Time with media screens (television, computers, videogames, cell phones, and tablets) is the primary activity of youth, second only to sleeping, and represents a major risk factor for cardiovascular diseases (CVD). Additionally, the populations with highest rates of screen time are also those most at risk of CVD from genetic predisposition (i.e., Blacks, Hispanics). The purpose of this descriptive, correlational study, based on cross-sectional analysis of archived data from the 2009 - 2010 NHANES for United States youth, newborn to 20 years old, was to determine whether the combination of media screen time with genetic background is a better predictor of CVD than either factor alone. The theoretical framework was the social ecological theory of disease distribution. The relationship between media screen time, genetic background, and CVD risk factor was determined using binary logistic regression. Results of this study indicated that the relationship between ethnicity, gender, and type/duration of exposure to media screen is important to predict the CVD risk factors C-reactive protein (CRP), triglycerides, and diastolic blood pressure. Interventions that limit exposure total screen time will reduce the risk of increased blood pressure among all races. However, culturally relevant intervention should be designed specifically for non-Hispanic Blacks, other Hispanics, and other race. These ethnicities have the highest propensity to increase in blood pressure, CRP, and triglycerides and also spend the largest amount of time in front of the media screen. Results from this study may help to promote policies and initiatives to limit screen time that are culturally relevant and more focused.

Background dependence is a ubiquitous attribute of eukaryotic gene systems that modulates the phenotypic effects of a mutant allele due to segregating genetic variation among different wildtype strains. Despite the wealth of literature demonstrating the presence of genetic background effects, very little is known about how they functionally or mechanistically contribute to the relationship between genetic variation and phenotypic expression. It has been postulated that background dependent effects may be highly specific to the activity of individual alleles or genes. A recent examination of mutant alleles in two interacting genes in the Drosophila wing network demonstrated the magnitude of phenotypic effect of a mutant allele may predict it’s sensitivity to the genetic background. To further understand this, I examined the background dependence of many alleles for genes across the regulatory network of Drosophila wing development in many inbred strains. Our goal was to understand whether effects of the genetic background are an attribute of individual alleles, alleles of the same gene, or genes with similar phenotypes or developmental roles. Our analysis suggests that background dependence is highly positively correlated among alleles of the same gene, especially between alleles with similar magnitudes of phenotypic effect. Similarly, the background dependence of genes within the same regulatory network were also positively correlated. Alleles from different genes, but of the same magnitude of phenotypic effect, generally demonstrated the highest degree of intergenic correlation. However, the background dependence of mutant alleles were generally not well correlated with the wildtype allele. Interestingly, we also found no recovery of any lethal alleles, despite thousands of individuals screened and evident suppression of mutant effects in some strains. We also analyzed the magnitude of intra-line variance in among a subset of our genes. This demonstrated a strong positive relationship between the magnitude of intra-line variation and the severity of phenotypic effects, regardless of the identity of the mutant allele. However, we show no correlation between intra-line variability in the wildtype and the magnitude of perturbation for a given mutant allele. To confirm the quantitative estimates of mean wing size accurately reflected subtle perturbations to wing tissue, we conducted a semi-quantitative analysis and compared it to our quantitative estimates. We demonstrate a high degree of correlation between the quantitative and semi-quantitative approaches, indicating semi-quantitative analysis is a useful way to capture subtle phenotypic effects. In addition, we repeated the quantitative analysis with a subset of the genes and inbred strains from the original data. Importantly, results of the repeated study largely recapitulate our original results. / Thesis / Master of Biological Science (MBioSci)

