIC), un métabolite de la leucine. Au test de tolérance au pyruvate, une mesure de la gluconéogenèse, les souris HLLKO ne présentent pas la réponse hyperglycémique normale. L'hyperammoniémie et l'hypoglycémie, des signes classiques de plusieurs erreurs innées du métabolisme (EIM) des acyls-CoA, surviennent de façon spontanée chez des souris HLLKO et sont inductibles par l'administration de KIC. Une charge en KIC augmente le niveau d'acyls-CoA reliés à la leucine et diminue le niveau d'acétyl-CoA. Les mitochondries des hépatocytes des souris HLLKO traitées avec KIC présentent un gonflement marqué. L'hyperammoniémie des souris HLLKO répond au traitement par l'acide N-carbamyl-L-glutamique. Ce composé permet de contourner une enzyme acétyl-CoA-dépendante essentielle pour l’uréogenèse, le N-acétylglutamate synthase. Ceci démontre un mécanisme d’hyperammoniémie lié aux acyls-CoA. Dans une deuxième EIM des acyls-CoA, la souris SCADD, déficiente en déshydrogénase des acyls-CoA à chaînes courtes. Le profil des acyls-CoA hépatiques montre un niveau élevé du butyryl-CoA particulièrement après un jeûne et après une charge en triglycérides à chaîne moyenne précurseurs du butyryl-CoA. / Most conditions detected by expanded newborn screening result from deficiency of one of the enzymes that degrade acyl-CoA esters in mitochondria. The role of acyl-CoAs in the pathophysiology of these disorders is poorly understood, in part because CoA esters are intracellular and samples are not generally available from human patients. We created a mouse model of one such condition, deficiency of 3-hydroxy-3-methylglutaryl-CoA lyase (HL), in liver (HLLKO mice). HL catalyses a reaction of ketone body synthesis and of leucine degradation. Chronic HL deficiency and acute crises each produced distinct abnormal liver acyl-CoA patterns, which would not be predictable from levels of urine organic acids and plasma acylcarnitines. In HLLKO hepatocytes, ketogenesis was undetectable. Measures of Krebs cycle flux diminished following incubation of HLLKO mitochondria with the leucine metabolite 2-ketoisocaproate (KIC). HLLKO mice also had suppression of the normal hyperglycemic response to a systemic pyruvate load, a measure of gluconeogenesis. Hyperammonemia and hypoglycemia, cardinal features of many inborn errors of acyl-CoA metabolism, occurred spontaneously in some HLLKO mice and were inducible by administering KIC. KIC loading also increased levels of several leucine-related acyl-CoAs and reduced acetyl-CoA levels. Ultrastructurally, hepatocyte mitochondria of KIC-treated HLLKO mice show marked swelling. KIC-induced hyperammonemia improved following administration of carglumate (N-carbamyl-L-glutamic acid), which bypasses an acetyl-CoA-dependent reaction essential for urea cycle function, thus demonstrating an acyl-CoA-related mechanism for this complication. In a second animal model of an inborn error of acyl-CoA metabolism, short chain acyl-CoA dehydrogenase (SCAD)-deficient mice, the main finding in liver acyl-CoAs is increased butyryl-CoA, particularly during fasting or after enteral loading with medium chain triglyceride precursor of butyryl-CoA.

<p>Volatile organic compounds (VOCs) are endogenous byproducts of metabolic pathways that can be altered by a disease or condition, leading to an associated and unique VOC profile or signature. Current methodologies for VOC detection include canines, gas chromatography-mass spectrometry (GC-MS), and electronic nose (eNose). Some of the challenges for canines and GC-MS are cost-effectiveness, extensive training, expensive instrumentation. On the other hand, a significant downfall of the eNose is low selectivity. This thesis proposes to design a breathalyzer using chemiresistive gas sensors that detects VOCs from human breath, and subsequently create an interface to process and deliver the results via Bluetooth Low Energy (BLE). Breath samples were collected from patients with hypoglycemia, COVID-19, and healthy controls for both. Samples were processed, analyzed using GC-MS and probed through statistical analysis. A panel of 6 VOC biomarkers distinguished between hypoglycemia (HYPO) and Normal samples with a training AUC of 0.98 and a testing AUC of 0.93. For COVID-19, a panel of 3 VOC biomarkers distinguished between COVID-19 positive symptomatic (COVID-19) and healthy Control samples with a training area under the curve (AUC) of receiver operating characteristic (ROC) of 1.0 and cross-validation (CV) AUC of 0.99. The model was validated with COVID-19 Recovery samples. The discovery of these biomarkers enables the development of selective gas sensors to detect the VOCs. </p><p><br></p><p>Polyethylenimine-ether functionalized gold nanoparticle (PEI-EGNP) gas sensors were designed and fabricated in the lab and metal oxide (MOX) semiconductor gas sensors were obtained from Nanoz (Chip 1: SnO₂ and Chip 2: WO₃). These sensors were tested at different relative humidity (RH) levels, and VOC concentrations. Contact angle which measures hydrophobicity, was 84° and the thickness of the PEI-EGNP coating was 11 µ m. The PEI-EGNP sensor response at RH 85% had a signal 10x higher than at RH 0%. Optimization of the MOX sensor was performed by changing the heater voltage and concentration of VOCs. At RH 85% and heater voltage of 2500 mV, the performance of the sensors increased. Chip 2 had higher sensitivity towards VOCs especially for one of the VOC biomarkers identified for COVID-19. PCA distinguished VOC biomarkers of HYPO, COVID-19, and healthy human breath using the Nanoz. A sensor interface was created to integrate the PEI-EGNP sensors with the printed circuit board (PCB) and Bluno Nano to perform machine learning. The sensor interface can currently process and make decisions from the data whether the breath is HYPO (-) or Normal (+). This data is then sent via BLE to the Hypo Alert app to display the decision.</p>