Thesis (Ph. D.)--University of Wisconsin--Madison, 1968. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliography.

Thesis (Ph.D.)--Bowling Green State University, 2006. / Document formatted into pages; contains viii, 97 p. Includes bibliographical references.

Thesis (M.A.) -- Central Connecticut State University, 2007. / Thesis advisor: Thomas R King. "... in partial fulfillment of the requirements for the degree of Master of Arts in Biomolecular Sciences." Includes bibliographical references (leaves 29-30). Also available via the World Wide Web.

No description available.

Abstract Type IX collagen is a quantitatively minor component of cartilage collagen fibrils. Although a few mutations have been associated with multiple epiphyseal dysplasia, recent evidence suggests involvement of type IX collagen in a wider spectrum of phenotypes. The functional role of this molecule remains undetermined, in part due to difficulties in obtaining high amounts of intact protein. To facilitate more efficient mutation screening and comparison of the genomic organization of the human genes encoding the α1(IX), α2(IX) and α3(IX) polypeptides, their genomic structures were characterized. Complete nucleotide sequences were determined for the COL9A2 and COL9A3 genes along with sequences for all the exon boundaries in the COL9A1 gene. Putative transcription control elements were identified and the alternative promoter region was characterized in the human and mouse COL9A1 genes. Mutation screening was performed for the COL9A3 gene and two apparently neutral 9-bp deletions within the COL1 domain were identified. These are the first deletions within a triple-helical domain of any collagen that are not associated with a disease phenotype. An insect cell expression system with an exogenous source of prolyl 4-hydroxylase was used to produce heterotrimeric human type IX collagen. The recombinant protein consisted of the three a chains in a 1:1:1 ratio and showed correct folding and high thermal stability. Up to 10 mg of secreted protein could be purified from a litre of culture medium. The expression system was used to analyze the chain association of type IX collagen in cellulo. Although the chains are capable of homotrimerization, a preference for heterotrimer formation was noted.The neutral deletion was characterized further using the insect cell system. Mutant α3(IX) chains carrying a deletion of one Gly-X-Y triplet were shown to form correctly folded heterotrimers with the wild-type α1(IX) and α2(IX) chains. The results suggest a function for the NC2 domain in neutralizing the effect of the deletion. This work provides a novel means for the analysis of type IX collagen mutations and their protein-level effects, and should enable future studies to be made of the structure-function relationship in type IX collagen.

Big Blue® lacI transgenic mice and rats carry the E. coli lacI gene integrated as a tandem array of approximately 40 copies integrated at a single site in chromosome 4. This mutationally well-characterized gene is highly sensitive to base substitution mutations and is readily recovered from virtually any tissue of the transgenic host, facilitating the in vivo study of mutation. The Big Blue® assay was used to investigate spontaneous and induced mutation, with an emphasis on dietary influences on mutational frequency and specificity. The effects of ageing and dietary restriction on spontaneous mutation in the lacI transgene were determined in mice, permitting evaluation of several well established theories of ageing. Mutation frequencies were found to increase with age in tissues that proliferate (bladder and liver, but not brain), validating a principle tenet of the somatic ageing theory. However, the unexpected lack of a change in mutational specificity in ageing mice suggests that theories of ageing based on oxidative damage, or a reduction in DNA repair efficiency, may not be seminal to the ageing process, at least until more advanced age. Similarly, dietary restriction, which is known to extend lifespan in rodents and was predicted to decrease oxidative DNA-damage, had no appreciable effect on either the frequency or the specificity of spontaneous mutation in liver of younger (6 month old) and older (12 month old) mice. Dietary influences on induced mutation were examined following treatment with aflatoxin B₁ (AFB₁) and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) powerful animal carcinogens which demonstrate tissue, species and sex-linked differences in rats and mice. As expected, AFB₁ was found to be potently mutagenic in rat but not mouse liver, in agreement with rodent carcinogenicity studies that found F344 rats to be highly susceptible to AFB₁-induced hepatocarcinogenesis, while C57BL/6 mice are highly resistant. PhIP was found to be potently and equally mutagenic in colon of both male and female rats. The result in female colon was surprising since PhIP predominantly induces colon cancer in male rats but mammary tumors in female rats. Therefore, the progression of PhIP-induced colon cancer in the rat colon is likely due to factors acting at a later stage in the tumorigenic process, following the damage and mutation of DNA. Rat prostate tissue, another tumor target tissue in PhIP-treated rats, was also found to be highly susceptible to PhIP-induced mutagenesis. Lastly, the PhIP studies were extended to an additional transgene target located in the shuttle vector construct from Big Blue® rodents, the bacteriophage λ-derived cII gene. These studies validated the use of the λ cII gene as an alternative mutational target for use in the Big Blue® assay, while the analyses of mutation in the lacI and the λcII transgenes serves as a paradigm for mutational studies which compare mutational responses in different genes. Collectively, these studies demonstrate the utility of the lacI (λ cII) transgenic mutagenicity assay for the in vivo investigation of mutational processes as a function of age, diet, sex, species, and target tissue specificity with respect to sites of mutation and cancer. / Graduate