When investigating the developmental roles of genes on phenotypic expression it may seem reasonable to assume that a mutation would result in consistent phenotypic change. However, increasing evidence has shown this is not often the case, and the “wild-type” genetic background of an individual plays a large role in phenotypic expression of mutations and severity of genetic mediated diseases. Previous work has demonstrated that degree of genetic background effects shows a non-linear relationship with severity of mutational effects. This relationship is characterized by alleles of moderate phenotypic expressivity showing the relatively greatest degree of background dependence and between genotype variability in comparison with alleles of severe and modest phenotypic expressivity. Our previous work has shown this relationship for Drosophila melanogaster wing size through a scalloped (sd) allelic series crossed to naturally derived strains from the Drosophila Genetics Reference Panel (DGRP). I explored these effects with a miniature (m) allelic series where the results from our experiment suggest a vastly different response. m when compared to sd is characterized by a more linear relationship, whereby alleles of moderate phenotypic effect do not show increased background dependence nor increased variability within and between strains. Furthermore, our results suggest a strong correlation across DGRP strains with respect to m mutational severity and that the effect m has on wing shape is not largely due to wing size. Our working hypotheses for why this might be occurring is due to the increased interaction of sd with other aspects of wing development relative to that of m, the differences in when the genes are playing active roles in wing development, or the effects the mutations have on the wing to affect size. To add to our previous results employingutilizing sd, I am beginnings to elucidate the non-linear relationship of genetic background effects with severity of mutational effects at a gene expression level. I am accomplishing this through crossing autilizing a sd allelic series crossed to six naturally derived DGRP strains used in previous experiments involving wing size. Preliminary results agree with previous work on genetic background effects, displaying a non-linear relationship with the severity of mutational effect. I aim to continue to explore this relationship including more genotypes and investigating more genes to better elucidate the mechanistic causes of genetic background effects. / Thesis / Master of Science (MSc) / When investigating the roles of genes on phenotype it may seem intuitive that a mutation affecting gene function would display a consistent change in phenotype. Increasing evidence has asserted that this may not always be the case and genetic background effects may affect the genotype-phenotype relationship affecting experimental design, disease treatment and evolutionary trajectories. Here, we investigate the mechanisms involved in these genetic background effects utilizing Drosophila melanogaster wing tissue. We outline a change from the typically observed non-linear relationship between genotype and phenotype and for the first time quantify shape change effects by the miniature mutation.

Many insects form symbioses with maternally inherited, intracellular bacteria, which can have major effects on the ecology and evolution of the insect host. Here I investigated the interaction between a global agricultural pest, Bemisia tabaci (the sweetpotato whitefly), and a Rickettsia bacterial symbiont. Rickettsia had previously been tracked sweeping through field populations of B. tabaci in the southwestern USA and had been shown to dramatically increase whitefly fitness under laboratory conditions. In contrast, the Rickettsia present in whiteflies in Israel has few observable fitness effects and is declining in frequency in field populations. I explored the population dynamics of Rickettsia in B. tabaci field populations in the USA and Israel, and assessed the genetic diversity of the Rickettsia in these populations. In laboratory experiments, there was no observable effect of Rickettsia on the heat shock or constant temperature tolerance of USA B. tabaci. Instead, whitefly genetic background appears to influence the effects of Rickettsia. Lastly, analysis of the genome sequence of Rickettsia provided insights into the mechanism of the fitness benefit and evolutionary history of the bacterium. Taken together, these integrated ecological, physiological and genomic studies provide some explanation for the contrasting and wide-ranging phenotypes associated with whitefly Rickettsia, and provide support for the hypothesis that the fitness benefit provided by Rickettsia is context dependent. The Rickettsia symbiosis exhibits geographically distinct population dynamics, is affected by whitefly genotype, and may involve manipulation of host plants and/or defense against pathogens rather than nutritional supplementation. Overall, these results highlight the important role that microbial symbionts may play in the adaptation of invasive species to changing environments.

Spinal muscular atrophy (SMA) is a genetic disorder characterized by muscle weakness and atrophy and death of motor neurons in humans. Although almost all cases of SMA occur due to mutations in a gene called survival motor neuron 1 (SMN1), SMA patients present with a wide range of severities of the symptoms. The most severe cases never achieve any developmental motor milestone and die within a few years after birth. On the other hand, mild cases of SMA have a normal life span and show trivial motor deficits. This suggests the role of other factors (rather than the function of SMN1) in the outcome of the disease. Indeed, the copy number of an almost identical gene, called SMN2, is the main determining factor for the severity of SMA. In addition, a few other genes (e.g. Plastin 3) are proposed as disease modifiers in SMA. SMN1 is a housekeeping gene, but due to unknown reasons, the most prominent pathologies in SMA are atrophy of myofibers and death of motor neurons. However, recent studies showed that some other cell types are also affected in the course of SMA disease. We investigated the alterations of central synapses in Smn2B/- mice, a model of SMA. We did not observe any degeneration of central synapses in these mice until a post symptomatic stage. However, mass spectrometry (MS) analysis on isolated synaptosomes from spinal cords of these animals revealed widespread alterations in the proteome of their central synapses at a presymptomatic stage. Functional cluster analysis on MS results suggested that several molecular pathways are affected within synapses of spinal cords of Smn2B/- mice prior to the onset of any obvious pathology in their motor units. The affected molecular pathways are involved in basic cell biological functions including energy production, protein synthesis, cytoskeleton regulation and intracellular trafficking. We showed that the levels of several proteins involved in actin cytoskeleton regulation are altered in synaptosomes isolated from spinal cords of Smn2B/- mice. More investigations are required to determine the exact functional abnormalities of affected pathways in central synapses of these mice. We also generated congenic Smn2B/- mice in two different mouse genetic backgrounds; FVB and BL6. Using a systematic approach, we showed that congenic Smn2B/- mice in the FVB background show a more severe SMA phenotype than Smn2B/- mice in a BL6 background. Smn2B/- mice in the FVB background had a shorter survival, higher rate of weight loss, earlier and more severe pathologic changes compared to Smn2B/- mice in the BL6 background. We investigated the levels of several actin binding proteins in spinal cords of these animals and found higher induction of plastin 3 in Smn2B/- mice in the BL6 background. More investigations are underway to determine the role of plastin 3 in the severity of the phenotype of Smn2B/- mice, and to find other possible SMA modifier genes in these animals.

