Aquaculture is an essential developing sector for world food production, however the attainment of sexual maturity during commercial on-growing is a major bottleneck to industry expansion. Sexual maturation brings a commercial loss due to reduced growth performance as well as reduced immune function. Furthermore, serious concerns exist over potential genetic interaction with native stocks through broadcast spawning or spawning interaction by escapees. In the north Atlantic region, the Atlantic cod (Gadus morhua) and Atlantic halibut (Hippoglossus hippoglossus) are key aquaculture species in which industry expansion is limited by pre-harvest sexual maturation. However, through a species specific combination of modern technologies and refinement in management practices it is possible that this sexual maturation can be controlled and on-growing potential enhanced. Thus the overall aim of this thesis was to conduct novel research that will improve our understanding of the underlying mechanisms that regulate sexual maturation, whilst also advancing the optimisation of technologies for the management of maturation in cod and halibut. In Atlantic cod, owing to the inconsistent inhibition of maturation in commercial conditions, ever increasing intensities of light and in some cases narrow spectrum technologies are being used to try to combat this problem. Firstly, this PhD project investigated the potential welfare impacts of high intensity artificial lighting which have not been studied to date (Chapter 2). The work specifically investigated the effect of traditional metal halide and novel green cathode lighting on the stress response, innate immunity, retina structure, feeding activity and light perception of Atlantic cod. Results indicated that although acute responses to light were observed, there were no clear significant long term effects of any of the lighting treatments on these parameters. Regarding light perception, interestingly even when subjected to high intensity constant lighting (metal halide mean tank intensity: 16.6 watts m-2), cod still demonstrated a day/night rhythm in melatonin release which suggests perception of the overlying ambient photoperiod. The second trial of this PhD project investigated the efficacy of shading of ambient photoperiod in addition to constant lighting to inhibit maturation of cod outdoors (Chapter 3). This aimed at improving the performance of artificial lighting regimes in the open cage system during commercial on-growing by reducing the relative difference between day/night light intensities. The trial was conducted over a one year period where a low and high shade treatment were tested in outdoor tanks. Shading increased the relative night time illumination to 6.6% and 31.3% of daytime levels respectively, compared to <2% in an unshaded set-up. Both shading treatments were effective at suppressing sexual development in cod as confirmed through measurements of gonadosomatic index, histological analysis of gonadal development, oocyte diameter measurements and sex steroid profiles as well as measurements of growth. In addition to research at the applied level in Atlantic cod, this thesis has also extended to the fundamental level and explored one of the potential mechanisms relaying photoperiod signal to the endogenous regulation of sexual maturation in cod, namely the kisspeptin system (Chapter 4). Partial sequences for the signal peptide Kiss2 and its receptor Kissr4 were isolated and described showing similarity to other teleost species such as the medaka, Oryzias latipes and stickleback, Danio rerio. Novel molecular qPCR assays were designed and developed to measure the expression of both genes in male and female cod over a maturation cycle and compared to cod under constant lighting which remained immature. Interestingly, expression patterns of kiss2 and kissr4 did not reveal any clear association with season or photoperiod treatment. However, pituitary expression of gonadotropins (FSH, follicle stimulating hormone; LH, luteinising hormone) did show a differential expression in relation to treatment from early winter approximately 4-6 months after the photoperiod change. These new results are in contradiction with the hypothesis that the kisspeptin system would be involved in the initiation of gametogenesis, as shown in mammals. However, the FSH/LH data defines a window during which time kisspeptin or another GnRH stimulating mechanism must be active, this compels the need further investigation. In Atlantic halibut farming, all-female production removes the concerns of production losses through sexual maturation. Accordingly, this thesis investigated the potential/feasibility of generating monosex populations by FACS (fluorescence activated cell sorting) semen sexing based on cellular DNA content, as proven in terrestrial agriculture. Results however did not show any clear differences between the DNA of sperm in a range of species tested (Atlantic halibut, cod, sea bass, perch) suggesting that this technique may not be applicable in such species. The project also focussed on the production of a population of sex reversed halibut broodstock (neomales) that will generate, in the long term, a basis for traditional monosex population generation in the UK. Two in feed MDHT (17α-methyldihydrotestosterone) treatments were tested with the aim to reduce the use of hormone. Results were very successful with a hormone treatment of 5ppm MDHT generating a 97% phenotypic male population thus suggesting the presence of sex-reversed halibut which can be used for future monosex production. Overall, this work aimed to develop and/or refine potential remediation techniques for sexual maturation in two key commercially important farmed marine fish species, cod and halibut, as well as further our understanding on the regulation of puberty. The knowledge gained from this work provides a means to optimise the techniques employed in the industry and has the potential to increase production and profitability without compromising farmed animal welfare, thus ultimately promoting the sustainable expansion of the Atlantic cod and halibut aquaculture.

Photoperiod manipulations are widely used throughout the Atlantic salmon (Salmo salar) farming industry as a means of producing a product of uniform quality all-year round. However, farmers still remain sceptical over their effectiveness to regulate growth and maturation during the on-growing stage. Furthermore, reports of a characteristic growth-dip following light exposure suggest that light may negatively affect the physiological performance of fish in the short-term. Thus, this thesis investigates the effects of light characteristics (spectral quality, intensity and photoperiod) on growth and maturation of salmonid fish and addresses some of the uncertainties surrounding photoperiod use currently reported within the industry. Rainbow trout (Oncorhynchus mykiss) are seemingly an ideal model species for examining photoperiod effects on growth. Consequently, the application of constant light exposure (LL) at two different intensities (28W and 16W) during two different thermal conditions (summer and winter) was examined on individually tagged fish. Feed intake and growth appeared to be related to the ambient water temperature and did not appear to be affected by intensity or photoperiod, although the onset of constant light did appear to initially affect growth rate. This may indicate that LL has a limiting effect on the growth of trout or that the prevailing water temperature at which light is applied may override the photoperiodic effect. Furthermore, the lack of enhanced growth in trout exposed to LL, unlike that demonstrated for other salmonids, suggest that there may be a species-specific response to environmental variables. Thus, questions regarding photoperiod effects should be limited to the species in question. The main source of variation in results observed under photoperiod manipulations stems from the salmon industry. Atlantic salmon post-smolts were reared in seawater tanks and either maintained under a natural photoperiod (NP) or exposed to a simulated natural photoperiod (SNP), constant light superimposed on the natural light (NPLL) or constant light only (LL). Artificial light onset, irrespective of photoperiod, resulted in an apparent trend for a reduced appetite lasting up to 60 days. Furthermore, the onset of constant light resulted in a significant chronic elevation of plasma cortisol levels and changes to growth and thyroid hormone levels, providing direct evidence that constant light exposure induces stress. In addition, fish exposed to SNP failed to exhibit a stress response despite a low feed intake. However, differences in the plasma melatonin levels during twilight times, as compared to NP, suggest that gradual changes in the natural light intensity throughout the day, particularly around dawn and dusk, may be important for synchronizing daily events. No differences in growth were observed between the NP and NPLL regimes, although fish reared in an enclosed regime (SNP and LL) exhibited a significantly lower weight gain than fish in an open environment (NP and NPLL). This further highlights the impact that the rearing environment has on the growth performances of fish and the need for commercially run trials. Advances in lighting technologies and a greater understanding of how light is transformed through the water column have focussed research on the spectral sensitivity of fish. Therefore the lighting efficiency of novel blue narrow bandwidth LED lighting units through the water column and their effects on growth and maturation performances of salmon reared in commercial production cages were compared against the standard metal halide units currently utilized throughout the industry. LL application, irrespective of intensity or spectrum, reduced the numbers of fish maturing as compared to fish reared under a natural photoperiod. However, this was greatest under the standard metal halide units reflecting a greater light penetration and perception as determined by plasma melatonin levels. The metal halide groups exhibited the greatest relative weight gain over the trial period as compared to control fish. No evidence was observed for a growth-dip under metal halide light, although blue lit treatments exhibited an initial significant reduction in food consumption, suggesting a possible welfare issue. Nevertheless, the prototype blue LED units showed possible potential for commercial application by penetrating the water depth at half the distance of the metal halide units for only one eighth the power and one fifth the brightness. However, further tests of these prototype spectral units are required to examine the potential welfare and physiological growth and reproductive effects. These studies have shown that the efficacy of artificial light regimes is largely dependent upon the effectiveness of the light source through the underwater environment and its perception by fish, providing a sufficient intensity is emitted exceeding the physiological threshold level for the species cultured. Moreover, whilst the onset of artificial light may elicit a stress response and demonstrate a trend for a suppression of appetite for salmon reared in experimental tanks, no compelling evidence for a suppression of appetite or growth was found under normal commercial cage conditions. This suggests that the growth-dip observed within the industry may in part be a combination of a physiological response to the onset of light further exaggerated by the farmer’s perception and altered judgement in feeding. In addition, the results obtained from this study have helped to standardize the use of light regimes within the industry. Nevertheless, further studies are necessary to fully elucidate the underlying mechanisms which may govern growth and maturation in fish following the onset of light exposure.

