by Wong Kwan Po, Gary. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 132-153). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Abbreviations --- p.vi / Abbrevation Table for Amino Acids --- p.ix / List of Figures --- p.x / List of Tables --- p.xiii / Table of Contents --- p.xiv / Chapter Chapte r One --- General Introduction --- p.1 / Chapter 1.1 --- Structures of PRL --- p.1 / Chapter 1.2 --- PRL receptor and its mechanism of action --- p.7 / Chapter 1.3 --- Biosynthesis of PRL --- p.11 / Chapter 1.4 --- Biological functions of PRL --- p.13 / Chapter 1.5 --- Organization and regulation of PRL gene --- p.16 / Chapter 1.6 --- Aims of this study --- p.25 / Chapter Chapter Two --- PCR Cloning of gfPRL Gene --- p.26 / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials and Methods --- p.27 / Chapter 2.2.1 --- Buffers and Reagents --- p.27 / Chapter 2.2.2 --- Methods --- p.30 / Chapter 2.2.2.1 --- PCR of the 5'-flanking region of gfPRL gene --- p.30 / Chapter 2.2.2.2 --- Genomic PCR of gfPRL gene --- p.31 / Chapter 2.2.2.3 --- Spectrophotometric quantification and qualification of DNA and RNA --- p.31 / Chapter 2.2.2.4 --- Agarose gel electrophoresis of DNA --- p.31 / Chapter 2.2.2.5 --- DNA radioactive labeling by random priming --- p.32 / Chapter 2.2.2.6 --- Vacuum transfer of DNA fragments to a nylon membrane --- p.32 / Chapter 2.2.2.7 --- Southern blot analysis --- p.33 / Chapter 2.2.2.8 --- Molecular Imager Analysis --- p.33 / Chapter 2.2.2.8 --- Phosphorylation of PCR amplified DNA --- p.34 / Chapter 2.2.2.9 --- Ligation of DNA fragment to linearized vector --- p.34 / Chapter 2.2.2.10 --- Preparation of Escherichia coli competent cells --- p.34 / Chapter 2.2.2.11 --- Bacterial transformation by heat stock --- p.35 / Chapter 2.2.2.12 --- Automated PCR sequencing with Sequencing Ready Reaction Kit --- p.35 / Chapter 2.2.2.13 --- Primer extension using reverse transcription --- p.36 / Chapter 2.3 --- Results --- p.38 / Chapter 2.3.1 --- Cloning of the 5'-flanking region of gfPRL gene --- p.38 / Chapter 2.3.2 --- PCR cloning of gfPRL gene --- p.43 / Chapter 2.3.3 --- Identification of the transcription initiation site --- p.47 / Chapter 2.4 --- Discussion --- p.51 / Chapter 2.4.1 --- Sequence analysis of the gfPRL gene --- p.51 / Chapter 2.4.2 --- Analysis of the exon-intron boundaries --- p.53 / Chapter 2.4.3 --- Analysis of the 5'flanking region of gfPRL gene --- p.53 / Chapter 2.4.4 --- Identification of the transcription initiation site --- p.54 / Chapter 2.5 --- Conclusion --- p.54 / Chapter Chapter Three --- Promoter Analysis of the gfPRL Gene --- p.55 / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Materials and Methods --- p.56 / Chapter 3.2.1 --- Preparation of Luciferase reporter constructs --- p.56 / Chapter 3.2.2 --- Preparation of frozen stock of culture cells --- p.56 / Chapter 3.2.3. --- Cell culture --- p.56 / Chapter 3.2.4 --- Transfection of mammalian cells for transient gene expression study --- p.57 / Chapter 3.2.5 --- Luciferase assay --- p.57 / Chapter 3.3 --- Results --- p.58 / Chapter 3.3.1 --- Tissue-specific transcription of gfPRL promoter --- p.58 / Chapter 3.3.2 --- Identification of regulatory regions of gfPRL gene promoter --- p.61 / Chapter 3.3.3 --- Inhibitory effect of DA on gfPRL promoter transcription activity --- p.63 / Chapter 3.3.4 --- GfPRL promoter sequences that specifically confer negative regulation by DA --- p.65 / Chapter 3.3.5 --- The action of TRH on gfPRL promoter --- p.67 / Chapter 3.3.6 --- Investigation of gfPRL promoter sequence responsiveness towards TRH --- p.69 / Chapter 3.4 --- Discussion --- p.71 / Chapter 3.4.1 --- Tissue-specific transcription of gfPRL promoter --- p.71 / Chapter 3.4.2 --- Identification of regulatory regions of goldfish prolactin gene promoter --- p.72 / Chapter 3.4.3 --- Dopamine inhibits gfPRL promoter activity --- p.73 / Chapter 3.4.4 --- TRH action on gfPRL promoter --- p.76 / Chapter 3.5 --- Conclusion --- p.78 / Chapter Chapter Four --- Seasonal Study on gfPRL and gfGH expression --- p.80 / Chapter 4.1 --- Introduction --- p.80 / Chapter 4.2 --- Materials and Methods --- p.81 / Chapter 4.2.1 --- Blood samples and radioimmunoassay --- p.81 / Chapter 4.2.2 --- Preparation of ribonuclease free reagents and apparatus --- p.81 / Chapter 4.2.3 --- Isolation of total RNA --- p.81 / Chapter 4.2.4 --- Formaldehyde agarose gel electrophoresis of RNA --- p.82 / Chapter 4.2.5 --- First strand cDNA synthesis --- p.82 / Chapter 4.2.6 --- RT-PCR --- p.83 / Chapter 4.2.7 --- Analysis of RT-PCR --- p.86 / Chapter 4.3 --- Results --- p.88 / Chapter 4.3.1 --- Tissue-specific expression of gfPRL transcript --- p.88 / Chapter 4.3.2 --- Sexual maturity of goldfish throughout the reproductive cycle --- p.90 / Chapter 4.3.3 --- Serum gfGH levels throughout the year of 2000 --- p.91 / Chapter 4.3.4 --- Serum gfPRL levels throughout the year of 2000 --- p.92 / Chapter 4.3.5 --- The variation of gfGHR and gfPRLR mRNA in the brain throughout the reproductive cycle --- p.93 / Chapter 4.3.6 --- The variation of gfGHR mRNA in the liver throughout the reproductive cycle --- p.94 / Chapter 4.3.7 --- The variation of gfGHR and gfPRLR mRNA in the kidney throughout the reproductive cycle --- p.95 / Chapter 4.3.8 --- The variation of gfGHR and gfPRLR mRNA in the gonads throughout the reproductive cycle --- p.96 / Chapter 4.4 --- Discussion --- p.98 / Chapter 4.4.1 --- Tissue-specific expression of gfPRL transcript --- p.98 / Chapter 4.4.2 --- Sexual maturity of goldfish throughout the reproductive cycle --- p.98 / Chapter 4.4.3 --- Serum gfGH and gfPRL level throughout the reproductive cycle --- p.99 / Chapter 4.4.4 --- The variation of gfGHR and gfPRLR mRNA in the brain throughout the reproductive cycle --- p.100 / Chapter 4.4.5 --- The variation of gfGHR mRNA in the liver throughout ´Øthe reproductive cycle --- p.101 / Chapter 4.4.6 --- The variation of gfGHR and gfPRLR mRNA in the kidney throughout the reproductive cycle --- p.102 / Chapter 4.4.7 --- The variation of gfGHR and gfPRLR mRNA in the gonads throughout the reproductive cycle --- p.102 / Chapter 4.5 --- Conclusion --- p.105 / Chapter Chapter Five --- Recombinant gfPRL Production --- p.106 / Chapter 5.1 --- Introduction --- p.106 / Chapter 5.2 --- Materials and Methods --- p.108 / Chapter 5.2.1 --- Buffers and Reagents --- p.108 / Chapter 5.2.2 --- Methods --- p.112 / Chapter 5.2.2.1 --- Recombinant protein expression --- p.112 / Chapter 5.2.2.2. --- Purification of the recombinant protein by XpressTM System Protein Purification (Invitrogen) --- p.112 / Chapter 5.2.2.3 --- SDS-PAGE preparation --- p.112 / Chapter 5.2.2.4 --- SDS-PAGE analysis of proteins --- p.113 / Chapter 5.2.2.5 --- Western blot analysis --- p.114 / Chapter 5.2.2.6 --- Protein refolding --- p.114 / Chapter 5.2.2.7 --- Alkaline Extraction --- p.115 / Chapter 5.2.2.8 --- Size Exclusion Chromatography --- p.115 / Chapter 5.2.2.9 --- ELISA analysis of the fractions --- p.115 / Chapter 5.2.2.10 --- Anion Exchange Chromatography --- p.116 / Chapter 5.3 --- Results --- p.117 / Chapter 5.3.1 --- Prokaryotic expression of recombinant gfPRL --- p.117 / Chapter 5.3.2 --- "Purification of reombinant gfPRL: SDS-PAGE, western blot and BCA analysis of purified recombinant gfPRL" --- p.119 / Chapter 5.3.3 --- Purification of native gfPRL and gfGH: Native hormone purification by size exclusion chromatography --- p.119 / Chapter 5.3.4 --- Native gfPRL purification by anion exchange chromatography --- p.122 / Chapter 5.3.5 --- Study the biological activity of refolded recombinant gfPRL --- p.126 / Chapter 5.4 --- Discussion --- p.127 / Chapter 5.4.1 --- Prokaryotic expression of recombinant gfPRL --- p.127 / Chapter 5.4.2 --- Purification of recombinant gfPRL --- p.128 / Chapter 5.4.3 --- Refolding of recombinant gfPRL --- p.129 / Chapter 5.4.4 --- Purification of native gfPRL --- p.130 / Chapter 5.4.5 --- Study the biological activity of recombinant gfPRL --- p.130 / Chapter 5.5 --- Conclusion --- p.131 / References --- p.132

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

