Dissertations / Theses on the topic 'Disease programming'

Before embarking on the work presented here I showed that certain activating signals, such as IFN-gamma and TNF-alpha programmed macrophages to develop distinct sets of properties in vitro, which included unresponsiveness to other types of activation. This raised the question whether macrophage programming occurs in passive and active renal inflammation and whether the macrophage programme could be biased by systemic administration of cytokines. The data presented here shows that macrophages infiltrating acutely inflamed glomeruli of rats with nephrotoxic nephritis display programmed behaviour: operationally they behave as though programmed by IFN-gamma, and maintain these characteristics despite systematic administration of anti-inflammatory cytokines such as IL-4 or TGF-beta. This triggered further studies using a model of mesangioproliferative nephritis that can be adapted to induce resolving or progressive glomerular injury. These show that glomerular localisation does not always induce macrophage programming and that whether macrophages become programmed or not depends on the nature of the injury. Furthermore the data shows that macrophages become committed to a particular programme shortly after entering a programming environment. These observations raise question about the factors that induce macrophage programming at early stages of inflammatory disease and its consequences for its outcome. It provides an important mechanistic insight into how macrophage functional development is influenced by the underlying disease process.

Building on a wealth of human and experimental data, these PhD studies present the hypothesis that oxidative stress underlies the molecular basis via which pregnancy complicated by prenatal hypoxia or prenatal under-nutrition contributes to the developmental programming of cardiovascular disease. An integrative approach was employed at the systems, isolated organ and molecular levels using a combination of fetal and adult rat offspring and chronically cathetised, unanaesthetised fetal sheep preparations. The data show that pregnancy complicated by hypoxia or undernutrition promoted asymmetric intrauterine growth restriction with subsequent postnatal catch-up growth. Maternal treatment with melatonin increased the placental expression of antioxidant enzymes and restored birth weight in undernourished but not in hypoxic pregnancy. Hypoxic but not undernourished pregnancy resulted in thickening of the fetal aortic wall with no alterations to the morphology of the fetal heart. In marked contrast, by adulthood offspring of hypoxic pregnancy showed dilated cardiomyopathy with increased vasoconstrictor reactivity in mesenteric arteries, but no changes in aortic structure. Pregnancy complicated by undernutrition did not affect the heart or the aorta in adult offspring but it did programme an increase in vascoconstrictor reactivity to ET-1 in mesenteric arteries. Maternal treatment with melatonin relieved all of the adverse cardiovascular consequences in adult offspring of hypoxic and undernourished pregnancy. Experiments in ovine pregnancy indicate that the xanthine oxidase pathway contributes to the increased generation of reactive oxygen species in hypoxic pregnancy. Combined, the work offers the potential for therapeutic targets for clinical intervention against a developmental origin of cardiovascular disease in complicated pregnancy.

Fetal exposure to excess glucocorticoid is associated with low birth weight and increased cardiovascular disease risk in first generation offspring. Such phenotypes can be produced experimentally through the administration of the synthetic glucocorticoid dexamethasone (Dex) to pregnant rats during the last week of gestation. These ‘programmed effects’ can be transmitted to a second generation through both maternal and paternal lines. The overall hypothesis for this thesis was that the transmission of programmed effects through the male line may result from alterations in fetal germ cells, which form sperm in adulthood. Epigenetic reprogramming of germ cells is characterised by the genome-wide erasure and subsequent re-establishment of 5-methylcytosine (5mC), however this process has not previously been described for the rat. Furthermore, the involvement of more recently identified cytosine modifications; 5-hydroxymethylcytosine (5hmC), 5- formylcytosine (5fC) and 5-carboxylcytosine (5caC), has not been characterised during germ cell ontogeny. Using immunofluorescence to study DNA modifications during late gestation I identified that 5hmC, 5fC and 5caC were present between e14.5 and e16.5 but absent thereafter. In contrast, 5mC was absent during this time but remethylation was noted from e19.5 onwards. Prenatal Dex exposure was associated with the presence of significantly more 5mC-positive germ cells at e19.5 relative to controls. This difference did not persist at e20.5 suggesting that Dex exposure promotes premature global remethylation. The mechanisms for this are unclear since there were no differences between groups in the localisation of the DNA methyltransferases DNMT3a and 3b, or in markers of normal testis maturation. To enable the study of gene-specific changes in DNA methylation in the germline a colony of Germ Cell Specific-Enhanced Green Fluorescent Protein (GCS-EGFP) rats was established and characterised. GCS-EGFP rats had a transgenerational decrease in pup weight with Dex exposure, as in Wistar rats. The expression of both established and novel candidate genes was compared between strains. Multiple genes across different pathways had altered expression, with some affected in both Wistar and GCS-EGFP rats, whilst other differences were strain-specific. Enhanced Reduced Representation Bisulfite Sequencing was performed on liver and fetal germ cells from males exposed to Dex in utero to explore effects on DNA methylation. These studies confirm that epigenetic reprogramming occurs in the rat and that this process may be susceptible to modification by prenatal Dex exposure. GCS-EGFP rats also exhibited a Dex programming phenotype, with decreased pup weight and altered liver gene expression. The use of this unique strain of rats will permit dissection of the mechanisms for the transmission of programmed phenotypes across generations.

Whether innate immune cells may be adapted into potential memory states has becoming an important question in the field of immunity. Although previous conceptual paradigm failed to acknowledge this important question, emerging clinical and basic observations have started to shed intriguing clues to shake the previous dogma regarding innate immunity of being "simple", "raw", "first-line defense with no memory". We have aimed to further address this fundamental issue in this dissertation work, under the close guidance of Dr. Liwu Li. We have chosen to use the model system of Toll-Like-Receptor (TLR) signaling networks within primary monocytes. TLRs play fundamental roles in sensing pathogen-associated molecular patterns (PAMPs) and modulation of innate immunity. Lipopolysaccharide (LPS), an endotoxin found on the cell membrane of gram-negative bacteria, is the ligand of TLR4 and induces a range of inflammatory as well as anti-inflammatory responses. Higher dosages of LPS were known to cause robust yet transient expression of pro-inflammatory mediators. On the other hand, the effects of super-low dose LPS, commonly manifested in humans with adverse health conditions, have been largely ignored in the basic research field. Super-low dose LPS may skew host immune environment into a mild non-resolving pro-inflammatory state, which is a risk factor for inflammatory diseases such as atherosclerosis, compromised wound healing, and elevated risks for sepsis. Our central hypothesize is that monocytes may be adapted by super-low dose LPS into a non-resolving low-grade inflammatory state conducive for the pathogenesis of inflammatory diseases. We have employed both in vitro cell culture system as well as in vivo disease models to test this hypothesis. For the in vitro system, we have cultured primary murine monocytes with increasing signal strength of LPS. Monocyte phenotypes such as the expression of key inflammatory mediators including cytokines, chemokines, and cellular surface markers were studied. Potential molecular and cellular mechanisms were examined. We revealed a novel low-grade inflammatory monocyte phenotype termed ML adapted by super-low dose LPS, mediated through IRF5. For the in vivo system, we have employed both acute and chronic models of inflammation. For the chronic model, we have tested the effects of super-low dose LPS on monocyte polarization in vivo, as well as its contribution to the pathogenesis of atherosclerosis. Furthermore, we have tested the effects of programmed monocytes on wound healing. For the acute model, we have tested the effects of pre-conditioning with super-low dose LPS on the subsequence risks of sepsis elicited by cecal ligation and puncture. We have demonstrated aggravated atherosclerosis, compromised wound healing, and increased sepsis mortality in mice pre-conditioned with super-low dose LPS. Taken together, our findings reveal that monocytes can be differentially programmed into distinct states, depending on the signal strength of LPS. The differential programming and adaptation of monocytes can occur both in vitro and in vivo, and may bear profound pathological consequences.
Ph. D.

Previous epidemiological evidence from a number of studies supports the hypothesis that the risk of essential hypertension, coronary heart disease and non-insulin dependent diabetes is, in part, programmed by intrauterine nutritional status. An increasing number of human studies indicate that the developing kidney is particularly vulnerable to the adverse effects of fetal growth retarding influences. In animals growth retarding diets or other insults, which have an impact on the development of cardiovascular functions, also appear to impact upon nephron number. In this study, the feeding of a 9% casein diet to pregnant rats, a mild protein restriction, reduced nephron number in the offspring, which progressively declined with age compared to those exposed to an 1 8% control diet. At weaning low-protein exposed offspring had hypertension and evedence of renal insufficiency. On natural death, the kidneys from aged male rats exposed to both low-protein and control maternal diets had a higher incidence glornerulosclerosis and renal disruption than females. Supplementing the maternal 9% casein diet with 3% glycine, 1.5% urea and 3% alanine in the rat normalised nephron number in the offspring. Only the addition of glycinc in the maternal low- protein diet prevented the appearance of high blood pressure in the offspring. In this study it has been demonstrated that in humans, those of a low birth weight or ponderal index, a marker of fetal undernutrition, had evidence of increased glomerular permeability, but not elevated blood pressure at age 10. This association was not evident at age 12 or in a separate cohort of young adults. It is possible that hypertension and a reduced nephron reserve are not causally associated. The evidence from this thesis suggest that prenatal undernutrition may programme renal structure in later life, but that renal programming is not one of the primary mechanisms leading to hypertension

Local public health units in the province of Ontario, Canada, are often the primary source of health promotion and health education resources, but many do not provide programming specific to the Indigenous population. As of January 2018, modernization of the Ontario Public Health Standards requires public health units to work with the Indigenous population in providing culturally appropriate programs and services. The practice question guiding the capstone project was to determine what chronic disease and injury prevention programs exist that are culturally appropriate for the Indigenous population. The purpose of this project was to do an environmental scan and compile an inventory of existing health promotion programming that is culturally appropriate to the Indigenous population. In total, 72 Indigenous-specific programs were identified from the 26 organizations that were included in the environmental scan. Of the 26 organizations, 3 were public health units, 7 were Aboriginal health access centers, 7 were Indigenous friendship centers, 5 were Indigenous health organizations, and 4 were non-Indigenous organizations with an Indigenous component. Results from the capstone project will inform public health units of available, culturally appropriate programs that can be adapted to their local context, thereby addressing a significant gap in the current public health system. This doctoral project aligns with the design of a new model of care in the Ontario public health system and has the potential to address a gap in practice at both the local and provincial level by providing culturally appropriate guidance in the effective delivery of CDIP programming specific to the Indigenous population. This positive social change would impact the health status of this underserved population.

