<p> The type 1 transmembrane receptor tyrosine kinase (RTK), Neu/ErbB2 is a member of the Epidermal Growth Factor Receptor (EGFR) family that functions as a potent mediator of normal cell-growth and development. However, the aberrant expression of RTKs has also been implicated in many human cancers. For example, Neu over-expression has been implicated in human breast and ovarian cancers, and is correlated with poor clinical prognosis. At least one pTyr residue in Neu, Let-23, and PDGFR-B receptor tyrosine kinase is reported to have negative signaling capability. The intrinsic negative signaling behaviour of any of these pTyr residues, such as pTyr at 1028 of Neu (NeuYA) has yet to be fully exploited with a view to better understanding of RTK signaling, leading to more precise knowledge in human cancer development and disease treatment. </p> <p> Here we aimed to determine the role, specificity and the signaling pathway components of rat-NeuYA in Drosophila melanogaster. Specifically, we asked whether pTyr 1028 of Neu could affect signaling from heterologous RTKs. If so, what would be the pathway components? Are they already known or novel? Using a targeted misexpression system, such as the GAL4-Upstream Activating Sequence (GAL4-UAS) system, we generated graded phenotypes of various Neu alleles in adult Drosophila eye and wing tissues, suitable for dosage sensitive modifier screening. Taking the advantage of these graded phenotypes, we sought to identify and evaluate the signaling characteristics of Neu/ErbB2. NeuYA, in particular, suppressed the rough-eye phenotype of other 'add-back' Neu alleles, suggesting an inhibitory role in RTK signaling. The dosage sensitive modifier screen has also shown that the signal attenuating steps, such as receptor-mediated endocytosis, receptor recycling, and lysosomal degradation work in a YA-independent manner. To identify the components of the NeuYA signaling pathway in RTK signal attenuation, a genome-wide dominant modifier screen was undertaken to screen over 60,000 F1 progeny either for suppression or enhancement of the rough-eye phenotype of GMR-NeuYAE adults. Using deficiency mapping, we isolated and identified that one complementation group of suppressors to be alleles of lilliputian. Additionally, we narrowed down several groups of enhancers to certain deficiency regions, uncovering 10-30 genes- previously not known to the receptor tyrosine kinase signaling pathways. Collectively, here we report several novel NeuYA-interactors in RTK signal attenuation in Drosophila. </p> / Thesis / Doctor of Philosophy (PhD)

Yes / Human identification from biological material is largely dependent on the ability to characterize genetic polymorphisms in DNA. Unfortunately, DNA can degrade in the environment, sometimes below the level at which it can be amplified by PCR. Protein however is chemically more robust than DNA and can persist for longer periods. Protein also contains genetic variation in the form of single amino acid polymorphisms. These can be used to infer the status of non-synonymous single nucleotide polymorphism alleles. To demonstrate this, we used mass spectrometry-based shotgun proteomics to characterize hair shaft proteins in 66 European-American subjects. A total of 596 single nucleotide polymorphism alleles were correctly imputed in 32 loci from 22 genes of subjects’ DNA and directly validated using Sanger sequencing. Estimates of the probability of resulting individual non-synonymous single nucleotide polymorphism allelic profiles in the European population, using the product rule, resulted in a maximum power of discrimination of 1 in 12,500. Imputed non-synonymous single nucleotide polymorphism profiles from European–American subjects were considerably less frequent in the African population (maximum likelihood ratio = 11,000). The converse was true for hair shafts collected from an additional 10 subjects with African ancestry, where some profiles were more frequent in the African population. Genetically variant peptides were also identified in hair shaft datasets from six archaeological skeletal remains (up to 260 years old). This study demonstrates that quantifiable measures of identity discrimination and biogeographic background can be obtained from detecting genetically variant peptides in hair shaft protein, including hair from bioarchaeological contexts. / The Technology Commercialization Innovation Program (Contracts #121668, #132043) of the Utah Governors Office of Commercial Development, the Scholarship Activities

