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Thesis (MMed (Obstetrics and Gynaecology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Study objective: To investigate whether an educational intervention in the Gynaecology Department of Tygerberg Hospital (TBH) was effective in improving the accuracy of the diagnosis and appropriateness of treatment options offered to women with suspected Ectopic Pregnancy (EP). Methods: A retrospective cross-sectional before-and-after study was performed, including 335 consecutive patients with suspected EP before (1/3 - 30/6/2008) and after (1/9 - 31/12/2008) “the intervention”. From the gynaecological admissions register all pregnant patients with symptoms potentially compatible with EP were selected and these were cross referenced with beta-hCG requests, entries in the theatre register for surgery for possible EP and methotrexate prescriptions for EP in these time periods. “The intervention” consisted of a formal lecture presented to the registrars and consultants regarding the latest evidence-based guidelines concerning the diagnosis and management of EP. An algorithm based on this information was introduced in the emergency unit and ultrasound unit together with a prescribed ultrasound reporting form containing all the pertinent information required to follow the algorithm. Clinical decisions were left to the registrar and consultant on duty. Primary outcomes: Time from presentation to treatment, number and appropriateness of special investigations, surgical procedures or medical management. Secondary outcomes: Number of in-patient days and visits, adherence to the algorithm. Results: There was a non-significant trend towards improved reporting of the uterine content and significantly less reports of definite signs of an intrauterine pregnancy (IUP) (p<0.001, RR 0.46, 95% CI 0.31-0.70) due to stricter ultrasound criteria being followed. There was a significant change in the spectrum of uterine findings (p=0.001), the spectrum of adnexal findings (p=0.006) and the spectrum of free fluid noted (p=0.05). There was a reduction in the total number of beta-hCG levels requested at presentation (patients with no beta-hCG: 24 vs 34, p=0.05, RR 1.60, 95% CI 0.99-2.59) with a significant reduction in the number of inappropriate beta-hCG requests (77 vs 40, p<0.001, RR 0.60, 95% CI 0.43-0.81). There was a significant difference in the spread of the number of beta-hCG tests per patient with less repeat tests in the study group (p=0.021). Significantly less manual vacuum aspirations (MVAs) were performed (47 vs 21, p=0.003, RR 0.51, 95% CI 0.32-0.81) but there was no change in the other treatment modalities offered nor in the time from presentation to treatment, number of visits or in-patient days. Adherence to the algorithm was poor (59 %). Conclusions: Except for a significant decrease in the MVAs performed, with possibly less interrupted early intrauterine pregnancies, the improvement in the use of special investigations after “the intervention” did not translate into fewer inappropriate diagnoses and management. This could be due to frequent non-adherence to the algorithm, and widespread implementation of the algorithm as well as continuous audits would be necessary before a future study could be attempted to assess the efficacy of the algorithm. / AFRIKAANSE OPSOMMING: Studiedoelwit: Die hoofdoel van hierdie studie is om te ondersoek of „n opvoedkundige intervensie in die Ginekologiese afdeling van Tygerberg Hospitaal (TBH) doeltreffend sou wees in die verbetering van die akkuraatheid van diagnose en die gepastheid van behandelingsopsies wat aan vroue gebied word met „n vermoedelike ektopiese swangerskap (ES). Metodes: „n Retrospektiewe, kruisdeursnee voor-en-na studie rakende 335 opeenvolgende pasiënte wat ‟n vermoedelike ES het voor (1/3/2008 – 30/6/2008) en na (1/9/2008 – 31/12/2008) “die intervensie”. Swanger pasiënte is uit die ginekologiese toelatingsregister geselekteer indien hulle simptome gehad het wat moontlik verbind kon word met ES. Hulle is kruisverwys met die beta-hCG‟s aangevra, inskrywings in die teaterregister vir chirurgie vir moontlike ES en ginekologie-pasiënte wat metotrexate vir ES binne hierdie tydperke ontvang het. “Die intervensie” het bestaan uit „n formele lesing aan die kliniese assistente en konsultante ten opsigte van die jongste bewysgebaseerde riglyne rakende die diagnose en hantering van ES. „n Algoritme gegrond op hierdie inligting is in die noodeenheid en ultraklank-afdeling ten toon gestel asook „n voorgeskrewe ultraklank rapporteringsvorm met al die toepaslike inligting wat vereis word om die algoritme te volg. Kliniese