Dans le but d’étudier le rôle fonctionnel des neuropeptides chez la drosophile, nous avons essayé d’utiliser le récepteur µ aux opioïdes (MOR) afin d’inhiber temporairement la libération de neuropeptides. Cependant, quand nous l’avons exprimé dans les cellules endocrines produisant l’hormone adipokinétique (AKH), le récepteur MOR a présenté une activité constitutive empêchant tout contrôle dans le temps. Nous proposons d’utiliser les récepteurs RASSL (Receptors Activated Solely by Synthetic Ligands) développés chez les vertébrés. Ces récepteurs, activés uniquement par des ligands synthétiques, ont été modifiés afin qu’ils soient dénués d’activité constitutive. Afin de tester rapidement l’efficacité d’un récepteur RASSL inhibiteur chez la drosophile, nous avons cherché une alternative à la mesure des taux de tréhalose et de glycogène (démontrant la libération d’AKH) qui en plus d’être laborieuse produit des résultats variables. L’hormone GPA2/GPB5, récemment découverte, est particulièrement intéressante, en effet, elle semble avoir un rôle antidiurétique. Nous avons généré des lignées transgéniques exprimant la protéine Gal4 dans les cellules GAP2/GPB5 et nous avons montré que l’ablation génétique de ces cellules compromet la survie suggérant qu’elles pourraient représenter un bon système pour tester l’efficacité du RASSL inhibiteur. Ensuite nous nous sommes intéressés au contrôle de la libération du neuropeptide AKH qui a un rôle fonctionnel homologue à celui du glucagon des vertébrés. Malgré quelques résultats encourageants, nous n’avons pas pu confirmer le rôle inhibiteur de l’AstA sur la libération de l’AKH. Enfin nous nous sommes intéressés au réseau qui contrôle le comportement de cour. Nous avons généré des lignées transgéniques exprimant la protéine Gal4 sous la dépendance du promoteur potentiel du récepteur du neuropeptide SIFamide (SIFR) et nous avons démontré que ce neuropeptide contrôle le comportement de cour via son récepteur SIFR en agissant, du moins en partie, sur les neurones fruitless. / In order to study the functional roles of neuropeptides in Drosophila, I attempted to use the µ opioïd receptor (MOR) to temporarily inhibit liberation of neuropeptides. However, when expressed in the AKH (adipokinetic hormone) endocrine cells of Drosophila, MOR turned out to be constitutively active, making it impossible to use it as envisioned. Others have encountered and subsequently solved similar problems for the so-called RASSL (Receptors Activated Solely by Synthetic Ligands) in vertebrates. As the bioassay for testing release of AKH is cumbersome, time-consuming and variable, I searched for an alternative neuroendocrine system to test the efficiency of an inhibitory RASSL in Drosophila. The recently discovered GPA2/GPB5 seemed particularly attractive, as it is likely an antidiuretic hormone. Transgenic flies expressing Gal4 in the GPA2/GPB5 were produced and genetic ablation of these cells severely compromised survival, suggesting that this might indeed be good model system. Attempts so demonstrate a physiological role of allatostatin A in the release of AKH yielded inconclusive results. In order to study the role of the neuropeptide SIFamide, which modulates sexual behavior in Drosophila, I generated transgenic flies expressing Gal4 under dependence of potential promoter of this neuropeptide receptor (SIFR) and showed that SIFamide controls courtship at least in part by acting directly on fruitless neurons.