All-male fry are preferred to prevent uncontrolled reproduction before harvest in intensive Nile tilapia (Oreochromis niloticus) aquaculture. Males also grow faster than females. An alternative approach to direct hormonal masculinisation of tilapia fry is to produce fry that are genetically male. However, sex determination system in tilapia is fairly complex. Recent developments have resulted in a linkage map and genetic markers that can be used to analyse the sex determination system. To analyse the genetic sex determination mechanism and to develop marker-assisted selection in the Stirling Nile tilapia population, a fully inbred line of clonal females (XX) was verified using test crosses and DNA markers (mostly microsatellites) to use as a standard reference line in sex determination studies. A series of crosses were performed involving this line of females and a range of males. Three groups of crosses were selected (each group consisted of three families) from progeny sex ratio distributions, and designated as type ‘A’ (normal XY males x clonal XX females), type ‘B’ (putative YY males x clonal XX females) and type ‘C’ (unknown groups of males x clonal XX females), for sex linkage study. For type ‘A’, inheritance of DNA markers and phenotypic sex was investigated using screened markers from tilapia linkage group 1 (LG1) to confirm the LG1-associated pattern of inheritance of phenotypic sex and the structure of LG1. Screened markers from LG1, LG3 and LG23 were used to investigate the association of markers with sex in families of type ‘B’ and ‘C’. In addition, a genome-wide scan of markers from the other 21 LGs was performed to investigate any association between markers and sex, in only families of cross type ‘B’. LG1 associated pattern of inheritance of phenotypic sex was confirmed by genotype and QTL analyses in families of cross type ‘A’. Analyses of genotypes in families of type ‘B’ and ‘C’ showed strong association with LG1 markers but no association with LG3 and LG23 markers. Genome wide scan of markers from all other LGs did not show any significant association between any markers and the sex. The allelic inheritance of two tightly linked LG1 markers (UNH995 and UNH104) in families of type ‘B’ and ‘C’ identified polymorphism in the sex determining locus: one of the alleles was associated mostly with male offspring whereas another allele was associated with both progeny (mostly males in type ‘B’ families, and approximately equal numbers in type ‘C’ families). This knowledge was used to identify and separate supermales (‘YY’ males) that should sire higher proportions of male progeny, reared to become sexually mature for use as broodstock. Two of them were crossed with XX females (one clonal and one outbred) to observe the phenotypic expression of the strongest male-associated allele in progeny sex. The observations of 98% male (99 males out of 101 progeny) and 100% male (N=75) from these two crosses respectively, suggest that a marker-assisted selection (MAS) programme for genetically male Nile tilapia production could be practical. This study also suggests that the departures from the sex ratios predicted using a “simple” XX/XY model (i.e., YY x XX should give all-male progeny) were strongly associated with the XX/XY system, due to multiple alleles, rather than being associated with loci in other LGs (e.g., LG3, LG23). This study also tentatively names the allele(s) giving intermediate sex ratios as “ambivalent” and emphasizes that the presence and actions of such allele(s) at the same sex-determining locus could explain departures from predicted sex ratios observed in some earlier studies in Nile tilapia.

