published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Molecular Biology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Biological Sciences / Master / Master of Philosophy

The Mac-T cell system was used to investigate the role of the prolactin (PRL) receptor in cell proliferation and the regulation of milk protein synthesis. This study was designed to investigate whether overexpressing the PRLR in the Mac-T cell line resulted in a change in its growth rate and an enhancement of its ability to produce milk proteins. To accomplish these goals, Mac-T cells were stably transfected with the rabbit prolactin receptor gene. Fifteen clones and a pool of transfectants were obtained. Of these, one clone and the pool were positive for the PRL receptor expression. The clone (S15) and pool (SP) cells were sorted into high (H), medium (M), and low (L) expressors, of the PRLR. The high expressors were used for all subsequent experiments. The presence of high levels of the PRLR on the surface of S15 and SP cells was further confirmed by receptor binding assay and Western Blot. Following the establishment of these cell lines, the cells were used to investigate the effect of increased levels of PRLR on cell proliferation and milk protein synthesis. / It was found that the growth rate of parental cells was depressed in the presence of 5 mug/ml of PRL. In contrast, the growth rate of the transfectants was enhanced by the addition of 5 mug/ml PRL to the culture medium. In addition, both "SP" and "S15" cells produced higher levels of STAT5 upon long-term (48 h) PRL stimulation. No effect on the synthesis of alpha S1- and beta-caseins was noted. It is likely that no differences in protein synthesis were observed because the cells have lost the ability to differentiate, even when cultured on collagen gels in the presence of lactogenic hormones.

A turkey prolactin (PRL) cDNA, encoding a 199 amino acid turkey PRL (tPRL), was cloned from a pituitary library. The mature PRL shared about 70% homology with mammalian PRLs and about 30% with fish PRLs. Areas of highest homology to other PRLs were located in the carboxyl terminus of the tPRL. Prolactin mRNA analyses, during the reproductive life of the turkey hen, confirmed that the high pituitary and plasma levels of PRL measured during the incubation phase are due to enhanced transcription of the PRL gene. Furthermore, tPRL mRNA levels were highly correlated with pituitary levels of tPRL. Recombinant tPRL (rctPRL), biologically and immunologically similar to pituitary tPRL, was purified from Escherichia coli cultures hosting an expression vector carrying the tPRL cDNA. Polyclonal antibodies raised against purified rctPRL behaved similar as antibodies raised against pituitary derived tPRL, in immunoblotting and immunocytochemistry experiments. Three tPRL isoforms (with estimated molecular weights of 27 kDa, 25 kDa and 24 kDa) were identified in turkey pituitary extracts. The relative proportion of the 27 kDa isoform increased while that of the 25 kDa decreased with increasing levels of total pituitary tPRL, during the reproductive life of the turkey hen. The partition of the immunoreactivity of tPRL into the three isoforms perhaps provides an additional control of the multitude functions of PRL.

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No description available.

published_or_final_version / abstract / Biological Sciences / Doctoral / Doctor of Philosophy

