Incretin-based therapy for type 2 diabetes mellitus is promising for treating neurodegenerative diseases

Incretin hormones include glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Due to their promising action on insulinotropic secretion and improving insulin resistance (IR), incretin-based therapies have become a new class of antidiabetic agents for the treatment of type 2 diabetes mellitus (T2DM). Recently, the links between neurodegenerative diseases and T2DM have been identified in a number of studies, which suggested that shared mechanisms, such as insulin dysregulation or IR, may underlie these conditions. Therefore, the effects of incretins in neurodegenerative diseases have been extensively investigated. Protease-resistant long-lasting GLP-1 mimetics such as lixisenatide, liraglutide, and exenatide not only have demonstrated promising effects for treating neurodegenerative diseases in preclinical studies but also have shown first positive results in Alzheimer’s disease (AD) and Parkinson’s disease (PD) patients in clinical trials. Furthermore, the effects of other related incretin-based therapies such as GIP agonists, dipeptidyl peptidase-IV (DPP-IV) inhibitors, oxyntomodulin (OXM), dual GLP-1/GIP, and triple GLP-1/GIP/glucagon receptor agonists on neurodegenerative diseases have been tested in preclinical studies. Incretin-based therapies are a promising approach for treating neurodegenerative diseases

Computational approaches for the characterization of the Dipeptidyl Peptidase IV inhibition: Applications to drug discovery, drug design and binding site similarity

La inhibició de l'enzim dipeptidil peptidasa IV (DPP-IV) ha emergit durant les últimes dècades com un dels tractaments més efectius per a la diabetis mellitus tipus II gràcies al seu baix risc hipoglucèmic i al manteniment del pes corporal. Els estudis d'anàlisi de relació estructura-activitat i els protocols de cribratge virtual s'han fet servir per explicar com els lligands interactuen amb el lloc d'unió de la DPP-IV i cercar en extenses bases de dades de compostos de baix pes molecular per tal de trobar nous inhibidors de DPP-IV. Per tant, la tesi doctoral s'ha centrat en: (a) la caracterització de la inhibició de DPP-IV amb l'objectiu de suggerir com els cribratges virtuals podrien ser millorats per a afavorir la identificació d'inhibidors de DPP-IV potents i selectius o bé per cercar noves molècules de partida; (b) el disseny d'una estratègia computacional adequada per identificar nous compostos de partida en bases de dades de molècules comercials que presentin baixa (o nul·la) similitud amb els actius existents; (c) la demostració que almenys de forma parcial, l'efecte antidiabètic descrit per a extractes de diferents espècies d'Ephedra és el resultat de l'activitat inhibitòria de DPP-IV per part dels compostos d'efedrina i derivats d'efedrina trobats en aquests mateixos extractes; i (d) l'anàlisi de les característiques fisico-químiques compartides pels llocs d'unió de DPP-IV i del receptor adrenèrgic β2 i comparar-los amb l'objectiu d'avaluar si és possible que un lligand pugui presentar activitat dual com a inhibidor de DPP-IV i β-bloquejant. És important destacar que el nostre treball aporta una nova hipòtesi sobre l'efecte cardiosaludable associat a la inhibició de DPP-IV i obre la porta al disseny d'un únic tractament dirigit simultàniament per a la diabetis mellitus tipus II i les malalties cardiovasculars, ambdues involucrades en la síndrome metabòlica.La inhibición de la enzima dipeptidil peptidasa IV (DPP-IV) ha surgido durante las últimas décadas como uno de los tratamientos más efectivos para la diabetes mellitus tipo II gracias a su bajo riesgo hipoglucémico y al mantenimiento del peso corporal. Los estudios de análisis de relación estructura-actividad y los protocolos de cribado virtual se han usado para explicar cómo los ligandos interactúan con el lugar de unión de la DPP-IV y buscar en extensas bases de datos de compuestos de bajo peso molecular para identificar nuevos inhibidores de DPP-IV. Por lo tanto, la presente tesis doctoral se ha centrado en: (a) la caracterización de la inhibición de DPP-IV con el objetivo de sugerir cómo los cribados virtuales podrían mejorarse para favorecer la identificación de inhibidores de DPP-IV potentes y selectivos o bien como buscar nuevas moléculas de partida; (b) el diseño de una estrategia computacional adecuada para identificar nuevos compuestos de partida en bases de datos de moléculas comerciales que presenten baja (o nula) similitud con los activos existentes; (c) la demostración de que al menos de forma parcial, el efecto antidiabético descrito para los extractos de diferentes especies de Ephedra es el resultado de la actividad inhibitoria de DPP-IV por parte de las moléculas de efedrina y derivados de ésta encontrados en estos mismos extractos; y (d) el análisis de las características fisico-químicas compartidas por los lugares de unión de DPP-IV y del receptor adrenérgico β2 y compararlos con el objetivo de evaluar si es posible que un ligando pueda presentar actividad dual como inhibidor de DPP-IV y β-bloqueante. Es importante destacar que nuestro trabajo aporta una nueva hipótesis sobre el efecto cardiosaludable asociado a la inhibición de DPP-IV y abre la puerta al diseño de un único tratamiento dirigido simultáneamente para la diabetes mellitus tipo II y las enfermedades cardiovasculares, ambas involucradas en el síndrome metabólico.The inhibition of dipeptidyl peptidase-IV (DPP-IV) enzyme has emerged over the last decade as one of the most effective treatments for type II diabetes mellitus with low risk for hypoglycemia and weight gain. Structure-activity relationship analyses and virtual screening protocols have been used to explain how ligands interact with the DPP- IV binding site and to mine large databases of small molecules searching for new DPP-IV inhibitors. The present doctoral thesis has been therefore focused on: (a) the characterization of DPP-IV inhibition in order to suggest how virtual screening protocols may be improved either to favor the identification of potent and selective DPP-IV inhibitors or to look for new lead molecules; (b) the design of a computational strategy suitable for identifying new lead compounds with very low (or no) similarity to known actives in purchasable databases; (c) the demonstration that, at least partly, the described antidiabetic effect of different Ephedra species extracts is the result of the DPP-IV inhibitory bioactivity by ephedrine and the ephedrine-derivatives found in these extracts and (d) the analysis of the physico-chemical features shared by the DPP-IV and β2-adrenergic receptors binding sites and their comparison in order to evaluate if small molecules with dual bioactivity as DPP-IV inhibitors and β-blockers are possible. It is noteworthy that our work provides a new hypothesis about the cardioprotective effect associated with DPP-IV inhibition and opens the door to a single treatment focused toward type II diabetes mellitus and cardiovascular diseases involved in the metabolic syndrome

