Epidemiology and genetic architecture of blood pressure: a family based study of Generation Scotland

Hypertension is a major risk factor for cardiovascular disease and mortality, and a growing global public health concern, with up to one-third of the world’s population affected. Despite the vast amount of evidence for the benefits of blood pressure (BP) lowering accumulated to date, elevated BP is still the leading risk factor for disease and disability worldwide. It is well established that hypertension and BP are common complex traits, where multiple genetic and environmental factors contribute to BP variation. Furthermore, family and twin studies confirmed the genetic component of BP, with a heritability estimate in the range of 30-50%. Contemporary genomic tools enabling the genotyping of millions of genetic variants across the human genome in an efficient, reliable, and cost-effective manner, has transformed hypertension genetics research. This is accompanied by the presence of international consortia that have offered unprecedentedly large sample sizes for genome-wide association studies (GWASs). While GWAS for hypertension and BP have identified more than 60 loci, variants in these loci are associated with modest effects on BP and in aggregate can explain less than 3% of the variance in BP. The aims of this thesis are to study the genetic and environmental factors that influence BP and hypertension traits in the Scottish population, by performing several genetic epidemiological analyses. In the first part of this thesis, it aims to study the burden of hypertension in the Scottish population, along with assessing the familial aggregation and heritialbity of BP and hypertension traits. In the second part, it aims to validate the association of common SNPs reported in the large GWAS and to estimate the variance explained by these variants. In this thesis, comprehensive genetic epidemiology analyses were performed on Generation Scotland: Scottish Family Health Study (GS:SFHS), one of the largest population-based family design studies. The availability of clinical, biological samples, self-reported information, and medical records for study participants has allowed several assessments to be performed to evaluate factors that influence BP variation in the Scottish population. Of the 20,753 subjects genotyped in the study, a total of 18,470 individuals (grouped into 7,025 extended families) passed the stringent quality control (QC) criteria and were available for all subsequent analysis. Based on the BP-lowering treatment exposure sources, subjects were further classified into two groups. First, subjects with both a self-reported medications (SRMs) history and electronic-prescription records (EPRs; n =12,347); second, all the subjects with at least one medication history source (n =18,470). In the first group, the analysis showed a good concordance between SRMs and EPRs (kappa =71%), indicating that SRMs can be used as a surrogate to assess the exposure to BP-lowering medication in GS:SFHS participants. Although both sources suffer from some limitations, SRMs can be considered the best available source to estimate the drug exposure history in those without EPRs. The prevalence of hypertension was 40.8% with higher prevalence in men (46.3%) compared to women (35.8%). The prevalence of awareness, treatment and controlled hypertension as defined by the study definition were 25.3%, 31.2%, and 54.3%, respectively. These findings are lower than similar reported studies in other populations, with the exception of controlled hypertension prevalence, which can be considered better than other populations. Odds of hypertension were higher in men, obese or overweight individuals, people with a parental history of hypertension, and those living in the most deprived area of Scotland. On the other hand, deprivation was associated with higher odds of treatment, awareness and controlled hypertension, suggesting that people living in the most deprived area may have been receiving better quality of care, or have higher comorbidity levels requiring greater engagement with doctors. These findings highlight the need for further work to improve hypertension management in Scotland. The family design of GS:SFHS has allowed family-based analysis to be performed to assess the familial aggregation and heritability of BP and hypertension traits. The familial correlation of BP traits ranged from 0.07 to 0.20, and from 0.18 to 0.34 for parent-offspring pairs and sibling pairs, respectively. A higher correlation of BP traits was observed among first-degree relatives than other types of relative pairs. A variance-component model that was adjusted for sex, body mass index (BMI), age, and age-squared was used to estimate heritability of BP traits, which ranged from 24% to 32% with pulse pressure (PP) having the lowest estimates. The genetic correlation between BP traits showed a high correlation between systolic (SBP), diastolic (DBP) and mean arterial pressure (MAP) (G: 81% to 94%), but lower correlations with PP (G: 22% to 78%). The sibling recurrence risk ratio (λS) for hypertension and treatment were calculated as 1.60 and 2.04 respectively. These findings confirm the genetic components of BP traits in GS:SFHS, and justify further work to investigate genetic determinants of BP. Genetic variants reported in the recent large GWAS of BP traits were selected for genotyping in GS:SFHS using a custom designed TaqMan® OpenArray®. The genotyping plate included 44 single nucleotide polymorphisms (SNPs) that have been previously reported to be associated with BP or hypertension at genome-wide significance level. A linear mixed model that is adjusted for age, age-squared, sex, and BMI was used to test for the association between the genetic variants and BP traits. Of the 43 variants that passed the QC, 11 variants showed statistically significant association with at least one BP trait. The phenotypic variance explained by these variant for the four BP traits were 1.4%, 1.5%, 1.6%, and 0.8% for SBP, DBP, MAP, and PP, respectively. The association of genetic risk score (GRS) that were constructed from selected variants has showed a positive association with BP level and hypertension prevalence, with an average effect of one mmHg increase with each 0.80 unit increases in the GRS across the different BP traits. The impact of BP-lowering medication on the genetic association study for BP traits has been established, with typical practice of adding a fixed value (i.e. 15/10 mmHg) to the measured BP values to adjust for BP treatment. Using the subset of participants with the two treatment exposure sources (i.e. SRMs and EPRs), the influence of using either source to justify the addition of fixed values in SNP association signal was analysed. BP phenotypes derived from EPRs were considered the true phenotypes, and those derived from SRMs were considered less accurate, with some phenotypic noise. Comparing SNPs association signals between the four BP traits in the two model derived from the different adjustments showed that MAP was the least impacted by the phenotypic noise. This was suggested by identifying the same overlapped significant SNPs for the two models in the case of MAP, while other BP traits had some discrepancy between the two sources