Obesity induced, non-alcoholic fatty liver disease (NAFLD), describes the spectrum from steatosis-to-cirrhosis. NAFLD is now the commonest cause of chronic liver disease in affluent populations. Its prevalence is increasing in tandem with rising obesity rates. However, given the exponential rise in obesity and NAFLD disease prevalence, availability of cheap energy dense foods may not alone be the sole determinant of these rising rates: maternal obesity – through developmental programming - may also be involved. A growing body of epidemiological evidence suggests that perinatal factors may contribute to chronic disease in adulthood via programming. Programming is the process whereby an insult/stimulus during a critical period of development induces permanent structural and/or physiological changes. The role of developmental programming in NAFLD is unknown. The aims, therefore were to determine if maternal obesity during gestation and/or lactation transmits a predisposition to NAFLD in offspring, to evaluate the natural progression of such programmed disease, to examine the relative contributions of maternal obesity and the post-natal environment on offspring NAFLD and to investigate the mechanisms therein. Using a rodent diet induced obesity-programming model, our initial studies confirmed that maternal obesity, through developmental programming, was indeed involved in the pathogenesis of NAFLD with the lactation period most susceptible to these programming effects. Moreover, there was an interaction between in utero exposure to an obesogenic environment and a post-weaning obesogenic environment to cause exacerbated offspring NAFLD. Mechanistically, perturbed hepatic innate immune function and disrupted peripheral circadian rhythms were observed in offspring with NAFLD programmed by maternal obesity. Finally, these results also provide a novel and uniquely pathophysiologically relevant model of NAFLD.

Chlamydia Trachomatis and Neisseria Gonorrhoeae rank as the two most commonly reported sexually transmitted diseases (STDs) in the United States. Under limited budget, publicly funded clinics are not able to screen and treat the two diseases for all patients. They have to make a decision as to which group of population shall go through the procedure for screening and treating the two diseases. Therefore, we propose a cubic integer programming model on maximizing the number of units of cured diseases. At the same time, a two-step algorithm is established to solve the cubic integer program. We further develop a web-server, which immediately make recommendation on identifying population groups, screening assays and treatment regimens. Running on the empirical data provided by the Centers for Disease Control and Prevention, our program gives more accurate optimal results comparing to MS Excel solver within a very short time.

For one week, six subjects with Parkinson Disease (PD) attended a boot camp at the Parkinson Wellness Recovery (PWR!) Gym. Each experienced a unique symptom of PD known as freezing of gait (FOG). Previous research supports task-specific exercise for improving PD symptoms; this study focuses on task-specific exercise to reduce FOG. Reducing the severity of hypokinetic and bradykinetic movement was addressed during the week through large amplitude training that was integrated into interval training during cardiovascular exercise, agility and postural control exercises, and real world environments. Five out of six subjects improved on a Freezing of Gait Assessment (FOGA) and four out of six subjects improved on the Pull Backwards Assessment. Task-specific improvement for all six individuals was achieved, with regards to typical freezing triggers-- narrow spaces, crowds, timed events, and more. These data suggest that techniques learned throughout the week can appear to be reflected into real-life. In addition, FOG specific training showed improvement in balance, a very important finidng in people that fall almost daily. Overall, specific exercise techniques assisted these six subjects in improving their quality of life, freezing severity, and postural control.

Polycystic ovary syndrome (PCOS) is a common and complex endocrine condition with reproductive and metabolic complications, affecting up to 10% of reproductive-age women. Hyperandrogenemia, ovulatory dysfunction, and luteinising hormone hypersecretion are characteristic traits of PCOS however, it seems that the most concerning long-term key issues are metabolic problems associated with the syndrome, such as hyperinsulinemia, insulin resistance, obesity, dyslipidaemia and non-alcoholic liver disease. Despite the numerous studies on PCOS, its origin and pathophysiology are still not fully understood. However, there is increasing evidence that the adult PCOS phenotype is programmed in fetal life by androgen excess. Exposure to increased levels of testosterone in utero in rodents, sheep and monkeys result in adult reproductive and metabolic pathologies that parallel those seen in PCOS women. Since hyperandrogenemia is a hallmark of PCOS and daughters of PCOS mothers have elevated levels of androgens at birth, it is likely that prenatal androgenisation during early life predispose to the future development of PCOS. Animal models of PCOS provide an opportunity to examine the developmental aetiology and molecular mechanisms underlying the pathogenesis of this condition. Over last 10 years our lab has successfully utilised a well-established ovine model of PCOS, where pregnant ewes were treated with testosterone propionate (TP) through mid-gestation. From this model, we had a large sample bank of fixed and frozen tissues from the fetal, lamb and adolescent prenatally androgenised animals that allowed to carry a broad range of experiments. In addition, a new cohort of prenatally androgenised adult sheep enabled additional in vivo analysis. Past research documented that prenatal androgenisation result in hyperinsulinemia with altered pancreas structure and function, and early fatty liver without difference in body weight in adolescent sheep. This thesis examines the effects and consequences of increased in utero androgen exposure on metabolic dysregulation in adolescent and adult female sheep. During puberty, but not fetal or early life, there was decreased adipogenesis in subcutaneous adipose tissue (SAT), but not visceral adipose tissue (VAT), accompanied by decreased circulating concentrations of fibroblast growth factor 21 (FGF21), leptin and adiponectin, and increased concentrations of fasting free fatty acids (FFA) in prenatally androgenised sheep. This was countered by upregulated expression of FFA transporters in liver. As adults, TP-exposed animals had increased body weight, elevated fasting insulin and FFA concentrations but normal FGF21, leptin and adiponectin levels. Histological analysis revealed that adult TP-exposed animals had SAT hypertrophy, which was associated with increased expression of inflammatory markers and correlated with increased fasting FFA. Therefore, it is likely that impaired preadipocyte differentiation in SAT during adolescence resulted in hypertrophy and inflammation of adult SAT. This consequently lowered capacity of SAT to safely store fat and potentially explains metabolic perturbations observed in PCOS-like female sheep. To further investigate potential causes of obesity in adult PCOS-like sheep postprandial thermogenesis (PPT), an important constituent of energy expenditure, was measured through implantation of datalogger thermometers into interscapular adipose tissue. Adult prenatally androgenised sheep had decreased amplitude of PPT, without difference in basal body temperature, despite receiving the same caloric intake, and independent of obesity. These findings indicate that adult PCOS-like sheep have reduced capacity for energy expenditure, which is mirrored in women with PCOS. This reduced capacity for postprandial thermogenesis was correlated with hyperinsulinemia decreased noradrenaline levels and reduced thermogenic potential of brown and/or beige adipose tissue. This suggests that women with PCOS might be prenatally programmed to become obese. In summary, findings documented in this thesis provide better understanding into the pathophysiology of PCOS from puberty to adulthood and give opportunities for early clinical intervention to ameliorate the metabolic phenotype of PCOS.

This study proposes a performance comparison between two Java applications with two different programming approaches, machine learning, and traditional programming. A case where both machine learning and traditional programming can be applied is a classification problem with numeric values. The data is heart disease dataset since heart disease is the leading cause of death in the USA. Performance analysis of both applications is carried to state the differences in four main points; the development time for each application, code complexity, and time complexity of the implemented algorithms, the classification accuracy results, and the resource consumption of each application. The machine learning Java application is built with the help of WEKA library and using its NaiveBayes class to build the model and evaluate its accuracy. While the traditional programming Java application is built with the help of a cardiologist as an expert in the field of the problem to identify the injury indications values. The findings of this study are that the traditional programming application scored better performance results in development time, code complexity, and resource consumption. It scored a classification accuracy of 80.2% while the Naive Bayes algorithms in the machine learning application scored an accuracy of 85.51% but on the expense of high resource consumption and execution time.

A strong body of evidence suggests that environmental insults from the point of fertilisation to birth and neonatal life can shape the health of the individual for many years to come. Adverse exposures, such as maternal overnutrition, in the early life environment increase the risk of traditionally adult-onset diseases such as cardiovascular disease and type 2 diabetes adding greatly to the next generation's burden of disease. Studies in animal models provide strong evidence that these effects are mediated by non-genetic programmed mechanisms. This is of particular concern, as recent studies in the UK suggest that over half of women are now overweight or obese during pregnancy. Current preventative strategies for adult cardiovascular disease have, thus far, focused on reducing an individual's modifiable risk factors. However, given growing evidence that risk of cardiovascular disease is determined in utero, there is strong rationale that disease risk from mother to child could be reduced prior to birth, through targeted interventions in the mother before and during pregnancy. Using an established murine model of maternal diet-induced obesity during pregnancy, the first aim of this thesis was to characterise potential programming factors in the obese mother and identify those that were targeted by a treadmill exercise intervention. Through feeding of an obesogenic diet, dams became heavier, with increased fat mass, and showed insulin resistance at weaning. Previous work has shown the intervention improved maternal insulin sensitivity during pregnancy (E19) and data from this thesis revealed that this was not accompanied by any changes to body composition. Previous data using this model showed that male offspring born to obese dams have pathological cardiac hypertrophy and ex vivo cardiac dysfunction. A second aim of this thesis was to establish if exercise intervention in obese dams was protective to the cardiovascular health of the offspring. These studies revealed that maternal exercise intervention during obese pregnancy had a positive impact by preventing pathological left ventricular cardiac hypertrophy and in vivo dysfunction, but did not prevent programmed hypertension in the male offspring. This demonstrates that offspring cardiac hypertrophy and dysfunction can be programmed independently of hypertension by maternal diet-induced obesity. The third aim of this thesis was to establish how female offspring were impacted by maternal obesity. The results demonstrated that female offspring born to obese dams were hypertensive and displayed right ventricular cardiac hypertrophy. However, there was no observable effect of maternal obesity on cardiac function in female offspring at this age. This highlights the potential sexually dimorphic effects of developmental programming by maternal obesity. A final aim of this thesis was to assess the immediate consequences of maternal obesity on the fetal heart and whether maternal exercise had any impact. This showed that in late gestation (E19), cardiac remodelling were already present in the male fetuses of obese dams, and the exercise intervention did not fully prevent this adverse finding. In conclusion, this thesis highlights that the cardiovascular health legacy of an individual is determined by maternal nutrition before birth and by the intrauterine environment. Just a small improvement in offspring risk could have important implications for the future prevalence of cardiovascular disease worldwide. Importantly this thesis highlights a potential need for combination intervention strategies to tackle the epidemic of obesity in pregnancy, as maternal exercise alone was not sufficient to reduce all aspects of the future burden of cardiovascular disease.

The American vaccine pricing market has many actors, making it a complex system to model. Because of this, previous papers have chosen to model only vaccine manufacturers while leaving out the government. However, the government is also an important actor in the market, since it buys over half of vaccines produced. In this work, we aim to introduce the government into vaccine pricing models to better recommend pricing strategies to the Centers for Disease Control and Prevention.

Nurses teach to maintain health and prevent disease. Rapport and good communication skills are especially required when teaching such sensitive subjects as prevention of sexually transmitted diseases, including acquired immune deficiency syndrome (AIDS). Neurolinguistic programming (NLP) is a communication technique that proposes to enhance rapport. Rapport is enhanced by the use of a combination of verbal and nonverbal techniques where the individual is matched and mirrored by the interviewer and verbal communication follows the sensory system most preferred by the speaker. The study investigated the effect of NLP as a rapport builder and teaching technique in one-on-one nurse-client teaching transactions including client satisfaction with the relationship and retention of knowledge of AIDS prevention information. A quasi-experimental design was used. Volunteer nurses were trained to teach AIDS prevention. Their adult volunteer clients were the treatment group. The control group of clients were taught by the nurses using the basic AIDS prevention curriculum. The two groups were compared according to the results of pre-test/post-test knowledge scores and satisfaction ratings for the nurse teacher. Data was analyzed using analysis of covariance and analysis of variance. There were no statistically significant differences between the two groups. Qualitative A data was collected after the completion of the teaching that supported usefulness of the techniques for teaching. Further studies were recommended.
Ed. D.

Public health data show the tremendous economic and societal impact of pandemic influenza in the past. Currently, the welfare of society is threatened by the lack of planning to ensure an adequate response to a pandemic. This preparation is difficult because the characteristics of the virus that would cause the pandemic are unknown, but primarily because the response requires tools to support decision-making based on scientific methods. The response to the next pandemic influenza will likely include extensive use of antiviral drugs, which will create an unprecedented selective pressure for the emergence of antiviral resistant strains. Nevertheless, the literature has insufficient exhaustive models to simulate the spread and mitigation of pandemic influenza, including infection by an antiviral resistant strain. We are building a large-scale simulation optimization framework for development of dynamic antiviral strategies including treatment of symptomatic cases and chemoprophylaxis of pre- and post-exposure cases. The model considers an oseltamivir-sensitive strain and a resistant strain with low/high fitness cost, induced by the use of the several antiviral measures. The mitigation strategies incorporate age/immunitybased risk groups for treatment and pre-/post-exposure chemoprophylaxis, and duration of pre-exposure chemoprophylaxis. The model is tested on a hypothetical region in Florida, U.S., involving more than one million people. The analysis is conducted under different virus transmissibility and severity scenarios, varying intensity of non-pharmaceutical interventions, measuring the levels of antiviral stockpile availability. The model is intended to support pandemic preparedness and response policy making.

Introdução: O modelo teórico, no qual perfis extremos de desigualdade coexistem num cenário complexo promovendo desfechos de saúde similares, denominado “similaridades nas desigualdades”, surgiu de evidências em humanos. O objetivo deste trabalho foi propor um modelo animal para refletir o fenômeno "similaridades nas desigualdades". Métodos: As ratas prenhes foram randomizadas pelo peso corporal, mantidas individualmente e no dia 10 de gestação foram divididas em três grupos: Controle (Cont), que recebeu ração padrão à vontade; Restrição Alimentar 50% (R50%), que recebeu 50% do consumo do grupo controle e Dieta Rica em Gordura (RG), que recebeu uma dieta rica em gordura à vontade. Essas dietas foram oferecidas a partir do dia 10 de gestação até o dia 21 de lactação. Em até 24 horas após o nascimento, todos os filhotes foram adotados por outras mães, formando os seguintes grupos: Cont_Cont, R50%_Cont, R50%_R50%, Cont_R50%, RG_Cont, RG_RG, Cont_RG. Peso corporal e consumo alimentar das genitoras, peso ao nascer, peso ao longo da vida e exercício físico voluntário foram comparados entre grupos por Equação de Estimação Generalizada (GEE), usando diferentes modelos estatísticos. ANOVA de duas vias foi usada para avaliar os desfechos de gordura abdominal e medidas bioquímicas. Resultados: O peso corporal das genitoras Cont e RG foi maior, comparado ao peso das genitoras R50%. Além de alguns efeitos isolados da exposição às dietas R50% ou RG durante momentos específicos perinatais (gestação e/ou lactação), o efeito das "similaridades nas desigualdades" foi observado no peso ao nascer (ambos filhotes R50% e RG foram mais leves do que os Cont) e na atividade física (os grupos extremos R50%_Cont e RG_Cont foram igualmente diferentes do grupo de referência Cont_Cont, sendo machos menos ativos e fêmeas mais ativas). O acompanhamento do peso corporal ao longo da vida mostrou que os machos pesaram mais que as fêmeas. Nenhum dos três modelos estatísticos evidenciou diferenças entre grupos no total de gordura abdominal. Conclusão: Este estudo contribui com a idéia de que as desigualdades em saúde estão relacionadas a resultados similares em saúde para ambos os extremos populacionais, e propõe um modelo animal para explorar ainda mais este efeito.
Introduction: We have previously proposed a theoretical model in which extreme unequal social backgrounds coexist in a complex scenario promoting similar health outcomes, named “Similarities in the inequalities”, and had evidence of this effect in humans. Our objective was to propose an animal model to reflect the “Similarities in the inequalities” phenomenon. Methods: Rats were time-mated and randomly allocated to: Control (Adlib), receiving an ad libitum diet of standard laboratory chow, 50% food restricted (FR), receiving 50% of the ad libitum-fed dam’s intake and high fat diet (HF), receiving a diet containing 45.0% fat. These diets were provided from day 10 of pregnancy throughout the 21-day of lactation. Within 24 hours after birth, all pups were cross-fostered to other dams, forming the following groups: Adlib_Adlib, FR_Adlib, FR_FR, Adlib_FR, HF_Adlib, HF_HF, Adlib_HF. Dam’s body weight and show consumption, pup’s birth weight, growth and physical activity in running wheels, was compared between groups through GEE, using different statistical models. Twoway ANOVA was used to evaluate abdominal fat and biochemical outcomes. Results: Body weight of Adlib and HF dams was higher compared to FR dams. Apart from some isolated effects of the exposure to the FR or HF diets during specific perinatal times (gestation and/or lactation), the “Similarities in the inequalities” effect was seen in birth weight (both FR and HF pups were smaller than Adlib pups) and physical activity (the extreme groups FR_Adlib and HF_Adlib were similarly different from the reference group Adlib_Adlib, being less active in males and more active in females). Body weight monitoring throughout life showed that males were heavier than females. None of the three statistical models showed differences between groups in total abdominal fat. Conclusion: Our study contributes to the idea that health inequalities are related to similar health outcomes for both populational extremes, and proposes an animal model to further explore this effect.

The study concerns the analysis of vocal cycle length perturbations in normophonic and dysphonic speakers.A method for tracking cycle lengths in voiced speech is proposed. The speech cycles are detected via the saliences of the speech signal samples, defined as the length of the temporal interval over which a sample is a maximum. The tracking of the cycle lengths is based on a dynamic programming algorithm that does not request that the signal is locally periodic and the average period length known a priori.The method is validated on a corpus of synthetic stimuli. The results show a good agreement between the extracted and the synthetic reference length time series. The method is able to track accurately low-frequency modulations and ast cycle-to-cycle perturbations of up to 10% and 4% respectively over the whole range of vocal frequencies. Robustness with regard to the background noise has lso been tested. The results indicate that the tracking is reliable for signal-to-noise ratios higher than 15dB.A method for analyzing the size of the cycle length perturbations as well as their frequency is proposed. The cycle length time series is decomposed into a sum of oscillating components by empirical mode decomposition the instantaneous envelopes and frequencies of which are obtained via AM-FM decomposition. Based on their average instantaneous frequencies, the empirical modes are then assigned to four categories (declination, physiological tremor, neurological tremor as well as cycle length jitter) and added within each. The within-category size of the cycle length perturbations is estimated via the standard deviation of the empirical mode sum divided by the average cycle length. The neurological tremor modulation frequency and bandwidth are obtained via the instantaneous frequencies and amplitudes of empirical modes in the neurological tremor category and summarized via a weighted instantaneous frequency probability density, compensating for the effects of mode mixing.The method is applied to two corpora of vowels comprising 123 and 74 control and 456 and 205 Parkinson speaker recordings respectively. The results indicate that the neurological tremor modulation depth is statistically significantly higher for female Parkinson speakers than for female control speakers. Neurological tremor frequency differs statistically significantly between male and female speakers and increases statistically significantly for the pooled Parkinson speakers compared to the pooled control speakers. Finally, the average vocal frequency increases for male Parkinson speakers and decreases for female Parkinson speakers, compared to the control speakers.
Doctorat en Sciences de l'ingénieur et technologie
info:eu-repo/semantics/nonPublished

A greater understanding of critical periods of body weight regulation, including pregnancy, may aid in efforts to optimize weight management strategies for the mother and her baby. The gestational period has been implicated to play, in the child, a vital role in the developmental origins of obesity and other cardiometabolic diseases later in life. Therefore, we initially examined existing literature on the role of maternal obesity and its link to pediatric obesity and documented the known underlying physiological mechanisms responsible for this relationship while suggesting potential intervention targets that may improve maternal-fetal outcomes. In a second paper, we aimed to quantify maternal predictors of large for gestational age (LGA) neonates in the Ottawa and Kingston (OaK) birth cohort with specific hypotheses verifying the independent contribution of maternal prepregnancy body mass index (BMI) and excessive gestational weight gain (GWG) to fetal overgrowth. This paper also highlights the clinical utility of the revised 2009 Institute of Medicine GWG guidelines and discusses the potential role of physiological factors underlying the observed associations between BMI, excessive GWG and LGA neonates. As a follow-up to our population-level analysis (i.e., OAK cohort), papers three and four highlight how the insulin-like growth factor (IGF) axis, a vital regulator of growth and development, may be compromised at the molecular level in cases of maternal obesity (paper 3) and excessive GWG (paper 4). In paper 3 we show that maternal obesity is associated with attenuated expression of IGF binding protein-4 (IGFBP4) in umbilical cord blood and discuss how this may preferentially promote fetal adipogenesis. The effects of excessive GWG on IGF axis protein expression are addressed in paper four where we show that excessive weight gain during pregnancy is associated with increased expression of IGFBP3 in maternal circulation in normoglycemic term pregnancies. In this paper we discuss the potential inhibitory role of IGFBP3 on adipogenesis and how it relates to glucose intolerance during pregnancy. Recognizing that both obesity and excessive GWG can alter physiological processes in mother and her baby, appropriate evidence-based interventions are warranted to best optimize outcomes. In paper five, we discuss the results of a study which sought to assess patient information channels and knowledge of nutrition and physical activity during pregnancy with the intent that these findings be applied to best design efficacious strategies that cater to the needs of our target group of pregnant women. In our analysis we show that the majority of pregnant women studied would be willing to participate in a lifestyle intervention for their own personal health and that of their child. Of great interest was the observation that most women were not informed of the importance of pregnancy-specific energy intake, or made aware of their own healthy GWG targets. Additionally, many of the respondents reported receiving no information pertaining to appropriate physical activity recommendations; despite the fact that the vast majority of participants consider this lifestyle modality to be safe during their pregnancy. Finally in paper six, we build on the results of our previous work and evaluate the risks and benefits of physical activity during pregnancy on maternal-fetal outcomes through a review of the literature and note that engaging in non-sedentary pursuits during gestation may aid in maternal weight regulation, protect against metabolic disorders and optimize neonatal birth weight and body composition. Overall, the collective nature of the papers presented in this dissertation provides qualitative and quantitative evidence to support not only the complexity of body weight regulation in the mother and her baby, but also highlights potential avenues for intervention that may improve maternal-fetal outcomes during this critical period.

Puerto Rico has the highest prevalence of type 2 diabetes, low birth-weight, and the second highest prevalence of preterm-birth in all the U.S. and its non-incorporated territories. These conditions are related. Birth-weight at both ends of the spectrum and preterm-birth are associated with an increased risk for developing type 2 diabetes and immune-inflammatory dysregulations. Maternal psychosocial stressors during pregnancy have also been recognized as potential risk factors for type 2 diabetes, and have been consistently associated with preterm-birth and low birth-weight across populations. Current evidence points toward epigenetic fetal metabolic-programming as the mechanism that underlies the increased risk for the previously mentioned morbidities. However, the particular psychosocial stressors that may contribute to the high prevalence of low birth-weight and preterm-birth in the population of Puerto Rico have not been well studied.

The present study assesses the relationships between particular psychosocial stressors, socioeconomic status, food insecurity, and birth outcomes. The results of this study show that low-risk pregnancy women were more likely to have babies with a higher ponderal index if they were exposed to stressors during gestation months 5, 6, and 7, or if exposed to "relationship stress" at any time during pregnancy. Women exposed to "financial difficulties" at any time during pregnancy were more likely to deliver babies at an earlier gestational age. Differences in birth outcomes between the exposed and non-exposed women were independent of maternal anthropometric measurements, maternal age at birth, number of previous births, and sex of the baby. Significant differences in birth outcomes were found between categories of father's self-identified and identified by others ethnicity, but sample size within categories was small. Although mothers with children at home had higher levels of food insecurity, and the level of food insecurity was correlated with higher levels of stress, no birth outcome measure was associated with food insecurity.

Some results are atypical in comparison with other populations, and therefore these findings may contribute to the understanding of population differences in the relationship between maternal stress during pregnancy and birth outcomes. The relatively small sample size and strict exclusion criteria of this study may limit the generalizability of the findings. Epidemiological similarities between Puerto Rico and other populations, and the possibility of a higher ponderal index increasing the risk for type 2 diabetes in the population of Puerto Rico need to be examined in future research.

Childhood obesity and overweight can often cause severe complications, including hypertension, dyslipidemia, insulin resistance, type 2 diabetes, and non-alcoholic fatty liver disease, amongst other disorders. Several studies have shown that early postnatal nutrition is of great importance in modulating newborn health outcomes. In this work, we have studied the role of nutrition during early stages of life in long-term metabolic health applying two different approaches: a) transgenerational transmission of impaired metabolic health induced by accelerated early weight gain caused by postnatal overnutrition and b) short and long-term metabolic effects on offspring of maternal diet supplementation with betaine. Rapid weight gain during early life has been associated with several components of the Metabolic Syndrome. Previously we developed a mouse model of neonatal overfeeding and rapid weight gain by litter size reduction. Neonatal overnutrition (ON) altered the metabolism of the exposed individuals (F0). Furthermore, offspring (F1) and grand-offspring (F2) of postnatal overfed male mice also developed metabolic complications during adulthood. In agreement, it has been shown that environmental exposure on males can affect health in subsequent generations. Here, we hypothesized that epigenetic modifications, including DNA methylation, histone modifications, and noncoding-RNA, might be involved in the inheritance of diabetes risk in our model. We analyzed sperm methylome of F0 and F1 generations, and in the liver of 8-day-old mice of F1 and F2 generations, observing significant changes in methylation of specific DNA regions. We found 912 probes differentially methylated when comparing control and ON mice throughout the three generations, between the two tissues. Our results suggest that methylation of the male germ line caused by nutritional challenges during early life may carry information that influence metabolism across multiple generations. We then analyzed gene expression by qPCR of these genes in the liver of 8-days-old mice finding differences in some genes. Breast milk composition is important in modulating growth and health of the infant. Amongst the many nutrients that breast milk contains one worth highlighting is glycine betaine (or betaine). In addition to decreasing levels of fat in the liver, previous data demonstrated that maternal supplementation with betaine during breastfeeding also improves glucose homeostasis and modulates offspring early-life gut microbiota composition. Gut microbiota in the newborn is defined at birth and during early nutrition. Breast milk also contains essential bacteria that can influence gut microbiota composition of the breastfed infant. Changes in the microbiome caused by antibiotic administration during early life were significantly correlated to higher adiposity and development of obesity during adulthood. We observed beneficial short and long-term metabolic effects of betaine on offspring and protection against adult diet-induced obesity. We have analyzed ilea and gut microbiota of mice supplemented with betaine, and with or without antibiotics at different stages of life. Analyzing the microbiome we found that microbial community composition was modulated by betaine supplementation in 2-week-old offspring. Antibiotic treatment annulled completely long-term betaine-induced effects on body weight. Moreover, glucose tolerance was no longer improved when combining antibiotics with betaine treatment.
L'obesitat i el sobrepès infantil poden causar sovint complicacions greus en la salut, incloent hipertensió, dislipèmia, resistència a la insulina, diabetis tipus 2 i esteatosis hepàtica no alcohòlica, entre d’altres. Diversos estudis han demostrat que la nutrició post-natal precoç és de gran importància en la modulació de la salut del nounat. En aquesta tesis, hem estudiat el paper de la nutrició durant les primeres etapes de la vida en la salut metabòlica a llarg termini aplicant dos enfocaments diferents: a) efectes metabòlics de suplementar de la dieta materna durant la lactància amb betaïna sobre la descendència a curt i llarg termini i b) transmissió transgeneracional del fenotip d’intolerància a la glucosa induïda per un augment accelerat de pes en etapes primerenques de la vida, causat per l'excés de nutrició post-natal. La composició de la llet materna és important per modular el creixement i la salut metabòlica de l'infant. Entre els nutrients que conté la llet materna, cal destacar la glicina betaïna (o betaïna). A més de disminuir els nivells de greix en fetge, diverses publicacions demostren que suplementar la dieta materna amb betaïna durant la lactància també millora l'homeòstasi de la glucosa i modula la composició de la microbiota intestinal del nounat. Al suplementar amb betaïna l’aigua de femelles durant la lactància vam observar efectes beneficiosos en la descendència a nivell metabòlic a curt i llarg termini. També vam poder observar que la betaïna protegia contra l'obesitat induïda per una dieta rica en greixos en l’etapa adulta. Se sap que la llet materna també conté bacteris essencials que poden influir en la composició de microbiota intestinal del lactant. S'ha analitzat la microbiota de l’ili i cec de ratolins suplementats amb betaïna, i amb o sense antibiòtics en diferents etapes de la vida. Analitzant el microbioma trobem que la composició de la comunitat microbiana dels ratolins de dues setmanes de vida estava modulada per la suplementació de betaina. Els canvis en el microbioma causats per l'administració d'antibiòtics durant la lactància estan significativament correlacionats amb una major adipositat i risc de desenvolupar obesitat durant l'edat adulta. El tractament amb antibiòtics en els nostres ratolins va anul·lar els efectes induïts per betaïna a llarg termini sobre el pes corporal. A més, la tolerància a la glucosa no estava millorarada quan es combinaven els antibiòtics amb el tractament amb betaïna. L'augment ràpid de pes durant les primeres etapes de la vida s'ha associat a diversos components de la Síndrome Metabòlica en l’adult. Prèviament en aquest laboratori hem desenvolupat un model murí de sobrealimentació neonatal i augment de pes ràpid a partir d’una reducció de la mida de la ventrada. L'excés d'alimentació neonatal (ON) va alterar el metabolisme dels mascles exposats (F0). A més, els fills (F1) i els néts (F2) dels ratolins exposats a la sobrenutrició també van desenvolupar un metabolisme alterat durant l'edat adulta. En acord, s'ha demostrat que l'exposició ambiental sobre els mascles pot afectar la salut de generacions posteriors. Així, ens vam plantejar que les modificacions epigenètiques, incloses la metilació de l'ADN, les modificacions de l'histona i l'ARN no codificant, podrien estar implicades en l'herència del risc de diabetis en el nostre model. Es va analitzar el metilma d’esperma de les generacions F0 i F1, i el metiloma de fetges de ratolins de 8 dies d'edat de les generacions F1 i F2, observant canvis significatius en la metilació de regions específiques d'ADN. Al comparar els ratolins control amb ON de cada generació i teixit, vam trobar 912 sondes diferentment metiladas. Els nostres resultats suggereixen que la metilació de la línia germinal masculina provocada per reptes nutricionals durant etapes primerenques de la vida pot portar informació que influeixi en el metabolisme en les següents generacions.

Mon projet de doctorat était de créer un pipeline pour taxono-génomique pour la comparaison de plusieurs génomes bactériens. Deuxièmement, je automatisé le processus d'assemblage (NGS) et annotation à l'aide de divers logiciels open source ainsi que la création de scripts de maison pour le laboratoire. Enfin, nous avons intégré le pipeline dans la description de plusieurs espèces bactériennes de laboratoire sur. Cette thèse est divisée principalement en Taxono- génomique et Microbiogenomics. Les avis de la section taxono-génomique, décrit sur les avancées technologiques en génomique et métagénomique pertinentes dans le domaine de la microbiologie médicale et décrit la stratégie taxono-génomique en détail et comment la stratégie polyphasique avec des approches génomiques sont reformatage de la définition de la taxonomie bactérienne. Les articles décrivent les bactéries cliniquement importantes, leur séquençage complet du génome et les études génomiques comparatives, génomiques et taxono-génomique de ces bactéries. Dans cette thèse, j'ai inclus les articles décrivant ces organismes: Megasphaera massiliensis, Corynebacterium ihumii, Collinsella massiliensis, Clostridium dakarense. Bacillus dielmoensis, jeddahense, Occidentia Massiliensis, Necropsobacter rosorum et Pantoea septica. Oceanobacillus
My PhD project was to create a pipeline for taxono-genomics for the comparison of multiple bacterial genomes. Secondly I automated the process of assembly (NGS) and annotation using various open source softwares as well as creating in house scripts for the lab. Finally we incorporated the pipeline in describing several bacterial species from out lab. This thesis is subdivided mainly into Taxono-genomics and Microbiogenomics. The reviews in taxono-genomics section, describes about the technological advances in genomics and metagenomics relevant to the field of medical microbiology and describes the strategy taxono-genomics in detail and how polyphasic strategy along with genomic approaches are reformatting the definition of bacterial taxonomy. The articles describes clinically important bacteria, their whole genome sequencing and the genomic, comparative genomic and taxono-genomic studies of these bacteria