Design, Synthesis, Characterization and Pharmacological Evaluation of DPP-IV Inhibitors for Antidiabetic Activity.

AIM: To develop novel, potent, selective and orally active inhibitors of Dipeptidyl Peptidase IV with Anti-Diabetic activity. OBJECTIVES: Identification of common Pharmacophore features responsible for inhibiting DPP-IV using Hiphop module of Catalyst® software 4.11 from Accelrys. Devlopment and validation of quantitative Pharmacophore hypothesis for series of DPP-IV receptor using Hypogen/Hyporefine module of Catalyst® software 4.11 from Accelrys. Generation of 10,000 scaffolds from the drug using scaffold hopping technique. Prediction of activity for designed molecules using the Hyporefine model and to identify novel and potent DPP IV receptor using Lipinski rule of five. The potent receptor inhibitors attained as results may be used as lead for drug development. From the lead molecule, the derivatives of the compounds which has higher score value were synthesised. Characterization of the synthesized compounds by UV, Infrared spectroscopy, Nuclear Magnetic Resonance spectroscopy and Mass Spectroscopy. In vivo anti diabetic activity of synthesised compounds. CONCLUSION: Twenty five molecules which passed Lipinski’s rule were docked against DPP-4. The molecule containing purine and pyridine nucleus was selected on the basis of synthetic feasibility. Four compounds with top scores were synthesized, purified, characterized using UV, IR and NMR spectroscopy and Mass spectrometry. These four compounds were subjected to acute toxicity studies to fix the LD50. The LD50 value of the title compounds (SR A- SR D) was expected to exceed 50mg/kg. All the synthesised compound so were subjected to, invivo experiment to determine antidiabetic activity and were found to decrease the blood glucose levels, but they did not fare better than the standard drug glibenclamide

