<p> This work incorporates information from two sources in order to examine the nature of natural selection acting on phenotypic characters in Drosophila melanogaster along a North South cline. Isofemale lines were established from flies collected along a North South cline extending from Winnipeg, Manitoba to Tampa Bay, Florida. Offspring from different lines within each position were then cultured under standardized conditions and used to examine phenotypic variation in 10 morphological characters along the cline. In a separate set of experiments, flies from Vineland, Ontario were mated in a half-sib design in order to estimate the genetic covariance of the set of 10 characters. The results from the clinal and heritability experiments were then combined using Lande's (1979) equation,&.= Gp-ls, to estimate the net selective differentials and net selection gradients for each adjacent set of populations. The study concluded that: 1) Clinal variation is non-linear, with larger flies in the middle latitudes and smaller flies in the north and south. 2) Selection appears to act primarily on body characters in the north (wing width and femur length) and head characters in the south (eye and face width). 3) Scutellum width and wing length generally moderate the prevalent trends in directional selection on the other characters through antagonistic correlated responses. 4) Clinal patterns of variation may not be at equilibrium, but instead dominated by seasonal responses to selection pressures. </p> / Thesis / Master of Science (MSc)

Malathion-resistance in Drosophila Melanogster was studied in susceptible laboratory strains (CS and OR), a heterogeneous intermediate-resistant strain (C1-39), and a more resistant, artificially selected (with malathion). strain (MH19) by comparing the in vitro metabolism of [¹⁴c]-malathion and [¹⁴c]-malaoxon in crude enzyme preparations made from adult flies. Extracts from all strains were found to contain two enzymatic activities that metabolized malathion and/or malaoxon. One activity degraded malathion to its α- and β-monocarboxylic acids and was designated as malathion-carboxylesterase activity (ME-activity). ME-activity was progressively lost in CS-extracts during reaction with [¹⁴c]-malathion due to inhibition of the enzyme(s) by a tightly bound [¹⁴c]-labeled molecule (not identified) that could not be removed by chromatography on Sephadex G-25. ME-activity, based on initial (0-1 min) rates with or without metyrapone present was similar in all strains and furthermore, the carboxylesterase inhibitors TPP and DEF did not synergize malathion toxicity in either resistant or susceptible strains. It was concluded that carboxylesterase-mediated degradation of malathion was not a factor in the resistance of the C1-39 and MH19 strains. A second enzyme system, microsomal mixed-function oxidases (MFO), converted malathion to malaoxon (activation) and degraded malaoxon to at least two products that were tentatively identified (malaoxon α-and β-monoacids and demethyl-malaoxon). The rate of conversion of malathion to malaoxon was highest in crude extracts of the most resistant MH19 flies, intermediate in C1-39 and could not be detected in the susceptible CS flies while the rate of malaoxon degradation was similar between MH19 and C1-39, but higher than that in the susceptible OR flies. Furthermore, malaoxon (but not malathion) toxicity was most strongly synergized by the MFO-inhibitor MTP in the more resistant strains (MH19 and C1-39) . These results were used along with a previous result that MH19 strain possesses a less sensitive form of the target enzyme, acetylcholinesterase (R.A. Morton, personal communication), to propose a biochemical mechanism that accounts for the increased malathion-resistance of the C1-39 and MH19 strains. / Thesis / Master of Science (MS)

Thesis / Master of Science (MS)

The period gene determines biological rhythmicity in Drosophila melanogaster. The X-linked gene is 7.4kb, containing 8 exons and 7 introns from which a 4.5kb message is translated. A striking feature of the protein encoded by per is a series of alternating threonine-glycine residues in the fifth exon. Moreover, this string of residues is polymorphic for length variation in natural populations, the most frequent variants having 17, 20 or 23 Thr-Gly pairs. In the present study, a geographic analysis of this polymorphism within North American populations was conducted, the results of which indicate significant variation of allele frequency with latitude. The use of spatial autocorrelation analysis and Mantel tests clearly show that the most common variant, encoding 