<p>Morfometrijske analize su ra&ntilde;ena merenjem krilne nervature 540 uzoraka krila<br />pčela sa 9 lokaliteta u Vojvodini. Izračunato je 16 uglova (A1, A4, B3, B4, D7, E9, G7,<br />G18, H12, J10, J16, K19, L13, M17, O26, Q21) koje zaklapa krilna nervatura i 4 indeksa<br />(Ci, Pci, Dbi, Ri), ukupno 20 mera. Izračunate su prosečne vrednosti i utvr&ntilde;ena je<br />statistička značajnost razlika izme&ntilde;u pčela iz regona Srema i Bačke i pčela iz regiona<br />Banata, tako&ntilde;e je izvr&scaron;eno i pore&ntilde;enje pčela svih lokaliteta sa DAWINO standardima za<br />5 rasa pčela (Apis mellifera carnica, Apis mellifera macedonica, Apis mellifera mellifera,<br />Apis mellifera ligustica i Apis mellifera caucasica).<br />Analizom varijanse izračunatih 20 osobina krilne nervature, utvr&ntilde;eno je da samo<br />kod osobine A4 nisu utvr&ntilde;ene statistički značajnie razlike izme&ntilde;u posmatranih<br />lokaliteta, dok su u 19 osobina utvr&ntilde;ene statistički značajne razlike.<br />Utvr&ntilde;ivanjem statističke značajnosti razlika, pčela iz regiona Srema i Bačke i pčela<br />iz regiona Banata, utvr&ntilde;eno je da 45% osobina ne pokazuju statistički značajne razlike,<br />dok 45% osobina pokazuje statistički vrlo značajne razlike (P&lt;0,01) i 10% osobina<br />pokazuje statistički značajne razlike (P&lt;0,05).<br />Upore&ntilde;ivanjem dobijenih vrednosti 20 parametera krilne nervature, pomoću ztesta,<br />sa DAWINO standardima za pet rasa pčela, utvr&ntilde;eno je da na bazi celog uzorka<br />statistički nema značajnih razlika kod osobina A4 i D7 sa A. m. carnica, kod osobina<br />H12, G18 i B4 sa A. m.macedonica i kod osobina J16 i B4 pore&ntilde;eno sa rasom A.<br />m.ligustica. Kod pčela iz regiona Srema i Bačke utvr&ntilde;eno je da statistički nema značajnih<br />razlika kod osobina A4, B3, D7 i G18 upore&ntilde;eno sa A.m. carnica, kod osobina H12 i B4<br />upore&ntilde;eno sa A. m.macedonica i kod osobina G18, K19, J16 i Q21 upore&ntilde;eno sa rasom<br />A. m.ligustica, dok kod pčela iz regiona Banata utvr&ntilde;eno je da statistički nema značajnih<br />razlika kod osobina A4, E9, D7 i J10 upore&ntilde;eno sa A.m. carnica, kod osobina H12, J10,<br />L13 i PCi upore&ntilde;eno sa A. m.macedonica i kod osobina B4, J16 i PCi upore&ntilde;eno sa<br />rasom A. m.ligustica.<br />Ocena genetičke povezanosti, unutar populacijska raznolikost i struktura<br />populacije, dva tipa pčela u Vojvodini, izračunata je na bazi varijacije alela 25 lokusa<br />mikrosatelita. Izvr&scaron;ena je genetska tipizacija sledećih mikrosatelita: A8, A14, A24, A29,<br />A43, A79, A88, A113, Ac11, Ac88, Ac139, Ac306, Ap15, Ap68, Ap85, Ap90, Ap223,<br />Ap224, Ap226, Ap249, Ap273, Ap274, Ap288, At168, At188. 92% ili 23 lokusa su se<br />pokazali kao polimorfni u uzorcima pčela iz Srema i Bačke, a 88% ili 22 lokusa su se<br />pokazali kao polimorfni u uzorcima pčela iz Banata. Izračunata heterozigotnost na nivou<br />cele populacije se nije statistički značajno razlikovala od očekivane heterozigotnosti.<br />Utvr&ntilde;eno je da dobijene genetičke razlike izme&ntilde;u analiziranih pčela iz regiona Srema i<br />Bačke i retgiona Banata nisu dovoljne da se ove dve populacije mogu smatrati<br />razdvojenim.</p> / <p> Morphometric analyses have been done by measuring the wing nervature in<br /> 540 samples of bees, collected from nine localities in Vojvodina. 16 angles<br /> formed by wing nervation have been calculated(A1, A4, B3, B4, D7, E9, G7,<br /> G18, H12, J10, J16, K19, L13, M17, O26, Q21) as well as four indexes (Ci, PCI,<br /> DBI , R), a total of 20 measures. The average values have been calculated and<br /> statistical significant differences in bees from Srem, Backa and Banat region<br /> determined. Five breeds of bees from these regions have been compared to<br /> Dawino standards.<br /> The analyses of the variance of calculated 20 features of wing nervature indicate<br /> that statistically significant differences in monitored localities have not been found<br /> only in A4, on the other hand in 19 properties significant differences have been<br /> discovered.<br /> Established statistically significant differences between breeds from Srem<br /> and Backa regions reveale that 45% properties do not show any statistically<br /> important differences, while 45% features show very important statistical<br /> differences (P&lt;0,01) and 10% show statistically important differences (P&lt;0,05).<br /> It has been established by comparing the obtained values of 20 parametres<br /> of wing nervature by means of z test to DAWINO standards for five breeds of<br /> bees that, based on the whole sample, there are no significant differences in<br /> features A4 and D7 in A.m. carnica, in features H12, G18 and B4 in A.m.