In vitro pharmacological screening of thiazolidinedione-derivatives on diabetes and Alzheimer’s potential therapeutic targets

There is an increased prevalence of diabetes and other non-communicable diseases in Sub-Saharan Africa and globally. In South Africa, the prevalence of type 2 Diabetes mellitus is currently estimated at 9.0% in people aged 30 and older and is expected to increase. Diabetes-related complications result in acute alterations in the mental state due to poor metabolic control as well as greater rates of decline in cognitive functioning with age, higher prevalence of depression and increased risk of Alzheimer’s disease. Alzheimer’s disease is the most common form of dementia in older adults and possibly contributes to 60 - 70% of cases. Alzheimer’s disease remains incurable, its progression inevitable with the currently available symptomatic therapies being palliative while the treatment of diabetes relies on insulin preparations and other glucose-lowering agents. Current treatment options have numerous side effects such as hypoglycaemia, diarrhoea, weight gain and abnormal liver function. This has geared the investigation of new generations of small molecules which exhibit improved efficacy and safety profiles. On this basis, several studies have shown that thiazolidinediones and their corresponding derivatives exhibit a broad spectrum of biological activities including anti-inflammatory, and antioxidant activities. Furthermore, recent evidence from experimental, epidemiological, and clinical studies highlight the utility of antioxidants for treating diabetes and its complications. Interestingly, there is increasing evidence that links diabetes and Alzheimer’s disease due to their pathophysiology and suppressing glycaemia has been shown to be beneficial in Alzheimer’s disease treatment. Accordingly, the aim of this study, was to evaluate the anti-diabetic and anti-Alzheimer’s properties of four novel synthesized thiazolidinedione-derivatives owing to their antioxidant properties. Methods The aim of this study was achieved through performing ferric reducing antioxidant power activity, 2,2’-Diphenyl-1-Picry Hydrazyl radical scavenging activity, α-amylase inhibition, α-glucosidase inhibition, aldose reductase inhibition, protein tyrosine phosphatase-1B inhibition, dipeptidyl peptidase-4 inhibition, acetylcholinesterase inhibition, matrix metalloproteinase-1 inhibition, and β-amyloid aggregation inhibition assays. In addition, peroxisome proliferator-activated receptor-γ activation was performed through docking studies. To establish physicochemical properties of TZD derivatives investigated, further in-silico studies were done using SwissADME tools. Results To this end, in-vitro and in-silico studies were successfully performed. In-silico ADME profiling predicted these derivatives to be drug-like with moderate to good solubility in the GI and not blood-brain barrier permeable. Furthermore, docking of these molecules against PPARγ predicted a similar mode of action to that of thiazolidinediones using Rosiglitazone as the standard drug with TZDD2 and TZDD4 forming equivalent conformations to that of Rosiglitazone in the same binding site and TZDD3 having an equivalent LBE to that of Rosiglitazone (-8.84 and -8.63kcal/mol respectively). In-vitro evaluation predicted a moderate antioxidant activity with TZDD2 and 3 exhibiting the highest FRAP activity and DPPH radical scavenging activity. Furthermore, enzymatic inhibition assays showed a relative inhibition activity with TZDD3 exhibited > 100% inhibition in concentrations ≥ 30 μg/mL and TZDD1, 2 and 4 exhibited ≥ 50% inhibition activity in all the concentrations (10, 20, 30, 40 and 50 μg/mL) in the α-amylase inhibition assay. Similarly, in the α-glucosidase inhibition assay, all the four derivatives exhibited a concentration dependent activity with TZDD3 showing the most activity. All the four derivatives exhibited ≥ 30% inhibition in the aldose reductase inhibition assay except TZDD1 at 10 μg/mL. TZDD4 exhibited a concentration dependent inhibition activity in the protein tyrosine phosphatase-1B inhibition assay. Interestingly, TZDD3 showed a decreasing inhibition activity as its concentration increased from 10 μg/mL through to 50 μg/mL. In the dipeptidyl peptidase–4 inhibition assay, TZDD2 and TZDD4 exhibited ≥ 20% inhibition activity across all the concentrations and in the acetylcholinesterase assay, TZDD1, 3 and 4 exhibited ≥ 25% across all the concentrations. Interestingly, in the matrix metalloproteinase-1 inhibition assay, some of these derivatives exhibited partial activation activity and partial inhibition with TZDD1 showing activation in concentrations 10 and 20 μg/mL and inhibition in concentrations 30, 40 and 50 μg/mL. TZDD4 showed activation in all the concentrations. In the β-amyloid aggregation assay, all the four derivatives showed inhibition activity ≥ 10% except TZDD1 at 50 μg/mL. Conclusions Diabetes mellitus and Alzheimer’s disease are a type of pathology of global concern, and several researchers worldwide have strived to search for novel therapeutic treatments and prevention for diabetes as well as Alzheimer’s disease. Recent studies provide a direct link v between diabetes mellitus and Alzheimer’s disease, and the need to find novel drugs that can mitigate these two is of increasing interest. In our search for antidiabetic and anti-Alzheimer’s activity, TZD derivatives (TZDD1, TZDD2, TZDD3 and TZDD4) exhibited good antioxidant activity, anti-hyperglycaemic activity and a relatively promising anti-Alzheimer’s activity. This was observed from the in vitro evaluation performed which included α – amylase, α – glucosidase, aldose reductase, PTP1B, DPP4, amyloid β aggregation, and AChE inhibition assays. Furthermore, docking of the derivatives against PPARγ predicted a similar molecular interaction to that of thiazolidinediones using Rosiglitazone as the standard drug. Furthermore, in silico ADME profiling predicted the derivatives to have moderate to good solubility in the GI (good GI bioavailability), and also exhibited excellent drug likeness. However, they are predicted not permeate the BBB. Further in silico studies and in vivo should be conducted to establish toxicities, as well as drug delivery to the brain for effective therapeutic effect against Alzheimer’s disease.Thesis (MSc) -- Faculty of Pharmacy, Pharmacology, 202

