Investigating the Effect of Histone H4 Sumoylation on Chromatin Structure and Function

Thesis (Ph.D.)--University of Washington, 2016-06

Mitochondrial control region diversity of the houbara bustard Chlamydotis undulata complex and genetic structure along the Atlantic seaboard of North Africa

The houbara bustard, Chlamydotis undulata, is a declining cryptic desert bird whose range extends from North Africa to Central Asia. Three subspecies are currently recognized by geographical distribution and morphology: C.u.fuertaventurae, C.u.undulata and C.u.macqueenii. We have sequenced 854 bp of mitochondrial control region from 73 birds to describe their population genetic structure with a particular sampling focus on the connectivity between C.u.fuertaventurae and C.u.undulata along the Atlantic seaboard of North Africa. Nucleotide and haplotypic diversity varied among the subspecies being highest in C.u.undulata, lowest in C.u.fuertaventurae and intermediate in C.u.macqueenii. C.u.fuertaventurae and C.u.undulata are paraphyletic and an average nucleotide divergence of 2.08% splits the later from C.u.macqueenii. We estimate that C.u.fuertaventurae and C.u.undulata split from C.u.macqueenii approximately 430 000 years ago. C.u.fuertaventurae and C.u.undulata are weakly differentiated (F-ST = 0.27, N-m = 1.3), indicative of a recent shared history. Archaeological evidence indicates that houbara bustards have been present on the Canary Islands for 130-170 000 years. However, our genetic data point to a more recent separation of C.u.fuertaventurae and C.u.undulata at around 20-25 000 years. Concordant archaeological, climatic opportunities for colonization and genetic data point to a scenario of: (i) initial colonization of the Canary Islands about 130 000 years ago; (ii) a period of secondary contact 19-30 000 years ago homogenizing any pre-existing genetic structure followed by; (iii) a period of relative isolation that persists today.

Mutation scanning analysis of Marteilia sydneyi populations from different geographical locations in eastern Australia

Marteilia sydneyi (Paramyxea) is the causative agent of QX disease in oysters. In spite of the economic impact of this disease, its origin and the precise reason(s) for its apparent spread in Australian waters are not yet known. Given such knowledge gaps, investigating the population genetic structure(s) of M. sydneyi populations could provide insights into the epidemiology and ecology of the parasite and could assist in its prevention and control. In this study, single strand conformation polymorphism (SSCP)-based analysis of a region (195 bp) of the first internal transcribed spacer (ITS-1) of ribosomal DNA was employed to investigate genetic variation within and among five populations of M. sydneyi from oysters from five different locations in eastern Australia. The analysis showed the existence of a genetic variant of M. sydneyi common to the Great Sandy Strait, and the Richmond and Georges Rivers, as distinct from variants at the Pimpama and Clarence Rivers. Together with historical and other information relating to the QX disease outbreaks in eastern Australia, the molecular findings support the proposal that the parasite originated in the Great Sandy Strait and/or Richmond River and then extended southward along the coast. From a technical perspective, the study demonstrated the usefulness of SSCP as a tool to study the population genetics and epidemiology of M. sydneyi. (C) 2003 Elsevier Ltd. All rights reserved.

Molecular systematics of the Philippine malaria vector Anopheles flavirostris

Allozyme and molecular sequence data from the malaria vector Anopheles flavirostris (Ludlow) (Diptera: Culicidae) were analysed from 34 sites throughout the Philippines, including the type locality, to test the hypothesis that this taxon is a single panmictic species. A finer-scaled allozyme study, of mainly Luzon samples, revealed no fixed genetic differences in sympatric sites and only low levels of variation. We obtained data from partial sequences for the internal transcribed spacer 2 (ITS2) (483 bp), the third domain (D3) (330 bp) of the 28S ribosomal DNA subunit and cytochrome c oxidase subunit I (COI) of mitochondrial DNA (261 bp). No sequence variation was observed for ITS2, only a one base pair difference was observed between Philippine and Indonesian D3 sequences and An. flavirostris sequences were unique, confirming their diagnostic value for this taxon. Sixteen COI haplotypes were identified, giving 25 parsimony informative sites. Neighbour-Joining, Maximum Parsimony, Maximum Likelihood and Bayesian phylogenetic analysis of COI sequences for An. flavirostris and outgroup taxa revealed strong branch support for the monophyly of An. flavirostris, thus confirming that Philippine populations of this taxon comprise a single separate species within the Minimus Subgroup of the Funestus Group. Variation in the behaviour of An. flavirostris is likely to be intraspecific rather than interspecific in origin. © 2006 The Royal Entomological Society.