Antibodies have become indispensable tools in diagnostics, research and as therapeutics. There are several strategies to generate monoclonal antibodies (mAbs) in order to avoid the drawbacks of polyclonal antibodies (pAbs) for therapeutic use. Moreover, the growing interest in precision medicine requires a well-characterized target and antibody to predict the responsiveness of a treatment. This thesis describes the use of epitope information and display technologies to generate and characterize antibodies. In Paper I, we evaluated if the epitope information of a well-characterized pAb could be used to generate mAbs with retained binding characteristics. In Paper II, the epitope on the complement protein C5 towards Eculizumab was mapped with surface display, the results of which explained the non-responsiveness of Eculizumab treatment among a patient group due to a mutated C5 gene. With this in mind, we showed efficacy in treatment of the mutated C5 variants using a drug binding to another site on C5, suggesting that our approach can be used to guide treatment in precision medicine. In Paper III, a Gram-positive bacterial display platform was evaluated to complement existing platforms for selection of human scFv libraries. When combined with phage display, a thorough library screening and isolation of nano-molar binders was possible. In Paper IV, a solid phase method for directed mutagenesis was developed to generate functional affinity maturation libraries by simultaneous targeting of all six CDRs. The method was also used to create numerous individual mutants to map the paratope of the parent scFv. The paratope information was used to create directed libraries and deep sequencing of the affinity maturation libraries confirmed the viability of the combination approach. Taken together, precise epitope/paratope information together with display technologies have the potential to generate attractive therapeutic antibodies and direct treatment in precision medicine. / <p>QC 20170418</p>

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

In vitro, meotic maturation of porcine oocytes and cumulus expansion are induced by FSH and EGF-like peptides AREG and EREG. FSH and EGF-like peptides induce expression of cumulus expansion-related genes (HAS2, PTGS2 and TNFAIP6). To define signaling pathways that control FSH- and AREG-induced cumulus expansion, porcine cumulus-oocyte complexes were treated with specific protein kinase inhibitors. Inhibitors of MAPK3/1, MAPK14 and ERBB1 significantly reduced both FSH- and AREG-induced expression of HAS2, PTGS2 and TNFAIP6. These inhibitors decreased FSH/LH-induced expression of AREG and EREG in mural granulosa cells. Surprisingly, inhibitor of PKA had no effect on AREG expression in cumulus-oocyte complexes but the inhibitor decreased expression of TNFAIP6 induced by AREG. Inhibitor of PI3K increased expression levels of AREG and PTGS2 but EREG, HAS2 and TNFAIP6 were reduced. Expression levels of the cumulus expansion-related genes were not affected by an analog of cGMP (8-CPT-cGMP). However, 8-CPT-cGMP blocked spontaneous in vitro meiotic maturation of porcine oocytes and its effect was abolished by FSH. Key words: cumulus expansion, cumulus expansion-related genes, meotic maturation, FSH, amphiregulin, cGMP