An update on peptide-based therapies for type 2 diabetes and obesity

Long-acting analogues of the naturally occurring incretin, glucagon-like peptide-1 (GLP-1) and those modified to interact also with receptors for glucose-dependent insulinotropic polypeptide (GIP) have shown high glucose-lowering and weight-lowering efficacy when administered by once-weekly subcutaneous injection. These analogues herald an exciting new era in peptide-based therapy for type 2 diabetes (T2D) and obesity. Of note is the GLP-1R agonist semaglutide, available in oral and injectable formulations and in clinical trials combined with the long-acting amylin analogue, cagrilintide. Particularly high efficacy in both glucose- and weight lowering capacities has also been observed with the GLP-1R/GIP-R unimolecular dual agonist, tirzepatide. In addition, a number of long-acting unimolecular GLP-1R/GCGR dual agonist peptides and GLP-1R/GCGR/GIPR triagonist peptides have entered clinical trials. Other pharmacological approaches to chronic weight management include the human monoclonal antibody, bimagrumab which blocks activin type II receptors and is associated with growth of skeletal muscle, an antibody blocking activation of GIPR to which are conjugated GLP-1R peptide agonists (AMG-133), and the melanocortin-4 receptor agonist, setmelanotide for use in certain inherited obesity conditions. The high global demand for the GLP-1R agonists liraglutide and semaglutide as anti-obesity agents has led to shortage so that their use in T2D therapy is currently being prioritized. [Abstract copyright: Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

Characterization and identification of hypotensive, immunomodulatory, and metabolic disorder benefiting peptides from Atlantic mackerel (Scomber scombrus) hydrolysate separated based on molecular weight, charge, and hydrophobicity