17 Thr-Gly pairs, exhibits a clinal pattern in its distribution along a north-south axis. Furthermore, DNA sequence analysis of several variants has uncovered a novel new variant which encodes 22 Thr-Gly pairs whose nucleotide sequence differs from any published data. Similar statistical analysis conducted on seven allozymes for populations collected along the same transect shows that several have monotonic clinal patterns in their allele frequency distributions which also show correlation with latitude. A previous study of morphological traits on the same populations showed the existence of a non-monotonic clinal pattern. Comparison of the results observed for the molecular and morphological markers indicates that they are subject to different evolutionary forces. The results highlight the importance of comparing patterns of geographic variation using different genetic elements. / Thesis / Master of Science (MS)

Insects accumulate various xenobiotics and toxic molecules through feeding and environmental exposure. This study examines the toxicology and regulation of a class of toxic molecules, organic cations, in Drosophila melanogaster. The results of this thesis demonstrate that transepithelial tetraethylammonium (TEA) secretion across the main segment of the Malpighian tubules is increased in response to diuretic factors. Both cAMP and cGMP, which increase transepithelial potential (TEP), as well as tyramine and LK-1, which decrease TEP, all enhanced TEA secretion. Both inc~eases and decreases ofTEP may enhance proton transport into the lumen of the tubule thus increasing the rate of organic cation/proton exchange across the apical membrane. These findings suggest that factors previously referred to as diuretic factors may in fact :let primarily or secondarily as stimulants of organic cation excretion. Haemolymrh concentrations of TEA increased linearly with the concentration of TEA in the diet and declined rapidly upon transfer of the larvae to TEA-free diet. The rate of decline was reduced by slowing the metabolic rate or by the addition of cimetidine to a diet containing TEA. Although larvae tolerated high levels of TEA in the diet, mortality increased when TEA was combined with either quinidine or cimetidine. It is suggested that inhibition of TEA transport by cimetidine or quinidine results in prolonged exposure to higher levels of TEA in the haemolymph and a consequent increase in toxicity. Surprisingly, TEA flux and fluid secretion rate were both reduced in Malpighian tubules isolated from adult flies raised on TEA-enriched diet. This suggests that the high concentration of TEA in the diet produced a non-lethal yet deleterious effect on the Malpighian tubules of Drosophila. / Thesis / Master of Science (MS)

Menopause, the complete cessation of menstrual cycles, apparently is a detrimental trait, yet all women experience it. Numerous theories have been proposed to explain the origin of menopause, but none has been satisfactory. In 2013, Morton et al. proposed a mate choice hypothesis to explain menopause and, using a computational model, showed how a bias in mating (i.e., older men preferring younger women) could have allowed such an otherwise detrimental trait to evolve neutrally through accumulation of female fertility-reducing mutations. To investigate whether biased mating could affect fecundity and fertility in a real system, two experimental populations were established using Drosophila melanogaster. Older males were mated with younger females and vice versa. Information was obtained, including data on fecundity, fertility, ovariole and matured egg chambers, and lifespan, for experimental, age-restricted-mating and control populations. A negative effect on the fecundity and fertility of the younger-mating sex was observed in restricted-mating compared to control populations. Age-restricted mating had no effect on longevity. Menopause could evolve according to the mate choice hypothesis. / Thesis / Master of Science (MSc) / All animals are expected to remain fertile until they die. Menopause is an enigma and an unsolved problem in evolutionary biology. Numerous theories have been proposed to explain menopause, but there is no clear understanding of how this fertility reducing trait evolved in the human population. It has been proposed that biased mate choice i.e., preference for younger females can lead to accumulation of fertility-reducing mutations and the evolution of menopause. In this study, mate choice theory was tested using altered (biased) mating schemes with Drosophila melanogaster and the results support the mate choice theory of menopause.

