De études récentes suggèrent une forte relation entre susceptibilité au paludisme et réponse allergique. Des niveaux élevés d’histamine plasmatique et tissulaire ont été associés à la sévérité de la maladie chez l’homme infecté par P. falciparum et dans de nombreux modèles animaux. Histamine releasing factor (HRF), une protéine pro-inflammatoire libérant l'histamine au cours des processus allergiques, est produite par le parasite au cours des infections palustres modérées et sévères, suggérant l’implication du HRF dans l’altération des réponses immunitaires et dans la pathogenèse. Les objectifs de ce travail consistaient à évaluer le rôle de la protéine parasitaire HRF dans le développement de la réponse immunitaire et à déterminer si son expression est associée à la sévérité de la maladie en étudiant deux parasites murins, PbANKA et PbNK65, déficients pour la protéine HRF (hrfΔ). Les souris infectées avec des sporozoïtes PbANKA-hrfΔ ont montré une diminution de la fréquence du neuropaludisme associée à un déficit du développement des parasites mutants au cours du stade hépatique et à une augmentation précoce systémique d’IL-6. En outre, l'infection par les parasites PbNK65-hrfΔ est caractérisée par l’élimination du parasite conduisant à une protection durable et au développement d’une mémoire immunitaire caractérisée par une augmentation d’IL-6, une diminution de l’expression de PD-1 sur les cellules T et une amélioration de la phagocytose dépendante des anticorps, confirmant l'importance de la protéine HRF dans la virulence du parasite. HRF est le premier gène de parasite Plasmodium dont l’effet direct sur la réponse immunitaire de l’hôte est démontré. / Recent findings have raised the hypothesis that clinical susceptibility to malaria may be related to allergy-type response. In human infection with P. falciparum, as well as in murine models of malaria, increased levels of histamine have been shown to be associated with disease severity. Histamine releasing factor (HRF), shown to be implicated in the release of pro-inflammatory histamine during late-phase allergy, was demonstrated to be produced by the parasite during mild and severe malaria infections suggesting that Plasmodium HRF may affect host immune responses and contributes to the pathogenesis. The objectives of this work were to evaluate the role of Plasmodium HRF in the development of the immune response and to determine whether its expression is associated with the severity of malaria disease by studying two HRF-deficient (hrfΔ) murine parasites (PbANKA and PbNK65). Infection with PbANKA-hrfΔ sporozoites showed a decrease in the frequency of ECM due to the impairment of the development of the mutant parasites in liver stages as a consequence of the up-regulation of IL-6. Infection with PbNK65-hrfΔ parasites confirmed the importance of HRF in enhancing the virulence of the parasite. Indeed, PbNK65-hrfΔ infection results in parasite clearance leading to a long-lasting protection and immune memory as reflected by an up-regulation of IL-6, a down-regulation of PD-1 expression on T cells and in the enhancement of Ab-mediated phagocytosis. HRF is the first parasite gene which directly modulates the host immune response.

The growth and development of an organism requires the coordinated actions of many factors. During development individual cells undergo proliferation, migration and differentiation to form the adult organism. Two structurally related members of the glucagon superfamily, growth hormone releasing hormone (GRF) and pituitary adenylate cyclase-activating polypeptide (PACAP), are thought to modulate vertebrate development. In mammals, GRF modulates the development of pituitary somatotrophs and the release of fetal growth hormone. In contrast, PACAP appears to have a more general role during development. PACAP may be involved in the patterning of the embryonic axis and in the development of the neural tube. The objectives of my study were to isolate GRF, PACAP and their receptors from the zebrafish, characterize their expression in the developing embryo and adult embryo and examine the role of PACAP during brain development. To study the role of GRF and PACAP, I isolated a genomic clone encoding the GRF and PACAP peptides from the zebrafish genomic library and characterized its gene copy number and adult tissue expression pattern. The GRF-PACAP gene isolated from the zebrafish was comprised of five exons with the GRF peptide encoded on the fourth exon and the PACAP peptide encoded on the fifth exon. This gene structure is similar to that found in other non-mammalian vertebrates and supports the hypothesis that the gene duplication leading to the encoding of the GRF and PACAP peptides on separate genes occurred later in evolution. In addition, the zebrafish genome was found to contain only one copy of the GRF-PACAP gene. The GRF-PACAP gene was widely expressed in the adult zebrafish in tissues developmentally derived from all three germ layers, suggesting that the gene may be widely expressed in the embryo as well. To examine the functional significance of the co-expression of GRF and PACAP in zebrafish, I isolated the GRF and PACAP receptors and characterized their expression pattern. I isolated three distinct cDNAs from zebrafish encoding the GRF receptor, the PACAP specific PAC1 receptor and the shared vasoactive intestinal peptide/PACAP receptor VPAC1. In addition, four isoforms of the PAC1 receptor were isolated from zebrafish including a novel isoform found in the gill. All three receptors were widely expressed in adult zebrafish and receptors for both GRF and PACAP were found in most tissues. This indicates that GRF and PACAP may modulate each other’s function. To determine the developmental role of GRF and PACAP, I characterized the expression pattern of the GRF-PACAP gene and the GRF, PAC1 and VPAC1 receptors in the zebrafish embryo. The GRF and PAC1 receptors are the earliest to be expressed in development starting at the cleavage stage. Later, the GRF-PACAP gene and the VPAC1 receptor are first expressed at the late blastula/early gastrula stage in the zebrafish and are expressed throughout the developmental period. Strong expression of the GRF, PACAP and their receptors during mid gastrulation indicates that these peptides may be involved in modulating the formation of the embryonic axis. During the segmentation period the GRF-PACAP gene is widely expressed in the zebrafish embryo and the PAC1 receptor short and hop isoforms are differentially expressed. Therefore, PACAP may regulate cell cycle exit or cell proliferation through activation of different PAC1 receptor isoforms during the segmentation stage. In the subsequent pharyngula period, the GRF-PACAP transcript is localized mainly to the hatching gland. However, expression is seen also in tissues that undergo differentiation during this stage. Therefore, the timing of the expression of the GRF-PACAP gene indicates that it may be involved in early patteming events and promoting cell cycle exit prior to differentiation. To investigate the role of GRF and PACAP in the developing brain, I localized the expression of GRF, PACAP and the PAC1 receptor in neuroblasts derived from an embryonic day 3.5 chick. PACAP was found to stimulate the cAMP pathway in these cells, indicating that PACAP may modulate brain development. This work indicates that GRF and PACAP play an important role in vertebrate development. / Graduate

