Program--Univ. Marburg (Novi prorectoris inaugurationem). / Bibliographical foot-notes.

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / The multifaceted phenotype of the B‐lymphocyte has a remarkably effective role in peptide derived pathogen clearance and the prevention of re‐infection. This mechanism of host tolerant defense can be attributed to the actions of particular cellular subsets that arise from Blymphocytes: memory cells and high‐affinity antibody secreting plasma cells. Notably B cell propagation does not commence without the help of follicular helper T cells (TFH), a specialized subset of CD4+ cells. TFH cells are involved in the maturation and differentiation of Blymphocytes after antigen stimulation with a thymus‐dependent peptide. With this specific stimulus the formation of germinal centers (GCs) within B‐cell follicles of secondary lymphoid organs is induced and it is within these centers that TFH cells are able to interact with B cells to facilitate immunoglobulin affinity maturation, somatic hypermutation, and isotype class switching. Importantly, these respective processes play a fundamental role in manufacturing high‐affinity antibodies for effective pathogen clearance. Conversely, by means not well understood, the occurrence of spontaneous GC formation and the mass production of high affinity autoreactive antibodies have been shown to occur simultaneously with the development of autoimmune diseases. By the same token this incident is of particular interest and could play a role in the destruction of pancreatic insulin secreting β cells consequently driving the pathogenesis of type I diabetes. Our objective is to identify a possible correlation between the evolution of type I diabetes and the proliferatory behavior of B‐lymphocytes and TFH cells within developing GCs of non‐obese diabetic (NOD) mouse models.

PAPP-A is a large glycoprotein with alpha2-electrophoretic mobility that is produced by the placenta during pregnancy. In this thesis a biochemical and molecular characterisation of PAPP-A was performed. The polyclonal antiserum (DAKO) directed against PAPP-A has been shown to also interact with proteins other than PAPP-A. These non-specific interactions were abolished by performing Western blotting immunodetection at a high salt concentration (0.6M NaCl). At this salt concentration a single band of 195 kDa was immunodetected and this corresponded to the monomeric PAPP-A molecule. It was also discovered that a subset of paratopes in this antiserum reacted, under the described high salt concentration conditions, with the glycan component of PAPP-A. A placental cDNA library was screened using this antibody for the PAPP-A cDNA but this did not yield a clone for PAPP-A. A possible explanation is that the interaction with this antibody requires carbohydrate components to be present on the PAPP-A molecule. It is known that proteins expressed in bacterial systems are not post-translationally modified. Therefore another approach to the isolation of the PAPP-A cDNA clone was adopted, but this required some primary amino acid sequence of this protein that was unavailable at the time. To generate this information, PAPP-A was purified using its previously unpublished affinity for L-arginine in combination with the already described procedures of ammonium sulphate precipitation, ion exchange and gel filtration. Final purification of PAPP-A was achieved by SDS-PAGE electrophoresis. The isolated monomeric PAPP-A gave a unique single N-terminal amino acid sequence: N-EARGATEEPS. The N terminal sequence combined with the sequence obtained from limited proteolytic digestion of PAPP-A were used to design oligonucleotide primers specific for PAPP-A. These primers were used in a PCR reaction that produced 500 and >1200 bp fragments using the cDNA library as DNA template; thus demonstrating that PAPP-A is synthesised in the placenta. PAPP-A was shown to have O and N-linked carbohydrate chains. Enzymatic deglycosylation demonstrated that the N-linked chains were 8% (w/w) of the molecule. The O-linked groups were extensively modified with the presence of oligomers of N-acetyl-glucosamine. It was also shown that it was these groups the PAPP-A antibodies bind to at high salt concentration. A physical interaction of PAPP-A with endoproteinase Arg-C (EGF-BP) was observed. It was seen that they form a 1:1 (PAPP-A: endoproteinase) sub-unit complex that was stable in SDS. A further investigation revealed that PAPP-A interacted with the endoproteinase Arg-C and this resulted in a 30% inhibition of the esterolytic activity of this enzyme.