besluite is aan die kliniese assistent en konsultant aan diens oorgelaat. Primêre uitkomste: Tydsduur vanaf aanmelding tot behandeling, aantal en gepastheid van spesiale ondersoeke, chirurgiese prosedures en mediese hantering. Sekondêre uitkomste: Die aantal binnepasiëntdae en besoeke, nakoming van die algoritme. Resultate: Daar was „n nie-betekenisvolle neiging tot beter rapportering van die uteriene-inhoud en betekenisvol minder rapportering van definitiewe tekens van „n intra-uteriene swangerskap (IUS) (p<0.001, RR 0.46, 95% CI 0.31-0.70) as gevolg van strenger ultraklankstandaarde gevolg. Daar was „n betekenisvolle verandering in die spektrum van uteriene bevindinge (p=0.001), die spektrum van die adneksale bevindinge (p=0.006) en die spektrum van die vrye vog aangeteken (p=0.05). Daar was „n vermindering in die totale aantal beta-hCG-vlakke aangevra met aanmelding (pasiënte met geen hCG: 24 vs 34, p=0.05, RR 1.60, 95% CI 0.99-2.59) met „n betekenisvolle vermindering in die aantal onvanpaste beta-hCGs aangevra (77 vs 40, p<0.001, RR0.60, 95% CI 0.43-0.81). Daar was „n betekenisvolle verskil in die verspreiding van die aantal beta-hCG-toetse per pasiënt, met minder herhalende toetse in die studiegroep (p=0.021). Betekenisvol minder manuele vakuum aspirasies (MVAs) is uitgevoer (47 vs 21, p=0.003, RR 0.51, 95% CI 0.32-0.81), maar geen verskil in ander behandelingsmodaliteite is aangebied nie, asook geen verskil in die tydsduur vanaf aanmelding, die aantal besoeke of die aantal binnepatiëntdae nie. Nakoming van die algoritme was swak (59%). Gevolgtrekkings: Behalwe vir „n betekenisvolle afname in die MVAs uitgevoer, met moontlik minder onderbroke vroeë IUS, het die verbetering in die gebruik van spesiale ondersoeke ná “die intervensie” nie minder onvanpaste diagnoses en hantering tot gevolg gehad nie. Dit kan die gevolg wees van gereelde nie-nakoming van die algoritme, en uitgebreide implementering van die algoritme asook voortdurende oudits sal nodig wees voor „n verdere studie aangepak kan word om die doeltreffendheid van die algoritme te bepaal.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2009. / Härtill 4 uppsatser.

The aim of this thesis was to investigate the effects of dietary fatty acids on body composition and ectopic fat in humans, with emphasis on the role of the omega-6 polyunsaturated fatty acid (PUFA) linoleic acid (18:2n-6) and the saturated fatty acid (SFA) palmitic acid (16:0). The overall hypothesis was that linoleic acid would be beneficial compared with palmitic acid during overfeeding, as previously indicated in animals. Papers I, II and IV were double-blinded, randomized interventions in which different dietary fats were provided to participants and Paper III was a cross-sectional study in a community-based cohort (PIVUS) in which serum fatty acid composition was assessed as a biomarker of dietary fat intake. In Paper I, overfeeding with sunflower oil (n-6 PUFA) for 7 weeks caused less accumulation of liver fat, visceral fat and total body fat (as assessed by MRI) compared with palm oil (SFA) in young and lean subjects despite similar weight gain among groups. Instead, sunflower oil caused a larger accumulation of lean tissue. In Paper II, plasma from Paper I was analyzed with NMR-based metabolomics, aiming to identify metabolites differentially affected by the two dietary treatments. Acetate decreased by PUFA and increased by SFA whereas lactate increased by PUFA and decreased by SFA. In Paper III, the proportion of linoleic acid in serum was inversely associated with contents of visceral-, subcutaneous- and total body adipose tissue whereas the proportion of palmitic acid was directly associated with visceral- and total body adipose tissue in 70-year old men and women. In Paper IV, overfeeding with sunflower oil for 8 weeks caused less accumulation of liver fat compared with palm oil also in overweight and obese subjects. SFA increased visceral fat in men only. Accumulation of lean tissue was similar between groups. In conclusion, SFA (palmitic acid) from palm oil promotes marked liver fat accumulation in both normal-weight and overweight/obese subjects during overeating, whereas n-6 PUFA (linoleic acid) from sunflower oil prevents such liver fat accumulation. Diverging effects of SFA and PUFA on visceral adipose tissue and lean tissue may only be applicable in some groups and/or circumstances. These results imply that negative effects associated with weight gain (e.g. fatty liver) may be partly counteracted by the type fat in the diet, overall supporting a beneficial role of diets higher in unsaturated fat compared with saturated fat for preventing liver fat accumulation.