Genetic research of complex diseases is a challenging, but exciting, area of research. The early development of the research was limited, however, until the completion of the Human Genome and HapMap projects, along with the reduction in the cost of genotyping, which paves the way for understanding the genetic composition of complex diseases. In this thesis, we focus on the statistical methods for two aspects of genetic research: phenotype definition for diseases with complex etiology and methods for identifying potentially associated Single Nucleotide Polymorphisms (SNPs) and SNP-SNP interactions. With regard to phenotype definition for diseases with complex etiology, we firstly investigated the effects of different statistical phenotyping approaches on the subsequent analysis. In light of the findings, and the difficulties in validating the estimated phenotype, we proposed two different methods for reconciling phenotypes of different models using Bayesian model averaging as a coherent mechanism for accounting for model uncertainty. In the second part of the thesis, the focus is turned to the methods for identifying associated SNPs and SNP interactions. We review the use of Bayesian logistic regression with variable selection for SNP identification and extended the model for detecting the interaction effects for population based case-control studies. In this part of study, we also develop a machine learning algorithm to cope with the large scale data analysis, namely modified Logic Regression with Genetic Program (MLR-GEP), which is then compared with the Bayesian model, Random Forests and other variants of logic regression.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
O objetivo do presente estudo foi avaliar se o Bezafibrato, um agonista PAN-PPAR, é capaz de aliviar a doença não alcoólica do fígado gorduroso (NAFLD) na prole de machos de mães C57BL/6 obesas. Fêmeas virgens foram alimentadas com uma dieta HL (hiperlipídica, 49% de lipídios) ou uma dieta C (controle, 10% de lipídios) por oito semanas antes do acasalamento e durante os períodos de gestação e lactação. A prole de machos foi subdividida em quatro grupos: C (dieta controle para as mães e filhotes); C/BZ (dieta controle para as mães e filhotes com tratamento com Bezafibrato[100mg/Kg]); HL (dieta HL para as mães e dieta controle para os filhotes); e HL/BZ (dieta HL para as mães e dieta controle para os filhotes com tratamento com Bezafibrato [100mg/Kg]). O tratamento com Bezafibrato começou na 12 semana e se manteve por três semanas. Análise do metabolismo, bioquímica, estereológica e por western-blotting foram realizadas. A dieta HL causou um fenótipo de sobrepeso nas mães e acarretou em uma intolerância oral à glicose com aumento da glicemia de jejum. A prole HL apresentou hiperfagia, ganho de massa corporal, altos níveis de triglicerídeo hepático e plasmático, esteatose hepática e aumento da expressão de proteínas lipogênicas concomitante com diminuição do receptor ativador de proliferação peroxissomal alfa (PPARα), que é responsável pela β-oxidação e aumento do receptor ativador de proliferação peroxissomal gama (PPARγ) e do elemento regulador de esterol ligante da proteína 1 (SREBP-1c) proteínas envolvidas na lipogênese hepática. Por outro lado, o tratamento com o Bezafibrato reverteu o quadro da programação metabólica no fígado, com uma melhora dos parâmetros morfológicos, bioquímicos e moleculares do fígado dos animais, com um aumento da ativação de PPARα em associação a uma diminuição do PPARγ e não alterando a expressão de SREBP-1c. Em conclusão, nós demonstramos que o tratamento com Bezafibrato melhora a NAFLD causada pela obesidade materna.
The aim of the present study was to evaluate whether Bezafibrate , a PAN-PPAR agonist, could attenuate non-alcoholic fatty liver disease (NAFLD) of male offspring from obese C57BL/6 dams. Dams were fed on a HF (high-fat, 49% lipids) diet or SC (standard chow; 10% lipids) diet for 8 weeks before mating and during gestation and lactation periods. Male offspring were subdivided into 4 groups: SC (standard-chow for dams and offspring); SC/BZ [standard-chow for dams and offspring with treatment with BZ (100mg/Kg)]; HF (high-fat diet for dams and standard-chow for offspring); HF/BZ [high-fat diet for dams and standard-show for offspring with treatment with Bezafibrate (100mg/Kg)]. Treatment with Bezafibrate started at 12th week and was maintained for 3 weeks. Metabolic measurements, biochemical analysis, stereological tools and western-blotting were performed. The HF diet yielded an overweight phenotype and an increase in oral glucose tolerance and fasting glucose of dams. The HF offspring presented hyperphagia, body mass gain, high levels of plasmatic and hepatic triglycerides, impairment of glucose metabolism, hepatic steatosis and high expression of lipogenic proteins concomitant to decreased expression of PPARα, which is responsible for β-oxidation. On the other hand, treatment with Bezafibrate reverted hepatic outcomes of metabolic programming, with an improvement of morphological, biochemical and molecular parameters of animals livers, with an increase of PPARα activation in association with a decrease of PPARγ expression and no changes in SREBP-1c expression. In conclusion, we demonstrated that treatment with Bezafibrate improved NAFLD caused by maternal obesity.

Introduzione: la BroncopneumoPatia Cronica Ostruttiva (BPCO) è ora la quarta principale causa di morte nel mondo e l'Organizzazione mondiale della sanità si aspetta che diventi la terza causa di morte più comune al mondo entro il 2020. Una cura efficace, continua e personalizzata può aiutare i pazienti a mantenere una buona salute. Lo scopo di questa revisione è indagare l'efficacia di programmi di educazione ed autogestione, tarati su esigenze specifiche del paziente-utilizzatore e basati su piattaforme elettroniche, sulla qualità della vita, tolleranza all'esercizio e dispnea nei pazienti con diagnosi di BPCO. Metodi: è stata sviluppata una revisione sistematica che include trial clinici randomizzati che confrontano l'efficacia degli interventi di programmi personalizzati di educazione ed autogestione basati su piattaforme elettroniche rispetto all'assistenza standard nei pazienti con diagnosi di BPCO. È stata condotta una ricerca elettronica su database medici PubMed e PEDro. Risultati: in 2 studi su 3 la tolleranza all'esercizio aumenta nel breve termine (3 mesi) ; vi è un impatto positivo degli interventi sulla qualità della vita e sulla tolleranza all'esercizio fisico rispetto alla tradizionale assistenza nel lungo termine (12 mesi). In 1 studio su 3 la dispnea a 3 mesi riporta risultati peggiori, mentre lo stesso outcome in un altro studio mostra risultati positivi anche a 12 mesi. Conclusioni: sono necessarie ulteriori ricerche su larga scala per stabilire con maggiore chiarezza l'impatto effettivo dell'autogestione supportata dalle tecnologie digitali e come queste tecnologie possano essere sfruttate per migliorare l'efficacia degli interventi virtualmente erogati anche nel lungo termine.

Thesis (Ph.D)--Industrial and Systems Engineering, Georgia Institute of Technology, 2010.
Committee Chair: Keskinocak, Pinar; Committee Member: Ergun, Ozlem; Committee Member: Goldsman, David; Committee Member: Hupert, Nathaniel; Committee Member: Swann, Julie. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Introduction A growing body of evidence, from epidemiological studies in humans and animal models, indicate that maternal health and nutritional status during gestation and lactation can program the propensity to develop obesity in their offspring. Huge efforts are now being directed toward understanding the molecular mechanisms underlying this developmental programming. Identification of these mechanisms could give some clues about potential strategies to prevent or revert programmed propensity to develop obesity, and may help in the identification of early biomarkers. Therefore, the main aim of this PhD-Thesis has been: To characterize in rats the programming effects of moderate maternal energy restriction during pregnancy or lactation on the metabolic health of their offspring in terms of obesity and related metabolic alterations, as well as to identify new preventing strategies against programmed obesity and early biomarkers of metabolic health. Investigation content We have characterized an animal model previously described to exhibit higher propensity to develop obesity and related metabolic alterations ― the offspring of rat dams exposed to moderate food restriction during gestation (CRG) ― to find out some of the potential mechanisms underlying their negative outcomes. Expression levels of key energy homeostasis-related genes in the hypothalamus and adipose tissue, and the measurement of some circulating parameters, showed that these animals were programmed, already from early ages, for a lower capacity to respond to insulin and to central leptin action. This could explain the hyperphagia observed in these animals and the greater propensity for obesity, manifested particularly in males. Some of these programmed disturbances, such as the impaired insulin and leptin sensitivity, and the increased systolic blood pressure, characteristic of CRG-animals, were reverted by enhancing hepatic fatty acid oxidation at early ages, through adeno-associated virus-mediated gene transference of the cDNA of Cpt1am (encoding for a permanently active form of CPT1A insensitive to its physiological inhibitor malonyl-CoA). Unlike calorie restriction during gestation, we show here that moderate calorie restriction in rat dams during lactation protects their offspring (CRL) against diet-induced obesity and related metabolic alterations, such as dyslipidemia, insulin resistance and hyperleptinemia. This condition during lactation determines early changes at gene expression level in WAT and liver, affecting lipogenic and oxidative capacity and increasing their sensitivity to the peripheral effects of leptin and insulin. Some of these adaptations were partially maintained in adulthood. Adult CRL-animals showed gender-dependent changes at gene expression level in adipose tissue and hypothalamus, suggesting that males were more protected against high-fat diet induced peripheral insulin resistance and they also showed improved capacity to respond centrally to leptin, while CRL-females were programmed for a better sensitivity to the peripheral actions of leptin and to the central action of insulin. We used this animal model to identify early transcriptome-based biomarkers of improved metabolic health by whole-genome microarray analysis in peripheral blood mononuclear cells (PBMCs). Concerning the factors potentially involved in the benefits of maternal calorie restriction during lactation on their offspring, the diminished content of markers of protein damage by oxidation and glycation found in breast milk from CRL-dams, in comparison to milk from controls, could be relevant. Conclusion Maternal nutrition during the perinatal period may be an important determinant of insulin and leptin sensitivity in their offspring. While calorie restriction during pregnancy programs their offspring for a lower capacity to respond to insulin and to central leptin action, the offspring of calorie restricted lactating dams show enhanced sensitivity to these hormones. Expression levels in blood cells of a set of genes are proposed as potential early biomarkers of metabolic health, and hence may provide a valid biological readout for the study of metabolic processes in humans.
Introducción Numerosas evidencias, procedentes de estudios epidemiológicos en humanos y de modelos animales, indican que la salud materna y su estado nutricional durante la gestación y la lactancia pueden programar la propensión a desarrollar obesidad en la descendencia. Se están llevando a cabo grandes esfuerzos para entender los mecanismos moleculares responsables de dicha programación metabólica. La identificación de los mecanismos responsables podría dar ciertas pistas para el desarrollo de estrategias que permitan prevenir o revertir dicha propensión a desarrollar obesidad, y también podría ayudarnos en la identificación de biomarcadores tempranos de salud metabólica. Por consiguiente, el principal objetivo de esta tesis doctoral ha sido: Caracterizar en ratas los efectos de una restricción energética materna moderada durante la gestación o la lactancia sobre la salud metabólica de la descendencia, en relación a la obesidad y las alteraciones metabólicas asociadas, así como también identificar nuevas estrategias de prevención frente la programación de la obesidad y nuevos biomarcadores tempranos de salud metabólica. Contenido de la investigación Hemos caracterizado un modelo animal, que previamente se había descrito que presentaba una mayor propensión a desarrollar obesidad y alteraciones metabólicas asociadas – las crías de ratas sometidas a una restricción calórica moderada durante la gestación (CRG) – para identificar algunos de los mecanismos potencialmente responsables de sus efectos negativos. Los niveles de expresión de genes claves relacionados con la homeostasia energética en el hipotálamo y el tejido adiposo, y el análisis de ciertos parámetros circulantes, revelaron que estos animales estaban programados, ya desde edades tempranas, para una menor respuesta a la insulina y a la acción central de la leptina. Esto podría explicar la hiperfagia observada en estos animales y la mayor propensión a la obesidad, que presentan particularmente los machos. Algunas de estas alteraciones programadas, tales como la alteración de la sensibilidad a la insulina y a leptina, y la elevada presión sistólica, característica de los animales CRG, se vieron revertidas al favorecer el incremento de la oxidación hepática de ácidos grasos, en edades tempranas, a través de la transferencia génica, mediada por vectores virales adeno-asociados, del ADNc de la Cpt1am (que codifica para una forma permanentemente activa de la CPT1A, insensible a su inhibidor fisiológico malonil-CoA). A diferencia de la restricción calórica durante la gestación, observamos que la restricción calórica moderada en ratas madre durante la lactancia protege a su descendencia (CRL) frente al desarrollo de obesidad inducida por la dieta y frente al desarrollo de alteraciones metabólicas asociadas, tales como la dislipidemia, la resistencia a la insulina y la hiperleptinemia. Esta condición durante la lactancia determina cambios tempranos a nivel de expresión génica en el tejido adiposo y el hígado, afectando la capacidad lipogénica y oxidativa, e incrementando la sensibilidad a la acción periférica de la insulina y la leptina. Algunas de estas adaptaciones se mantuvieron parcialmente en edad adulta. Los animales CRL adultos mostraron cambios a nivel de expresión génica en el tejido adiposo y en el hipotálamo que fueron dependientes del sexo, sugiriendo que los machos estaban más protegidos frente a la resistencia periférica a la insulina inducida por una dieta hiperlipídica, así como también mostraron una capacidad mejorada para responder a la leptina a nivel central; en cambio, las hembras CRL estaban programadas para una mejor sensibilidad a la acción periférica de la leptina y a la acción central de la insulina. Utilizamos este modelo animal para identificar marcadores tempranos de transcripción indicadores de salud metabólica mejorada mediante el análisis por microarray de células mononucleares de sangre periférica (PBMCs). Con respecto a los factores que podrían estar potencialmente implicados en los efectos beneficiosos de la restricción calórica durante la lactancia sobre las crías, el menor contenido de marcadores de daño proteico por oxidación y glicación hallado en la leche de madres CRL, en comparación con la leche de madres control, podría ser relevante. Conclusión La nutrición materna durante el periodo perinatal puede ser un determinante importante de la sensibilidad a la insulina y la leptina de sus crías. Mientras que la restricción calórica durante la gestación programa a las crías para una menor capacidad de respuesta a la inulina y a la acción central de la leptina, las crías de madres expuestas a una restricción calórica moderada durante la lactancia muestran una sensibilidad mejorada a estas hormonas. Se han identificado un grupo de genes cuyos niveles de expresión en células sanguíneas podrían considerarse como potenciales marcadores tempranos de salud metabólica, pudiendo proporcionar una herramienta biológica válida para el estudio de procesos metabólicos en humanos.
Introducció Nombroses evidències, procedents d’estudis epidemiològics en humans i de models animals, indiquen que la salut materna i el seu estatus nutricional durant la gestació i la lactància poden programar la propensió de la descendència a desenvolupar obesitat. S’estan duent a terme grans esforços per entendre els mecanismes moleculars responsables d’aquesta programació metabòlica. La identificació d’aquests mecanismes podria donar-nos certes pistes per al desenvolupament d’estratègies que permetin prevenir o revertir la propensió programada a desenvolupar obesitat, i també podria ajudar-nos en la identificació de biomarcadors primerencs de salut metabòlica. Per això, el principal objectiu d’aquesta tesis doctoral ha estat: Caracteritzar en rates els efectes d’una restricció energètica moderada a les mares durant la gestació o la lactància sobre la salut metabòlica de la descendència, en relació a l’obesitat i les alteracions metabòliques associades, així com també identificar noves estratègies de prevenció enfront a la programació de l’obesitat i nous biomarcadors primerencs de salut metabòlica. Contingut de la investigació Hem caracteritzat un model animal, que prèviament se va veure que exhibia una major propensió a desenvolupar obesitat i alteracions metabòliques associades – les cries de rates sotmeses a una restricció calòrica moderada durant la gestació (CRG) – per identificar alguns dels mecanismes potencialment responsables dels seus efectes negatius. Els nivells d’expressió de gens clau relacionats amb la homeòstasi energètica a l’hipotàlem i al teixit adipós, i l’anàlisi de certs paràmetres circulants, varen mostrar que aquests animals estaven programats, ja des d’etapes primerenques de la vida, per una menor resposta a la insulina i a la acció central de la leptina. Això podria explicar la hiperfàgia observada en aquests animals i la major propensió a l’obesitat, que presenten particularment els mascles. Algunes d’aquestes alteracions programades, com ara l’alteració de la sensibilitat a la insulina i a la leptina, i l’elevada pressió sistòlica, característica dels animals CRG, es van veure revertides en afavorir l’increment de la oxidació hepàtica dels àcids grassos, a edats primerenques, a través de la transferència gènica, mitjançant vectors virals adeno-associats, de l’ADNc de la Cpt1am (que codifica per una forma permanentment activa de la CPT1A, insensible al seu inhibidor fisiològic, el malonil-CoA). A diferència de la restricció calòrica durant la gestació, observarem que la restricció calòrica moderada en rates mare durant la lactància, protegeix a la seva descendència (CRL) enfront del desenvolupament d’obesitat induïda per la dieta i el desenvolupament d’alteracions metabòliques associades a l’adultesa, com ara la dislipèmia, la resistència a la insulina i la hiperleptinèmia. Aquesta condició durant la lactància determina canvis primerencs a nivell d’expressió gènica en el teixit adipós i en el fetge, afectant la capacitat lipogènica i oxidativa, i incrementant la sensibilitat a la acció perifèrica de la insulina i la leptina. Algunes d’aquestes adaptacions se varen mantenir parcialment en edat adulta. Els animals adults CRL varen mostrar canvis en els nivells d’expressió gènica en el teixit adipós i a l’hipotàlem, que varen ser dependents del sexe, suggerint que els mascles estaven més protegits enfront a la resistència perifèrica a la insulina induïda per una dieta hiperlipídica, i també mostraren una capacitat millorada per respondre a la leptina a nivell central; en canvi, les femelles CRL estaven programades per a una millor sensibilitat a l’acció perifèrica de la leptina i a la acció central de la insulina. Utilitzarem aquest model animal per a la identificació de biomarcadors primerencs de transcripció, indicadors de salut metabòlica millorada, mitjançant l’anàlisi per microarray en cèl•lules mononuclears de sang perifèrica (PBMCs). Pel que fa als factors que podrien estar potencialment implicats en els efectes beneficiosos de la restricció calòrica materna durant la lactància sobre la descendència, el menor contingut de marcadors de dany proteic per glicació i d’oxidació trobat a la llet de mares CRL, en comparació amb la llet de mares control, podria ser rellevant. Conclusió La nutrició materna durant el període perinatal pot ser un determinant important de la sensibilitat a la insulina i a la leptina de les seves cries. La restricció calòrica durant la gestació programa a les cries per a una menor resposta a la insulina i a la acció central de la leptina; en canvi, les cries de mares exposades a una restricció calòrica moderada durant la lactància mostren una major sensibilitat a aquestes hormones. S’han identificat un grup de gens, els nivells de expressió dels quals en cèl•lules sanguínies se poden considerar com a potencials marcadors primerencs de salut metabòlica, podent proporcionar una eina biològica vàlida per a l’estudi dels processos metabòlics en humans.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro
O desenvolvimento da programação fetal é considerado um importante fator de risco para doenças não-transmissíveis da vida adulta, incluindo doença cardíaca coronariana. Com o objetivo de investigar a associação entre nutrição materna e o desenvolvimento das artérias coronárias (AC) em embriões de camundongos estadiados; embriões de camundongos C57BL/6 nos estádios de 16-23 foram retirados de mães alimentadas com dietas de proteína normal (NP) ou de baixa proteína (LP), e as AC foram estudadas. Embora os embriões LP possuam massa corporal menor, entretanto tinham taxas de crescimento cardíaco maior, quando comparados com os embriões NP. O Plexo subepicárdico foi observado no início do período pós-somítico (estádio 16) de embriões NP, enquanto que nos embriões LP apenas no estádio 17 (P <0,01), persistindo até o estádio 18 (P <0,01). As artérias coronárias foram detectadas inicialmente no estádio18 dos embriões NP, já nos embriões LP foram encontradas a partir do estádio 19 (P <0,01). Núcleos apoptóticos foram observados em torno do anel aórtico peritruncal no estádio 18 em embriões NP e LP. Células FLK1+ (Fetal Liver Kinase 1 = VEGFr2 = Vascular Endothelial Growth Factor Receptor 2) apresentaram uma distribuição homogênea nos embriões NP já no estádio 18, enquanto uma distribuição semelhante nos embriões LP foi visto apenas nos estádios 22 e 23. A restrição proteica materna em camundongos leva a um atraso no crescimento do coração no período embrionário modificando o desenvolvimento do plexo peritruncal subepicárdica e diminuindo a taxa de apoptose na região do futuro orifício coronariano.
Programming of fetal development is considered to be an important risk factor for non-communicable diseases of adulthood, including coronary heart disease (CHD). Aiming to investigate the association between maternal nutrition and the development of the coronary arteries (CA) in staged mice embryos, C57BL/6 mice embryos from stages 16 to 23 were taken from mothers fed a normal protein (NP) or low protein (LP) diet, and the CA were studied. Although the LP embryos had lower masses, they had faster heart growth rates when compared to the NP embryos. The subepicardial plexuses were observed earlier in the NP embryos (stage 20) than in the LP ones (stage 22) (P<0.01). Apoptotic nuclei were seen around the aortic peritruncal ring beginning at stage 18 in the NP and LP embryos. FLK1+ (fetal liver kinase 1 = VEGFr2 or vascular endothelial growth factor receptor 2) cells had a homogeneous distribution in the NP embryos as early as stage 18, whereas a similar distribution in the LP embryos was only seen at stages 22 and 23. Maternal protein restriction in mice leads to a delay in the growth of the heart in the embryonic period modifying the development of the subepicardial peritruncal plexus and the apoptosis in the future coronary orifice region.

