Innate immunity operates independently of prior exposure to pathogens. There are several signal transduction pathways that play a key role in inflammatory and immune responses. Critical signaling cascade in the interest of my research is type-1 interferon (IFN) signaling pathway in response to infection with Salmonella Typhimurium (ST). The role of type-I interferons is well established in the context of a viral infection; however, their role in bacterial infections is not clear. In my thesis I aimed to understand the role of type-1 IFNs in bacterial pathogenesis, and scrutinize the mechanism adopted by various components of type-1 IFN signaling, especially ISGF3 complex in response to Salmonella Typhimurium. My results indicate that type-I IFN signaling is detrimental to host survival. I further investigated the mechanism through which type-1 IFN signaling results in host susceptibility against Salmonella. My results indicated that the three transcription factors downstream of IFNAR1 have different impacts in mounting an innate immune response against ST. IRF9 and STAT2 promote susceptibility against ST whereas STAT1 through IFNAR1-signaling, promotes enhanced expression of pro inflammatory cytokines and protection against ST. I also observed that the monocytes/macrophages lineage in Ifnar1⁻ᐟ⁻ mice is responsible for conferring the enhanced resistance against ST. Furthermore, my work determined that expression of type-I IFN signaling compromises the fitness of macrophages by reducing mitochondrial respiration, glycolysis and myelopoiesis.

Background From a global perspective, around 8.4 million individuals live with diabetes type 1, which is a chronic disease for which complex medical treatment. The disease can lead to complications and women with type 1 diabetes have a higher risk of mental illness. Women with a medical disability also violate society's norms. Competence and understanding of the basic nurse can prevent ill health and alleviate suffering. Aim: The purpose of this literature review was to investigate women's experience of living with type 1 diabetes during reproductive age. Method: A literature study based on twelve articles of qualitative method were analyzed and a new result built up. Through analysis with the support of Friberg's five-step process, three main themes and ten subthemes emerged. Results: The result showed that the feeling of inadequacy in the form of physical, sexual and psychological limitations and lack of knowledge was described. The women also experienced a sense of sadness characterized by worry and fear for the future. To overcome this disease, support and motivation were important. Conclusion: The preventive work can prevent women with type 1 diabetes from risking exclusion, sexual ill health, necessary suffering and contracting mental illness. In order to promote women's health and help them reach acceptance, knowledge, support and confirmation from the health and medical services and relatives were required. By being able to make demands and question society's norms, work can be done for a more equal illness experience. / Syftet med denna litteraturstudie var att undersöka kvinnors upplevelse av att leva med diabetes typ 1 under reproduktiv ålder. I resultatet framkom en del skillnad i upplevelser beroende på vart i livet under den reproduktiva perioden kvinnorna befann sig, men även många likheter identifierades. Resultatet visade att kvinnor bar känslor som oro och rädsla, otillräcklighet samt en känsla av att vara annorlunda. Sjukdomen innebar begränsningar i vardagen som beskrevs som betungande. Frustration och besvikelse uttrycktes hos kvinnorna kring sjukdomens komplexitet och de beskrev känslor som att inte kunna ta en paus från sjukdomen, att bli berövad av sin spontanitet och frihet samt att inte duga till trots väl valda strategier att kontrollera sjukdomen. Acceptans kring att sjukdomen var en del av kvinnornas personlighet förknippades med bättre egenvård och motivation till att kontrollera sjukdomen. Förståelse och acceptansen erhölls främst genom stöd och hopp från hälso- och sjukvårdspersonal samt närstående. Även känslan av samhörighet och att inte vara ensam med sjukdomen var betydelsefull. Examensarbetet belyser två vårdvetenskapliga begrepp; människa och lidande. Denna litteraturstudie har en kvalitativ ansats då kvinnors upplevelser efterfrågas. Av tolv artiklar som analyserades framkom tre teman samt tio subteman som beskriver resultatet. Kompetens hos sjuksköterskor kring kvinnors upplevelse av att leva med diabetes typ 1 under reproduktiv ålder kan främja hälsa, identifiera ohälsa och lindra lidande. Diskussionen och slutsatsen i detta arbete lyfter normer kring att vara kvinna med en kronisk sjukdom samt konsekvenser som de upplevda känslorna kan leda till framför allt för de enskilda kvinnorna, men också ur ett hållbarhetsperspektiv för samhället.