The accumulation of leukocytes in non-lymphoid tissues and their structural organization into tertiary lymphoid structures (TLS), a process known as ectopic lymphoid neogenesis (ELN), is observed in response to chronic inflammation and in the target organ of several autoimmune diseases. TLS strongly resemble secondary lymphoid organs with specialised high-endothelial venules (HEV), segregated B/T cell areas and presence of follicular dendritic cells (FDC) networks promoting in situ affinity maturation of the antibody response. TLS have been associated with a growing number of autoimmune conditions and usually their presence is prognostic for undesirable disease progression. In Sjögren’s syndrome (SS), an autoimmune disease affecting the salivary and lachrymal glands leading to exocrine dysfunction, TLS develop in the salivary glands (SG) of around one-third of the patients. The immunobiology of the SG and the pathogenesis of SS have been poorly clarified and to date a robust and reproducible inducible animal model of SS and TLS in the SG is still absent. In my PhD, I developed and validated a novel inducible model of ELN in murine SG that also reproduces several features of SS. The retrograde administration of a replication-deficient adenovirus (AdV) in the SGs of wild-type C57Bl/6 mice was able to induce within three weeks fully formed TLS that displayed B/T cell segregation, FDC networks, HEVs and were positive for markers of germinal centres. Moreover, the AdV-treated mice showed a significant reduction of salivary flow and in 75% of the cases development of anti-nuclear antibodies.

DNA damage occurs through endogenous and exogenous sources, and can lead to stalled replication forks, genetic disorders, cancer, and cell death. Homologous recombination (HR) is a relatively fast and error-free repair pathway for damaged DNA, which can occur through a gene conversion event or through a crossing-over event with the exchange of genetic material. Homologous recombination occurs most frequently in the G2 phase of the cell cycle and utilizes the sister chromatid as the repair template. When the sister chromatid is unavailable, the homologous chromosome or a homologous sequence in an ectopic location can be used to repair the lesion; the latter of which is referred to as ectopic homologous recombination (EHR). Rad51 is a key protein involved in HR, and to test its role in EHR, variant Rad51 proteins were expressed in murine hybridoma cells. These Rad51 variants were assayed for their effects on EHR. Excess wild-type Rad51 as well as a deficiency of wild-type Rad51 decreased EHR from the background level found in these cell lines. Thus, Rad51 is necessary for EHR, but there may be an optimal amount of Rad51 required for efficient EHR. Expression of the Rad51 catalytic mutants Rad51K133A and Rad51K133R was found to have an inhibitory effect on EHR, as expected based on the loss of ATP binding and ATP hydrolysis, respectively, in these variants. Excess wild-type Rad51 was verified in this study to increase HR via a gene targeting assay. MMC treatment, but not ionizing radiation, leads to an increase in EHR in the presence of excess wild-type Rad51. Thus, endogenous levels of Rad51 are sufficient to maintain EHR, but in the presence of excess wild-type Rad51, the level of EHR can increase in response to certain DNA damaging agents and in response to gene targeting.

It is a well established phenomenon that the epithelial lining of the urinary bladder (urothelium) when implanted into skeletal muscle induces ectopic ossification. However, despite numerous observations, this reaction is poorly understood. This research further studied this reaction by - (a) demonstrating the reaction in a suitable small animal model; (b) attempting to induce the reaction by implanting urothelial cells purified by cell culture techniques; and (c) comparing the bone forming reaction induced by implanted urothelium to the reaction induced by implanting Bone Marrow Stem Cells (BMSC's) and Osteophyte Stem Cells (OSC's). By demonstrating newly formed bone after the implantation of guinea pig urothelium into the skeletal muscle of a Severe Combined Immuno-Deficient Mouse (SCID-Mouse) this research demonstrated that a suitable small animal model had been established. This is despite inherent difficulties (particularly bacterial contamination) associated with establishing a primary cell culture of guinea pig urothelial cells. Additionally, the intramuscular ectopic osteoinductive potential of human BMSC's (hBMSC's) in the SCID-mouse has also been demonstrated. Confirming that the injection of cultured cells in suspension is an adequate intramuscular delivery technique, this research demonstrates that hBMSC's induce ectopic ossification by non-immunological means. This research has demonstrated a number of differences between urothelium induced ectopic ossification and ectopic ossification induced by BMSC's, suggesting they are two separate processes. This is important because the chemotaxis and subsequent osteogenic differentiation of BMSC's has previously been one of the more popular postulated mechanisms of urothelium induced ectopic ossification. Finally, this research has demonstrated the ectopic osteoinductive potential of stem cells isolated from the marrow of human osteophytes (human Osteophyte Stem Cells, hOSC's). This observation has not been previously reported, and will hopefully provide a valuable contribution to a body of knowledge that has important ramifications in both the treatment of osteoarthritis, and the use of BMSC's in tissue engineering.