Optimal static and sequential design : a critical review

The aim of this thesis is to review and augment the theory and methods of optimal experimental design. In Chapter I the scene is set by considering the possible aims of an experimenter prior to an experiment, the statistical methods one might use to achieve those aims and how experimental design might aid this procedure. It is indicated that, given a criterion for design, a priori optimal design will only be possible in certain instances and, otherwise, some form of sequential procedure would seem to be indicated. In Chapter 2 an exact experimental design problem is formulated mathematically and is compared with its continuous analogue. Motivation is provided for the solution of this continuous problem, and the remainder of the chapter concerns this problem. A necessary and sufficient condition for optimality of a design measure is given. Problems which might arise in testing this condition are discussed, in particular with respect to possible non-differentiability of the criterion function at the design being tested. Several examples are given of optimal designs which may be found analytically and which illustrate the points discussed earlier in the chapter. In Chapter 3 numerical methods of solution of the continuous optimal design problem are reviewed. A new algorithm is presented with illustrations of how it should be used in practice. It is shown that, for reasonably large sample size, continuously optimal designs may be approximated to well by an exact design. In situations where this is not satisfactory algorithms for improvement of this design are reviewed. Chapter 4 consists of a discussion of sequentially designed experiments, with regard to both the philosophies underlying, and the application of the methods of, statistical inference. In Chapter 5 we criticise constructively previous suggestions for fully sequential design procedures. Alternative suggestions are made along with conjectures as to how these might improve performance. Chapter 6 presents a simulation study, the aim of which is to investigate the conjectures of Chapter 5. The results of this study provide empirical support for these conjectures. In Chapter 7 examples are analysed. These suggest aids to sequential experimentation by means of reduction of the dimension of the design space and the possibility of experimenting semi-sequentially. Further examples are considered which stress the importance of the use of prior information in situations of this type. Finally we consider the design of experiments when semi-sequential experimentation is mandatory because of the necessity of taking batches of observations at the same time. In Chapter 8 we look at some of the assumptions which have been made and indicate what may go wrong where these assumptions no longer hold.

NIR-spectroscopy for bioprocess monitoring & control

The Ingold port adaption of a free beam NIR spectrometer is tailored for optimal bioprocess monitoring and control. The device shows an excellent signal to noise ratio dedicated to a large free aperture and therefore a large sample volume. This can be seen particularly in the batch trajectories which show a high reproducibility. The robust and compact design withstands rough process environments as well as SIP/CIP cycles. Robust free beam NIR process analyzers are indispensable tools within the PAT/QbD framework for realtime process monitoring and control. They enable multiparametric, non-invasive measurements of analyte concentrations and process trajectories. Free beam NIR spectrometers are an ideal tool to define golden batches and process borders in the sense of QbD. Moreover, sophisticated data analysis both quantitative and MSPC yields directly to a far better process understanding. Information can be provided online in easy to interpret graphs which allow the operator to make fast and knowledge-based decisions. This finally leads to higher stability in process operation, better performance and less failed batches.