<br /> macedonica and features J16 and B4 compared to A.m. ligustica. As for bees from<br /> Srem and Backa region,there are statistically no significante differences in<br /> features A4, B3, D7 and G18 compared to A.m. carnica, features H12 and B4<br /> compared to A.m. macedonica and features G18, K19, J16 and Q21 compared to<br /> A.m. ligustica, while in bees from Banat region, statistically there are no<br /> significant differences in features A4, E9, D7 and J10 compared to A.m. carnica,<br /> features H12, J10, L13 and Pci compared to A.m. macedonica and features B4,<br /> J16 and Pci compared to A.m. ligustica.<br /> The evaluation of genetic correlation, the diversity of bees population and<br /> population structure of two types of bees in Vojvodina have been established on<br /> the basis of allels variations in 25 locus microsatelites.The following<br /> microsatelites have been standardized &ndash; A8,A14,A24,A29, A43, A79, A88, A113,<br /> Ac11, Ac88, Ac139, Ac306, Ap15, Ap68, Ap85, Ap90, Ap223, Ap224, Ap226,<br /> Ap249, Ap273, Ap274, Ap288, At168, At188. 92% or 23 locus have shown as<br /> polymorphs in bees from Srem and Backa and 88% or 22 locus samples have<br /> shown as polzmorphs in bees samples from Banat and Backa region. The whole<br /> population calculated heterozygosity has not shown statistically significant<br /> differrence from expected heterozygisity. It has been established that the obtained<br /> genetic differences between the analysed bees from Srem and Backa region and<br /> Banat region are not significant to indicate two populations.</p>

Orientador: Jeffrey Frederico Lui / Banca: Humberto Tonhati / Banca: Selma de Fátima Grossi / Resumo: A ocorrência de híbridos entre javalis e suínos, tanto na natureza como em cativeiro, é bastante comum. Assim, tem-se detectado polimorfismo em javalis, variando o número de cromossomos de 36 a 38. Nesse sentido, objetivou-se, com este trabalho, caracterizar geneticamente javalis (Sus scrofa scrofa) puros e híbridos criados no Brasil, por meio de loci de microssatélites (STRs) do suíno doméstico (Sus scrofa domestica). Para efeito de classificação, os animais foram agrupados, segundo análise de pedigree e número diplóide (2n), em 5 grupos genéticos: grupo I, constituído de 59 suínos domésticos; grupo II, formado por 46 javalis puros de origem; grupo III, constituído de 3 híbridos, com 2n=36, provenientes de acasalamentos entre híbridos e retrocruzamentos; grupo IV, representando 30 híbridos com suíno doméstico de ploidia igual a 37 cromossomos; e grupo V, constituídos de 10 híbridos também com o doméstico, porém com 2n=38, conhecidos popularmente como Javaporcos, devido à similaridade cariotípica e fenotípica com o suíno doméstico. O DNA genômico foi extraído e, posteriormente, amplificou-se, pela técnica de PCR, os fragmentos desses microssatélites - IGF1, ACTG2, TNFB -, os quais foram desenvolvidos para a subespécie Sus scrofa domestica. As condições de amplificação foram padronizadas para as amostras de javali realizadas em um termociclador, com as temperaturas de anelamento variando para cada primer. Ao final das amplificações, os produtos dos microssatélites foram colocados em um seqüenciador capilar modelo ABI 3100 Avant (Applied Biosystems). A partir dos resultados obtidos no presente trabalho, concluiu-se que os microssatélites IGF1, ACTG2 e TNFB, usados em suínos, são eficiente na amplificação heteróloga e podem ser aplicados em javali. Os javalis puros se diferenciam geneticamente dos suínos e dos híbridos...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The occurrence of crossbred animals between wild boar and pigs, both in nature and in captivity, is quite common. Thus, polymorphism has been detected among wild boar, varying chromosomes from 36 to 38. Considering this, the objective of the present work was to perform a genetic characterization of wild boar and crossbred boars raised in Brazil, through. the microsatellites loci (STRs) of the domestic pig (Sus scrofa domestica). For classification purposes, the animals were grouped according to pedigree analysis and diploid number (2n) into 5 genetic groups: group I, composed of 59 domestic pigs with 2n = 38; group II, composed of 46 wild boar with 2n = 36 imported from France in 1997; group III, composed of 3 crossbred animals with 2n = 36 from the crossing between crossbred and backcrossing