<p>Every living cell faces a multitude of DNA threats in its lifetime because damage to DNA is intrinsic to life itself. A double-strand break (DSB) is the most cytotoxic type of DNA damage and is a potent inducer of chromosomal aberrations. Defects in DSB repair are a major driver of tumorigenesis and are associated with numerous developmental, neurological and immunological disorders. To counteract the deleterious effects of DSBs, organisms have evolved a homologous repair (HR) mechanism that is highly precise. The key to its error-free nature lies in its use of a homologous template in restoring the DSB and its preferential occurrence during late S and G2 phase of the cell cycle when identical sister chromatids are available as templates for repair. However, HR can also engage homologous chromosomes and ectopic substrates that share homology, resulting in mitotic loss-of-heterozygosity (LOH) and unwanted chromosomal aberrations. In this case, understanding of the underlying mechanisms and molecular factors that influence accurate sequence transfer and exchange between two homologous substrates becomes crucial. </p><p>The focus of this dissertation is examination of the genetic factors and molecular processes occurring at early intermediate steps (DNA end resection and DNA synthesis) of mitotic recombination in Saccharomyces cerevisiae. To model DSB repair, we established a unique plasmid-based assay with a small 8-base pair (bp) gap in the middle of an 800-bp plasmid substrate. To delineate the molecular structures of strand exchange intermediates during HR, we used a 2% diverged plasmid substrate relative to a chromosomal repair template to generate mismatch-containing heteroduplex DNA (hetDNA) intermediates. The assay was performed in a mismatch repair (MMR)-defective background allowing hetDNA to persist and to segregate into daughter cells at the next round of replication. Unexpectedly, even when MMR was inactivated, sequence analysis of the recombinants revealed patches of gene conversion and restoration reflecting mismatch correction within hetDNA tracts. We showed that, in this system, MMR and nucleotide excision repair (NER) correct mismatches via two different mechanisms. While mispairing of nucleotides triggers MMR, NER is recruited by the subtle 6-methyladenine mark on the plasmid substrate, leading to coincident correction of mismatches. The methylation marks on the plasmid were acquired from the bacterial host’s native restriction-modification system during plasmid propagation. </p><p>Formation of hetDNA occurs when a plasmid substrate engages the chromosomal template for repair, forming a D-loop intermediate. D-loop extension requires DNA synthesis by DNA polymerase/s. Translesion synthesis (TLS) polymerases have been implicated in HR in both chicken DT40 cells and fruit fly, but not in yeast. This class of polymerases is known for its low fidelity due to a lack of exonuclease domain and is commonly used for lesion bypass and in extending ends with mismatches. We reported for the first time a requirement of Polζ-Rev1 and Polη (TLS polymerases in S. cerevisiae) for completing gap repair. Moreover, gap-repair efficiency suggested that these two polymerases function independently. We concluded that TLS polymerases are involved in either extending the invading 3’ end and/or in the gap-filling process that completes recombination. </p><p>DNA resection of a DSB serves as a primary step to generate a 3’ single-stranded DNA (ssDNA) for subsequent homologous template invasion, but this process has mostly been studied in the absence of a repair template or when downstream HR steps are disabled. To analyze the individual contributions of identified nucleases to DSB resection in the context of repair, we established a chromosomal assay; the substrate size was increased to 4 kilobases (kb) and 85 SNPs were present at ~50 bp intervals. In this chromosomal assay, resection and DNA synthesis influence the length of hetDNA tracts in the final recombinants, allowing these two steps to be analyzed. We specifically focused on synthesis-dependent strand annealing (SDSA) events, where hetDNA reflects DNA synthesis and extent of resection. Our main conclusions are as follows. DNA end resection on the annealing end of NCO products generated by SDSA is not as extensive as one might expect from resection measured in single-strand annealing (SSA) assays. In addition, although the two long-range resection pathways (Sgs1-Dna2 and Exo1) can support recombination in a redundant manner, hetDNA was significantly reduced upon loss of either. End processing of DSBs is predominantly 5’ to 3’, but we also observed loss of sequences (greater than 8 nt but less than 40 nt) at the 3’ termini. We have tested and ruled out the involvement of Mre11 and Polε proofreading activity. Lastly, Pol32 functions as a subunit of Polδ to promote extensive repair synthesis during SDSA. hetDNA tract lengths were significantly shorter in the absence of the Pol32 subunit of Polδ, providing direct evidence that Polδ extends the invading end during HR. Together, this work advances our understanding of how resection nucleases and DNA polymerase/s function to regulate mitotic recombination outcome and influence the molecular patterns of NCOs.</p> / Dissertation