L'hypertension artérielle est l'un des principaux facteurs de risque de syndrome cardiométabolique. En outre, l'inflammation chronique de bas grade joue également un rôle important dans la pathogenèse du syndrome. Il existe un lien entre l'apparition de la résistance à l 'insuline et l'hypertension qui peut initier le diabète de type 2 et l'obésité. Des peptides bioactifs terrestres et marins, des biomolécules de poissons en particulier, ont démontré des effets immunomodulateurs puissants ainsi que des effets hypotenseurs potentiels dans le traitement de ces facteurs de risque et de leurs complications associées. Notre hypothèse était que les fractions d'hydrolysat protéique de maquereau de l’Atlantique (Scromber scombrus) possèdent une activité biologique bénéfique sur l'hypertension. Le but de notre travail était de fractionner et d'identifier des peptides antihypertenseurs, immunomodulateurs et antiMetS basés sur diverses caractéristiques moléculaires de charge, de polarité et de taille. Les fractions ont été produites en utilisant la technique d'extraction en phase solide chromatographique (SPE), l'ultrafiltration baromembranaire (UF) et l'électrodialyse conditions expérimentales étaient l’utilisation des pH avec membrane UF (EDUF). Les 3, 6, 9, et des membranes de seuils de coupure (MWCO) de < 20 kDa pour l’EDUF et de MWCO de < 1 kDa pour l’UF. Selon nos résultats, parmi toutes les fractions hydrophobes obtenues par SPE il y avait une fraction ayant un effet antihypertenseur important, de 5 µg, et possédant des peptides antidémontrant une inhibition de 50% à une quantité inflammatoires, ayant une inhibition de 17% à une quantité de 10 µg de protéines. Par rapport au témoin négatif (Lipopolysaccharide) les peptides anioniques et cationiques des fractions d’EDUF à pH3 ainsi que l'hydrolysat de maquereau ont démontré des effets pro antiACE et anti-- inflammatoires significatifs jusqu'à 27%. La fraction inflammatoire la plus puissante était riche en acides aminés à chaîne latérale ramifiée et hydrophobe mais avait moins d’acides aminés chargés par rapport aux fractions proinflammatoires EDUF. Toutes les séquences possibles identifiées dans cette fraction sont courtes et ont des valeurs GRAVY positives. Cependant, l'hydrolysat et la fraction positivement du pH3 n'ont pas exercé d'effet antichargée MetS significatif chez les souris nourries au régime hypercalorique. En conclusion, la polarité et la charge de la fraction du maquereau de l’Atlantique sont les facteurs les plus importants pour l' immunomodulation et l'activité hypotensive des peptides. De plus, ces facteurs n'étaient pas suffisants pour réguler les déficiences métaboliques chez les souris obèses et résistantes à l'insuline induites par le régime alimentaire. Par conséquent, la compréhension du mécanisme d'action et la caractérisation approfondie des fractions bioactives seraient nécessaires pour une conclusion définitive et une application clinique du matériel.While, on the one hand, high blood pressure is one of the main risk factors of cardiometabolic syndrome, on the other hand, chronic low-grade inflammation similarly plays a significant role in the pathogenesis of the syndrome. Moreover, there is a link between the occurrence of insulin resistance and hypertension consequently initiating type 2 diabetes and obesity. Interestingly, terrestrial, and marine bioactive peptides, in particular fish biomolecules, have been reported as potent immunomodulators and or hypotensive material in the treatment of these risk factors and associated complications. Hence, our hypothesis was that Atlantic mackerel (Scomber scombrus) protein hydrolysate fractions possess beneficial biological activity on hypertension, inflammation, and other Metabolic Syndrome (MetS) factors. The aim of our work was to fractionate and identify antihypertensive, immunomodulating and anti-MetS peptides based on various molecule characteristics of charge, polarity, and size. Fractions were produced using chromatographic Solid Phase Extraction technique (SPE), pressure driven-Ultra Filtration (UF) and Electrodialysis with UF membrane (EDUF) under experimental conditions of pH 3, 6 and 9 with MWCO of < 1 kDa and < 20 kDa, respectively. According to the results of our in-vitro analysis the highly hydrophobic fraction of SPE was a potent antihypertensive, 50% inhibition at 5 µg, and anti-inflammatory product, 17% inhibition at 10 µg of protein, among all. Furthermore, in comparison to the negative control (Lipopolysaccharide), anionic and cationic peptides of pH3 EDUF as well as mackerel hydrolysate demonstrated significant pro-inflammatory effects up to 27%. The most potent anti-ACE and anti-inflammatory fraction was rather branched chain and hydrophobic amino acid rich with lesser charged amino acids compared to the EDUF pro-inflammatory fractions. All the identified possible sequences of the same fraction had rather small molecular mass with positive GRAVY values. Selected material, the hydrolysate and positively charged fraction of pH3, however, did not exert any significant anti-MetS effect in hypercaloric diet fed obese insulin resistant rats. In conclusion, polarity and charge of a fraction were the most important factors for immunomodulation and hypotensive activity of Atlantic mackerel peptides. Nevertheless, those factors were not sufficient enough to regulate metabolic impairments in diet-induced obese and insulin resistant rats. Accordingly, understanding the mechanism of action and thorough characterization of bioactive fractions would be required for a definite conclusion and clinical application of the material

Evolution of the Molecular Biology of Brain Tumors and the Therapeutic Implications

A dramatic increase in knowledge regarding the molecular biology of brain tumors has been established over the past few years. In particular recent new avenues regarding the role of stem cells and microRNAs along with further understanding of the importance of angiogenesis, immunotherapy and explanations for the resistance of the tumors to chemotherapeutic agents and radiation therapy has been developed. It is hopeful that this new information will lead to efficacious treatment strategies for these tumors which remain a challenge. In this book a review of the latest information on these topics along with a variety of new therapeutic treatment strategies with an emphasis on molecular targeted therapies is provided

Bioinformatic studies of the disposition pathways and targets of synthetic drugs and herbal medicines

Drugs are pharmaceutical compounds administered to treat or diagnose disease. In the human body, drugs have specific reactions with molecular structures or drug targets to produce an effect. Some major concerns of current drugs include undesired side effects or toxicity and poor therapeutic response. Drug discovery is a crucial step to produce new and improved drugs with better efficacy and safety profiles to benefit patients. Generally, drug discovery can be improved in 3 aspects: better understanding of diseases, search for new target classes and the use of new and improved biological and chemical tools. Using advanced bioinformatics tools, this project aimed to study: a) genotype-phenotype relations of Phase II drug metabolising enzyme uridine 5’-diphospho-glucuronosyltransferase (UGT); b) disposition pathways and targets of synthetic drugs and herbal medicines; and c) to design a comprehensive drug and herb target database. We used 2 algorithms, Sorting Intolerant From Tolerant (SIFT) and Polymorphism Phenotyping (Polyphen) to predict the genotype-phenotype relationship of UGTs. Results showed that SIFT and Polyphen are good prediction tools with correct prediction rates of 57.1% and 66.7 %, respectively. Using this method, we can screen for polymorphisms of various genes that may potentially cause diseases and altered drug response or toxicity. Further to this, we used Protein ANalysis THrough Evolutionary Relationships (PANTHER) to classify human therapeutic targets of rheumatoid arthritis and non-insulin-dependent diabetes mellitus (NIDDM), and to identify which classes of molecular targets are most targeted by current medications for these common conditions. The results give us a focus on chief target classes and can be useful in the future to identify new therapeutic targets. Then, we explored targets that are associated with herbal compounds. Using berberine as an example, we collected available human target data and via PANTHER analysis, identified its major target classes. Together with this data and known clinical effects of berberine, we discussed the identification of new therapeutic targets and the development of new drugs from herbal medicines. Finally, we summarised key databases for drug and herbal targets and propose to construct a database containing comprehensive data on drug and herbal targets. Initial stages of the database design are discussed. Findings from these studies suggests that: predicting the phenotypic consequence of nsSNPs in human UGTs using computational algorithms may provide further understanding of genetic differences in susceptibility to diseases and drug response and would be useful information for further genotype-phenotype studies; and b) therapeutic targets of rheumatoid arthritis, NIDDM and berberine can be investigated using a computational approach and has important implications in potential target discovery. Furthermore, the study of current therapeutic targets of drugs and herbal medicine can lead a new direction of future targets identification. Our studies show a promising future for the use of bioinformatics tools in the identification of new therapeutic targets and the exploration of novel drugs. This is valuable for the drug discovery and development process and ultimately, improving drug efficacy and safety profiles to benefit patients

Investigating the biological importance of Dipeptidyl Peptidase 9 enzymatic activity using a mouse model

Dipeptidyl peptidase 9 (DPP9) is an atypical post-proline serine protease of the DPP4 enzyme family which has ubiquitous expression and DPP4-like enzymatic activity. It is localised intracellularly and has roles in antigen processing and epidermal growth factor signalling. It interacts with H-Ras and small ubiquitin-like modifier-1 and influences cellular interactions with extracellular matrix. Our lab made the first DDP9 gene knock-in (gki) mouse with a DPP9 active site (S729A) mutation which results in a lack of DPP9 enzymatic activity. This thesis investigated the biological properties of DPP9 using this genetically modified mouse model. Mice lacking DPP9 proteolytic activity die in the early neonatal stage. This study validated that the DPP9 protein, while present in the gki mouse, is enzyme-inactive and the DPP9-S729A protein and wild-type DPP9 have similar subcellular localisation. A range of investigations were undertaken to determine if obvious histological and/or physiological differences exist between early neonate DPP9S729A/S729A homozygotes from their heterozygous and wild-type littermates and involved analysis of organ morphology, histology and function and investigation of autophagy. The novel findings of this study show that DPP9 enzymatic activity is essential for early neonatal survival in mice and suggest that there is dysregulated autophagy in the DPP9-enzyme-activity-deficient neonatal mice that may contribute to the lethal phenotype. Other members of the DPP4 protein family are implicated in wound healing with fibroblast activation protein expressed in the granulation tissue of healing wounds and DPP4-positive T cells involved in regulation of granulation tissue formation. As there is a heterozygous effect on survival to weaning and DPP9 immunoreactivity was shown in the skin of both WT and DPP9S729A/S729A neonates, the skin of adult mice was studied using a model of cutaneous wound healing. This involved analysis of wound closure rates, tensile strength and collagen levels of both wounded and steady state tissue in adult DPP9wt/S729A mice and their wild-type littermates. It was found that adult heterozygous DPP9-GKI mice display reduced skin collagen levels compared to their WT littermates, but that the rate of skin wound healing appeared unaffected. DPP9 has in vivo expression in normal immunological tissues and major lymphocyte populations that alters with chronic liver injury, suggesting a role in immune function. To investigate the importance of DPP9 in immune regeneration and function, primary and secondary mixed chimeric mice were created using DPP9S729A/S729A and WT fetal liver cells and adult bone marrow, respectively, injected into irradiated recipients. The process of regeneration of the immune system in these mice was assessed at several time-points and the immune cells present in the regenerated immune systems were compared between DPP9S729A/S729A-origin and WT-origin chimeras. These chimeric mice were then subjected to an immune challenge by influenza virus infection. The novel findings of this study suggest that DPP9-enzyme-activity-deficient secondary chimeric mice may have an enhanced ability to recover from an immune challenge with influenza virus. Therefore, DPP9 appears to influence immune function in that viral infection. Together, these studies underline the biological significance of DPP9 in vivo in both neonatal and adult mice. These data contribute to the expanding knowledge of the role of DPP9, building upon recent discoveries in vitro of its involvement in multiple biological processes. By providing physiologically relevant results, this study has enhanced the knowledge of DPP9 enzyme function and the overall biological roles of DPP9

Search for biomarkers related to rhinitis and different asthma phenotypes by serum proteomics and immunoassays

sthma is a heterogeneous disease with several clinical phenotypes and molecular endotypes. However, the specific connection between asthma phenotypes and the underlying pathological features is difficult to explain. Thus, the overall aim of this thesis was to search for biomarkers associated with rhinitis and different phenotypes (allergic and non-allergic) and severities (intermittent-mild and moderate-severe) of asthma. To achieve this aim, we studied the role of the immune system through the analysis of certain biomarkers previously related to this disease: CD14 (innate immune system) and CD26/CD126 (adaptive immune system). In addition, in the second part of the present thesis, a prospective, non-target proteomic study aimed to identify new biomarkers associated with different phenotypes of this disease was also performed. This study consisted of the analysis of low abundance serum proteome through iTRAQ-LC-MS/MS and allowed us the identification of several potential biomarkers for allergic (e.g., IGFALS, protein AMBP, or HSPG2) and non-allergic asthma (e.g., CFI), as well as disease severity (e.g., IGFALS)