animals; group IV, composed of 30 crossbred animals with domestic pig with ploidy equal to 37 chromosomes and group V, composed of 10 crossbred animals also with domestic pig, but with 2n = 38, popularly known as "Boarpigs" due to their karyotypic and phenotypic similarity with the domestic pig. The genomic DNA was extracted and, after that, the fragments of these microsatellites - IGF1, ACTG2, TNFB - were amplified through the PCR technique, which were developed for the Sus scrofa domestica species. The amplification conditions were standardized for wild boar samples and performed in a thermocycler with the annealing temperatures varying for each primer. At the end of amplifications, the products of microsatellites were placed in a genetic analyzer model ABI 3100 Avant (Applied Biosystems). Considering the results of this research, the microsatellites IGF1, ACTG2 and TNFB used for pigs, were considered to be efficient on the heterologous amplifications and can also be applied on wild boar. The wild boar differs genetically from pigs and crossbreds... (Complete abstract, click electronic access below) / Mestre

O relógio biológico é um mecanismo molecular autossustentado gerador de ritmos. Ele integra vias de percepção das condições ambientais com um oscilador central para gerar respostas fisiológicas rítmicas em escalas diária e sazonal. Nas plantas, o relógio biológico está associado a vias metabólicas e fisiológicas importantes, como fotossíntese. Na cana-de-açúcar, uma gramínea de grande interesse econômico, estudos realizados em condições circadianas mostraram que o relógio biológico tem uma influência superior àquela vista em outras plantas. Assim, este trabalho visa a compreender os mecanismos de funcionamento do oscilador central do relógio biológico da cana-de-açúcar crescida em campo. Para tanto, foram investigados o transcriptoma de diferentes órgãos da cana-de-açúcar; a expressão de isoformas alternativas e de múltiplos alelos dos genes do relógio biológico da cana; e o efeito do sombreamento mútuo das plantas em campo sobre o funcionamento do relógio biológico. Os resultados obtidos sugerem que o relógio biológico é funcional e sincronizado entre os diferentes órgãos da cana-de-açúcar analisados. Os transcritos regulados sinergicamente pelo relógio biológico e pelo ambiente flutuante pertencem a vias metabólicas, fisiológicas e de regulação gênica e epigenéticas todas essenciais à produtividade da cana-de-açúcar. O sombreamento mútuo observado em campo parece alterar a fase de expressão de genes do relógio biológico da cana-de-açúcar. Além disso, eventos de splicing alternativo foram observados nos genes do relógio biológico em condições de baixa temperatura e múltiplos alelos dos genes do relógio biológico são expressos e a regulação de sua expressão parece ser sazonal. / The circadian clock is a self-sustaining molecular mechanism that generates rhythms. It perceives the environmental conditions and connects this pathway with its central oscillator, generating daily and seasonal rhythms of physiological responses. In plants, the circadian clock is associated with major metabolic and physiological pathways. In sugarcane, an economically important grass, previous studies showed that the circadian clock has the largest influence on plants seen so far under circadian conditions. This work aims to understand how the central oscillator of the circadian clock works in field-grown sugarcane. Thus, the transcriptome from different sugarcane organs; the expression of alternative isoforms and multiple alleles of circadian clock genes; and the effect of mutual shading in the field on the circadian clock function were analyzed. The results suggest that there is a functional and synchronized circadian clock in the different sugarcane organs. The transcripts regulated synergistically by the circadian clock and the variable environment are related to metabolic, physiological, genetic or epigenetic pathways, all important to sugarcane productivity. Mutual shading observed in the field seems to change the phase of expression of the sugarcane circadian clock. Besides, alternative splicing events have been reported for circadian clock genes under low temperature conditions and multiple alleles of circadian clock genes are expressed and their expression is likely to be seasonally regulated.