Les petites protéines de choc thermique (sHsps) ont été découvertes initialement chez Drosophila. Les membres de cette famille sont des chaperons moléculaires sont présentsdans la plupart des organismes eucaryotes et procaryotes et certains virus. En plus d’être induites en réponse à la plupart des stresseurs dont un choc thermique, elles sont également exprimés en absence de stress. Les sHsps forment des structures dynamiques s'assemblant en oligomères et elles sont essentielles durant les conditions de stress en empêchant l'agrégation des protéines dénaturées et en favorisant leur repliement par des chaperons moléculaires dépendants de l'ATP. Le génome de Drosophila melanogastercode pour 12 sHsp, qui ont des profils d'expression développementaux, des localisations intracellulaires diverses et des spécificités de substrats distincts. DmHsp22 est jusqu'à présent la seule sHsp localisée dans les mitochondries avant et après un choc thermique. Elle est préférentiellement régulée lors du vieillissement et en réponse à la chaleur et aux stress oxydants. La surexpression de DmHsp22 augmente la durée de vie et la résistance au stress et sa régulation négative est préjudiciable. C'est un chaperon efficace, qui pourrait être impliqué dans la réponse mitochondriale au dépliement protéique (UPRMT). Cependant, le mécanisme exact de son action est mal compris. Structurellement, DmHsp22 forme une population d'oligomères semblable aux nombreux sHsps de métazoaires et différente deDmHsp27. L'alignement des séquences de la région ACDde DmHsp22 avec des sHsp de drosophile et d'autres organismes a démontré la présence de trois résidus d'arginine hautement conservés dans ce domaine. Une forte conservationde ces résidus suggère leur implication possible dans la structure et la fonction de DmHsp22. La substitution des résidus d'arginine hautement conservés dans les sHsps de mammifères est associée à certaines pathogenèses et déclenche des changements de conformation des protéines ainsi que l'agrégation des protéines intracellulaires. La mutation de l'arginine en glycine au niveau de trois résidus hautement conservés d'ACD dans DmHsp22 (R105, R109, R110) résulte en une population oligomérique qui, dans le cas de R110G, perturbe la structure et provoque la formation de petits oligomères. Bien que DmHsp22 ainsi que les mutants aient été caractérisés comme des chaperons efficaces in vitro, les mécanismes d'action exacts dans les mitochondries et l'information sur le comportement protecteur nécessitent la détermination du réseau d’interaction in vivo. Nous avons utilisé la technique capture d’immunoaffinité (CIA) pour récupérer 60 protéines qui interagissent spécifiquement avec DmHsp22 in vivo pendant le traitement normal et thermique, dans le surnageant des cellules de mammifères exprimant la DmHsp22. L’CIA effectuée sur la fraction mitochondriale a permis d’identifies 39 protéines qui interagissent spécifiquement avec DmHsp22. La combinaison de l’IAC avec l'analyse par spectroscopie de masse de mitochondries de cellules HeLa transfectées avec DmHsp22 a conduit à l'identification de partenaires de liaison à DmHsp22 dans des conditions de normales et de choc thermique. L'interaction entre DmHsp22 et deux autres chaperons mitochondriaux a été validée par immunobuvardage. Notre approche a montré que les cellules HeLa exprimant DmHsp22 augmentent la consommation d'oxygène mitochondrial et les teneurs en ATP, ce qui confère un nouveau rôle à DmHsp22 dans les mitochondries. En outre, l'activité d’une luciférase exogène a légèrement augmenté dans les cellules HeLa exprimant DmHsp22 après que l'activité enzymatique ait été réduite à la suite de l'exposition à la chaleur. En résumé, ce projet a permis de caractériser la structure oligomérique de DmHsp22 et un certain nombre de mutants dans le domaine alpha cristallin tout en fournissant un rôle potentiel mécanistique dans l’homéostase mitochondriale. La détermination du réseau mitochondrial de DmHsp22 suggère son importance dans cette organelle non seulement en tant que chaperon moléculaire, mais aussi en tant que protéine impliquée dans plusieurs fonctions cellulaires significatives. / The small heat shock proteins (sHsps) were first discovered in Drosophila. Members of this family are molecular chaperones and are present in most eukaryotic and prokaryotic. Although, they are induced in response to most of the stressors including heat shock, they are also expressed in absence of stress. SHsps for mdynamic structures that assemble into oligomers which are essential during stress conditions by preventing aggregation of denatured proteins and promoting their folding by ATP dependent molecular chaperones. Drosophila melanogaster genome encodes 12 sHsps, that have developmental expression patterns, diverse intracellular localizations and distinct substrate specificities. DmHsp22 is up to now the only sHsp localized in mitochondria before and after heat shock. It is preferentially regulated during ageing and in response to heat and oxidative stresses. Over-expression of DmHsp22 increases lifespan and resistance to stress and its down-regulation is detrimental. It is an efficient chaperone and could be involved in the mitochondrial unfolding protein response (UPRMT). However, the exact mechanism of its action is poorly understood. Structurally, DmHsp22 forms one population of oligomers similar to the many metazoan sHsps but DmHsp27. Sequence alignment of DmHsp22 with sHsps in Drosophilaand other organisms at the alpha crystalline domain (ACD) region demonstrated the presence of three highly conserved arginine residues in this domain. Strong conservation of these residues suggest their possible involvement in structure and function of DmHsp22. Substitution of highly conserved arginine residues in mammalian sHsps is associated with some pathogenesis and triggers protein conformational changes as well as intracellular protein aggregation. Mutation of arginine to glycine at three highly conserved residues of ACD in DmHsp22 (R105, R109, R110) results in one oligomeric population as well which in the case of R110G disrupts the structure and causes formation of smaller oligomers. Although DmHsp22 as well as mutants have been characterized as effective in vitro chaperones, the exact mechanism(s) of action in mitochondria and information about protective behavior requires defining of in vivoprotein interacting network. We have used immunoaffinity conjugation (IAC) technique to recover 60 proteins that specifically interact with DmHsp22 in vivo during normal and heat treatment using cell extract of mammalian cells expressing DmHsp22. The IAC performed on mitochondrial fraction identified 39 proteins that specifically interact with DmHsp22. Combination of IAC with mass spectroscopy analysis of mitochondria of HeLa cells transfected with DmHsp22 resulted in identification of DmHsp22-binding partners under normal andunder heat shock conditions. Interaction between DmHsp22 and two other mitochondrial chaperones was validated by immunoblotting. Our approach showed that HeLa cells expressing DmHsp22 increase maximal mitochondrial oxygen consumption and ATP contents which provides a new mechanistic role for DmHsp22 in mitochondria. Further more, exogenous luciferase activity slightly increased in HeLa cells expressing DmHsp22 after the enzyme activity reduced as a result of exposure to heat. In summary, this project has characterized the oligomeric structure of DmHsp22 and a number of mutants inthe alpha crystalline domain while providing a potential mechanistic role in mitochondrial homeostasis. Determining mitochondrial network of DmHsp22 suggest its importance in this organelle not only as a molecular chaperone but also as a protein involved in several significant cellular functions.

La petite protéine de choc thermique Hsp27 chez Drosophila melanogaster est localisée au noyau. Dans le but d'identifier la séquence peptidique responsable de cette localisation, la mutagenèse dirigée d'un signal potentiel de localisation nucléaire bipartite situé entre les acides aminés 42 et 59 a été effectuée, et les vecteurs obtenus ont été transfectés dans des cellules de mammifères HeLa. Il s'est avéré que la mutation des arginines 54, 55 et 56 de la protéine occasionne une diminution importante de la localisation nucléaire, bien qu'on observe toujours des résidus nucléaires localisés au niveau de structures granulaires. La localisation au niveau de structures granulaires est également observée avec la protéine sauvage. Des analyses avec des marqueurs de corps nucléaires ont donc été effectuées afin de déterminer si ces granules contenant Hsp27 correspondaient à des corps nucléaires connus. Il a été observé que la protéine colocalise avec le facteur d'épissage SC35 au niveau des "speckles" nucléaires. Ces observations s'avéreront utiles dans l'identification de fonctions pour la protéine Hsp27.

La polarité apico-basale des cellules épithéliales est indispensable au maintien de l’intégrité des tissus et sa dérégulation contribue à la progression tumorale. L’interaction antagoniste des protéines basolatérales Lethal giant larvae (Lgl) et Yurt (Yrt) avec les composants apicaux Crumbs (Crb) et la protéine kinase C atypique (aPKC) permet de définir le domaine apical et latéral. Crb et aPKC sont cruciales à la croissance du domaine apical. Les domaines apical et latéral sont délimités par les jonctions adhérentes (JA), une structure impliquée dans la cohésion cellulaire et la polarité. La protéine Girdin est essentielle à la morphogenèse des tissus épithéliaux chez Drosophila melanogaster et régule la stabilité des JA. Notre objectif est de déterminer si Girdin joue un rôle dans la polarité épithéliale et, si oui, via quels mécanismes. Nous avons montré que la diminution du niveau de Girdin, mais pas celle de composantes majeures des JA, amplifie l’apicalisation des cellules épithéliales dans les mutants zygotiques lgl ou yrt. L’activité résiduelle de Lgl et Yrt est donc réprimée suite à la réduction du niveau de girdin, possiblement suite à une sur-activation de leur répresseur aPKC. De plus, nous avons observé qu’une réduction de l’activité de girdin restaure partiellement l’intégrité épithéliale des embryons déplétés en aPKC ou mutants hypomorphes crb, un substrat apical d’aPKC, ce qui suggère que Girdin réprime l’activité d’aPKC et de Crb. Nous avons constaté que l’absence de Girdin entraîne une augmentation du niveau de phosphorylation de Yrt et Lgl, deux substrats basolatéraux d’aPKC. Girdin pourrait donc, tout en régulant les JA, moduler l’activité d’aPKC en s’y liant directement ou en s’associant à Yrt ou Lgl, deux protéines qui répriment aPKC et Crb. Nos résultats indiquent que Girdin agit comme régulateur négatif d’aPKC. Ceci place Girdin au coeur de la régulation de la polarité épithéliale, la morphogenèse tissulaire et la progression tumorale. / Apical-basal polarization of epithelial cells is indispensable for maintaining tissue integrity, and its deregulation contributes to tumor progression. The antagonistic interactions between the basolateral proteins Lethal giant larvae (Lgl) and Yurt (Yrt) and the apical components Crumbs (Crb) and the atypical protein kinase C (aPKC) define apical and basolateral domains. Crb and aPKC are essential for apical membrane growth. The apical and lateral domains are segregated by adherens junctions (AJ), a structure involved in cell-cell cohesion and polarity. The protein Girdin is essential for epithelial tissues morphogenesis in Drosophila melanogaster and mediates AJ stabilization. Our objective is to determine if Girdin has a role in regulating epithelial polarity. We have shown that reducing Girdin levels, but not the levels of other major AJ components, exacerbates the apicalization of epithelial cells in lgl or yrt zygotic mutants. Residual activity of Lgl and Yrt is thus repressed when girdin dosage is reduced, probably due to an over-activation of their repressor aPKC. Moreover, we observed that a reduction of girdin activity partially rescues the epithelial integrity of aPKC-depleted or hypomorphic crb mutant embryos, Crb being an apical substrate of aPKC. This suggests that Girdin represses aPKC and Crb activity. We noticed that the absence of Girdin leads to an increase of the basolateral aPKC substrates Yrt and Lgl phosphorylation levels. Therefore, in parallel to regulating the AJ complex, Girdin may modulate aPKC activity by either interacting directly with the kinase or by associating to Yrt or Lgl, two repressors of aPKC and Crb. Our results indicate that Girdin acts as a negative regulator of aPKC. This places Girdin as a hub in epithelial polarity regulation, tissue morphogenesis and tumor progression.