Plusieurs aspects de la réponse immunitaire sont dérégulés de manière irréversible au cours de la phase aiguë de l'infection par le virus de l’immunodéficience de type 1 (VIH-1) menant à une activation immunitaire élevée et une inflammation généralisée impliquées dans l’épuisement du système immunitaire et la progression de la maladie. Nos travaux précédents ont mis en évidence une production plus élevée de vésicules extracellulaires (VE) appelées exosomes par les cellules dendritiques (CD) exposées au VIH-1. Ces exosomes ayant un effet pro-apoptotique sur les lymphocytes T CD4 (LT CD4), ils peuvent ainsi accentuer la déplétion de ces cellules, déjà initiée par l’infection elle-même. Produites par une grande variété de cellules et libérées dans de nombreux fluides corporels, les VE sont le reflet de leur cellule d’origine et peuvent transporter des protéines mais aussi du matériel génétique (ARN, microARN) aux cellules voisines faisant d’elles des messagers essentiels à la communication intercellulaire. De par leur contenu, les VE sont impliquées dans différents processus biologiques dont la modulation de la réponse immunitaire. En s’appuyant sur ces données et sur l’implication des microARN dans la modulation des réponses immunitaires, l’hypothèse du rôle des VE dans la mise en place et l’entretien des perturbations immunitaires associée à la pathogenèse au VIH-1, notamment via leur contenu en microARN, a émergé. Mes travaux de doctorat ont montré un profil en VE plasmatiques spécifique associé au statut clinique des sujets infectés par le VIH-1. La quantité et la taille des VE retrouvées dans le plasma de sujets infectés non traités sont plus élevées que chez les individus contrôles et corrèlent avec la déplétion en LT CD4 ou la baisse du ratio CD4/CD8, considérés comme des marqueurs de l’évolution de la maladie, soulignant le potentiel biomarqueur des VE dans la progression de l’infection. De plus, ces données révèlent un enrichissement des VE en microARN, dont le miR-155, auparavant décrit dans la modulation de différentes réponses immunitaires. L’inoculation subséquente d’une production virale enrichie en VE miR-155-positives dans un modèle de souris humanisées a mis en évidence la contribution du miR-155 vésiculaire dans le développement des perturbations immunitaires et la progression de la pathogenèse associée à l’infection par le VIH-1. Ainsi, une meilleure compréhension des mécanismes participant à l’enrichissement du miR-155 dans les VE permettrait éventuellement un contrôle de l’épuisement immunitaire caractéristique de l’infection par le VIH-1 et, à plus long terme, ouvrirait de nouvelles perspectives pour le développement de thérapies. / Several aspects of the immune response are irreversibly dysregulated during the acute phase of HIV-1 infection leading to a generalized immune activation and inflammation which are involved in immune cells depletion and disease progression. Our previous work revealed a higher production of extracellular vesicles (EVs) called exosomes by HIV-1 pulsed dendritic cells (DCs). These exosomes displayed pro-apoptotic effect on CD4 T lymphocytes (CD4 TL), and thus increase the depletion of these cells, already initiated by the infection itself. Produced by a wide variety of cells and released into body fluids, EVs, which reflect cellular origin, are believed to function as messengers in intercellular communication delivering proteins and genetic material (RNA, microRNA) to neighboring cells. By their content, EVs are involved in various biological processes including the modulation of immune responses. Based on these data and the involvement of microRNAs in immune response modulation, the hypothesis assuming the role of EVs in the establishment and maintenance of immune disruptions during HIV-1 pathogenesis, notably through their microRNA content, has emerged. My doctoral research showed a specific profile of plasma EVs associated with clinical status of HIV-1 infected patients. The amount and size of EVs present in the plasma of HIV-1- infected patients’ naïve for treatment were higher than those from healthy individuals and correlate with CD4 TL depletion or CD4/CD8 ratio decrease, which are considered markers of disease progression. These data highlight the biomarker potential of EVs. Furthermore, microRNAs are enriched in EVs, including miR-155, previously described in different immune responses. Subsequent inoculation of EV-borne miR-155-enriched-virus in a humanized mouse model revealed that vesicular miR-155 can contribute to the development of immune disruptions and the progression of HIV-1-associated pathogenesis. To conclude, a greater understanding of the mechanisms involved in miR-155 enrichment in EVs would help to control the immune exhaustion characteristic of HIV-1 infection and, on long-term, would open new perspectives for therapies.