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The human prolactin-inducible protein (PIP) or gross cystic disease fluid protein -15 (GCDFP-15) is a 15 kD protein secreted by human breast cancer cells and is abundant in fluids from gross cystic breast disease. Previous results from our laboratory showed that PIP KO mice had significantly lower numbers of CD4+ T cells in their secondary lymphoid organs, and these cells are impaired in their ability to differentiate into Th1 cells in vitro and in vivo leading to failure to control Leishmania major infection. In the present study, we further assessed the role of PIP in adaptive immunity by comparing cytokine production and intracellular signaling events in macrophages from WT and PIP KO mice following IFN-γ and lipopolysaccharide (LPS) stimulation. We show that although the expressions of IFN-γR and TLR4 on macrophages from KO and WT mice were comparable, PIP KO macrophages were significantly impaired in producing proinflammatory cytokines following IFN-γ and LPS stimulation. This was associated with impaired phosphorylation of mitogen-activated protein kinases (MAPKs) and signal transducers of activation of transcription (STATs) proteins in IFN-γ and LPS-stimulated macrophages from PIP KO mice. Interestingly, the expression of suppressors of cytokine signaling (SOCS) 1 and 3 proteins, known to suppress IFN-γ and LPS signaling, was higher in PIP KO macrophages compared to those from WT mice. Collectively, our studies clearly show that deficiency of PIP significantly affects intracellular signaling events leading to proinflammatory cytokine production in macrophages, and further confirm a role for PIP as important immunoregulatory protein involved in host defense. / October 2015

The morphological and physiological effects of pineal gland activity on the prolactin-secreting cells of the anterior pituitary were examined in blind-anosmic male and female rats. Prolactin synthesis was measured by the ability of anterior pituitaries to incorporate ³H-leucine into prolactin in vitro. Pituitary storage of prolactin was assessed by measuring radioimmunoassayable prolactin levels in the pituitaries in vivo and the total amount of immunoassayable prolactin in vitro. The effects of the activated pineal on prolactin release were estimated by monitoring radioimmunoassayable serum prolactin levels. Finally, the morphology of the prolactin cells was analyzed by both light microscopic immunocytochemistry and transmission electron microscopy. Eight weeks after blinding and olfactory bulbectomy in prepubertal male and female rats, prolactin synthesis, storage and release were all significantly decreased compared to unoperated control values. Pinealectomy in blind-anosmic rats completely prevented these effects. Similar results were obtained four weeks after treatment, but not after only one week. Furthermore, the reductions in prolactin synthesis, storage and release were not a consequence of the pineal-induced gonadal atrophy in these animals, since these effects persisted in ovariectomized-blind-anosimic rats. The pineal also elicited these effects in female rats rendered blind-anosmic after puberty, though to a lesser degree than when immature animals were used. Concomitant with these alterations in prolactin synthesis, storage and release were regressive changes in the morphology of individual prolactin cells and in the number of these cells in the pituitary. Anterior pituitaries from blind-anosmic rats were approximately half the weight of glands from intact animals and contained a third less DNA. This loss of cell number was largely accounted for by a reduction in the number of prolactin cells, as shown by immunocytochemistry. Additionally, each prolactin cell was smaller in size in blind-anosmic female rats and showed scant endoplasmic reticulum, a small Golgi complex, few secretory granules and rare exocytosis patterns. From these data I conclude that the pineal gland exerts a strong inhibition on the prolactin cells of blind-anosmic rats.