Growth hormone-releasing hormone (GRF) and pituitary adenylate cyclase-activating polypeptide (PACAP) are members of the PACAP/Glucagon superfamily. The family is proposed to have developed from an ancestral PACAP-like molecule in invertebrates. Through successive exon, gene and genome duplications the family has grown to include seven other members. In mammals GRF and PACAP are located on different genes, but in fish, amphibians and birds they are located on the same gene. The main function of GRF is the release of growth hormone (GH) from the pituitary. Also, during development GRF influences the fetal pituitary and stimulates GH release during late gestation. In contrast, the functions of PACAP are extremely varied. PACAP is the newest member of the superfamily and there is still much work to be done before its actions are well understood. Like GRF, PACAP is a releasing hormone acting on the pituitary and in addition, the adrenal gland, pancreas and heart, as well as other organs. Also, PACAP regulates smooth muscle in the vascular system, gut, respiratory tract and reproductive tract During development PACAP affects proliferation, differentiation and apoptosis. GRF and PACAP are expressed throughout development in fish, beginning during the blastula period in rainbow trout and at the end of gastrulation in zebrafish (earliest stage examined). In rainbow trout the grf/pacap gene is expressed as two transcripts, a short and a long transcript. The short transcript is produced by alternative splicing of the gene and does not include the fourth exon which codes for GRF. The long transcript includes the coding regions for both GRF and PACAP. By this means PACAP can be regulated separately from GRF. With the extensive role PACAP appears to play in development, separate regulation of the hormone may be necessary. Expression of the grf/pacap gene in zebrafish is widespread early in development and gradually becomes localized. Of particular interest is the expression of the grf/pacap transcript in regions associated with the prechordal plate, an important organizing center in development. Although it is not yet confirmed, there is evidence to suggest GRF and PACAP are expressed in the prechordal plate and its derivatives in the gut and hatching gland. In addition, expression of the grf/pacap transcript is observed in the neuroectoderm (eye, brain and spinal cord) and the developing heart. Considering the expression pattern of GRF and PACAP, I propose that one of both of these hormones may be involved in patterning during vertebrate embryogenesis. The evolution of gene families is thought to occur through successive exon, gene and genome duplications. Duplicate exons or genes become differentiated and eventually gain new functions or become functionless. During evolution of the grf/pacap lineage, several duplication events have occurred. Analysis of rainbow trout leads me to think that this fish and other salmonids possess two copies of the grf/pacap gene. This is not unexpected considering the tetraploid nature of salmonids. Present day mammals encode GRF and PACAP on separate genes. At some point during the evolution of this lineage a duplication event has occurred, possibly in early mammals or prior to the divergence of birds. The study of multigene families is a useful way to understand evolutionary processes. To this end I examined three members of multigene families from sockeye salmon. Therefore, in addition to the evolutionary mechanisms and pathways that directed grf/pacap gene evolution, I examined the ferritin-H subunit, the alpha-tubulin subunit and the beta-globin subunit. These cDNA sequences are similar to their counterparts in other teleost. The evolution of the ferritin gene family is particularly interesting because it involves the addition or deletion of DNA sequences that affect regulation and cytosolic location. / Graduate