by Ng Yuen Keng. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 141-148). / Table of contents --- p.1 / List of figures --- p.5 / List of tables --- p.12 / List of abbreviations --- p.13 / Abbreviation table for amino acids --- p.16 / Chapter Chapter 1 --- Literature Review --- p.17 / Chapter 1.1 --- Introduction --- p.17 / Chapter 1.2 --- The Hematopoietin/cytokine receptor superfamily --- p.20 / Chapter 1.3 --- The PRLR protein --- p.22 / Chapter 1.3.1 --- The receptor size --- p.22 / Chapter 1.3.2 --- Primary structure --- p.22 / Chapter 1.3.3 --- Structure of the extracellular domain --- p.26 / Chapter 1.3.4 --- Structure of the cytoplasmic domain --- p.30 / Chapter 1.3.5 --- Characteristics of specific PRL binding to PRLR --- p.32 / Chapter 1.5 --- The PRLR gene --- p.33 / Chapter 1.6 --- Heterogeneity of PRLR --- p.33 / Chapter 1.7 --- Signal transduction of PRLR --- p.35 / Chapter 1.7.1 --- JAK: a novel family of cytoplasmic protein tyrosine kinases --- p.35 / Chapter 1.7.2. --- Interaction between JAK2 and PRLR --- p.37 / Chapter 1.7.3 --- STAT proteins: mediators of PRL-dependent gene transcription --- p.37 / Chapter 1.7.4 --- Other signaling pathways of PRLR --- p.38 / Chapter 1.7.5 --- Future prospects on PRLR signaling --- p.38 / Chapter 1.8 --- Regulation of PRLR gene expression --- p.39 / Chapter 1.9 --- Objective of cloning the PRLR cDNA in male Syrian golden hamster --- p.42 / Chapter Chapter 2 --- PCR cloning of hamster PRLR cDNA fragment from adult male hamster liver --- p.44 / Chapter 2.1. --- Introduction --- p.44 / Chapter 2.2. --- Materials and Methods --- p.45 / Chapter 2.2.1 --- Primer design and PCR strategy --- p.45 / Chapter 2.2.2 --- Collection of liver --- p.46 / Chapter 2.2.3 --- Reverse transcription of polyadenylated RNA --- p.46 / Chapter 2.2.4 --- Nested PCR --- p.47 / Chapter 2.2.5 --- Southern analysis of the PCR products --- p.48 / Chapter 2.2.6 --- Subcloning of PCR product --- p.49 / Chapter 2.2.7 --- Sequence determination of the positive recombinant clone --- p.49 / Chapter 2.2.8 --- Sequence alignment and homology comparison --- p.50 / Chapter 2.3 --- Results --- p.55 / Chapter 2.3.1 --- Nucleotide sequence alignment and primer design --- p.55 / Chapter 2.3.2 --- Nested PCR --- p.55 / Chapter 2.3.3 --- Subcloning of the PCR product --- p.56 / Chapter 2.3.4 --- Analysis of nucleotide and predicted amino acid sequences --- p.56 / Chapter 2.4 --- Discussion --- p.66 / Chapter Chapter 3 --- Nucleotide sequence determination of the 5' and the 3' ends of hamster PRLR cDNA --- p.69 / Chapter 3.1 --- Introduction --- p.69 / Chapter 3.2 --- Materials and Methods --- p.71 / Chapter 3.2.1 --- Collection of liver --- p.71 / Chapter 3.2.2 --- Total RNA preparation and poly (A) + RNA isolation --- p.72 / Chapter 3.2.3 --- Double stranded cDNA synthesis --- p.73 / Chapter 3.2.4 --- Adaptor ligation --- p.74 / Chapter 3.2.5 --- 5´ة and 3' RACE PCR --- p.74 / Chapter 3.2.6 --- Cloning of the RACE PCR products --- p.76 / Chapter 3.2.7. --- Sequence determination of the RA CE PCR products --- p.77 / Chapter 3.2.8. --- Sequence analysis of the RACE PCR products --- p.78 / Chapter 3 .2.9 --- Northern blot analysis of hamster PRLR mRNA in male hamster tissues --- p.79 / Chapter 3.3 --- Results --- p.79 / Chapter 3.1.1 --- RNA preparation and double stranded cDNA synthesis --- p.79 / Chapter 3.3.2 --- RACE PCRfor the 5' and the 3' ends of hamster PRLR cDNA --- p.84 / Chapter 3.3.3 --- Cloning of the 5' and 3'RACE PCR products --- p.92 / Chapter 3.3.4 --- Sequence determination of the RACE PCR products --- p.92 / Chapter 3.3.5 --- Nucleotide sequence analysis of hamster PRLR full length cDNA --- p.101 / Chapter 3.3.6 --- Northern blot analysis of hamster PRLR --- p.101 / Chapter 3.4 --- Discussion --- p.106 / Chapter Chapter 4 --- Attempts to study the PRLR gene expression in male hamster tissues --- p.113 / Chapter 4.1 --- Introduction --- p.113 / Chapter 4.2 --- Materials and Methods --- p.115 / Chapter 4.2.1 --- Collection of tissues --- p.115 / Chapter 4.2.2 --- Total RNA preparation and poly (A)+ RNA isolation --- p.116 / Chapter 4.2.3 --- Reverse Transcription --- p.116 / Chapter 4.2.4 --- Polymerase chain reaction for detecting the presence of hamster PRLR cDNA in various tissues --- p.117 / Chapter 4.2.5 --- Nested PCR for detecting heterogeneity in PRLR cDNA sizes in various tissues --- p.117 / Chapter 4.2.6 --- Analysis and quantitation of PCR products --- p.118 / Chapter 4.3 --- Results --- p.119 / Chapter 4.4 --- Discussion --- p.134 / Chapter Chapter 5 --- General Discussion --- p.137 / References --- p.141 / Appendices --- p.149 / Chapter I. --- "Stock solution preparation (Sambrook et al., 1989)" --- p.149 / Chapter II. --- List of primers --- p.152 / Primers for sequence determination --- p.152 / "Primer for first strand cDNA synthesis and 3' RACE PCR (Frohman et al., 1988 and Loh et al.,1989)" --- p.152 / "Primers for amplifying the actin cDNA fragment (Chan et al.,1995)" --- p.152 / Primers used for PCR-cloning and semi-quantitative analysis of hamster PRLR cDNA --- p.153 / Chapter III. --- "First strand cDNA synthesis primer, cDNA adaptor and adaptor primers used in the 5' and3' end sequence determinations of hamster PRLR cDNA" --- p.154 / Chapter IV. --- "Multiple cloning sites of the pCRII (Invitorgen), pUC 18 (Pharmacia) and pBluescript SK+ vectors (Clontech)" --- p.155 / Chapter VI. --- Nucleic acid molecular weight size markers --- p.158