Validation of a generalized transfer function to noninvasively derive central blood pressure during exercise

Exercise brachial blood pressure ( BP) predicts mortality, but because of wave reflection, central ( ascending aortic) pressure differs from brachial pressure. Exercise central BP may be clinically important, and a noninvasive means to derive it would be useful. The purpose of this study was to test the validity of a noninvasive technique to derive exercise central BP. Ascending aortic pressure waveforms were recorded using a micromanometer-tipped 6F Millar catheter in 30 patients (56 +/- 9 years; 21 men) undergoing diagnostic coronary angiography. Simultaneous recordings of the derived central pressure waveform were acquired using servocontrolled radial tonometry at rest and during supine cycling. Pulse wave analysis of the direct and derived pressure signals was performed offline (SphygmoCor 7.01). From rest to exercise, mean arterial pressure and heart rate were increased by 20 +/- 10 mm Hg and 15 +/- 7 bpm, respectively, and central systolic BP ranged from 77 to 229 mm Hg. There was good agreement and high correlation between invasive and noninvasive techniques with a mean difference (+/- SD) for central systolic BP of -1.3 +/- 3.2 mm Hg at rest and -4.7 +/- 3.3 mm Hg at peak exercise ( for both r=0.995; P < 0.001). Conversely, systolic BP was significantly higher peripherally than centrally at rest (155 +/- 33 versus 138 +/- 32mm Hg; mean difference, -16.3 +/- 9.4mm Hg) and during exercise (180 +/- 34 versus 164 +/- 33 mm Hg; mean difference, -15.5 +/- 10.4 mm Hg; for both P < 0.001). True myocardial afterload is not reliably estimated by peripheral systolic BP. Radial tonometry and pulse wave analysis is an accurate technique for the noninvasive determination of central BP at rest and during exercise.

The clinical assessment of systemic hypertension in optometric practice

This thesis sets out to examine in detail the condition of systemic hypertension (high Blood Pressure) in relation to optometric practice in the United Kingdom. Systemic hypertension, which is asymptomatic in the early stages, is diagnosed from the Blood Pressure (BP) measurement recorded by a sphygmomanometer and/or from the complications that have developed in target organs. Optometric practice based surveys revealed that diagnosed systemic hypertension was the most prevalent cardiovascular medical condition (20.5%). Measurement of BP of patients in this sample revealed that if an optometrist included sphygmomanometry into the sight examination then at least one patient each day would be referred for suspect systemic hypertension. Optometric opinion felt that the measurement of BP in optometric practice would advance the profession, being appreciated by both patients and General Practitioners (GPs), but was felt to be an unnecessary routine procedure. The present sight examination for the systemic hypertensive is similar to that of the normotensive patient, but may involve an altered fundus examination and a visual field test. The GPs were in favour of optometric BP measurement and a future role in the share care management of the systemic hypertensive. The application of a new pictorial grading scale for the grading of vascular changes associated with pre-malignant systemic hypertension was found to be both accurate and reliable. Clinical trial of the grading scale in optometric practice found positive correlations between BP and increasing severity of the retinal vascular features. The subtle pre-malignant vascular changes require reliable accurate detection and analysis to assist in the management of the systemic hypertensive patient. Vessel width was shown to decrease with increasing age. Image analysis of the A/V ratio, arteriolar tortuosity and focal calibre changes revealed a positive correlation to the patient's BP (p<0.001). The retinal vasculature is relatively stable longitudinally with only minor changes in response to early disease states. Age and elevated BP increased a patient's risk of developing systemic medical conditions over a two-year period. The application of the pictorial grading scale to optometric practice and training the optometrist in the use of sphygmomanometry would improve the management of the systemic hypertensive patient in optometric practice. Future advances in image analysis hold substantial benefits for the detection and monitoring of subtle vascular changes associated with systemic hypertension.

Is measurement of blood pressure worthwhile in the diabetic eye clinic?