Low birth weight is associated with an increased risk of impaired glucose tolerance and type 2 diabetes and with signs of increased hypothalamic pituitary adrenal axis activity in later life (1, 2). Low birth usually weight reflects a reduction in fetal growth, which largely depends on an adequate supply of nutrients and oxygen. Variations in supply modify the metabolic and neuroendocrine characteristics of the fetus, which in turn modulate the pattern of functional development as well as growth (3). An adverse fetal environment, evident as low birth weight, is therefore proposed to alter functional development with long term effects for the function and risk of disease in the individual later in life (4, 5). Increased HPAA impairs metabolic homeostasis and could therefore mediate effect of prenatal challenge on later metabolic control (6). It was therefore hypothesised that restriction of fetal growth, increases circulating cortisol and/or alters sensitivity to cortisol, which increases fasting blood glucose, and impairs glucose tolerance in the young adult. Large litter size in the guinea pig is characterised by reduced placental and fetal growth, reduced size at birth and insulin resistance in offspring in later life, providing a suitable model to test this hypothesis. Spontaneous restriction of fetal growth in the guinea pig, evident as small size at birth, was associated with increased salivary cortisol, in both sexes but at different stages of postnatal life. In males, salivary cortisol was increased with small size at birth in early and adult life, but reduced later with ageing. In females however, salivary cortisol was increased in juveniles and in aged adults, possibly reflecting the impact of the oestrus cycle on cortisol production in mature cycling females. Altered activity of the HPGA, which can influence that of the HPAA, has also been reported to be programmed by prenatal restriction. In the guinea pig, salivary testosterone in males increased with age and small size at birth in juveniles, young and aged adults. In females, salivary progesterone increased with age up to 300 days, and decreased with size at birth in the young guinea pig. Although testosterone inhibits HPAA activity, in males, mean salivary cortisol correlated positively with mean salivary testosterone at 100 and 300 days of age. In contrast, progesterone may enhance HPAA activity, and consistent with this, in females, mean salivary progesterone correlated with mean salivary cortisol at 400 days of age. Therefore, salivary testosterone in the male and salivary progesterone in the female guinea pig changes with maturation and has previously reported in this or other species, but small size at birth increases salivary testosterone in males with modest effects in early life in females. This together with the unexpected positive associations of salivary cortisol with testosterone in males, suggests that programming of the HPAA makes little contribution to that of the HPAA as indicated by salivary cortisol. Here we show that low birth weight is associated with increased fasting blood glucose and impaired glucose tolerance in both male and female young adult guinea pigs aged 100 days. Fasting and mean (during IVGTT) plasma cortisol was reduced in low birth weight female adult guinea pigs, and is not vary with size at birth at this age in males. This suggests that circulating cortisol does not contribute to the impaired glycaemia associated with small size at birth in the guinea pig. Glucose tolerance was increasingly impaired in males but not females, as mean plasma cortisol increased. This is consistent with cortisol impairing glycaemia in the guinea pig as in other species, in males at least. To assess the role of cortisol in prentally programmed impairment of glycaemia directly, metyrapone or vehicle containing 24% ethanol was administered to young adult guinea pigs for 3 days. Treatment with the latter impaired fasting blood glucose and glucose tolerance in females and the latter in males compared to a previous IVGTT and this was exacerbated in low birth weight females. Metyrapone prevented this impairment of fasting glycaemia and glucose tolerance in the low birth weight adult female guinea pig and in the male guinea pig regardless of birth weight class. Neither vehicle or metyrapone altered plasma cortisol, before or during a second IVGTT. Limited numbers of animals, particularly females, limited this study however and additional investigation is required. Nevertheless this shows for the first time that inhibition of glucocorticoid synthesis in the guinea pig improves glucose control. Furthermore this suggests that the low birth weight guinea pig may be more sensitive to cortisol, have increased cortisol synthesis or reduced inactivation of cortisol in peripheral tissues, leading to increased local cortisol action. In conclusion, alterations in peripheral HPAA activity in the guinea pig due to restricted fetal growth may contribute to their prenatally programmed development of impaired glucose tolerance as young adults, but the extent of that contribution may vary with age and gender.
Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

In this thesis, health care policy issues for prevention and cure of cervical cancer have been considered. The cancer is typically caused by Human Papilloma Virus (HPV) for which individuals can be tested and also given vaccinations. Policymakers are faced with the decision of how many cancer treatments to subsidize, how many vaccinations to give and how many tests to be performed in each period of a given time horizon. To aid this decision-making exercise, a stochastic dynamic optimal control problem with feedback was formulated, which can be modeled as a Markov decision process (MDP). Solving the MDP is, however, computationally intractable because of the large state space as the embedded stochastic network cannot be decomposed. Hence, an algorithm was proposed that initially ignores the feedback and later incorporates it heuristically. As part of the algorithm, alternate methodologies, based on deterministic analysis, were developed, Markov chains and simulations to approximately evaluate the objective function. Upon implementing the algorithm using a meta-heuristic for a case study of the population in the United States, several measures were calculated to observe the behavior of the system through the course of time, based on the different proposed policies. The policies compared were static, dynamic without feedback and dynamic with feedback. It was found that the dynamic policy without feedback performs almost as well as the dynamic policy with feedback, both of them outperforming the static policy. All these policies are applicable and fast for easy what-if analysis for the policymakers.

Tese de doutoramento em Biociências, na especialidade de Toxicologia, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra
Early-life malnutrition results in structural alterations to fetal kidney and heart, predisposing offspring to later life cardio-renal dysfunction. Epidemiologic studies link low birth weight to predisposition to cardiovascular disease (CVD) later in life with both sex and diet impacting the incidence of CVD. Kidneys of adults who suffered from growth restriction at birth have substantial variation in nephron endowment. Animal models suggest cardio-renal structural and functional consequences in the offspring exposed to sub-optimal intrauterine nutrition. Mitochondrial bioenergetics plays a key role in cardiac and renal energy metabolism, growth and function. In this relevant work, we hypothesized that moderate maternal nutrient reduction (MNR) would adversely impact fetal cardio-renal mitochondrial metabolism in a well-established non-human primate model which produces intra-uterine growth reduction at term. Female pregnant baboons were fed normal chow diet or 70% of control diet (maternal nutrient reduction, MNR). Cesarean sections were performed at 0.9 gestation (165 days gestation) under anesthesia. Maternal fasting blood was drawn from the femoral vein in the morning before cesarean section and before the fetus was exteriorized from the uterine cavity. Umbilical vein blood was also sampled. The mother, the placenta and the fetus were analyzed for morphometric measurements and tissue sampling. Fetal kidneys and heart were rapidly harvested and appropriately processed, flash frozen or fixed, for posterior analyses. Biochemical and amino acid analyses were performed in the maternal and fetal blood samples. Analysis of mitochondrial DNA was performed by quantitative real-time PCR, and Human Mitochondrial Energy Metabolism and Human Mitochondria Pathway PCR Arrays were used to analyze mitochondrial relevant mRNA. In situ protein content was detected by immunohistochemistry and semi-quantification was performed by Western blot. Enzymatic activity of mitochondrial proteins was determined by alterations in the absorbance of specific substrates or products. Adenine nucleotide levels and energy charge were determined by HPLC, as well determination of vitamin E and reduced and oxidized glutathione contents. Other indicators of oxidative state, as malondialdehyde content (MDA), glutathione peroxidase and glutathione reductase activities were determined spectrophotometrically. Ultimately, transmission electron microscopy was use to assess mitochondrial morphology. MNR until 0.9 gestation decreases maternal weight gain and placental weight, being the effects more severe in MNR mothers carrying a male fetuses. Despite the smaller overall fetal size, fetal kidney weight-to-body weight or the heart weight-to-body weight ratios were not affected. MNR caused adjustments in the protein metabolism reflected in altered maternal amino acids concentrations and impaired glucose metabolism, with MNR mothers displaying higher levels of cortisol and glucose in blood circulation. Regarding the fetal kidney, we demonstrated fetal gender-specific differential mRNA expression encoding mitochondrial metabolite transport and dynamics proteins. MNR-related differential gene expression was more evident in female fetuses, with 16 transcripts significantly altered, including 14 downregulated and 2 upregulated. MNR impacted 10 transcripts in male fetuses, with 7 downregulated and 3 upregulated. Alteration in mRNA levels was accompanied by a decrease in mitochondrial protein cytochrome c oxidase subunit VIc. In conclusion, transcripts encoding fetal renal mitochondrial energy metabolism proteins are nutrition sensitive in a gender-dependent manner. For the fetal cardiac left ventricle, we found that MNR increased mtDNA content and the transcription of key mitochondrial genes involved in mitochondrial dynamics and oxidative phosphorylation (OXPHOS), resulting in increased content of several mitochondrial proteins, namely components of the mitochondrial respiratory chain (NDUFB8, UQCRC1 and cytochrome c) and ATP synthase. However, the activity of OXPHOS enzymes was significantly decreased in MNR fetuses, possibly contributing to a decreased ATP content and an increased oxidative stress in the cardiac left ventricle tissues reported by augmented levels of the lipid peroxidation marker, MDA. Microscopy of the fetal cardiac left ventricles reflected the disturbance induced by MNR, revealing mitochondria with sparse and disarranged cristae. These checkpoints suggest that MNR orchestrated a serial of events that ultimately resulted in an impaired capacity of fetal cardiac left ventricle tissue to produce energy through the OXPHOS system. The present study provides for the first time evidence of an association between MNR and mitochondrial remodeling in the fetus. Although the MNR fetal manifestation were tissue and gender specific, the overall scenario point to and impairment in mitochondrial function in the fetal tissues analyzed. We speculate that these differences lead to decreased mitochondrial fitness that contributes to cardio-renal dysfunction in later life. Our work has a translational application in human health, showing that control of maternal health during pregnancy may reduce long term disease risk in the offspring with greatest benefit for the individual and for national health care systems.
FCT - SFRH/BD/64694/2009

Research Doctorate - Immunology/Microbiology
Asthma is a chronic inflammatory disease of the airways that is characterised by activation of CD4+ T-helper 2 type (Th) cells and eosinophils. The cause of this aberrant Th2 response is unknown but lack of early life infection is thought to play a significant role. The timing of infection and the type of pathogen may be critical to programming the immune response to a protective Th1, or destructive Th2, phenotype. The immune responses to infection with Salmonella typhimurium and Mycobacterium bovis Bacille Calmette Guerin (BCG) have been identified as targets for reprogramming or preventing the development of asthma. However, the role of these infections in contributing to a Th2-Th1switch or suppression of this response remains limited. In this investigation ovalbumin (OVA) T-cell receptor (TCR) transgenic (Tg) mice in combination with these bacterial strains expressing OVA have been used to specifically track the affects of each infection as well as OVA exposure on the T-cell response and the development of allergic airways disease (AAD) in the mouse model. BCG infection as an adult and a neonate prior to OVA challenge induced significant reductions in eosinophils in broncho-alveolar lavage fluid (BALF) and lung tissue compared to sham-infected mice that received OVA challenge. However, high levels of both Th1 (interferon gamma (IFN-γ)) and Th2 (interleukin (IL)-4, IL-5, IL-13) cytokines from supernatants of cultured peri-bronchial lymph node (PBLN) cells and splenocytes were found in all groups examined. Further studies tracking the development of the immune system after BCG infection at birth without OVA exposure revealed significant decreases in lung tissue eosinophils and decreased immunoglobulin (Ig) G1, IgG2a and IgE levels from serum compared to sham-infected controls. This coincided with decreased numbers of CD4+ and CD8+ T-cells in the spleens and PBLN cells. Levels of cytokines in splenocytes and PBLN cell cultures failed to show significant trends toward either a polarised Th1 or Th2, leaving a mixed Th1/Th2 phenotype. Infection with S.typhimurium lowered eosinophil levels in BALF, and mucous secreting cell (MSC) and eosinophil number in lung tissue after challenge with 23 OVA, compared to sham-infected mice challenged with OVA. In mice infected as neonates and adults prior to OVA challenge increased levels of IFN-γ from splenocyte culture supernatants were found, compared to sham-infected OVA challenged controls. Decreased levels of IL-5 from splenocyte culture supernatants was found in neonates but not adult mice infected with S.typhimurium prior to OVA challenged compared to sham-infected OVA challenged controls. High levels of both Th1 and Th2 cytokines were present in splenocyte and PBLN culture supernatants from all groups tested, indicating a mixed Th1/Th2 phenotype rather than a profound switch to Th1 immune response. Further studies showed that infection with S.typhimurium at birth without OVA exposure causes changes to the development of the neonatal immune system resulting in decreased eosinophil numbers in BALF and lung tissue, decreased levels of serum IgG1 and IgG2a, and a shift from Th2 to a mixed Th1/Th2 cytokine profile. These changes were found in samples examined up to 9-weeks post infection. This investigation demonstrates that infection with BCG or S.typhimurium can alter the immune system resulting in attenuation of various immunological and patho-physiological features of asthma. Infection with BCG or S.typhimurium as a neonate appears to produce the most pronounced modification in the subsequent immune responses to OVA. These findings provide important insights into possible modified vaccination regimes at birth and during childhood, which may have the potential to prevent the development of asthma and allergic inflammatory disorders in adulthood.