OBJECTIVE: To evaluate the incidence of chromosomal abnormalities in ectopic pregnancy chorionic villi. METHODS: A prospective study of patients with the diagnosis of ectopic pregnancy was conducted, with chorionic villi obtained at the time of surgical therapy cultured and analyzed for karyotype. Review of the patient's medical record and ultrasound evaluation was then completed and findings correlated with karyotype results. RESULTS: Twenty- two patients undergoing surgery for the diagnosis of ectopic pregnancy yielded chorionic villi for culture. Successful culture was performed in 21 patients, with 3 (14%) revealing abnormal karyotypes. Review of the medical record showed ultrasound results consistent with fetal development or a gestational sac in 15 of 18 patients with normal chromosomal analysis. Three of 6 patients without fetal development yielded abnormal chromosomal findings. CONCLUSION: Our results confirm that a high degree of success can be achieved in the karyotype analysis of ectopic pregnancy chorionic villi and that these conceptuses have a rate of abnormality similar to that reported for intrauterine gestations. Our data further suggest that when a gestational sac or fetal pole is identified by ultrasound, there is usually a normal karyotype.

No description available.

Mammalian telomeric DNA consists of tandem repeats of the sequence TTAGGG associated with a specialized set of proteins, known collectively as Shelterin. These telosomal proteins protect the ends of chromosomes against end-to-end fusion and degradation. The objective of this project was to investigate whether expression of Shelterin and Shelterin-associated proteins are altered, and influence the protection and maintenance of telomeres, in breast cancer cells. Initial findings showed that most of the Shelterin and Shelterin-associated genes were significantly down-regulated (at the mRNA expression level) in a panel of ten breast cancer cell lines. Epigenetic alterations to DNA (methylation at CpG Islands) and histones can result in altered expression of genes. Further investigations showed that the promoter region of POT1 was partially methylated in the breast cancer cell line, 21NT. To support these observations, a DNA methylation inhibitor, 5-aza-CdR, and a histone deacetylation inhibitor, TSA, were used in an attempt to reactivate the expression of silenced genes. This work generated novel findings. Treatment with 5-aza-CdR and TSA resulted in the highest recovery of TIN2 and POT1 mRNA levels at both short-term (48 and 72 hours) and long-term (3 weeks) treatment of the breast cancer cell line, 21NT cells. In addition, POT1 promoter methylation was analysed before and after treatment of 21NT cells. Bisulphite sequencing data were consistent with the mRNA expression results, showing up-regulation of POT1, as all methylation sites were demethylated after the treatment of 21NT cells with 5-aza-CdR. These studies also showed for the first time that both the short-term (72 hours) and 3 weeks treatment of 21NT cells with 5-aza-CdR was able to increase telomere lengths (using four measurement methods, i.e. TRF, q-PCR, flow-FISH and iQFISH). Breast cancer cell lines expressed low levels of several telosomal mRNAs and that this down-regulation was found to be due in part to promoter methylation. Methylation was shown to be relieved through treatment of the cells with 5-aza-CdR and TSA; specifically, POT1 was shown to be up-regulated to a higher extent compared with other Shelterin genes. Given that previous studies involved over-expression of POT1 in telomerase-positive cells to demonstrate telomere length elongation, we addressed the possibility that over-expression of POT1 may affect telomere length in 21NT breast cancer cells. The results showed that the average telomere length of the POT1 over-expressing clones was increased by 2 to 3 kb compared with 21NT non-transfected and empty vector controls. The study also demonstrated that increased telomere length (by ectopic over-expression of POT1) is not due to a direct effect of telomerase enzyme activity. One explanation for this could be that POT1 may induce a negative regulator of telomerase activity to maintain telomere length. Taken together, the results generated in this project suggest that POT1 may control a localised activation of telomerase enzyme at the telomere end, and regulate stability of the Shelterin complex.

Background Obesity is increasing worldwide and is a major contributor to morbidity and mortality. Notably, abdominal (central) obesity carries a high risk of obesity-related diseases, while peripheral fat accumulation can act in a protective manner. A redistribution of fat from peripheral to central depots is seen after the menopause and is associated with an increasing prevalence of diabetes and cardiovascular disease. A key mediator may be ectopic fat accumulation in the liver. Our hypothesis was that a Palaeolithic-type diet (PD) consumed ad libitum improves body composition and metabolic risk markers, including liver fat and insulin sensitivity, in obese postmenopausal women. Methods In study I the study subjects (n=10) used a PD during 5 weeks. In study II and III (n=70) the effect of a Palaeolithic-type diet (PD) was compared to a diet according to the Nordic Nutrition Recommendations diet (NNR) during a 2-year randomized clinical trial (RCT). Food records and nitrogen excretion in urine validated food intake. Anthropometric measurements were performed in a standardized manner. Body composition was calculated using Dual Energy X-ray Absorptiometry (DXA). Total energy expenditure was calculated by accelerometry (Actiheart®) in combination with indirect calorimetry. Liver and muscle fat content was estimated by magnet resonance spectroscopy (1H-MRS). Insulin sensitivity was measured either with hyperinsulinemic euglycemic clamps (paper I) or oral glucose tolerance tests (OGTTs) (paper III). Results In study I a significant weight loss, linked to improved lipid and blood pressure levels, was associated with a 49% decrease in liver fat. Concomitantly, hepatic insulin sensitivity improved, while peripheral insulin sensitivity (and muscle fat) was unaltered. In study II/III both groups had a significant and sustained weight loss after 2 years. The PD was more effective than the NNR diet regarding loss of weight and fat mass after 6 months, but not after 24 months. Serum triglyceride levels were significantly lower at 24 months in the PD group. Liver fat decreased throughout the study in both groups. Hepatic insulin sensitivity improved during the first 6 months of the study, while peripheral insulin sensitivity did not change. Hepatic insulin sensitivity was associated with liver fat at baseline, but not during the diet intervention. Energy expenditure did not change in any of the study groups. Conclusion Ad libitum diets can have sustained beneficial effects on weight and body composition in obese postmenopausal women, a PD being more effective on short-term than a diet according to the NNR. This is associated with a reduction in liver fat that may reduce the risk of future diabetes and cardiovascular disease. Further studies are needed in order to explore the association between liver fat and metabolic dysfunction, including insulin sensitivity.

Understanding the mechanisms plants utilize for seed storage protein (SSP) synthesis, transport and deposition have the potential rewards of enabling high yields of modified or foreign proteins. Hayashi et al. (1999) indicated that the machinery devoted to the synthesis of protein storage vacuoles in cotyledon cells can be induced in vegetative tissue by the constitutive expression of a pumpkin 2S albumin phosphinothricin-acetyl-transferase gene fusion (pumpkin 2S-PAT) resulting in the biogenesis of precursor-accumulating (PAC) vesicles in Arabidopsis leaves. This discovery was the impetus behind the work described which sought to examine this phenomenon further by ectopically evoking SSP trafficking and vesicle biogenesis machinery in leaves. With the aim of elucidating the mechanisms necessary to evoke PAC vesicle biogenesis, a suite of constructs including the pumpkin 2S-PAT and analogous napin-PAT and napin-GFP variants were synthesized. Analysis of these transgenes in Arabidopsis revealed that the pumpkin 2S albumin has a capacity unique from napin peptides to result in fusion protein accumulation. Further, the truncated pumpkin 2S albumin peptide and the pumpkin 2S albumin C-terminus were found to direct deposition to vesicles; however, the C-terminus alone was not enough to direct deposition to vesicles unless combined with a significantly shortened napin peptide. An increased ER protein throughput was correlated to trafficking of the fusion protein by Golgi-independent mechanisms resulting in stable accumulation of the unprocessed protein whereas less ER throughput indicated passage through the Golgi-dependent pathway resulting in accumulation of a processed variant. At the level of gene expression, as examined by a microarray study, both inducible and constitutive ectopic expression of pumpkin 2S-PAT resulted in substantial perturbations of the endomembrane system affecting protein folding, flowering time and ER-associated biosynthetic functions which indicated that modulation of flowering time and photoperiodism are highly dependent on protein trafficking and vacuolar biogenesis mechanisms and that high ER protein throughput occurs at the expense of biosynthesis and cessation of ER functioning.

Thesis (M.S.)--University of Michigan, 1962.

Thesis (Ph. D.)--Washington State University. / Includes bibliographical references.