Purpose - The UK Prospective Diabetic Study has confirmed the importance of blood pressure (BP) as a major risk factor for diabetic retinopathy (DR). We wanted to investigate whether measuring the BP in the diabetic eye clinic could identify new hypertensive patients and monitor control in existing ones. Patients and methods - We compared BP in patients attending the diabetic eye clinic with home blood pressure measurement (HBPM) and ambulatory BP measurement (ABPM). In all, 106 patients attending a diabetic eye clinic were selected at random from clinic attendees. BP measurement (on an Omron 705 CP) was performed in the eye clinic and also compared to HBPM three times per day with an Omron 705 CP machine, and was compared to diabetic clinic measurements. In addition, 11 randomly chosen patients had 24 h ABPM to validate the above techniques. Results - In all, 106 patients (70 male and 36 female) were recruited for the study, of which 71 were known to be hypertensive on antihypertensive medication. Of the total, 75 patients (70.8%) had BP>140/85 in the eye clinic, of which 51 (68%) were known to be hypertensive on treatment and this was confirmed in 46 (90%) on HBPM. A total of, 24 patients (22.6%) were newly diagnosed as hypertensive in the eye clinic, which was confirmed by HBPM in 22 patients (92%). The mean BP of the measurements performed in the eye clinic was significantly higher than that carried out in the diabetic clinic (P<0.01). Tropicamide 1% and phenylephrine 2.5% eye drop instillation had no effect on BP. In 11 randomly chosen patients, 24 h ABPM validated both diabetic eye clinic and home BP measurements. Conclusion - Attendance at the diabetic eye clinic is an important chance to detect both new patients with systemic hypertension and those with inadequate BP control. Ophthalmologists should be encouraged to measure BP in their diabetic patients attending diabetic eye clinics, as it is an important risk factor for DR. On the basis of our findings, good BP control is a goal yet to be achieved in diabetic patients with retinopathy.

(Table 1) Radiocarbon ages of samples taken from five sites in north-eastern European Russia

Aim: Concepts about patterns and rates of post-glacial tree population migration are changing as a result of the increasing amount of palaeobotanical information being provided by macroscopic plant remains. Here we combine macrofossil, pollen and stomata records from five sites in north-eastern European Russia and summarize the results for the late-glacial-early Holocene transition. The late-glacial-early Holocene transition encompasses the first indications of trees (tree-type Betula, Picea abies, Abies sibirica and Larix sibirica) and subsequent forest development. Considerable time-lags between the first macrobotanical and/or stomata finds of spruce (Picea abies) and the establishment of a closed forest are reconsidered. Location: Pechora basin, north-eastern European Russia. Methods: We used plant macrofossil, stomata, pollen and radiocarbon analyses to reconstruct late-glacial and early Holocene tree establishment and forest development. The data were derived from lake sediment and peat archives. Results: Palaeobotanical data reveal an early Holocene presence (11,500-10,000 cal. yr bp) of arboreal taxa at all five sites. One site presently located in the northernmost taiga zone, shows the presence of spruce and reproducing tree birch during the late-glacial. Given the current view of post-glacial population dynamics and migration rates, it seems likely that the source area of these early tree populations in north-eastern European Russia was not located in southern Europe but that these populations had local origins. Results thus support the emerging view that the first post-glacial population expansions in non-glaciated regions at high latitudes do not reflect migration from the south but were a result of an increase in the size and density of small persisting outlying tree populations. Main conclusions: Results suggest that the area east of the margin of the Scandinavian ice sheet to the Ural Mountains had isolated patches of trees during the late-glacial and early Holocene and that these small populations acted as initial nuclei for population expansion and forest development in the early Holocene.