The robust behavioural courtship ritual displayed by Drosophila melanogaster males is governed by their underlying nervous system (NS). Two key genes of the Sex Determination Hierarchy, fruitless (fru) and doublesex (dsx), determine most neuronal substrates for sexual behaviour. In this study we aim to better understand the role fru plays in determining these neural substrates, as a means of better understanding the relationships between brain, behaviour and genes, and thus how the development of neuronal networks shape innate and species-specific behaviours. Fru has two major functions: control of male sexual behaviour, and viability in both sexes. Alternative splicing of fru produces transcription factors driven by four promoters: P1 transcripts are sex-specifically spliced (only viable in the male), and P2-4 transcripts are crucial to both sexes survival. The resulting proteins contain a BTB protein-protein interaction domain at the N-terminus, and one of four C-terminal zinc-finger (ZnF) DNA binding domains. Male-specific proteins (FruM) contain an additional 101 amino acid N-terminal domain, and one of three alternative C2H2 ZnF domains (FruMA, FruMB, FruMC). These male-specific isoforms are expressed in the central nervous system (CNS) beginning in the late stage larvae (L3), peaking during pupation and on into adulthood. Little is known, however, about the roles of the individual isoforms, and no clear transcriptional targets have been identified. The central aims of this thesis are to document the wild-type expression patterns of Fru isoforms throughout development in the CNS, create and characterise isoform-specific mutants, and to identify and evaluate putative transcriptional targets of Fru. Combining these findings will lead to a better understanding of the underlying molecular functions of individual FruM isoforms, as a means to understanding their roles in sexual behaviour. Expression analysis of FruM isoforms throughout development in the NS is described. To further characterise the role of individual FruM isoforms, isoform-specific mutants in fruA and fruB exons were generated using site-specific homologous recombination (HR). These novel mutants were validated by PCR and Fru isoform-specific antibody stainings. Mutants were analysed in fruM- and fru-null genetic backgrounds, to distinguish the roles of sex-specific vs. common isoforms. These analyses included: fertility, viability and morphology. FruA was found to have a role in wing extension with possible repercussions for song production. FruB was found to be developmentally lethal, in addition to having defects in male courtship behaviour. To understand the role of fru in the NS, downstream transcriptional targets of FruM isoforms were identified. DNA adenine methyltransferase identification (DamID) was used to identify putative transcription targets of FruM isoforms. The Dam protein methylates DNA in Drosophila in a sequence-specific manner allowing targets of Fru to be isolated. Candidate genes were identified using computational analysis (including gene ontology, peak analysis and motif analysis) along with a biologically significant connection with fru. The relationship between fru and six candidate genes were characterised using RNAi. The results of these studies advance our knowledge of how FruM isoforms influence the development and physiology of the NS underlying male sexual behaviour in Drosophila.

Submitted by Aelson Maciera (aelsoncm@terra.com.br) on 2017-05-16T18:52:20Z No. of bitstreams: 1 DissMRS.pdf: 2504914 bytes, checksum: 996d118fd641f9218c27c0deeefbb963 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-05-18T20:19:58Z (GMT) No. of bitstreams: 1 DissMRS.pdf: 2504914 bytes, checksum: 996d118fd641f9218c27c0deeefbb963 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-05-18T20:20:07Z (GMT) No. of bitstreams: 1 DissMRS.pdf: 2504914 bytes, checksum: 996d118fd641f9218c27c0deeefbb963 (MD5) / Made available in DSpace on 2017-06-01T13:02:45Z (GMT). No. of bitstreams: 1 DissMRS.pdf: 2504914 bytes, checksum: 996d118fd641f9218c27c0deeefbb963 (MD5) Previous issue date: 2011-10-28 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / The fruit flies (Anastrepha sp) are organisms endemic to the Neotropics that are similar morphologically but present some behavioral, reproductive and molecular variations. Among