Bone mineral density (BMD) is a reflection of the action of osteoblasts compared to osteoclasts. An imbalance in the activity of osteoblasts or osteoclasts, results in bone disease such as osteoporosis caused by overactive osteoclasts. BMD is influenced by genetic and environmental factors as demonstrated through twin studies, association studies and linkage analysis (Ralston, 1999). Several polymorphisms involved in the determination of BMD have been identified, with Vitamin D receptor and Collagen Type 1 showing reproducible associations. To identify genes influencing BMD two distinct strategies have been employed: 1) To determine if DNA polymorphism within the runt related transcription factor (RUNX2) gene is a determinant of BMD and fracture in women. 2) The identification of RANKL target genes in osteoclastogenesis. RUNX2 is a runt domain transcription factor (Werner et al., 1999) essential for osteoblast differentiation (Lee et al., 1997). RUNX2 gene knock-out mice have no osteoblasts due to a failure in osteoblast differentiation and consequently unmineralised skeletons, (Komori et al., 1997; Otto et al., 1997). In humans, mutations in RUNX2 cause cleidocranial dysplasia (CCD), a disorder characterised by hypoplasia or aplasia of the clavicles, short stature, supernumerary teeth, patent fontanelles and other changes in skeletal patterning and growth (Mundlos et al., 1997). RUNX2 contains a poly-glutamine poly-alanine (polyQ/polyA) repeat where mutations causing cleidocranial dysplasia have been observed. BMD has not been routinely examined in CCD, two studies have identified CCD patients with lower BMD with one fracture case identified (Quack et al., 1999; Bergwitz et al., 2001). The central role of RUNX2 in determining osteoblast differentiation makes RUNX2 a prime candidate gene for regulating adult bone density. To determine if polymorphism was present in the polyQ/polyA tract the repeat was amplified within the upper and lower deciles of femoral neck (FN) BMD in the Geelong Osteoporosis study (GOS). The upper and lower deciles of FN BMD acted as a surrogate for genotyping the entire cohort. This study identified two common variants within the polyA repeat: an 18 base pair deletion (11Ala) and a synonymous alanine codon polymorphism with alleles, GCA and GCG (noted as A and G alleles, respectively). The 11Ala and SNP polymorphism are found on codon 64 and 66 respectively (RUNX2 MRIPV variant). A allele frequencies were significantly different in a comparison of the upper and lower deciles of FN BMD (p=0.019). In 495 randomly selected women of the Geelong Osteoporosis Study (GOS), the A allele was associated with higher BMD at all sites tested. The association was maximal at the ultra-distal radius (p=0.001). In a separate fracture study, the A allele was significantly protective against Colles' fracture in elderly women but not spine and hip fracture. The 11Ala polymorphism was not related to BMD in GOS. To further decipher the role of the RUNX2 A allele we genotyped 992 women from a Scottish cohort. The alleles of RUNX2 within the glutamine/alanine repeat were determined by MspA1I restriction digest. To examine the possible influence on estrogen related therapies or estrogen status on the potential genetic effect conferred by RUNX2, we divided the cohort by menopausal and hormone replacement therapy status. Within postmenopausal Scottish women the RUNX2 A allele was associated with significantly higher FN BMD (p=0.028, n=312) but not lumbar spine (LS) BMD. The A allele was associated with higher FN BMD (p=0.035) within a postmenopausal subgroup of the population (n=312). To investigate the effect of weight on the RUNX2 alleles the Scottish cohort was segregated into thin/normal (BMI ≥ 25 kg/m2) and overweight /obese (BMI > 25 kg/m2). RUNX2 A allele showed a stronger effect on FN BMD in postmenopausal women above the median BMI. The 11Ala RUNX2 deletion allele was significantly associated with decreased LS BMD (p=0.018) within overweight/obese women (n=546). The 11Ala allele was significantly associated with increased levels of pyridinoline (p=0.014) and deoxypyridinoline (p=0.038) in the HRT treated subgroup of the population (n=492). Glutamine variants and an alanine insertion were identified within the group. These data suggest that the RUNX2 11Ala and A alleles exert differing affects on BMD showing preference for different skeletal sites in a weight dependent manner. We genotyped 78 individuals from an osteoarthritic population to elucidate the role of the RUNX2 alleles on markers of bone turnover and inflammation. The RUNX2 11Ala allele was significantly associated with decreased osteocalcin (OC) serum levels (p = 0.01). The RUNX2 A allele was significantly related to reduced tumor necrosis factor alpha (TNF-alpha) serum levels (p = 0.004). RUNX2 is known to bind to the OC promoter. An OC promoter polymorphism is found 7bp upstream from a putative RUNX2 binding site. We hypothesized that OC polymorphism may effect the RUNX2 transactivation of the OC gene and thus affect OC serum levels. OC promoter polymorphism was not related to OC serum levels (n=78). These data present a novel link between RUNX2 alleles and OC