Maintaining reproductive health and diagnosing and treating conditions of infertility in stud dogs is important in canine theriogenology. However, there is still a great deal to be learned about reproductive physiology and factors that affect reproductive organs and semen quality in dogs. This thesis is an investigation of two factors in the male dog; serum 25-hydroxy Vitamin D (25OHVD) and prolactin (PRL) concentrations, and their possible associations with benign prostatic hyperplasia (BPH), prostate volume and/or sperm morphology and motility characteristics. 28 (Vitamin D Study) and 29 (28 plus one for the Prolactin study) client dogs of various breeds from the Ontario Veterinary College and Graham Animal Hospital in Southwestern Ontario, Canada were enrolled in the study from March to December 2009. Of these dogs 22 were successfully collected for semen. BPH was diagnosed using prostate volume measured by ultrasound, as well as clinical signs including blood in the ejaculate. Semen analysis was performed using manual microscopic techniques for morphology and computer assisted sperm analysis equipment for motility. In the vitamin D study, no associations were found between BPH and serum 25OHVD concentrations. In contrast, several sperm motility (motility, progressive motility, beat cross frequency (BCF), distance average path (DAP), curvilinear distance (DCL), linear distance (DSL), average path velocity (VAP), curvilinear velocity (VCL) and straight line velocity (VSL), amplitude lateral head displacement (ALH) and average orientation change (AOC)) and morphology characteristics (percentage normal sperm, head defects and detached heads) had desirable outcomes with 25OHVD concentrations between 120-180 nmol/l. Using bivariable analysis, positive associations were observed with 25OHVD and some semen quality characteristics from 4 to 8 years of age (motility, progressive motility, BCF, DCL, VCL, ALH, AOC) and at transformed prostate volumes smaller than or equal to 4.5 (motility, progressive motility, DCL, VCL, and normal morphology) while negative associations of these semen parameters were found at ages greater than 8 years and transformed prostate volumes greater than or equal to 5.5. Head defects were negatively associated with 25OHVD. Vitamin D may have an impact on spermatogenesis and normal sperm physiology that warrants further research. The prolactin study showed no statistically significant associations between serum PRL and BPH and serum PRL and sperm motility characteristics. However, two sperm morphology characteristics (percentage proximal droplets and percentage midpiece defects) had significant negative associations with PRL concentrations. Age interaction with PRL was also a factor in the percentage of midpiece defects with desirable outcomes associated at 4 years of age compared with older ages. Overall, undesirable outcomes occurred at PRL concentrations less than 2.5 ng/ml. In conclusion, both 25OHVD and PRL may have important roles in spermatogenesis and normal sperm physiology in the dog. / Ontario Veterinary College Pet Trust

Changes in the levels of expression of the prolactin receptor (PRLR) mRNA in the pituitary gland, hypothalamus, liver, pancreas, kidney and gonad from embryonic day (ED) 15 and ED21 to 1 day post-hatch, respectively, in chickens and turkeys were measured by real-time PCR. In both species, PRLR mRNA increased from low levels during the last week of ED to reach maxima at the peri-hatch period. Similarly, circulating levels of prolactin (PRL) also increased during this interval and were correlated with the observed increases in tissue content of PRLR mRNA. This suggested that PRL was up-regulating its own receptor during late embryogenesis. In support of this, in vitro stimulation of the pituitary gland of turkeys with VIP on ED24 resulted in a 4 fold and 3 fold increase in PRL and PRLR, respectively. Stimulation with VIP of either the hypothalamus or gonad had no effect on either levels of the PRLR transcript. This suggests that VIP acts indirectly through increased PRL to upregulate the number of receptors. In order to investigate the transcription of genes that may be induced/suppressed by PRL, suppressive subtractive hybridization (SSH) libraries from control or VIP stimulated ED24 turkey pituitary glands were constructed. Stimulation with VIP resulted in a 5.7 and 2.8 fold increase in media and pituitary content of PRL, respectively. The changes in PRL were consistent with endogenous levels of PRL observed just prior to hatch. Following sequence analysis of random clones (n=96) from each library, a total of 145 non-redundant putative genes were obtained. About 51% of the putative genes have as yet no assigned function, whereas, 15% were housekeeping genes and 34% had known functions within various pathways. Real-time PCR was used to confirm the differential expression of 21 of these genes in VIP treated and control pituitaries. Since the majority of these genes were expressed at levels consistent with the direction of subtraction, these data suggest that these libraries may be useful to study the direct and indirect effects of increasing levels of PRL on anterior pituitary function at about the time of hatch.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2006. / Vita. Includes bibliographical references.