Studies of antihypertensive drug persistence and adherence in the Glasgow Blood Pressure Clinic

Hypertension (HTN) is a major risk factor for cardiovascular diseases including stroke, coronary heart disease (CHD), chronic renal failure, peripheral vascular disease, myocardial infarction, congestive heart failure and premature death. The prevalence of HTN in Scotland is very high and although a high proportion of the patients receive antihypertensive medications, blood pressure (BP) control is very low. Recommendations for starting a specific antihypertensive class have been debated between various guidelines over the years. Some guidelines and HTN studies have preferred to start with a combination of an antihypertensive class instead of using a single therapy, and they have found greater BP reductions with combination therapies than with monotherapy. However, it has been shown in several clinical trials that 20% to 35% of hypertensive patients could not achieve the target BP, even though they received more than three antihypertensive medications. Several factors were found to affect BP control. Adherence and persistence were considered as the factors contributing the most to uncontrolled hypertension. Other factors such as age, sex, body mass index (BMI), alcohol intake, baseline systolic BP (SBP), and the communication between physicians and patients have been shown to be associated with uncontrolled BP and resistant hypertension. Persistence, adherence and compliance are interchangeable terms and have been used in the literature to describe a patient’s behaviour with their antihypertensive drugs and prescriptions. The methods used to determine persistence and adherence, as well as the inclusion and exclusion criteria, vary between persistence and adherence studies. The prevalence of persistence and adherence have varied between these studies, and were determined to be high in some studies and low in others. The initiation of a specific antihypertensive class has frequently been associated with an increase or decrease in adherence and persistence. The tolerability and efficacy of the initial antihypertensive class have been the most common methods of explaining this association. There are also many factors that suggest a relationship with adherence and persistence. Some factors in previous studies, such as age, were frequently associated with adherence and persistence. On the other hand, relationships with certain factors have varied between the studies. The associations of age, sex, alcohol use, smoking, baseline systolic blood pressure (SBP) and diastolic BP (DBP), the presence of comorbidities, an increase in the number of pills and the relationship between patients and physicians with adherence and persistence have been the most commonly investigated factors. Most studies have defined persistence in terms of a patient still taking medication after a period of time. A medication possession ratio (MPR) ≥ 80 has been used to define compliance. Either of these terminologies, or both, have been used to estimate adherence. In this study, I used the same definition for persistence to identify patients who have continued with their initial treatment, and used persistence and MPR to define patients who adhered to their initial treatment. The aim of this study was to estimate the prevalence of persistence and adherence in Scotland. Also, factors that could have had an effect on persistence and adherence were studied. The number of antihypertensive drugs taken by patients during the study and factors that led to an increase in patients being on a combination therapy were also evaluated. The prevalence of resistance and BP control were determined by taking the BP after the last drug had been taken by persistent patients during five follow-up studies. The relationship of factors such as age, sex, BMI, alcohol use, smoking, estimated glomerular filtration rate (eGFR), and albumin levels with BP reductions for each antihypertensive class were determined. Information Services Division (ISD) data, which includes all antihypertensive drugs, were collected from pharmacies in Scotland and linked to the Glasgow Blood Pressure Clinic (GBPC) database. This database also includes demographic characteristics, BP readings and clinical results for all patients attending the GBPC. The case notes for patients who attended the GBPC were reviewed and all new antihypertensive drugs that were prescribed between visits, BP before and after taking drugs, and any changes in the hypertensive drugs were recorded. A total of 4,232 hypertensive patients were included in the first study. The first study showed that angiotensin converting enzyme inhibitor (ACEI) and beta-blockers (BB) were the most prescribed antihypertensive classes between 2004 and 2013. Calcium channel blockers (CCB), thiazide diuretics and angiotensin receptor blockers (ARB) followed ACEI and BB as the most prescribed drugs during the same period. The prescription trend of the antihypertensive class has changed over the years with an increase in prescriptions for ACEI and ARB and a decrease in prescriptions for BB and diuretics. I observed a difference in antihypertensive class prescriptions by age, sex, SBP and BMI. For example, CCB, thiazide diuretics and alpha-blockers were more likely to be prescribed to older patients, while ACEI, ARB or BB were more commonly prescribed for younger patients. In a second study, 4,232 and 3,149 hypertensive patients were included to investigate the prevalence of persistence in the Scottish population in 1- and 5-year studies, respectively. The prevalence of persistence in the 1-year study was 