and TNF serum levels, providing putative mechanisms of action for the RUNX2 alleles. Further studies in larger populations are required to confirm these findings. Ten individuals within the GOS and the Scottish cohort were found to carry rare mutations of the polyQ/polyA repeat. All polyQ variants had a normal polyA repeat (17 amino acids) and were heterozygous for a normal 23Q/17A allele. Variants observed were 15, 16, 24 and 30Q. One individual was observed with an extended polyA repeat (24A). Patient records indicated otherwise unremarkable clinical history except for fracture in 4/10 individuals from GOS (hip and spine). BMD data from the LS and the FN were expressed as T-scores, a measure that relates BMD in terms of standard deviations below the young normal value. In addition, BMD data were also expressed as Z-scores around the age-mean. Under the null hypothesis, where RUNX2 Q repeat variation has no effect on BMD, Z scores would be expected to be distributed around a mean of zero. However, when all variants were pooled the BMD was significantly lower than expected. This effect persisted when deletion variants were considered alone. The effect was stronger on FN BMD (p=0.001) rather than LS BMD (p=0.096), reflecting either difference in precision of BMD measurements at these sites or perhaps a differential genetic effect on different skeletal sites. These data suggest that polyQ and polyA variants are associated with significantly lower BMD, and may be an important determinant for fracture. Glutamine variants exist at high frequency (~0.7%): this rate of mutation could be important when considering large populations at risk of age related osteoporosis. Considering that these subjects are heterozygous for a normal allele, it suggests that a more severe phenotype might be expected in rare subjects homozygous for glutamine repeat variants. In summary, this study investigated the role of novel polymorphisms and rare variants of the RUNX2 gene in influencing BMD, fracture and markers of bone turnover. Two common polymorphisms were identified within the polyA repeat: an 18 base pair deletion and a synonymous alanine codon polymorphism with alleles, A and G. The A allele was associated with increased BMD and was protective against a common form of osteoporotic fracture within a Geelong population. To verify these findings the RUNX2 alleles were genotyped in 992 women from a Scottish cohort. The magnitude and the direction of the effect of the A allele was maintained in the Scottish cohort. Interestingly, the A allele was shown to exert a menopause specific effect, with postmenopausal women showing the strongest effect. On re-analysis of the GOS data the post-menopausal women were found to drive the significance identified in the cohort. The magnitude of the effect of the A allele on BMD was greater in overweight/obese postmenopausal women indicating a gene-weight interaction for RUNX2. The RUNX2 11Ala allele showed a significant relationship with decreased LS BMD in overweight/obese Scottish women. The 11Ala allele was also associated with higher levels of urinary PYD and DPD in women treated with HRT, indicating higher levels of bone turnover in carriers of the 11Ala allele. In contrast to the Scottish cohort, no significant association with heterozygous carriers of 11Ala was observed in GOS, although a significant association was detected for homozygous carriers and LS BMAD. The 11 Ala RUNX2 allele was significantly associated with decreased serum osteocalcin levels and the A allele was significantly associated with TNF in OA patients. Glutamine variants and an alanine insertion were identified within Geelong and Scottish cohorts, which showed low Z and T scores suggesting that RUNX2 variants may be related to genetic effects on BMD and osteoporosis. Polymorphism of the polyQ/polyA region of RUNX2 were identified within this study were shown to associate with significant differences in BMD. The A allele showed a significant association with increased BMD in postmenopausal women from a Geelong and Scottish cohort, with a decreased frequency of the A allele observed in Colles' fracture patients from Geelong. The 11Ala deletion allele was significantly associated with decreased LS BMD and increases in markers of bone turnover in the Scottish cohort. A significant decrease in OC serum levels was observed in OA patients suggesting a direct effect of the allele on the transactivation of the RUNX2 gene. Rare variants of RUNX2 were identified which showed low BMD. These studies have provided insight into the role of RUNX2 in influencing BMD, further studies are required to verify the role of the A allele on BMD and fracture, the role of the rare variants and to identify the precise mechanisms behind the observed changes in BMD. - 2) The identification of RANKL target genes in osteoclastogenesis. Osteoclastogenesis is regulated in vivo by the action of osteoblast/stromal cells that express membrane bound, receptor activator of NF-kB ligand (RANKL). Monocytes treated in vitro with a soluble form of RANKL and macrophage colony stimulating factor (M-CSF) differentiate to osteoclasts, whereas monocytes treated with M-CSF alone differentiate