Population genetic of three species of Clupeidae family Sardinella sindensis, Sardinella albella, Dussumieria acuta in the Persian Gulf & Oman Sea

Sardines and other Microfilidae have very important ecological role in marine ecosystems because they are first consumers in marine food chain and they are the main food of valuable species as tuna. So decries in their population will decline fishing of these spices. There are 10 genus of Clupeidae in south of Iran and Sardinella is the one of the most abundant of them. In this study we investigated about morphological and genetically differences in population of 3 species: Sardinella sindensis, Sardinella abella, Dussomieria acuta. About 65 specimens of Sardinella sindensis, 61 specimens of Sardinella albella and 63 specimens of, Dussomieria acuta from three regions of their distribution: Jask (Oman Sea), Qeshm (Hormoz) and Lengeh (Pearsian Gulf) have been collected. Morphological research of their characters and statistical studies were done. To determine the genetically structure of specie's population we sequenced 500 bp of mitochondrial control region. Genetical studies determine meaningful difference in alleles and heterozigosity frequency of Sardinella sindensis. This must be the result of divergence in population of this species. Morphological investigation of Sardinella albella shows the meaningful difference. But detailed studies diffused it. Genetical studies show a meaningful variance in allele and heterosigosity frequency. This may be an aspect of sardine tendency to live in estuaries. Morphological research of Dussomieria acuta in Jask and Lengeh show a meaningful variance in these regions. Such a situation might be result of Monsoon, upwelling and better weather which occur in Oman Sea in spite of Persian Gulf.

ROC curves of obesity indicators have a predictive value for children hypertension aged 7-17 years

Objective: The aim of the study is to examine the distribution of integrated covariate and its association with blood pressure (BP) among children in Anhui province, China, and assess the predictive value of integrated covariate to children hypertension. Methods: A total of 2,828 subjects (1,588 male and 1,240 female) aged 7-17 years participated in this study. Height, weight, waistline, hipline and BP of all subjects were measured, obesity and overweight were defined by an international standard, specifying the measurement, the reference population, and the age and sex specific cut off points. High BP status was defined as systolic blood pressure (SBP) and/or diastolic blood pressure (DBP) > 95th percentile for age and gender. Results: Our results revealed that the prevalence of children hypertension was 11.03%, the SBP and DBP of obesity group were significantly higher than that of normal group. Anthropometric obesity indices such as body mass index (BMI) were positively correlated with SBP and DBP. Integrated covariate had a better performance than the single covariate in the receiver-operating characteristic (ROC) curve, the cut-off value; the sensitivity and the specificity of the integrated covariate were 0.112, 0.577, 0.683, respectively. Conclusion: Integrated covariate is a simple and effective anthropometric index to identify childhood hypertension.

The development of an algorithm to track blood pressure accurately based on the pulse transit time method

Oscillometric blood pressure (BP) monitors are currently used to diagnose hypertension both in home and clinical settings. These monitors take BP measurements once every 15 minutes over a 24 hour period and provide a reliable and accurate system that is minimally invasive. Although intermittent cuff measurements have proven to be a good indicator of BP, a continuous BP monitor is highly desirable for the diagnosis of hypertension and other cardiac diseases. However, no such devices currently exist. A novel algorithm has been developed based on the Pulse Transit Time (PTT) method, which would allow non-invasive and continuous BP measurement. PTT is defined as the time it takes the BP wave to propagate from the heart to a specified point on the body. After an initial BP measurement, PTT algorithms can track BP over short periods of time, known as calibration intervals. After this time has elapsed, a new BP measurement is required to recalibrate the algorithm. Using the PhysioNet database as a basis, the new algorithm was developed and tested using 15 patients, each tested 3 times over a period of 30 minutes. The predicted BP of the algorithm was compared to the arterial BP of each patient. It has been established that this new algorithm is capable of tracking BP over 12 minutes without the need for recalibration, using the BHS standard, a 100% improvement over what has been previously identified. The algorithm was incorporated into a new system based on its requirements and was tested using three volunteers. The results mirrored those previously observed, providing accurate BP measurements when a 12 minute calibration interval was used. This new system provides a significant improvement to the existing method allowing BP to be monitored continuously and non-invasively, on a beat-to-beat basis over 24 hours, adding major clinical and diagnostic value.