72.9%, while it was only 62.8% in the 5-year study. Those patients taking ARB and ACEI showed high rates of persistence and those taking diuretics and alpha blockers had low rates of persistence. The association of persistence with clinical characteristics was also investigated. Younger patients were more likely to totally stop their treatment before restarting their treatment with other antihypertensive drugs. Furthermore, patients who had high SBP tended to be non-persistent. In a third study, 3,085 and 1,979 patients who persisted with their treatment were included. In the first part of the study, MPR was calculated, and patients with an MPR ≥ 80 were considered as adherent. Adherence rates were 29.9% and 23.4% in the 1- and 5-year studies, respectively. Patients who initiated the study with ACEI were more likely to adhere to their treatments. However, patients who initiated the study with thiazide diuretics were less likely to adhere to their treatments. Sex, age and BMI were different between the adherence and non-adherence groups. Age was an independent factor affecting adherence rates during both the 1- and 5-year studies with older patients being more likely to be adherent. In the second part of the study, pharmacy databases were checked with patients' case notes to compare antihypertensive drugs that were collected from the pharmacy with the antihypertensive prescription given during the patient’s clinical visit. While 78.6% of the antihypertensive drugs were collected between clinical visits, 21.4% were not collected. Patients who had more days to see the doctor in the subsequent visit were more likely to not collect their prescriptions. In a fourth study, 3,085 and 1,979 persistent patients were included to calculate the number of antihypertensive classes that were added to the initial drug during the 1-year and 5-year studies, respectively. Patients who continued with treatment as a monotherapy and who needed a combination therapy were investigated during the 1- and 5-year studies. In all, 55.8% used antihypertensive drugs as a monotherapy and 44.2% used them as a combination therapy during the 1-year study. While 28.2% of patients continued with treatment without the required additional therapy, 71.8% of the patients needed additional therapy. In all, 20.8% and 46.5% of patients required three different antihypertensive classes or more during the 1-year and 5-year studies, respectively. Patients who started with ACEI, ARB and BB were more likely to continue as monotherapy and less likely to need two more antihypertensive drugs compared with those who started with alpha-blockers, non-thiazide diuretics and CCB. Older ages, high BMI levels, high SBP and high alcohol intake were independent factors that led to an increase in the probability of patients taking combination therapies. In the first part of the final study, BPs were recorded after the last drug had been taken during the 5 year study. There were 815 persistent patients who were assigned for this purpose. Of these, 39% had taken one, two or three antihypertensive classes and had controlled BP (controlled hypertension [HTN]), 29% of them took one or two antihypertensive classes and had uncontrolled BP (uncontrolled HTN), and 32% of the patients took three antihypertensive classes or more and had uncontrolled BP (resistant HTN). The initiation of an antihypertensive drug and the factors affecting BP pressure were compared between the resistant and controlled HTN groups. Patients who initiated the study with ACEI were less likely to be resistant compared with those who started with alpha blockers and non-thiazide diuretics. Older patients, and high BMI tended to result in resistant HTN. In the second part of study, BP responses for patients who initiated the study with ACEI, ARB, BB, CCB and thiazide diuretics were compared. After adjusting for risk factors, patients who initiated the study with ACEI and ARB were more respondent than those who took CCB and thiazide diuretics. In the last part of this study, the association between BP reductions and factors affecting BP were tested for each antihypertensive drug. Older patients responded better to alpha blockers. Younger patients responded better to ACEI and ARB. An increase in BMI led to a decreased reduction in patients on ACEI and diuretics (thiazide and non-thiazide). An increase in albumin levels and a decrease in eGFR led to decreases in BP reductions in patients on thiazide diuretics. An increase in eGFR decreased the BP response with ACEI. In conclusion, although a high percentage of hypertensive patients in Scotland persisted with their initial drug prescription, low adherence rates were found with these patients. Approximately half of these patients required three different antihypertensive classes during the 5 years, and 32% of them had resistant HTN. Although this study was observational in nature, the large sample size in this study represented a real HTN population, and the large pharmacy data represented a real antihypertensive population, which were collected through the support of prescription data from the GBPC database. My findings suggest that ACEI, ARB and BB are less likely to require additional therapy. However, ACEI and ARB were better tolerated than BB in that they were more likely to be persistent than BB. In addition, users of ACEI, and ARB have good BP response and low resistant HTN. Linkage patients who participated in these studies with their morbidity and mortality will provide valuable information concerning the effect of adherence on morbidity and mortality and the potential benefits of using ACEI or ARB over other drugs.