Title: The Level of Postural Maturation of Children in Athletic-preparatory Schools Author: Terezie Janečková Objective: There are more and more athletic-preparatory schools in athletic units and clubs nowadays. Athletic-preparatory school means a group of five or six-year-old children with one or two training lessons per week. Czech athletic association wants to spread an interest in athletics among parents and their children, to find athletic talents as soon as possible and develop them by this way. The recruitment of children at this age already exists in many sports, but it is new in athletics in the Czech Republic. But that does not go for everyone that this sport training at preschool age is the appropriate and healthy way for a child motor development. I would like to evaluate the influence of athletic-preparatory schools on the child motor development. Due to the fact that there are no tests to apply for this purpose in literaure, we have to compose our own set of tests, which are used in physiotherapy. The theoretic part of the work is a bibliographic search of accessible information about postural maturation and motor development. The experimental part of the work is an evaluation of the level of postural maturation of children in athletic-preparatory schools before their start of regular...

4 Abstract The antral follicle provides a specialized microenvironment or niche, which is necessary for production of high quality oocyte. The developmental competence of bovine oocyte is influenced by the follicle size. Oocytes originated from medium or larger follicles (≥6 mm) have greater developmental competence (ability to develop to the blastocyst stage). The changes in cytoplasmic factors, for example mRNAs, could explain differences in oocyte developmental potential. Using Bovine Oligonucleotide microarrays the differences in gene expression profiles of oocytes at germinal vesicle and MII stages from medium (MF, 6-10 mm) or small (SF, 2-5 mm) follicles were characterized. The aim was to find differencies between oocytes diverse developmental competence. The expression fold change between the two experimental groups was in 61 genes. Subsets of 15 differentially expressed genes were validated by quantitative RT-PCR. Before maturation, significant differences were confirmed at the level of ATP5C1, MAP3K13, MTRF1L, TAF1A and UBL5. Subpopulations of oocytes were classified according to atresia of cumulus cells and follicle size. We determined the level of 12 individual transcripts after maturation. ATP5F1 remained stable in all experimental groups of oocytes. The level of BRD7 transcript remained stable...