Replicating satellite RNA induces sequence-specific DNA methylation and truncated transcripts in plants

Tobacco plants were transformed with a chimeric transgene comprising sequences encoding β-glucuronidase (GUS) and the satellite RNA (satRNA) of cereal yellow dwarf luteovirus. When transgenic plants were infected with potato leafroll luteovirus (PLRV), which replicated the transgene-derived satRNA to a high level, the satellite sequence of the GUS:Sat transgene became densely methylated. Within the satellite region, all 86 cytosines in the upper strand and 73 of the 75 cytosines in the lower strand were either partially or fully methylated. In contrast, very low levels of DNA methylation were detected in the satellite sequence of the transgene in uninfected plants and in the flanking nonsatellite sequences in both infected and uninfected plants. Substantial amounts of truncated GUS:Sat RNA accumulated in the satRNA-replicating plants, and most of the molecules terminated at nucleotides within the first 60 bp of the satellite sequence. Whereas this RNA truncation was associated with high levels of satRNA replication, it appeared to be independent of the levels of DNA methylation in the satellite sequence, suggesting that it is not caused by methylation. All the sequenced GUS:Sat DNA molecules were hypermethylated in plants with replicating satRNA despite the phloem restriction of the helper PLRV. Also, small, sense and antisense ∼22 nt RNAs, derived from the satRNA, were associated with the replicating satellite. These results suggest that the sequence-specific DNA methylation spread into cells in which no satRNA replication occurred and that this was mediated by the spread of unamplified satRNA and/or its associated 22 nt RNA molecules.

PTHR1 polymorphisms influence BMD variation through effects on the growing skeleton

We investigated whether polymorphisms in PTHR1 are associated with bone mineral density (BMD), to determine whether the association of this gene with BMD was due to effects on attainment of peak bone mass or effects on subsequent bone loss. The PTHR1 gene, including its 14 exons, their exon-intron boundaries, and 1,500 bp of its promoter region, was screened for polymorphisms by denaturing high-performance liquid chromatography (dHPLC) and sequencing in 36 osteoporotic cases. Eleven single-nucleotide polymorphisms (SNPs), one tetranucleotide repeat, and one tetranucleotide deletion were identified. A cohort of 634 families, including 1,236 men (39%) and 1,926 women (61%) ascertained with probands with low BMD (Z< -2.0) and the Children in Focus subset of the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort (785 unrelated individuals, mean age 118 months), were genotyped for the five most informative SNPs (minor allele frequency >5%) and the tetranucleotide repeat. In our osteoporosis families, association was noted between lumbar spine BMD and alleles of a known functional tetranucleotide repeat (U4) in the PTHR1 promoter region (P = 0.042) and between two and three marker haplotypes of PTHR1 polymorphisms with lumbar spine, femoral neck, and total hip BMD (P = 0.021-0.047). This association was restricted to the youngest tertile of the population (age 16-39 years, P = 0.013-0.048). A similar association was found for the ALSPAC cohort: two marker haplotypes of SNPs A48609T and C52813T were associated with height (P = 0.006) and total body less head BMD (P = 0.02), corrected for age and gender, confirming the family findings. These findings suggest a role for PTHR1 variation in determining peak BMD.

An N-ethyl-n-nitrosourea induced corticotropin-releasing hormone promoter mutation provides a mouse model for endogenous glucocorticoid excess

Cushing's syndrome, which is characterized by excessive circulating glucocorticoid concentrations, maybe due to ACTH-dependent or -independent causes that include anterior pituitary and adrenal cortical tumors, respectively. ACTH secretion is stimulated by CRH, and we report a mouse model for Cushing's syndrome due to an N-ethyl-N-nitrosourea (ENU) induced Crh mutation at -120 bp of the promoter region, which significantly increased luciferase reporter activity and was thus a gain-of-function mutation. Crh -120/+ mice, when compared with wild-type littermates, had obesity, muscle wasting, thin skin, hair loss, and elevated plasma and urinary concentrations of corticosterone. In addition, Crh-120/+ mice had hyperglycemia, hyperfructosaminemia, hyperinsulinemia, hypercholesterolemia, hypertriglyceridemia, and hyperleptinemia but normal adiponectin. Crh -120/+ mice also had low bone mineral density, hypercalcemia, hypercalciuria, and decreased concentrations of plasma PTH and osteocalcin. Bone histomorphometry revealed Crh-120/+ mice to have significant reductions in mineralizing surface area, mineral apposition, bone formation rates, osteoblast number, and the percentage of corticoendosteal bone covered by osteoblasts, which was accompanied by an increase in adipocytes in the bone marrow. Thus, a mouse model for Cushing's syndrome has been established, and this will help in further elucidating the pathophysiological effects of glucocorticoid excess and in evaluating treatments for corticosteroid-induced osteoporosis.