In order to facilitate a biophysical analysis of the carboxy terminal acidic transactivation domain (AAD) of Vmw65 from Herpes Simplex Virus Type 1 (HSV-1), an overexpression system in Escherichia coli was constructed and optimized to produce milligram quantities of this polypeptide. Purification of the polypeptide was facilitated by creating a fusion protein to glutathione S-transferase (GST) from Schizosoma japonicum using a commercially available vector. Upon thrombin digestion of the fusion protein, the carrier and AAD products were resolved by anion-exchange chromatography. With typically 15 mg of AAD available from a 12 litre culture, several biophysical studies were initiated. Circular dichroism and fluorescence spectroscopy both described a polypeptide with an extended structure reminicent of a random-coil; that is, it did not possess substantial quantities of known elements of secondary structure such as a-helicies and β-sheets under physiological conditions. A new structure high in α-helical content was induced upon addition of trifluoroethanol to mimic a hydrophobic milieu. Ultracentrifugation data supported the spectroscopic observations by describing an extended, monomeric polypeptide. The ultimate goal of the study, a teritiary structure, was sought by attempting to crystallize AAD with popular salts and organic solvents. Biologically, the described random-coil structure of AAD could be relevant to its role as a promoter and stablizer of the transcriptional pre-initation complex, the determining step in gene expression. A structurally labile domain would support AAD’s ability to interact with several targets including TFIID and TFIIB, though not necessarily by similar mechanisms. The requirement for a drastic conformational change such as a random-coil to α-helical transition currently remains unclear though observations made in this study of AAD in trifluoroethanol have shown that a conformational change is indeed possible. With a means of producing large quantities of AAD, the opportunity now arises to study its interaction with available cloned targets. The ensuing biophysical studies will then provide a greater understanding of AAD’s important role in gene expression. / Thesis / Master of Science (MSc)

Indiana University-Purdue University Indianapolis (IUPUI) / Previous studies have shown an increased growth of the tibial growth plate in rats infused with supraphysiologic doses of IGF-I. However, no one has demonstrated this effect on the TMJ in vivo. To determine the effect of a constant infusion of IGF-1 on the TMJ, 20 Sprague-Dawley rats were divided into three groups: (1) control, (2) surgical control, and (3) IGF-1 and placebo infused. IGF-I was delivered at a rate of 1 μg/day over the TMJ via osmotic minipumps. lntravital bone labels were administered at two-week intervals to monitor growth rate. Following sacrifice, seven mandibular (Mn) dimensions were measured anthropometrically. The mandibles were then imbedded in acrylic and stained with tetrachrome to visualize the Mn cartilage. Fluorescence microscopy was utilized to measure the Mn growth between bone labels and calculate growth rates. In addition, the hypertrophic cartilage layer thickness was measured photomicrographically. ANOVA showed no significant difference (P<0.05) in growth rates or cartilage thicknesses between the groups. However, multiple t tests did show an increase in several Mn dimensions (increase in length from gonion to the mental foramen; increase in length from condylion to the mental foramen; and increase in condylar head anterior-posterior length) in the experimental animals comparing the IGF-I infused side with the placebo infused control side. Therefore, it was concluded that IGF-I, when infused at a constant supraphysiologic dose, may increase mandibular growth in certain directions. The present study is not able to definitively demonstrate that these increases are due to direct effects on Mn cartilage growth.

Levels of circulating large extracellular vesicles (L-EVs) are increased in individuals with type 1 diabetes mellitus (T1DM) and associated with increased cardiovascular risk. T1DM in pregnancy induces vascular injury leading to adverse maternal and neonatal outcomes. Conversely, exercise has been shown to improve cardiovascular and metabolic health in pregnancy and may represent a non-pharmacological approach to improving pregnancy outcomes. Assessment of vascular health may aid in the identification of individuals at risk of complications and allow for intervention with strategies to improve the maternal vasculature. Unfortunately, there is a paucity of strategies for assessing vascular health in pregnant women. L-EVs are membrane-encapsulated particles released from stressed/injured cells. They are emerging biomarkers of vascular health. The purpose of this thesis was to assess the impact of T1DM and pregnancy on L-EV levels and protein composition, the relationship between L-EVs and pregnancy outcomes and the effect of exercise on L-EV levels. In aim #1, I observed that high levels of L-EVs are predictive of adverse pregnancy outcomes. In aim # 2, I examined the protein composition of circulating L-EVs in hypertensive, diabetic and healthy mice models. Diabetes-enriched proteins were involved in inflammation, SNARE signaling and NAD+ biogenesis. The changes were found in L-EV protein content were consistent with proteins associated with inflammation, cytoskeletal organization, and angiogenesis. Finally, in aim #3, I examined the changes in plasma L-EVs after an acute bout of moderate-intensity aerobic exercise in healthy pregnant and non-pregnant women. I observed that circulating L-EVs significantly decreased after the acute exercise only in non-pregnant individuals. Taken together, my thesis work advances knowledge on L-EVs in T1DM, pregnancy, and hypertension and sets the stage for future work on L-EVs as predictive biomarkers, for molecular profiling, and for monitoring of vascular health interventions in pregnancy.