to macrophage-like cells. The gene expression profile of human osteoclasts has not been extensively explored. Genes highly expressed by rabbit osteoclasts were identified through random sequencing of an osteoclast cDNA library (Sakai et al., 1995). Differential gene expression of mouse osteoclastogenesis was elucidated by array analysis (Cappellen et al., 2002). To identify genes important for human osteoclastogenesis, total RNA was isolated from monocytes treated for three weeks with either M-CSF alone or with RANKL and M-CSF. RANKL treatment for 3 weeks and 12 hours was investigated in this study, to complement previous data. Differential display was performed on RNA (12 hour treatment with RANKL) and differential gene expression profiles examined. The differential display products were pooled to generate a probe for screening a gene array system derived from a human osteoclast cDNA library. cDNA (3 week treatment with RANKL) hybridisation experiments against the array revealed additional regulated genes. Gene clones that showed significant regulation in M-CSF and RANKL treated cells compared M-CSF treated cells represent genes that are targets for RANKL-specific regulation. Osteopontin, creatine kinase and various mitochondrial genes were up regulated by the treatment of RANKL. Changes in gene expression observed in the array data were confirmed with real-time PCR using mRNA derived from in vitro induced osteoclasts. Cathepsin K gene expression was more than 300 fold greater in osteoclasts compared to macrophage-like cells after one week treatment with RANKL and M-CSF. Cystatin C expression showed a six-fold induction at two weeks of RANKL and M-CSF treatment and cystatin B showed a steady increase in expression. Some of these regulated genes may provide useful targets for influencing BMD.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2008. / Title from PDF title page (viewed on Sept. 17, 2009). Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains viii, 141 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The conservation of forest species requires the prior knowledge of genetic parameters belonging to the population, to help to design strategies for this purpose. This study aimed to evaluate genetic parameters and suggest indicators of sustainability in populations of Schinus terebinthifolius Raddi. aiming the conservation and maintenance of diversity in this species. In analysis of the study area, were selected 20 descriptors, and these suggested 20 indicators of sustainability for the area. Tender vegetal leaves were sampled in five populations located on the shores and islands of São Francisco River, and the material collected from 15 individuals for each population. In the DNA extraction it was used tender leaves of individuals and 2% CTAB method. We applied 20 primers of 10 bases of arbitrary sequence in amplification, the products were separated in agarose gel at 1% submitted to horizontal electrophoresis, stained with ethidium bromide and visualized in ultraviolet light. The presence and absence of bands were used to construct a binary matrix for the analysis of genetic parameters. In each population the percentage of polymorphic loci ranged from 32.92% to 45.34%. The average gene diversity of Nei was 0.37. The total genetic variation observed, 63.60% corresponded to variation among populations, and 36.40% within populations. The gene flow (Nm) estimated was 0.28. Thus the conclusion is the populations are genetically isolated, and analyzed the genetic parameters can be used as indicators of sustainability of the area. / A conservação de espécies florestais requer o conhecimento prévio de parâmetros genéticos pertencentes às populações, para se traçar estratégias para este fim. Assim, este trabalho teve como objetivo avaliar parâmetros genéticos e sugerir indicadores de sustentabilidade em populações de Schinus terebinthifolius Raddi. visando à conservação e manutenção da diversidade nesta espécie. Em análise da área de estudo, foram selecionados 20 descritores, e destes sugeridos 20 indicadores de sustentabilidade da área. As amostragens foram realizadas em cinco populações localizadas nas margens e ilhas do Rio São Francisco, sendo coletado material de 15 indivíduos para cada uma das populações. Na extração do DNA, empregou-se folhas tenras dos indivíduos e o método CTAB 2%. Foram usados 20 oligonucleotídeos de 10 bases de sequência arbitrária nas amplificações, cujos produtos foram separados em gel de agarose 1% submetidos à eletroforese horizontal, corados com brometo de etídio e visualizados em luz ultravioleta. A presença e a ausência de bandas foi usada para a construção de uma matriz de binária para a análise dos parâmetros genéticos. Em cada população a porcentagem de locos polimórficos variou de 32,92 a 45,34%. A diversidade média gênica de Nei foi de 0,37. Da variação genética total observada, 63,60% correspondeu à variação entre as populações; e, 36,40% dentro das populações. O fluxo gênico (Nm) estimado foi de 0,28. Desta forma conclui-se que as populações encontram-se isoladas geneticamente e os parâmetros genéticos analisados podem ser utilizados como indicadores de sustentabilidade da área.