Mycobacterium tuberculosis (Mtb) is the pathogen causing tuberculosis (TB), a disease most often affecting the lung. 1.5 million people die annually due to TB, mainly in low-income countries. Usually considered a disease of the poor, also developed nations recently put TB back on their agenda, fueled by the HIV epidemic and the global emergence of drug-resistant Mtb strains. HIV-coinfection is a predisposing factor for TB, and infection with multi-drug resistant and extremely drug resistant strains significantly impedes and lengthens antibiotic treatment, and increases fatality. Mtb is transmitted from a sick individual via coughing, and resident macrophages are the first cells to encounter the bacterium upon inhalation. These cells phagocytose intruders and subject them to a range of destructive mechanisms, aiming at killing pathogens and protecting the host. Mtb, however, has evolved to cope with host pressures, and has developed mechanisms to submerge macrophage defenses. Among these, inhibition of phagosomal maturation and adaptation to the intracellular environment are important features. Mtb profoundly alters its phenotype inside host cells, characterized by altered metabolism and slower growth. These adaptations contribute to the ability of Mtb to remain dormant inside a host during latent TB infection, a state that can last for decades. According to recent estimates, one third of the world’s population is latently infected with Mtb, which represents a huge reservoir for active TB disease. Mtb is also intrinsically tolerant to many antibiotics, and adaptation to host pressures enhances tolerance to first-line TB drugs. Therefore, TB antibiotic therapy takes 6 to 9 months, and current treatment regimens involve a combination of several antibiotics. Patient noncompliance due to therapeutic side effects as well as insufficient penetration of drugs into TB lesions are reasons for treatment failure and can lead to the rise of drug-resistant populations. In view of the global spread of drug-resistant strains, new antibiotics and treatment strategies are urgently needed. In this thesis, we studied the interplay of the primary host cell of Mtb, human macrophages, and different Mtb phenotypes. A low-burden infection resulted in restriction of Mtb replication via phagolysosomal effectors and the maintenance of an inactive Mtb phenotype reminiscent of dormant bacteria. Macrophages remained viable for up to 14 days, and profiling of secreted cytokines mirrored a silent infection. On the contrary, higher bacterial numbers inside macrophages could not be controlled by phagolysosomal functions, and intracellular Mtb shifted their phenotype towards active replication. Although slowed mycobacterial replication is believed to render Mtb tolerant to antibiotics, we did not observe such an effect. Mtb-induced macrophage cell death is dependent on ESAT6, a small mycobacterial virulence factor involved in host cell necrosis and the spread of the pathogen. Although well-studied, the fate of ESAT6 inside infected macrophages has been enigmatic. Cultivation of Mtb is commonly carried out in broth containing detergent to avoid aggregation of bacilli due to their waxy cell wall. Altering cultivation conditions revealed the presence of a mycobacterial capsule, and ESAT6 situated on the mycobacterial surface. Infection of macrophages with this encapsulated Mtb phenotype resulted in rapid ESAT6-dependent host cell death, and ESAT6 staining was lost as bacilli were ingested by macrophages. These observations could reflect the earlier reported integration of ESAT6 into membranes followed by membrane rupture and host cell death. In conclusion, the work presented in this thesis shows that the phenotype of Mtb has a significant impact on the struggle between the pathogen and human macrophages. Taking the bacterial phenotype into account can lead to the development of drugs active against altered bacterial populations that are not targeted by conventional antibiotics. Furthermore, deeper knowledge on Mtb virulence factors can inform the development of virulence blockers, a new class of antibiotics with great therapeutic potential.

This dissertation thesis focuses on creating tools for the analysis and potential therapeutic intervention in the biological processes regulated by proteolysis. I focus on two important proteolytic enzymes: HIV-1 protease, which is indispensable for the polyprotein processing of the nascent virus and thus for the development of infectious viral particle, and glutamate carboxypeptidase II, a tumor marker and a neuropeptidase from the prostate and central nervous system. Rational design of inhibitors of these therapeutically relevant enzymes serves two purposes: firstly, protease inhibitors were shown to be powerful drugs (HIV protease is in fact the example of successful drug development driven by structural biology). Secondly, and in the context of this thesis perhaps more importantly, inhibitors of medicinally relevant proteases might serve as tools for the elucidation of basic biological questions concerning regulation, timing and spatiotemporal control of such key processes as virus maturation or cancer development. The experimental work described in this thesis summarizes my results in both these areas. Human Immunodeficiency Virus Protease Human immunodeficiency virus (HIV), a causative agent of AIDS, has been estimated to kill close to 40 million people during the past four decades with 1.5...

During early embryonic development oocytes are arrested in prophase I of the first meiotic division, in which they can persist for years. After reaching sexual maturity and the luteinizing hormon surge resumption of meiosis and meiotic maturation occur. Oocytes are arrested again at metaphase of the second meiotic division. At this stage they are ovulated and waiting for a fertilisation. Oocytes are during their development exposed to factors that cause DNA damage, of which DNA double-strand breaks (DSBs) are the most serious threat. The maintaining of genome integrity is crucial for quality of oocytes, fertility and proper embryonic development. The mechanism of the oocyte response to DSBs presence is not fully understood and it seems to differ from somatic cells. We assume that DSBs are repaired during meiotic maturation probably by a mechanism of homologous recombination (HR). In this thesis we focuse on essencial recombinase RAD51, which participates in the repair by HR. We found that RAD51 inhibition leads to an increase of segregation errors in anaphase I. Using high resolution live cell imaging we observed chromosomal fragments and anaphase bridges. Immunofluorescence detection of DSBs-marker γH2AX showed increased amount of DSBs in prophase I and MII stage after RAD51 inhibition. Our data...