Mutations are generally thought to be targeted events. The distribution of mutations is based upon the initial original deposition of DNA damage and the fidelity and efficiency of repair of this damage. These factors are dependent on the primary site of DNA modification and the surrounding nucleotides (i.e., mutation is “context sensitive"). To better understand mutagenesis, I measured DNA damage and/or mutation at the DNA sequence level, then considered the impact of mutation location and the surrounding nucleotide environment. The selected mutagens, ultraviolet light (UV) and benzo(a)pyrene [B(a)P], were chosen because they produce well-characterized lesions and are environmentally relevant. UVC (254nm) light-induced DNA damage is well documented. UVC produces a characteristic spectrum of mutations. The predominant UV-induced mutations are C → T transitions occurring at TC or CC sites, as well as CC → TT tandem transitions. The latter class of mutation is considered the hallmark of UV mutagenesis. Quantitatively speaking, the primary types of UV-induced DNA lesions are cyclobutane pyrimidine dimers (CPDs) and the 6-4 pyrimidine/pyrimidone (64PyPy). These are also the suspected predominant pre-mutagenic lesions. Each lesion was independently measured at the DNA sequence level in a defined region of DNA. The pattern of UVC-induced DNA damage revealed a complex induction pattern. The flanking DNA nucleotides partially influenced the pattern of damage deposition. Sites where C → T transitions and CC → TT transitions were recovered at high frequencies were also frequently damaged. Thus, at these sites, mutation fixation was potentially more influenced by initial DNA damage than the rate of DNA repair. Two other components of the UV spectrum [UVB (290-320 nm) and UVA (320-400 nm)] are more environmentally relevant than UVC since UVB and UVA reach the surface of the earth.The results of UVC experiments were used as a guide to interpret the results obtained using UVB since direct light absorption by DNA has been shown to be one of the main biological effect at both wavelengths. The model that was chosen for the studies was an in vivo transgenic rodent mutagenesis assay. The research presented in the thesis represents one of the first studies to characterize UV-induced mutation at the DNA sequence level in rodent skin. The backs of female C57B1/6 lacl transgenic mice were shaved and exposed to either UVB or UVA light. UVB was found to be significantly more mutagenic than UVA. The UVB-induced mutation spectrum was characterized by C → T transitions at dipyrimidine sites, implicating CPD and/or 64PyPy lesions as premutational DNA lesions. The majority of UVA-induced mutations was C → T transitions at dipyrimidines sites and hence, as with UVB-induced mutation, attributed to CPDs and 64PyPy. In the UVA-dose response experiments, the induced mutant frequency was lower than expected at higher doses. A statistically significant increase in putative clonal expansion suggested that skin cells might have undergone cell killing followed by repopulation. In a final study, C57Bl/6 lacI transgenic male mice were intraperitoneally injected with the mutagen B(a)P at doses of 0, 62.5, 125, 250, and 500 mg/kg. This resulted in a linear increase in mutation frequency (4.8 to 53 × 10⁻⁵). All mutations increased at GC basepairs and not AT basepairs following B(a)P treatment. This was consistent with models suggesting guanosine adducts to be mutagenic lesions. In conclusion, the transgenic lacI mouse mutagenesis model was a sensitive target for in vivo mutagenesis from UVB, UVA and benzo(a)pyrene exposures. The system detected class-specific mutation frequency differences and increases in cell proliferation after mutagen exposure. With a further refinement of techniques, the correlation of DNA damage and mutation will allow even more exquisite studies. / Graduate

Software is all around us in our lives in the industrialized world, and we as a society and individuals need it to function correctly. Software testing fills the role of performing behavior audits, to guide the correction of the software to its intended behavior. The consequences of faulty software can range to the late arrival of trains, to nuclear meltdowns. This places quality requirements on the software of various levels. Program based mutation testing provides a high level of faultfinding capability. It does this by injecting many synthetic faults into the code under test, as described by mutation operators. These faults are used to search for testcases that would identify such faults, and consequently find real faults that the synthetic faults mimic. However, mutation testing is costly on three accounts; each mutant of the original code is compiled, each mutant should ideally have an associated testcase to reveal that fault the mutant contains, finally the testcases are analyzed thoroughly by looking the output of the original and mutants to reveal the error in behavior. In order to reduce cost while maintaining a high level of faultfinding, selective mutation testing is investigated, it uses a subset of all the available mutation operators. The investigation found that using Absolute value-, and Relational operator-, mutation reduces cost of mutation testing by 80%, while uncovering 83% of the injected faults.

Abstract Collagens IX and XI are quantitatively minor components of the collagen fibrils in cartilage. The spectrum of the phenotypes caused by mutations in the COL2A1 gene coding for collagen II, the main cartilage collagen, is relatively well defined, but there is little data on the phenotypes caused by collagen IX and XI mutations. The structure of the human COL11A1 gene coding for the 1 chain of collagen XI was characterized here. It was found to consist of 68 exons and span 160 kb, excluding introns 1 and 4. Over 50 kb of new intronic sequences were defined. The exon-intron organization coding for the major triple helical domain was found to be identical to that of the human COL11A2 gene, which codes for the 2 chain of collagen XI. The sensitivity of conformation sensitive gel electrophoresis (CSGE) for mutation detection was improved and tested with a large number of sequence variations in collagen genes. The sensitivity with the revised conditions was found to be close to 100%. In addition, CSGE was found to be a simple and practical method for analyzing large numbers of samples. Fifteen mutations in the COL11A1 gene and eight in the COL2A1 gene were found by CSGE in patients with Marshall or Stickler syndrome. The genotypic-phenotypic comparison indicated that mutations leading to a premature translation termination codon in the COL2A1 gene resulted in Stickler syndrome and splicing mutations of 54 bp exons in the C terminal half of the COL11A1 gene resulted in Marshall syndrome. The other COL11A1 mutations caused phenotypes overlapping both syndromes. In an analysis of the COL9A2 gene in 157 patients with intervertebral disc disease, six were found to have a tryptophan for glutamine substitution in the central collagenous domain of the collagen IX molecule. None of 174 control individuals had this substitution. The substitution cosegregated with the phenotype in the families studied, and linkage and linkage disequilibrium analyses supported the association of the locus and the disease with a joint lod score of over 11.

In 1980, the World Health Organization task force first sought to define and classify cardiomyopathies. They defined cardiomyopathies as "heart muscle diseases of unknown cause" with three main classifications including: hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and restrictive cardiomyopathy [1]. Over the next three decades it became patently obvious that this simple definition was not sufficient to describe the complex heterogeneity of diseases present in the patient population. More robust definitions were necessary for mechanistic links to be established and meaningful therapeutics to be developed. Since then the accepted definition of a cardiomyopathy has evolved and the classifications have greatly expanded. The most recent definition from the American Heart Association Council on Clinical Cardiology states: Cardiomyopathies are a heterogeneous group of diseases of the myocardium associated with mechanical and/or electrical dysfunction that usually (but not invariably) exhibit inappropriate ventricular hypertrophy or dilatation and are due to a variety of causes that frequently are genetic. Cardiomyopathies either are confined to the heart or are part of generalized systemic disorders, often leading to cardiovascular death or progressive heart failure–related disability [2]. This latest definition (2006) reflects the growing recognition of molecular genetics as a key factor in the development of cardiomyopathies and highlights the ever-growing complexity of disease classification. Today the genetic basis of HCM and DCM is widely recognized yet our understanding of the precise mechanisms underlying the disease remains unclear. To add to this disconnect, by the time patients become symptomatic, pathology has progressed past the initial phase, where meaningful treatment could occur, to advanced end-stage pathology. By this time often the only treatment options available become "blunt sword" therapeutics that are non-specific and used primarily for symptom management. In fact, over the last 3 decades there has been a marked decline in the innovation of cardiovascular pharmaceuticals owed partially to the vast complexity of disease presentation and progression [3]. In this dissertation, I will focus on a genetic sarcomeric DCM caused by a mutation in alpha-tropomyosin (Tm). Using novel accurate mouse models as a tool we will define the mechanism by which it leads to disease, investigate how disease severity due to the mutation is modified in an age-dependent manner, and examine what this mechanism could mean in the larger picture of cardiomyopathic disease progression. I hope to convince you that by using accurate models of this DCM at multiple levels of biological complexity to tease out the precise mechanisms of disease we can establish meaningful genotype-phenotype relationships that could lead to the development of specific novel therapeutics.