La protéine Girdin humaine est surexprimée dans divers types de cancers où elle contribue à la progression tumorale. Elle favorise notamment la migration et l’invasion cellulaires. Récemment, il a été montré que Girdin interagit directement avec Par-3. Cette interaction est essentielle à la polarisation des cellules en migration. Par-3 et son orthologue chez la drosophile Bazooka sont également impliquées dans l’établissement et le maintien de la polarité épithéliale et des jonctions d’adhérence (JA). La polarité épithéliale, caractérisée par la distribution asymétrique des composantes cellulaires, est nécessaire au maintien de l’intégrité des épithéliums. En effet, la perte de plusieurs régulateurs de la polarité épithéliale mène à la transition épithélio-mésenchymateuse. Nous avons donc émis l’hypothèse que Girdin est impliquée dans la polarité épithéliale et/ou l’adhésion cellule-cellule tout comme son partenaire d’interaction Par-3. Afin de vérifier notre hypothèse in vivo, nous avons généré des allèles mutants nuls de Girdin (orthologue de Girdin chez la drosophile) et établit un anticorps anti-Girdin spécifique. Tout d’abord, nous avons démontré que Girdin est essentiellement exprimée durant l’embryogenèse. Dans les épithéliums embryonnaires elle est enrichie aux JA. Ensuite, les embryons Girdin mutants présentent divers défauts morphogénétiques dont la formation de kystes ectopiques de cellules épithéliales et parfois l’ouverture de la ligne médiane ventrale, ce qui pourrait être attribuable à des défauts de cohésion cellulaire. De plus, l’association entre les composantes des JA, incluant Armadillo (β-Caténine) et DE-Cadhérine (DE-Cad), et le cytosquelette diminue dans les embryons Girdin mutants. Ces résultats suggèrent donc que Girdin est impliquée dans le maintien de la stabilité des JA. Nous nous sommes ensuite intéressés à l’implication de Girdin dans la polarité épithéliale. Bien que les mutants Girdin ne présentent aucun défaut de polarité épithéliale, nous avons pu mettre en évidence des interactions génétiques fortes entre Girdin et trois gènes codant pour des régulateurs de la polarité épithéliale : crb, yrt et lgl. Dans l’ensemble, nos résultats identifient Girdin comme un nouveau régulateur de la polarité épithéliale et de l’adhésion cellule-cellule. À l’avenir il sera essentiel de prendre en considération ces fonctions de Girdin pour évaluer s’il s’agit d’une cible thérapeutique appropriée contre le cancer. / The human Girdin protein is overexpressed in various cancers, and promotes cell migration and invasion. This suggests that Girdin contributes to tumor progression. Recently, it was shown that Girdin directly interacts with Par-3. This interaction is essential for cell polarization associated with directed cell migration. Par-3 and its Drosophila ortholog Bazooka are also known for their role in the establishment and maintenance of epithelial cell polarity and adherens junction (AJ) formation. Epithelial polarity, which is characterized by the asymmetric distribution of many cellular constituents, is necessary for epithelial tissue function and homeostasis. Indeed, loss of several epithelial polarity regulators leads to epithelial to mesenchymal transition. We thus hypothesized that Girdin plays a role in epithelial polarity and/or cell-cell adhesion, as reported for its binding partner Par-3. In order to test this premise in vivo, we generated null alleles of Girdin, which encodes the Drosophila ortholog of Girdin, and established specific anti-Girdin antibodies. First, we demonstrated that Girdin is mainly expressed during embryogenesis. In embryonic epithelial cells, it is predominantly associated with the plasma membrane and enriched at AJ. Girdin mutant embryos present several morphogenetic defects including the formation of ectopic epithelial cell cysts and opening of the ventral midline, suggesting that loss of Girdin weakens cell-cell adhesion. Consistent with this phenotype, the association between AJ components, including Armadillo (β-catenin) and DE-Cadherin (DE-Cad), and the cytoskeleton decreases in Girdin mutant animals. These results suggest that Girdin participates in AJ stability. We then investigated the implication of Girdin in epithelial polarity. Although we could not observe any epithlelial polarity defects in Girdin mutants, we found strong genetic interactions between Girdin and three genes encoding polarity regulators : crb, yrt and lgl. Together, our data identifies Girdin as a novel regulator of epithelial cell polarity and cell-cell adhesion. These results thus reveal unsuspected roles for Girdin related proteins. Comprehensive analysis of Girdin function is essential to evaluate whether it is an appropriate target to treat cancer.