It is only through hearing stories from others that I realise just how lucky I have been. Bev, Simon and Matt, firstly I can’t thank you enough for taking a chance and giving this PhD opportunity to someone fresh out of their undergraduate degree. Your support over the years has been overwhelming. The support with animal work, meticulous checking, constructive comments and advice on paper drafts, travel grants, conference abstracts, data analysis and presentations have shaped me into a better scientist and writer. The opportunities you have openly and generously supported to travel the world and to see so much more of what is in it have shaped me as person. Working under your supervision has only enhanced my desire to pursue a career in scientific research and I can honestly say I have enjoyed every moment of this PhD. I know how fortunate I have been to complete this PhD with that feeling. A huge thanks is also owed to all of the staff and colleagues at both the University of Nottingham and the University of Adelaide for creating such a supportive and welcoming environment to work in, there are simply too many of you to name. I have worked in two departments, both of which I have felt very lucky to have been a part of and I can only hope that this continues to be the case wherever I may end up in the future. I would like to take this opportunity to particularly thank Grace George, Zoe Daniel and Simon Welham. Grace, a rat wasn’t something I knew how to handle until you showed me. Zoe, a PCR plate wasn’t something I knew how to load until you taught me how. Simon, a PhD wasn’t something I ever thought I could achieve and I wouldn’t have attempted it if it wasn’t for the encouragement and confidence you instilled in me throughout a short summer placement. Special gratitude will always go to my family. No matter what I do in my professional career you will always provide me with laughter, perspective and purpose. Finally, I would like to respectfully acknowledge the contribution made by the laboratory rats for the completion of this research.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food & Wine, 2020

Dissertação de Mestrado em Bioquímica apresentada à Faculdade de Ciências e Tecnologia
O constante aumento da incidência de obesidade, especialmente em mulheres em idade fértil, tem sido um forte motivo de alarme. É estimado que 50% das grávidas têm excesso de peso ou são obesas. A obesidade materna predispõe o descendente para um risco acrescido de desenvolver inúmeras doenças crónicas, tais como obesidade, diabetes tipo 2 ou doença cardiovascular. A doença cardiovascular é, globalmente, a principal causa de morte para homens e mulheres. Não obstante, o risco de desenvolvimento de doença cardiovascular é diferente de acordo com o sexo. A obesidade e nutrição materna durante a gestação podem desencadear o desenvolvimento de doença cardiovascular na descendência, através de adaptações do sistema cardiovascular no útero. Isto pode levar à programação de mecanismos epigenéticos e de vias metabólicas, incluindo a função mitocondrial cardíaca. Atualmente, apesar de alternativas de suplementação serem indicadas nestes casos clínicos de modo a prevenir os efeitos deletérios da obesidade materna na função cardíaca dos descendentes, a falta de estratégias terapêuticas eficazes é notória. Este trabalho engloba uma estratégia inovadora propondo investigar de que forma é que o sistema cardiovascular de um descendente reage à prática materna de exercício físico durante uma gravidez obesogénica, a qual foi induzida através de uma dieta rica em açúcares e gorduras, em específico, como é que a prática de exercício físico materno durante uma gravidez obesogénica pode modular a função cardíaca dos descendentes. Ratos machos e fêmeas com 32 semanas de idade, descendentes de fêmeas rato da estirpe Sprague-Dawley, foram utilizados neste trabalho. Foi avaliado o efeito, a longo termo, da prática de exercício físico materno durante uma gravidez obesogénica na prevenção dos efeitos deletérios induzidos pela obesidade materna durante a gravidez na função cardíaca, em importantes vias metabólicas e na função mitocondrial de descendentes jovens-adultos. Apesar da evidente resposta dimórfica na fisiologia inata dos descendentes, a prática de exercício físico materno durante uma gravidez obesogénica induziu a modulação de parâmetros bioquímicos nos descendentes, traduzida em níveis alterados dos seguintes metabolitos no sangue: triglicerídeos, HDL e LDL, tendo esta resposta um evidente dimorfismo sexual. Estas alterações foram acompanhadas por uma remodelação metabólica do tecido cardíaco, que foi apreciada pelos níveis de proteínas-chave na via de sinalização da insulina, assim como por alteração nos níveis do transportador de ácidos gordos de cadeia curta, o CD36. Isto pode indicar que o transporte de ácidos gordos para o cardiomiócito encontrasse comprometido e, em conjunto com alterados níveis de metabolitos de lípidos, pode modular a função cardíaca mitocondrial de descendentes de mães obesas e exercitadas. De facto, a função cardíaca mitocondrial destes descendentes foi melhorada pela prática de exercício físico durante uma gestação obesogénica, com uma modulação positiva da bioenergética mitocondrial, em conjunto com uma possível modulação da dinâmica mitocondrial, através da observação de níveis alterados de proteínas envolvidas em eventos de fusão (MFN-1 e OPA1) e biogénese (PGC1α e TFAM) mitocondrial. Para além disso, resultados preliminares indicaram que o exercício materno praticado durante uma gravidez obesogénica pode prevenir o stress nitrosativo no tecido cardíaco. Concluindo, a prática de exercício físico durante uma gravidez obesogénica levou a uma modulação dos parâmetros bioquímicos, cardiometabólicos e da função mitocondrial cardíaca dos descendentes em idade jovem, com uma resposta específica de acordo com o sexo do descendente. Estas alterações podem ser benéficas o suficiente para melhorar a saúde cardiovascular dos descendentes, o que poderá levar à atenuação ou até mesmo à prevenção do desenvolvimento de doença cardiovascular numa fase mais tardia da vida.
Obesity incidence has been increasing at an alarming rate, especially in women of reproductive age. It is estimated that 50% of the total number of pregnancies occur in overweight or obese women. Maternal obesity (MO) predisposes the offspring to an increased risk of developing many chronic diseases in an early stage of life, including obesity, type 2 diabetes, and cardiovascular disease (CVD). CVD is the main cause of death worldwide among men and women. Despite this, CVD risk exhibits sexual dimorphism. Maternal diet and MO during gestation could prompt the offspring for CVD development through adaptations of the offspring’s cardiovascular system in the womb. This could lead to cardiac epigenetic and persistent metabolic programming of signalling pathways, including mitochondrial metabolic function, culminating in offspring’s increased predisposition to CVD development. Currently, despite diet supplementation alternatives being provided, effective therapeutical solutions to prevent the deleterious cardiac offspring function programming by obesogenic womb are lacking. This innovative work involves a novel approach to unravel how the offspring’s cardiovascular system reacts to maternal physical exercise practice during an obesogenic pregnancy, which was induced with a high fat/high sugar (HFHS) diet, and whether exercising during an obesogenic pregnancy (MOEx) can modulate the offspring cardiac function programming. Thirty-two-week-old offspring Sprague-Dawley rats exposed to MO and MOEx were used. The long-term therapeutic effect of maternal physical exercise during pregnancy in reversing the MO-induced effects on the cardiac function of young-adult offspring was evaluated by measuring hallmarks of cardiac metabolic impairment and mitochondrial dysfunction. Although the innate sex-specific response in the offspring’s cardiovascular physiology, MOEx induced biochemical modulation in the offspring, as indicated by the altered circulating levels of relevant molecules such as triglycerides, high-density lipoprotein (HDL), and low-density lipoprotein (LDL), despite the evident sexual dimorphism. These alterations were accompanied by cardiac metabolic remodelling, which was evaluated by measuring key-proteins involved in the insulin signalling pathway, along with alterations in the short-chain fatty acid transporter, CD36. This could indicate that MOEx affected cardiomyocyte fatty-acid uptake, and, along with the observed altered lipid metabolites levels, the MOEx offspring’s cardiac mitochondrial function could be modulated. Indeed, the offspring’s cardiac mitochondrial function seemed to be improved by MOEx comparing to MO, exhibiting a positive modulation of mitochondrial bioenergetics, along with possible mitochondrial dynamics modulation, which was observed through altered mitochondrial fusion- (MFN-1 and OPA1) and mitochondrial biogenesis-related proteins (PGC1α and TFAM). In addition, preliminary data indicated that MOEx prevents MO-induced nitrosative stress. Overall, maternal physical exercise practice during an obesogenic pregnancy leads to the modulation of the offspring’s biochemical, cardiac metabolic parameters and cardiac mitochondrial function, in a sex-specific way. These alterations may be favorable enough for the MO offspring’s cardiovascular health, which might result in the attenuation or even prevention of the development of CVD in MOEx offspring early life.
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Bibliography: leaves 179-190.
xii, 189 leaves : ill. ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
This thesis proposes a genetic-programming-based classifier system for the diagnosis of coronary artery disease. Based on genetic programming, a software system called Evolutionary Pre-Processor has been developed as a new method for the automatic extraction of non-linear features for supervised classification. Two different hybrid intelligent system techniques are presented; fuzzy systems integrated with genetic algorithms and genetic algorithms combined with back-propagation algorithms. All approaches were tested on a real-world problem of coronary artery disease data.
Thesis (Ph.D.)--University of Adelaide, Dept. of Applied Mathematics, 1998

Bibliography: leaves 179-190.
xii, 189 leaves : ill. ; 30 cm.
This thesis proposes a genetic-programming-based classifier system for the diagnosis of coronary artery disease. Based on genetic programming, a software system called Evolutionary Pre-Processor has been developed as a new method for the automatic extraction of non-linear features for supervised classification. Two different hybrid intelligent system techniques are presented; fuzzy systems integrated with genetic algorithms and genetic algorithms combined with back-propagation algorithms. All approaches were tested on a real-world problem of coronary artery disease data.
Thesis (Ph.D.)--University of Adelaide, Dept. of Applied Mathematics, 1998