Call number: LD2668 .T4 1984 S585 / Master of Science

A series of experiments were conducted to investigate the regulatory mechanisms governing the absence of and the initiation of pulsatile pituitary LH secretion during the infantile and prepubertal periods of development in the bull calf. In the first experiment, patterns of hypothalamic GnRH secretion into hypophyseal portal vessels were measured in sixteen Holstein bull calves at 2, 5, 8 and 12 weeks of age. The results of this study correlated the attainment of an hourly rate of pulsatile GnRH release to the onset of prepubertal LH secretion. In the second experiment, nine Holstein bull calves were infused with exogenous GnRH (200 ng) on an hourly basis from 1 to 6 weeks of age. From this experiment, we were able to demonstrate that an hourly rate of pulsatile GnRH release stimulates the age-associated changes in the hypothalamic-pituitary axis necessary for initiating pituitary LH secretion during the transition from the infantile to prepubertal period of development.

Pineal-mediated depressions in prolactin cell activity after light deprivation were studied in the male and female Golden Syrian hamster. Prolactin cell activity was determined by measuring radioimmunoassayable prolactin, newly synthesized prolactin and prolactin mRNA levels in the pituitary. Serum prolactin was also measured by radioimmunoassay. Use of the recombinant DNA plasmid, pPRL-1, which contains the rat prolactin complimentary DNA sequence, was validated in this dissertation for measuring prolactin mRNA in the hamster. Male hamsters blinded for 11, 21, or 42 days showed significant and progressively greater declines in prolactin mRNA levels which were completely prevented by pinealectomy. The decline seen after 11 days is the earliest depression in prolactin cell activity reported after light deprivation in the hamster. Female hamsters blinded for 28 days, however, showed no such decreases in prolactin cell activity if they continued to display estrous cyclicity. This supports the hypothesis that, unlike the male, there is not a gradual decline in prolactin cell activity after blinding in the female hamster and that loss of estrous cyclicity may precede or possibly accompany declines in prolactin cell activity. After 12 weeks of blinding, females were acyclic and had dramatically depressed levels of prolactin cell activity. However, pinealectomy did not completely prevent this decline due to blinding unless the females continue to display estrous cyclicity. Thus, when pinealectomy was ineffective in preventing the loss of estrous cyclicity due to blinding, it was also ineffective in preventing declines in prolactin cell activity. In ovariectomized females, blinding caused a decline in prolactin cell activity. Pinealectomy was not consistently effective in preventing this decline after 12 weeks of treatment, although, in females blinded for 4 weeks (at which time all animals were cycling) and then ovariectomized for an additional 4 weeks, pinealectomy completely prevented this decline in prolactin cell activity. In a separate study, significant changes in prolactin cell activity during the estrous cycle were seen in untreated normally cycling female hamsters. These changes in prolactin mRNA, prolactin synthesis, and radioimmunoassayable prolactin in the pituitary were measured in the morning, when, consistent with other reports, no differences in serum prolactin were observed.

Gonadotrophin-releasing hormone (GnRH) can inhibit proliferation of multiple reproductive tissue cancer cell lines through direct interaction with GnRH receptors (GnRHR) on tumour cells. GnRH analogues may therefore have a role in treating some cancers. The signalling pathways associated with these inhibitory effects are poorly defined, and characterising them may help to understand therapeutic sensitivity. To elucidate these pathways, transcriptomic and proteomic approaches were used to compare the effects of the GnRH agonist Triptorelin in responsive GnRHR-transfected HEK293 cells (SCL60) and unresponsive (HEK293) cells both in vitro for up to 24h and in vivo for up to 7 days. Gene expression profiling demonstrated that SCL60 gene expression was temporally regulated with Triptorelin treatment, with expression of some genes increased at one time point but decreased at another. Early and mid-phase gene expression changes comprised mainly transcription factors and late changes included the hormonal signalling component CGA. Pathway analysis implicated mitogen-activated protein kinase and cell cycle pathways, supporting the detection of G2/M arrest. Signalling effects within SCL60 xenografts, 4 and 7 days following Triptorelin treatment, were investigated using a phosphoproteomic antibody array. Changes included cell cycle and apoptosis regulators, as well as cell surface receptors and NFκB signalling pathway members. Reverse-phase protein arrays and western blotting also showed that pAkt was decreased and pNFκB-p65 was increased after Triptorelin treatment in vitro. An NFκB inhibitor enhanced the anti-proliferative effect of Triptorelin in SCL60 cells in vitro, suggesting that NFκB acts as a survival factor in the response to GnRHR stimulation. A range of GnRHR expression was observed in breast cancer tumours by immunohistochemistry, and on average GnRHR expression was significantly higher in the Triple Negative Phenotype (TNP) subgroup and in grade 3 tumours. A GnRHR-transfected breast cancer cell line, MCF7-h14, was developed. Despite this expressing a similar level of GnRHR to responsive SCL60 cells, MCF7-h14 cells were not inhibited by GnRHR activation, indicating that a high level of GnRHR is insufficient for the antiproliferative effects of Triptorelin.

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy