Dissertation (Ph.D.)--Auburn University, 2006. / Abstract. Vita. Includes bibliographic references.

Proefschrift Universiteit van Amsterdam. / Met lit.opg. en een samenvatting in het Nederlands.

München, Techn. Univ., Diss., 2010.

Industrial experiences have shown that powders can unexpectedly change from normal powder flow properties to exhibit liquid-like flow characteristics. This change in flow properties, known as flooding, can result in a major loss of a powder's containment. The prime objective of the research presented in this thesis is to develop a method which quantifies a powder's likelihood to flood, and to identify the conditions where the tendency to flood becomes important. A powder is known to exhibit liquid flow properties at high shear rates or when aerated at or above the minimum fluidisation velocity. The interaction of these two factors, however, is not fully understood. A new type of shear cell is developed which enables the measurement of the shear characteristics of an aerated powder. This shear cell is based on Couette geometry, where a powder sample is sheared between two concentric cylinders, while under controlled aeration conditions. Evaluation of the equipment with a variety of powders identifies that the transition to liquid-like flow properties can occur at low shear velocities and at an aeration substantially below fluidisation. The characterisation of a sample of flooded material shows that additional fine particles significantly increases the tendency for that material to flow like a liquid. The effect of additional fine particles on a selection of powders is studied in detail and powders with a narrow particle size distribution are shown to be most vulnerable to flooding. The quantities of fines required before a powder is likely to show liquid-like flow properties can be small, highlighting that the flooding problem can be significantly effected by segregation. The ability to characterise the effect of small quantities of additional fines on the likelihood to undergo liquid-like flow is an important step forward in understanding the apparent random nature of flooding.

Rhopalosiphum padi viras (RhPV) is one of several picorna-like viruses that infect insects; sequence analysis has revealed distinct differences between these agents and mammalian picornaviruses. RhPV has a single-stranded positive sense RNA genome of about 10kb; unlike the genomes of the Picornaviridae, however, this genome contains two long open reading frames (ORFs). ORF1 encodes the virus non- structural proteins, while ORF2 specifies the structural proteins. Both ORFs are preceeded by long untranslated regions (UTRs). The intergenic UTR is known to contain an internal ribosome entry site (IRES) that directs non-AUG-initiated, translation of ORF2. The 5' UTR of RhPV was assayed for IRES activity by translating synthetic bicistronic mRNAs containing this sequence in a variety of systems. The 5' UTR contains an IRES element that directs internal initiation of protein synthesis from an AUG codon in mammalian. Drosophila and plant in vitro systems and in vivo in an insect non-aphid cell line. In contrast, the encephalomyocarditis virus IRES only functions in mammalian in vitro and in vivo systems. The RhPV 5' IRES element has features in common with picornavirus IRES elements, in that no coding sequence is required for IRES function, but also with cellular IRES elements, as deletion analysis indicates that this IRES element does not have sharply defined boundaries. IRES activities of the 5' UTRs of other insect picorna-like viruses Acyrthosiphon pisum virus (APV) and the type 2 insect picorna-like virus Sacbrood viras (SBV) were also examined. Both viruses were shown to contain IRES elements that functioned in mammalian and plant in vitro systems and in vivo in an insect non-aphid cell line. Comparison of the IRES activities of RhPV with APV and SBV showed the RhPV 5' IRES element to be more efficient than both the APV and SBV IRES elements in mediating internal initiation of protein synthesis.

The hepatitis G virus and GBV-C are recently discovered variants of the same virus belonging to the family Flavivirus (HGV/GBV-C). Although initially thought to be a hepatitis virus, it has been shown to have no association with liver disease. No work has been performed on the prevalence or molecular characteristics of HGV/GBV-C in southern Africa. In addition, although it is clear that the liver is not the primary site of replication, there is no data on the sites of HGV/GBV-C replication in normal subjects. Thus, this study aimed to assess the prevalence of HGV/GBV-C carriage in the urban and rural adult Black communities of the Western and Eastern Cape Provinces of South Africa, and compare it to the prevalence of serological markers of the hepatitis viruses A-E. In addition, this study aimed to assess the molecular features of South African HGV/GBV-C isolates and demonstrate the organs where viral replication was present. The mean prevalences of antibodies to hepatitis A lgG, hepatitis B surface antigen and antibodies to hepatitis B surface antigen were 98%, 4.3% and 61.1 % respectively. The mean prevalence of antibodies to hepatitis C was 1.8%. No significant differences in prevalence were shown between the urban and rural regions for these viruses. The mean anti-hepatitis E prevalence varied from 5.8% to 19.1 % in the different regions. Those living in mud houses without access to chlorinated tap water had a significantly higher prevalence of antihepatitis E. No anti-hepatitis D positive samples were isolated. The overall prevalence of HGV/GBV-C was 26.9%, with rural communities having a significantly lower prevalence than urban communities. A significant relationship was observed between HGV/GBV-C infection with the use of illicit drugs, female gender, younger age and past blood transfusions. Phylogenetic analysis demonstrated a novel fourth South African HGV/GBV-C genotype, distinct from the previously described genotypes 1-3. In addition, certain isolates showed a major deletion in the highly conserved 5' non-coding region of HGV/GBV-C. Analysis of 23 tissue biopsies from infected cadavers suggested that the spleen and bone marrow were the primary sites of HGV/GBVC replication.

No description available.

類胰島素生長因子1(IGF1)是一種負責代謝、細胞生長、身體發展的多肽激素。微衛星和單核苷酸多態性(SNP), 與循環IGF1水平顯著相關。然而，目前沒有研究指出微衛星和SNPs的綜合影響，且這些遺傳變異對IGF1的調控機制仍是未知。本研究的假設是，微衛星和SNPs在啟動子區域可能有相互作用從而調控IGF1水平。因此，本研究的目的是找出影響IGF1表達的主要元素，並研究每個基因變異的作用。 / 在這項研究中，整個IGF1的基因，包括上游和下游的5萬鹼基對(bp)，可分為4個單體型區段，而IGF1的調控區在第3和第4個區段。與其它重復序列的微衛星相比，有21個重復序列的微衛星(IGF1上游969bp)與一套獨特的SNPs有關連。此外，有19個重復序列的微衛星有較低的循環IGF1。 / 功能性細胞分析進一步分析在生長激素(GH)依賴模型和GH獨立模型中，每個基因變異的角色。在GH獨立模型中，常見的單體型之間有不同的轉錄活性。與以前的研究結果相一致的是，有19個重複序列的單倍型轉錄活性最低。當單倍型為C-T-T，啓動子的轉錄活性受微衛星長度影響，較長的單體型有較低的轉錄活性。微衛星的長度效應或倚賴功能性SNP 1411C> T（rs35767）和叉頭蛋白A3（FOXA3）。以前研究發現在不同基因調控中，一個只結合C等位點並含有CCAAT /增強子結合蛋白delta（CEBPD）的轉錄激活複合物與FOXA3並存。因此，CEBPD可能與FOXA3相互作用從而調控IGF1的表達。而微衛星長度可能通過調節上游CEBPD轉錄複雜和下游FOXA3的相互作用從而影響IGF1的表達。單倍型T-C-A可能採取另一種調控機制，該機制或許被長約178鹼基對，含有“CA“部分的片段調控。GH依賴模型是模擬幼年時期IGF1的表達。在這個階段中，常見的單體型之間有不同的轉錄活性，但每個基因變異的調節作用均不強。 / 總括而言，IGF1的表達主要是由微衛星和SNPs組成的單體型調控。在幼年和成年，常見的單體型之間有差別顯著的轉錄活動。然而，GH獨立模型和GH依賴模型的調控機制是不同的。 / Insulin-like growth factor 1 (IGF1) is a polypeptide hormone responsible for metabolism, cell growth, and somatic development. Microsatellite and SNPs have been demonstrated to be significantly associated with circulating IGF1 level. However, no studies have ever investigated the combined effects of microsatellite and SNPs, and regulatory mechanisms of IGF1 expression by these genetic variants are yet unknown. The hypotheses of this study were that the microsatellite and SNPs may have certain regulatory functions in the promoter region, and interact with each other in the regulation. Therefore, the objectives were to identify the primary regulatory element in the regulation of IGF1 expression and to investigate the role of each genetic variant. / In this study, the whole IGF1 gene, including 50kb upstream and downstream, was divided into four haplotype blocks, in which the regulatory region of IGF1 lied in haploblock 3 and 4. Results of high-resolution melting analysis showed that a microsatellite (969bp upstream) with 21 repeats was associated with a different set of SNPs, compared to microsatellite with other repeat numbers. Also, haplotype with 19 CA repeats was significantly associated with a lower level of circulating IGF1. / Functional cellular assays were performed to further analyze the roles of each genetic variant in growth hormone (GH)-independent and GH-dependent models. In GH-independent model, it was found that common haplotypes showed differential transcriptional activities, and, consistent with previous findings, haplotype with 19 repeats was the least activated. On the background of haplotype C-T-T, transcriptional activity was regulated by microsatellite length, in which the haplotype with a longer microsatellite length tended to have a lower transcriptional activity. Further analysis showed that the microsatellite length effect depended on a functional polymorphism -1411C>T (rs35767) and forkhead box A3 (FOXA3), whose binding sites were several base pairs upstream of IGF1 transcription start site. Telgmann et al found a transcription activator complex containing CCAAT/enhancer binding protein delta (CEBPD) bound exclusively to the C allele and CEBPD often coexisted with FOXA3 in the regulation of various genes. Therefore, in the activation of IGF1, microsatellite length might regulate the interaction between the upstream CEBPD transcription complex and the downstream FOXA3. Haplotype T-C-A showed a yet unknown regulatory mechanism of IGF1 expression, which might be accounted for by the “C-A“ portion. In GH-dependent model, common haplotypes also showed differential transcriptional activities. However, further analysis revealed that the regulatory effects of each genetic variant alone (microsatellite or SNPs) were not strong. / To conclude, haplotype effect, which was contributed by both microsatellite and SNPs, played an important role in the regulation of IGF1 expression. Common haplotypes showed significantly differential transcriptional activities. However, the regulatory mechanisms were different in GH-independent model and GH-dependent model. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Yu Holly. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 127-140). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgement --- p.v / LIST OF ABBREVIATIONS --- p.vi / LIST OF FIGURES --- p.viii / LIST OF TABLES --- p.x / PUBLICATIONS --- p.xi / Chapter CHAPTER 1 --- INTRODUCTION TO INSULIN-LIKE GROWTH FACTOR 1 (IGF1) --- p.1 / Chapter 1.1 --- Production of IGF1 --- p.1 / Chapter 1.2 --- Other factors affecting IGF1 level --- p.5 / Chapter 1.2.1 --- Nutritional status --- p.5 / Chapter 1.2.2 --- Ethnicity --- p.5 / Chapter 1.2.3 --- Age --- p.8 / Chapter 1.2.4 --- Gender --- p.8 / Chapter 1.2.5 --- IGFBP --- p.9 / Chapter 1.2.6 --- Other growth factors --- p.10 / Chapter 1.3 --- Cellular functions of IGF1 --- p.10 / Chapter 1.4 --- Physiological functions of IGF1 --- p.13 / Chapter 1.4.1 --- Metabolism --- p.14 / Chapter 1.4.2 --- Somatic growth --- p.17 / Chapter 1.4.3 --- Longevity --- p.18 / Chapter CHAPTER 2 --- PATHOLOGY OF IGF1 --- p.20 / Chapter 2.1 --- IGF1 and cancer predisposition --- p.20 / Chapter 2.1.1 --- Evidences in genetic studies --- p.21 / Chapter 2.1.2 --- Evidences in lifestyle factors --- p.21 / Chapter 2.1.3 --- Evidences from population studies --- p.22 / Chapter 2.1.4 --- Miscellaneous evidence --- p.22 / Chapter 2.2 --- IGF1 and diabetes mellitus (DM) --- p.23 / Chapter 2.3 --- IGF1 and other diseases --- p.24 / Chapter CHAPTER 3 --- HYPOTHESES AND AIMS OF THE STUDY --- p.26 / Chapter 3.1 --- Hypotheses of the study --- p.26 / Chapter 3.2 --- Aims of the study --- p.26 / Chapter CHAPTER 4 --- RELATIONSHIP BETWEEN GENETIC VARIANTS AND IGF1 EXPRESSION --- p.28 / Chapter 4.1 --- Introduction --- p.28 / Chapter 4.2 --- Materials and methods --- p.31 / Chapter 4.2.1 --- Study subjects --- p.31 / Chapter 4.2.2 --- tagSNP selection and haplotype block construction --- p.32 / Chapter 4.2.3 --- Genescan analysis of the CA repeat microsatellite --- p.32 / Chapter 4.2.4 --- Genotyping assay of tagSNPs --- p.34 / Chapter 4.2.5 --- Statistical analysis --- p.36 / Chapter 4.3 --- Results --- p.37 / Chapter 4.3.1 --- Characteristics of the subjects --- p.37 / Chapter 4.3.2 --- Determination of haplotype blocks --- p.38 / Chapter 4.3.3 --- Selection of tagSNPs --- p.41 / Chapter 4.3.4 --- Genotyping analysis of tagSNPs --- p.43 / Chapter 4.3.5 --- Genescan analysis of -969bp CA repeat microsatellite --- p.46 / Chapter 4.3.6 --- Phased haplotype consisting of SNP / SNP and microsatellite --- p.48 / Chapter 4.3.7 --- Correlation between haplotypes in IGF1 promoter and circulating IGF1 level --- p.50 / Chapter 4.4 --- Discussion --- p.53 / Chapter CHAPTER 5 --- Transcriptional regulation of GENETIC VARIANTS IN different haplotypeS --- p.57 / Chapter 5.1 --- Introduction --- p.57 / Chapter 5.1.1 --- IGF1 gene structure --- p.57 / Chapter 5.1.2 --- Regulatory elements in IGF1 promoter --- p.58 / Chapter 5.1.3 --- Functional variant -1411C>T (rs35767) in IGF1 promoter --- p.60 / Chapter 5.1.5 --- Objectives of the study --- p.62 / Chapter 5.2 --- Materials and methods --- p.64 / Chapter 5.2.1 --- Comparative genomics --- p.64 / Chapter 5.2.2 --- Study subjects --- p.64 / Chapter 5.2.3 --- tagSNP selection and genotyping assay --- p.64 / Chapter 5.2.4 --- Primers and standard polymerase chain reaction (PCR) --- p.65 / Chapter 5.2.5 --- Enzyme digestion --- p.68 / Chapter 5.2.6 --- Ligation --- p.68 / Chapter 5.2.7 --- Transformation of DNA ligation products --- p.68 / Chapter 5.2.8 --- Preparation of E.coli supercompetent cells --- p.69 / Chapter 5.2.9 --- Construction of plasmids --- p.70 / Chapter 5.2.10 --- Cell lines --- p.71 / Chapter 5.2.11 --- Nucleic acid extraction --- p.72 / Chapter 5.2.12 --- Reverse transcription polymerase chain reaction (RT-PCR) --- p.73 / Chapter 5.2.13 --- Transient transfection --- p.73 / Chapter 5.2.14 --- Luciferase reporter assay --- p.73 / Chapter 5.2.15 --- Optimization of a saturated luciferase reporter system --- p.74 / Chapter 5.2.16 --- Electrophoretic mobility shift assay (EMSA) --- p.74 / Chapter 5.2.17 --- Western blot analysis --- p.74 / Chapter 5.2.18 --- Prediction of putative functional SNPs --- p.76 / Chapter 5.2.19 --- Statistical analysis --- p.77 / Chapter 5.3 --- Results --- p.77 / Chapter 5.3.1 --- Evolutionarily conserved region (ECR) --- p.77 / Chapter 5.3.2 --- Frequency distribution of haplotypes of IGF1 promoter in the Chinese population --- p.79 / Chapter 5.3.3 --- Optimization of luciferase reporter system --- p.81 / Chapter 5.3.3.1 --- Gene expression level of different cell lines --- p.81 / Chapter 5.3.3.2 --- Cell line selection --- p.81 / Chapter 5.3.3.3 --- Saturation of expression plasmids in the luciferase reporter system --- p.83 / Chapter 5.3.3.4 --- Western blot analysis of gene expression level after transfection --- p.86 / Chapter 5.3.4 --- Possible functional SNPs in IGF1 regulatory region beyond ECR --- p.89 / Chapter 5.3.4.1 --- In silico analysis of putative functional SNPs --- p.89 / Chapter 5.3.4.2 --- Binding capacity of possible functional SNPs --- p.92 / Chapter 5.3.5 --- Transcriptional activities of common haplotypes and their derivatives --- p.95 / Chapter 5.3.5.1 --- GH-independent (GH-) model --- p.95 / Chapter 5.3.5.1.1 --- Common haplotypes --- p.95 / Chapter 5.3.5.1.2 --- Effect of microsatellite length on transcriptional activity of IGF1 promoter --- p.97 / Chapter 5.3.5.1.3 --- Effect of SNP on transcriptional activity of IGF1 promoter --- p.99 / Chapter 5.3.5.1.4 --- Summary --- p.101 / Chapter 5.3.5.2 --- GH-dependent (GH+) model --- p.101 / Chapter 5.3.5.2.1 --- Common haplotypes --- p.101 / Chapter 5.3.5.2.2 --- Effect of microsatellite length on transcriptional activity of IGF1 promoter --- p.103 / Chapter 5.3.5.2.3 --- Effect of SNP on transcriptional activity of IGF1 promoter --- p.105 / Chapter 5.3.5.2.4 --- Summary --- p.106 / Chapter 5.3.6 --- Putative mechanism of the interaction between microsatellite and SNPs --- p.106 / Chapter 5.3.6.1 --- Microsatellite length effect in C-T-T haplotype relied on rs35767 (-1411C>T) --- p.107 / Chapter 5.3.6.2 --- The interaction of SNP and microsatellite was dependent on FOXA3 --- p.110 / Chapter 5.3.6.3 --- Summary --- p.112 / Chapter 5.3.7 --- Serial deletion of IGF1 promoter fragment --- p.112 / Chapter 5.4 --- Discussion --- p.116 / Chapter 5.4.1 --- Distal regulatory mechanism of IGF1 expression --- p.116 / Chapter 5.4.2 --- Localized regulatory mechanism of IGF1 expression --- p.117 / Chapter CHAPTER 6 --- CONCLUSIONS AND FUTURE STUDIES --- p.125 / Chapter 6.1. --- Conclusions --- p.125 / Chapter 6.2. --- Future studies --- p.126 / Reference --- p.127

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Oct. 13, 2008). Includes bibliographical references (p. 56-64).

Gametogenesis is a highly complex process that requires stringent control of gene expression, in which translational regulation plays an essential role. Deleted in Azoospermia-like (DAZL) belongs to the DAZ family of RNA-binding proteins, which are restricted to germ cells, and regulate mRNA translation. Importantly, loss of function of these proteins results in infertility in both males and females in a wide variety of organisms. A model for the mechanism by which DAZL stimulates translation has been proposed based on work in Xenopus laevis (X. laevis) oocytes. In this model, DAZL functions by recruiting the translation initiation factor poly(A)-binding protein (PABP) to the 3’ untranslated region (UTR) of messenger RNAs. Simultaneous binding of PABP to Dazl and factors at the 5’ end confers a “closed-loop” mRNA conformation, which promotes translation initiation. To examine whether DAZL plays a similar role in mammals, co-expression of Dazl and PABP family members was investigated in fetal and adult mouse gonads. In contrast to X. laevis, mammals encode four cytoplasmic PABPs which share a similar domain organisation: PABP1, tPABP, ePABP and PABP4, of which PABP1 and PABP4 appear to be expressed in a wide range of tissues. Immunohistochemistry revealed that Dazl, Pabp1 and Pabp4 are all expressed in primordial germ cells (PGCs) but these show different expression patterns following germ cell sex differentiation. In adult testes Dazl is expressed in spermatogonia and spermatocytes, coinciding with the peak of Pabp4 expression. In contrast, the peak of Pabp1 expression occurs later than that of Dazl, with these proteins only being co-expressed in late pachytene and secondary spermatocyte phases. In adult ovaries, Pabp1, Pabp4 and Dazl are all expressed in the oocytes of primordial and primary follicles. Since both PABP family members are co-expressed with Dazl, the ability of DAZL to interact with PABP1 and PABP4 was investigated in vitro and in vivo. Surprisingly, these studies showed that DAZL discriminates between different PABP family members, only interacting with PABP1, providing the first report of a PABP-specific protein partner. Several putative DAZL mutations have been identified in patients with impaired fertility. Two of these mutations, I37A and R115G, are located in the RNA recognition motif (RRM), a domain which is found in many RNA-binding proteins and mediates both RNA and protein interactions. Thus, the role of these mutations in the ability of DAZL to stimulate translation was investigated. To this end, a translational target of human DAZL (hDAZL) was sought. The 3’UTR of growth differentiation factor 9 (hGDF9) mRNA was found to confer regulation by hDAZL and thus the ability of mutant DAZLs to stimulate reporter mRNAs containing this 3’UTR was examined. This revealed that both mutations compromised the ability of hDAZL to stimulate hGDF9 translation, suggesting a causative effect. These results were further confirmed in assays in which hDAZL is artificially tethered to mRNAs. The ability of mutant hDAZLs to stimulate translation in this assay was compromised suggesting that loss of function is, at least in part, due to impaired protein-protein interactions rather than altered RNA-binding. This work provides insights into the molecular mechanism by which DAZL stimulates the translation of specific mRNAs during mammalian gametogenesis and provides evidence that this function may play an important physiological role in human reproduction.