A evolução da vida na terra foi possível graças ao desenvolvimento de mecanismos temporais precisos capazes de ajustar processos fisiológicos que ocorriam no interior do organismo com os ciclos ambientais, promovendo assim, ganhos na capacidade adaptativa e comportamental desses indivíduos. A retina exerce função de suma importância nesse processo através da percepção da informação fótica que possibilita o ajuste dos ritmos circadianos. Nesse tecido, o glutamato apresenta um importante papel tanto na transmissão da informação fótica direcionada ao processo de formação de imagem quanto nos ajustes dos relógios biológicos. O objetivo desse trabalho foi avaliar como o glutamato, aplicado por períodos diferentes (6 e 12h), é capaz de modular a expressão dos genes de relógio na retina de Gallus gallus. Através de diferentes protocolos que envolveram a administração de glutamato na concentração de 100μM por 6 e 12 horas e em diferentes repetições (1 e 3 pulsos) avaliou-se através de PCR quantitativo a expressão dos genes Clock, Per2 e Bmal1. Os diferentes genes de relógio na retina de Gallus gallus apresentam diferentes respostas frente às trocas de meio e frente ao tratamento com o glutamato. O gene Clock responde com ativação da transcrição para ambos os tratamentos, de forma dependente da repetição dos estímulos. Já para o gene Per2 o tratamento com glutamato impõe uma oscilação de expressão com um ritmo ultradiano, enquanto que as trocas de meio não determinam alterações na transcrição. A expressão do gene Bmal1 não é afetada nem por trocas de meio, nem por glutamato. Novos estudos devem ser fomentados no sentido de se elucidar as vias pelas quais o glutamato leva ao perfil de oscilação observado e qual o mecanismo pelo qual a repetição de trocas de meio atua como sinalizador para o estabelecimento da sincronização celular / The evolution of life on earth was possible thanks to the development of precise temporal mechanisms to adjust physiological processes to environmental cycles, thus promoting gains in the individual adaptive and behavioral ability. The retina plays a very important role of paramount importance in this process through the perception of photic information that allows the adjustment of circadian rhythms. In this tissue, glutamate functions in the transmission of photic information directed to both image formation and biological clock entrainment. The aim of this study was to evaluate how glutamate, applied for different periods (6 and 12h), is able to modulate the expression of the clock genes in the retina of Gallus gallus. Using different protocols involving the administration of 100μM glutamate for 6 and 12 hours and with different repetitions (1 and 3 pulses) the expression of Clock, Per2 and Bmal1 genes was evaluated by quantitative PCR. Clock gene responds with activation of transcription to both treatments depending on the repetition of the stimulus. As for Per2 gene, glutamate treatment imposes an oscillation with an ultradian expression rhythm, whereas medium changes do not affect its transcription. The expression of Bmal1 gene is not affected by either medium changes or glutamate. Further studies should be encouraged in order to elucidate the pathways by which glutamate leads to observed oscillation profile, and which mechanism triggered by the repetition of medium changes acts as signal to establish cell synchronization