The objective of this thesis was to examine muscle function and myofiber physiology in skeletal muscles in those with type 1 diabetes (T1D) by investigating the effects of diabetic myopathy on these metrics of muscle health under various conditions: at rest, after exercise and with increasing age. These works recruited adults from surrounding communities with T1D and non-diabetic counterparts (i.e. controls) matched for age, sex, body mass index, and self-reported physical activity levels. We hypothesized that adults with T1D would exhibit decreased muscle function (i.e. lower maximal strength) and altered myofiber physiology in each of these conditions. At rest, we observed that those with T1D exhibited more fast-twitch fibers and fewer satellite cells. After exercise, T1D muscles recovered less strength, showed higher amounts of myofiber damage, and delayed satellite cell proliferation. With increasing age, adults with T1D exhibited exaggerated signs of muscular aging compared to age-matched controls in the form of more abundant hybrid fibers and type 1 fiber grouping. Finally, individuals with T1D exhibited higher baseline expression of myostatin, a negative muscle growth regulator, compared to controls. Overall, our work provides the first evidence in muscle dysfunction from humans with T1D at various ages and after damaging exercise. Our findings provide novel insights on muscle health and its contribution to overall health during this lifelong, debilitating disease. Our work aims to guide future clinical & exercise guidelines with the ultimate purpose of improving the lives of millions of individuals living with T1D. / Thesis / Doctor of Philosophy (PhD) / Type 1 diabetes (T1D) is a disease that affects millions of adults worldwide by harming their ability to manage blood sugar levels. Insulin therapy has allowed for longer and healthier lives but requires constant attention manage blood sugar levels. Over time, the quality of life declines because of complications from T1D. Muscle is able to control blood sugar levels through exercise, but little is known about muscle in those with T1D. Therefore, the purpose of this work was to examine skeletal muscle health in people with T1D who do not have other complications. We found that adults with T1D exhibit signs of aging in their muscles earlier than non-diabetic people. Also, we observed that muscle from young adults with T1D recovered slower from exercise. Finally, we learned that people with T1D have more of a muscle-shrinking protein. This is the first evidence of dysfunctional muscle fibers at rest, after exercise, and with age in adults with T1D. This work aims to improve future guidelines for millions of adults with T1D.

Background: Since March 2020, health systems around the world shifted to virtual care approaches as social distancing measures were recommended to stem the spread of SARS-COV-2, the virus responsible for the COVID-19 pandemic. For children and families living with type 1 diabetes, virtual consultations in pediatric diabetes care were rare prior to the pandemic but became the norm since the start of the pandemic. Data regarding glycemic outcomes and comorbidities in children living with type 1 diabetes mellitus (T1DM) during the pandemic are limited, and there is a need for these data to drive future care models design and delivery. Aim & Methods: The aim of this project was to assess the association of the COVID-19 pandemic with measures of glycemic control (HbA1c), hyperglycemia, hypoglycemia, diabetic ketoacidosis (DKA) and hospitalization for the period spanning March 2020-2021 at McMaster Children’s Hospital, a tertiary pediatric academic center in Hamilton, Ontario, Canada. Data from the onset of virtual care were compared with data from two years pre-pandemic. Results: The COVID-19 pandemic was not associated with changes in HbA1c (MD -0.14, p=0.058), hospitalization (OR 0.57, p=0.068), or hypoglycemia (OR 1.11, p=0.484), but was significantly associated with the increase in reported hyperglycemia (OR 1.38, p=0.003) and reduction in DKA presentation (OR 0.30, p=0.009). Conclusions: Glycemic control was stable during the early stages of the COVID-19 pandemic, when virtual and hybrid care models prevailed in diabetes care. These results suggest that patients and their families were able to adapt to the uncertain circumstances of the pandemic. Virtual consultations for pediatric diabetes did not hinder glycemic control, and likely aided in the maintenance of diabetes management. Longitudinal studies are necessary before virtual consultations should be recommended to replace in-person clinic visits, but the initial data seem encouraging. / Thesis / Master of Science (MSc) / The COVID-19 pandemic restricted face-to-face healthcare-based interactions to limit the spread of the virus. These restrictions posed as a challenge for children and youth with type 1 diabetes mellitus (T1DM), who relied exclusively on in-person clinic visits as part of their care regimen pre-pandemic. In this retrospective study, we assessed the association of the first year of the COVID-19 pandemic with measures of glycemic control (HbA1c), diabetic ketoacidosis (DKA), hospitalization, hyperglycemia, and hypoglycemia, compared to two years pre-pandemic. We determined that children living with type 1 diabetes had no deterioration of glycemic control measures, apart from an increase in hyperglycemia, during the first 12 months of the pandemic. This study provides insights into health outcomes of children living with T1DM in the early stages of the pandemic and offers a roadmap to guide the further avenues of exploration needed to assess the full impact of the pandemic on this population.

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