these insects we can highlight the fraterculus group, which consists of a set of cryptic species, between them Anastrepha oblique, that represents threats to national fruit culture. The effective control of this grouping requires the expansion of the few studies concerning to the biology and history of populations which has a very complex systematic. Previous works suggest a high rate of evolution of genes related to reproductive issues and recognition of partners due to the high selective pressures involved in that relationship, hence, we chose to work with the fruitless gene which is recognized for its role in sexual differentiation and male courtship behavior in dipteran insects. Since a small region of this gene was already been amplified in our laboratory, we amplified and described the flanking sequences by RACE-PCR technique which used primers constructed targeting the 5 'and 3' ends. We used cDNA libraries produced from the extraction of mRNAs from different stages of development of these organisms enabling the identification of different isoforms arising from alternative splicing and structural characterization of the coding region (CDS) common to both sexes. We compared the data generated with the corresponding sequences of the fruitless gene in other insect species identifying the rate of similarity for nucleotides and amino acids (identity ratio) which revealed that the éxons coding for specific domains such as BTB and Zinc Fingers tend to remain more conserved in relation to other regions and éxons from Connector Region. Subsequently, we performed positive selection tests through the Relaxed Branch-site test and Strict Branch-site test, which were contrasted by Likelihood Ratio Tests (LRT) which identified 25 new sites evolving under positive selection, corroborating the findings of Sobrinho e de Brito (2010) and suggesting the presence of these forces on specific regions of this important gene linked to reproductive aspects of several major groups of insects. / As moscas-das-frutas (Anastrepha sp) são organismos endêmicos da região neotropical que apesar de serem semelhantes morfologicamente distinguem-se por variações comportamentais, reprodutivas e moleculares. Entre esses insetos podemos destacar o grupo fraterculus o qual é composto por um conjunto de espécies crípticas, entre elas Anastrepha obliqua, que representam ameaças à fruticultura nacional. O controle eficiente deste agrupamento exige a ampliação dos ainda escassos estudos referentes à biologia e história das populações cuja sistemática tem se mostrado bastante complexa. Trabalhos anteriores sugerem uma alta taxa evolutiva agindo sobre genes relacionados a aspectos reprodutivos e reconhecimento de parceiros devido as elevadas pressões seletivas envolvidas nessa relação, para tanto, escolhemos trabalhar com o gene fruitless por sua reconhecida importância na diferenciação sexual e comportamento de corte nos machos nos insetos dípteros. Uma vez que dispúnhamos de uma pequena região já amplificada deste gene em nosso laboratório, amplificamos as sequências flanqueadoras através da técnica de RACE-PCR que utilizou primers construídos e orientados para as extremidades 5´ e 3´. Foram utilizadas bibliotecas de cDNAs produzidas a partir da extração de mRNAs de diferentes estágios de desenvolvimento desses organismos possibilitando a identificação de diferentes isoformas provenientes de splincing alternativo e caracterização estrutural da região codificadora (CDS) comum a ambos os sexos. Comparamos os dados gerados com as sequencias correspondentes do gene fruitless em outras espécies de insetos identificando sua taxa de identidade nucleotídica e de aminoácidos as quais revelaram que os éxons que codificam domínios específicos como BTB e Zinc Fingers tendem a se manter mais conservados em relação às demais regiões e aos éxons da Região Conectora. Posteriormente, realizamos testes de seleção positiva conhecidos como Relaxed Branch-site test e Strict Branch-site test, os quais foram contrastados pelo Likelihood Ratio Test e possibilitaram a identificação de 25 novos sítios evoluindo sob seleção positiva, corroborando as descobertas de Sobrinho e de Brito (2010) e sugerindo a atuação dessas forças sobre regiões específicas deste importante gene ligado aos aspectos reprodutivos de diversos grupos principais de insetos.

Orientation: Over the years, the increase in executive remuneration in both the private sector and state-owned entities (SOEs) has been the subject of intense discussions. The poor performance of some SOEs with highly remunerated executives begs the question whether chief executive officers (CEOs) in South African SOEs deserve the high levels of remuneration they receive. Research purpose: The main purpose of the study was to determine whether there is a relationship between CEOs’ remuneration and company performance in South Africa’s Schedule 2 SOEs. Motivation for the study: A greater understanding of the relationship between CEO remuneration and organisational performance would expand knowledge when developing optimal CEO remuneration systems to ensure sustainability of SOEs in the South African context. If a relationship exists, it could justify the high remuneration received by CEOs. Research design, approach, and method: This quantitative, longitudinal study, conducted over a nine-year period, collected secondary data from the annual reports of 18 Schedule 2 SOEs. The primary statistical techniques used in the study included were OLS multiple regression analysis and correlational analysis on a pooled dataset. Main findings/results: The primary finding was that there is a relationship between CEO remuneration and company performance (mainly an inverse relationship), with no consistent trend between the constructs. Turnover appears to be an important component, as it was the most stable measure of company performance during the study period. The results indicate that the CEOs’ remuneration continued to increase, even when the SOEs were performing poorly. Practical managerial implications: Since the study focused on the relationship between CEOs’ remuneration and company performance, it may aid policymakers in forming new rules and regulations that would help improve the country’s economic performance while attracting international investors. Contribution/value-add: The study provides new knowledge to the limited research available on SOEs in South Africa. Further, this research focused on three different components of CEOs’ remuneration, thereby shedding more light on the relationship between their remuneration and company performance. / Business Management

Magister Legum - LLM / Directors of companies are the forerunners in overseeing and strategically managing a company.1 The Companies Act 71 of 2008 (the Companies Act) gives the board of directors the legislative obligation for a company to be managed by or under the direction of the board of directors.2 The board of directors have a central role in the decision making and operation of a company; this position also applies to the board of directors of State owned Companies (SOC). This dissertation explores methods to hold directors of SOCs personally liable for irregular, wasteful and fruitless expenditure Irregular expenditure is defined as expenditure that does not comply with the provisions of the Public Finance Management Act 1 of 1999 (PFMA), the State Tender Board Act 86 of 1968 or any legislation that provides for provincial government procedure.3 Fruitless and wasteful expenditure is defined as ‘expenditure which was made in vain and would have been avoided had reasonable care has been exercised’.This research aims to analyse legislative mechanisms put in place that hold directors of SOCs personally liable for irregular, reckless, wasteful and fruitless expenditure. Section 77(2)(b) and 218(2) of the Companies Act contains the legislative basis for the personal liability of directors of SOCs for irregular, wasteful and fruitless expenditure.

Orientation: Over the years, the increase in executive remuneration in both the private sector and state-owned entities (SOEs) has been the subject of intense discussions. The poor performance of some SOEs with highly remunerated executives begs the question whether chief executive officers (CEOs) in South African SOEs deserve the high levels of remuneration they receive. Research purpose: The main purpose of the study was to determine whether there is a relationship between CEOs’ remuneration and company performance in South Africa’s Schedule 2 SOEs. Motivation for the study: A greater understanding of the relationship between CEO remuneration and organisational performance would expand knowledge when developing optimal CEO remuneration systems to ensure sustainability of SOEs in the South African context. If a relationship exists, it could justify the high remuneration received by CEOs. Research design, approach, and method: This quantitative, longitudinal study, conducted over a nine-year period, collected secondary data from the annual reports of 18 Schedule 2 SOEs. The primary statistical techniques used in the study included were OLS multiple regression analysis and correlational analysis on a pooled dataset. Main findings/results: The primary finding was that there is a relationship between CEO remuneration and company performance (mainly an inverse relationship), with no consistent trend between the constructs. Turnover appears to be an important component, as it was the most stable measure of company performance during the study period. The results indicate that the CEOs’ remuneration continued to increase, even when the SOEs were performing poorly. Practical managerial implications: Since the study focused on the relationship between CEOs’ remuneration and company performance, it may aid policymakers in forming new rules and regulations that would help improve the country’s economic performance while attracting international investors. Contribution/value-add: The study provides new knowledge to the limited research available on SOEs in South Africa. Further, this research focused on three different components of CEOs’ remuneration, thereby shedding more light on the relationship between their remuneration and company performance. / Business Management / PhD (Management Studies)