No description available.

Comme de nombreuses espèces naturelles, l’abeille européenne (Apis mellifera) est confrontée à une pression croissante de facteurs biotiques et abiotiques : environnement, diversité génétique, parasitisme, etc. Chacun de ces facteurs peut potentiellement influencer les communautés de micro-organismes qui constituent le microbiote de l’abeille et évoluent avec elle. L’objectif de cette thèse était de comprendre la dynamique et la structuration du microbiote intestinal de la lignée évolutive M de l’abeille européenne en fonction de facteurs biogéographiques, génétiques et temporels des colonies d’abeilles. Cette analyse a été réalisée dans sept conservatoires de cette lignée évolutive, répartis au Portugal (A. m. iberiensis), en Espagne et en France (A. m. mellifera), selon un gradient Nord-Sud et Est-Ouest. Dans un premier temps, mon travail a permis de décrire un facteur jusqu’ici mal connu : l’humidité dans les ruches. Celle-ci s’est révélée stable, entre 50 et 60 % d’humidité relative toute l’année, suggérant une hygrorégulation par les colonies d’abeilles, quelle que soit la période de l’année et la dynamique populationnelle au sein des nids (ici, la ruche). Par la suite, nous avons développé un protocole permettant le suivi spatio-temporel de la charge virale des abeilles par cytométrie en flux. Son application sur nos abeilles a montré que le temps influence moins la charge virale que le lieu géographique. Enfin, une analyse métagénomique sur un gène ciblé (ARNr 16s) a confirmé l’effet de la localisation géographique des ruches, cette fois sur la structuration des communautés bactériennes peuplant les intestins des abeilles qui appartiennent à la lignée évolutive M. Par ailleurs, cette dernière analyse a montré l’importance de prendre en compte l’interaction entre les facteurs, qui peuvent avoir plus d’impact pris ensemble que séparément. Enfin, des perspectives sont envisagées, telles que la réalisation d’un réseau d’inter-actants permettant de comprendre la part de chaque facteur sur les communautés bactériennes mais également les pathogènes. / Like many natural species, the European bee (Apis mellifera) faces a threat of biotic and abiotic factors: environment, genetic diversity, parasitism, etc. Each of these factors can potentially influence the communities of microorganisms that constitute the bee's microbiota and evolve with it. The aim of this work was to understand the dynamics and structure of the gut microbiota of the M evolutionary line of A. mellifera according to biogeographic, genetic and temporal factors of bee colonies. This analysis was carried out in the conservatories of this evolutionary line, located in Portugal (A. m. iberiensis), Spain and France (A. m. mellifera) along a North-South and East-West gradient. First, my work has described a hitherto barely understood factor: humidity in hives. It appeared to be stable in our beehives, between 50 and 60% relative humidity all year long, which suggests a hygroregulation by honeybee colonies whatever the periode of year and the populational dynamics inside the nest (i.e. the beehive). We developed a protocol allowing spatio-temporal monitoring of the viral load of bees by flow cytometry. After using it on our colonies, we showed that time influences less viral load than geographical location. Finally, a metagenomic analysis on a target gene (16s rRNA) confirmed the importance of the geographical location of beehives, this time on the structuring of bacterial communities living in guts of honeybees belonging to the M evolutive lineage. In addition, this last analysis has shown the importance to take into account the interaction between factors, which can have a bigger effect when taken together. Finally, perspectives are envisaged, such as the realization of a network of interactants allowing to understand the part of each factor on the bacterial but also pathogens’ communities.

Apesar de apenas 10% dos indivíduos infectados por Mycobacterium tuberculosis desenvolverem tuberculose, essa doença causa a morte de milhares de pessoas anualmente. Sabendo que o background genético do hospedeiro está relacionado com suscetibilidade/resistência à infecção por M. tuberculosis, nosso grupo vem avaliando diferenças na resposta imunológica entre camundongos C57BL/6 resistentes e BALB/c suscetíveis. No presente estudo, nós nos propusemos a avaliar populações de macrófagos M1 e M2 em animais com capacidade diferente de elaborar resposta imune celular nessa infecção. Animais C57BL/6, que elaboram resposta imune celular de maior magnitude, e BALB/c foram infectados com M. tuberculosis, e avaliados aos 30 e 70 dias de infecção. Observamos maior porcentagem de células CD11b+Ly-6C+, que caracterizam monócitos inflamatórios, no pulmão de animais BALB/c com 70 dias de infecção comparada à porcentagem detectada em animais C57BL/6 com mesmo período de infecção. Houve correlação positiva significativa entre número de bacilos e expressão gênica para iNOS aos 30 dias de infecção comparando ambas as linhagens. Ao contrário da nossa hipótese inicial, observamos maior porcentagem de células CD11b+F4/80+CD206+, que caracteriza o fenótipo de macrófagos M2, no pulmão de animais C57BL/6 com 70 dias de infecção comparada à porcentagem detectada em animais BALB/c com mesmo período de infecção, havendo correlação inversa significativa de células CD11b+F4/80+CD206+ e número de bacilos aos 70 dias de infecção. Não houve diferença na análise funcional de macrófagos M1 e M2 obtidos a partir de precursores da medula óssea, comparando as duas linhagens de camundongos. Dessa forma, experimentos com o propósito de estudar a função dessas células in vivo serão conduzidos para avaliar se os macrófagos M2 participam da resposta protetora que auxilia na eliminação dos bacilos ou na resposta protetora que auxilia no reparo tecidual do hospedeiro. / Despite only 10% of individuals infected with Mycobacterium tuberculosis develop tuberculosis, this disease causes millions of deaths annually. We know that the host genetic background is associated with susceptibility/resistance to M. tuberculosis-infection. Therefore, our group has studied differences in the immunologic response between resistant C57BL/6 mice and susceptible BALB/c mice. In the present study, our aim was to evaluate populations of M1 and M2 macrophages in mice that generate different magnitude of cellular immune response in this infection. C57BL/6 mice, which elaborate a higher immune cellular response, and BALB/c mice were infected with M. tuberculosis, and evaluated 30 and 70 days post infection. We observed higher percentage of CD11b+Ly-6C+ cells, which characterize inflammatory monocytes, in the lung of BALB/c mice with 70 days of infection compared to the percentage detected in C57BL/6 animals in the same period of infection. There was a significant positive correlation between CFU number and iNOS genic expression comparing both mouse strains 30 days post infection. On the contrary to our initial hypothesis, we observed higher percentage of CD11b+F4/80+CD206+ cells, which characterize M2 macrophage phenotype, in the lung of Day 70-infected C57BL/6 animals compared with the percentage detected in BALB/c mice at the same time of infection. There was a significant inverse correlation between CD11b+F4/80+CD206+ cells and bacillus number at 70 days post infection. There was no difference in the functional analysis of M1 and M2 macrophages obtained from precursors of bone marrow, comparing both mouse strains. Therefore, experimental assays with the aim to study whether M2 macrophages contribute for the protective immune response that induce bacillus clearance or contribute to the protective immune response that promote host tissue repair will be performed in an attempt to understand better the role of these cells in the M. tuberculosis-infection.

Apesar de apenas 10% dos indivíduos infectados por Mycobacterium tuberculosis desenvolverem tuberculose, essa doença causa a morte de milhares de pessoas anualmente. Sabendo que o background genético do hospedeiro está relacionado com suscetibilidade/resistência à infecção por M. tuberculosis, nosso grupo vem avaliando diferenças na resposta imunológica entre camundongos C57BL/6 resistentes e BALB/c suscetíveis. No presente estudo, nós nos propusemos a avaliar populações de macrófagos M1 e M2 em animais com capacidade diferente de elaborar resposta imune celular nessa infecção. Animais C57BL/6, que elaboram resposta imune celular de maior magnitude, e BALB/c foram infectados com M. tuberculosis, e avaliados aos 30 e 70 dias de infecção. Observamos maior porcentagem de células CD11b+Ly-6C+, que caracterizam monócitos inflamatórios, no pulmão de animais BALB/c com 70 dias de infecção comparada à porcentagem detectada em animais C57BL/6 com mesmo período de infecção. Houve correlação positiva significativa entre número de bacilos e expressão gênica para iNOS aos 30 dias de infecção comparando ambas as linhagens. Ao contrário da nossa hipótese inicial, observamos maior porcentagem de células CD11b+F4/80+CD206+, que caracteriza o fenótipo de macrófagos M2, no pulmão de animais C57BL/6 com 70 dias de infecção comparada à porcentagem detectada em animais BALB/c com mesmo período de infecção, havendo correlação inversa significativa de células CD11b+F4/80+CD206+ e número de bacilos aos 70 dias de infecção. Não houve diferença na análise funcional de macrófagos M1 e M2 obtidos a partir de precursores da medula óssea, comparando as duas linhagens de camundongos. Dessa forma, experimentos com o propósito de estudar a função dessas células in vivo serão conduzidos para avaliar se os macrófagos M2 participam da resposta protetora que auxilia na eliminação dos bacilos ou na resposta protetora que auxilia no reparo tecidual do hospedeiro. / Despite only 10% of individuals infected with Mycobacterium tuberculosis develop tuberculosis, this disease causes millions of deaths annually. We know that the host genetic background is associated with susceptibility/resistance to M. tuberculosis-infection. Therefore, our group has studied differences in the immunologic response between resistant C57BL/6 mice and susceptible BALB/c mice. In the present study, our aim was to evaluate populations of M1 and M2 macrophages in mice that generate different magnitude of cellular immune response in this infection. C57BL/6 mice, which elaborate a higher immune cellular response, and BALB/c mice were infected with M. tuberculosis, and evaluated 30 and 70 days post infection. We observed higher percentage of CD11b+Ly-6C+ cells, which characterize inflammatory monocytes, in the lung of BALB/c mice with 70 days of infection compared to the percentage detected in C57BL/6 animals in the same period of infection. There was a significant positive correlation between CFU number and iNOS genic expression comparing both mouse strains 30 days post infection. On the contrary to our initial hypothesis, we observed higher percentage of CD11b+F4/80+CD206+ cells, which characterize M2 macrophage phenotype, in the lung of Day 70-infected C57BL/6 animals compared with the percentage detected in BALB/c mice at the same time of infection. There was a significant inverse correlation between CD11b+F4/80+CD206+ cells and bacillus number at 70 days post infection. There was no difference in the functional analysis of M1 and M2 macrophages obtained from precursors of bone marrow, comparing both mouse strains. Therefore, experimental assays with the aim to study whether M2 macrophages contribute for the protective immune response that induce bacillus clearance or contribute to the protective immune response that promote host tissue repair will be performed in an attempt to understand better the role of these cells in the M. tuberculosis-infection.

The leptin receptor (Lepr) pathway is important for food intake regulation, energy expenditure, and body weight. Mutations in leptin and the Lepr have been shown to cause early-onset severe obesity in mice and humans. In studies with C57BL/6NCrl mice, we found a mouse with extreme obesity. To identify a putative spontaneous new form of monogenic obesity, we performed backcross studies with this mouse followed by a quantitative trait locus (QTL) analysis and sequencing of the selected chromosomal QTL region. We thereby identified a novel Lepr mutation (C57BL/6N-LeprL536Hfs*6-1NKB), which is located at chromosome 4, exon 11 within the CRH2-leptin-binding site. Compared with C57BL/6N mice, LeprL536Hfs*6 develop early onset obesity and their body weight exceeds that of Leprdb/db mice at an age of 30 weeks. Similar to Leprdb/db mice, the LeprL536Hfs*6 model is characterized by hyperphagia, obesity, lower energy expenditure and activity, hyperglycemia, and hyperinsulinemia compared with C57BL/6N mice. Crossing Leprdb/wt with LeprL536Hfs*6/wt mice results in compound heterozygous LeprL536Hfs*6/db mice, which develop even higher body weight and fat mass than both homozygous Leprdb/db and LeprL536Hfs*6 mice. Compound heterozygous Lepr deficiency affecting functionally different regions of the Lepr causes more severe obesity than the parental homozygous mutations.