Starch analysis as a method to discover subsistence strategies in prehistoric Finland and Estonia

Ancient starch analysis is a microbotanical method in which starch granules are extracted from archaeological residues and the botanical source is identified. The method is an important addition to established palaeoethnobotanical research, as it can reveal ancient microremains of starchy staples such as cereal grains and seeds. In addition, starch analysis can detect starch originating from underground storage organs, which are rarely discovered using other methods. Because starch is tolerant of acidic soils, unlike most organic matter, starch analysis can be successful in northern boreal regions. Starch analysis has potential in the study of cultivation, plant domestication, wild plant usage and tool function, as well as in locating activity areas at sites and discovering human impact on the environment. The aim of this study was to experiment with the starch analysis method in Finnish and Estonian archaeology by building a starch reference collection from cultivated and native plant species, by developing sampling, measuring and analysis protocols, by extracting starch residues from archaeological artefacts and soils, and by identifying their origin. The purpose of this experiment was to evaluate the suitability of the method for the study of subsistence strategies in prehistoric Finland and Estonia. A total of 64 archaeological samples were analysed from four Late Neolithic sites in Finland and Estonia, with radiocarbon dates ranging between 2904 calBC and 1770 calBC. The samples yielded starch granules, which were compared with the starch reference collection and descriptions in the literature. Cereal-type starch was identified from the Finnish Kiukainen culture site and from the Estonian Corded Ware site. The samples from the Finnish Corded Ware site yielded underground storage organ starch, which may be the first evidence of the use of rhizomes as food in Finland. No cereal-type starch was observed. Although the sample sets were limited, the experiment confirmed that starch granules have been preserved well in the archaeological material of Finland and Estonia, and that differences between subsistence patterns, as well as evidence of cultivation and wild plant gathering, can be discovered using starch analysis. By collecting large sample sets and addressing the three most important issues – preventing contamination, collecting adequate references and understanding taphonomic processes – starch analysis can substantially contribute to research on ancient subsistence in Finland and Estonia.

Efetividade da doxiciclina na profilaxia contra letptospirose

Leptospirosis is a worldwide zoonosis of considerable medical and economical importance that affects humans in both urban and rural contexts, as well as domestic animals and wild fauna. Leptospira interrogans is the causative agent and is transmitted to humans by indirect contact with contaminated soil or water. The clinical syndromes include sub clinical infection, self-limited anicteric febrile illness, and severe and potentially fatal illness, known as Weil´s syndrome. In developed countries, leptospirosis is related to occupational or recreational activities while in developing countries, outbreaks occur during floods. In those regions, traditional strategies to prevent the transmission are difficulties to be implemented because of costs and lack of community acceptance. In addition, no efficient vaccine is available for human use. Several studies have suggested that chemoprophylaxis with doxycycline pre and post-exposure may be effective to prevent leptospirosis. Leptospirosis has been reported in rural areas of the State of Rio Grande do Norte, Brazil since 1985 in rice farmers who present the anicteric illness. The disease cause great social and economics impact. The study was conducted in São Miguel where an epidemic of leptospirosis in rice farmers was reported. The main objective was to determine the efficacy of doxycycline in preventing Leptospira exposure. A taxa de soroprevalência de leptospirose na população estudada antes e após a colheita foi de 14,2% (n=22) e de 16.6% (n=27) respectivamente. Anti-Leptospira serology was determined for 61 subjects in two instances, pre and post-exposure to potential contaminated water. There was an increased risk of 29.0 per cent in acquiring infection for individuals that did not use doxycycline. In addition, an increased risk of 30.0 % observed in farmers who did not use protection when exposed to Leptospira. The adhesion to preventive chemoprophylaxis was 55.7%. Therefore doxycycline, under specific circunstances appears to be an effective alternative to protect against leptosprirosis infection. A large sample composed of individuals to adhere to preventive therapy is needed to define time, dosage and length of use of doxycycline in this area

Análise experimental do efeito da diversificação em carteiras de ações: a relação entre risco e quantidade de ativos sob a ótica da estratégia ingênua

The objective is to analyze the relationship between risk and number of stocks of a portfolio for an individual investor when stocks are chosen by "naive strategy". For this, we carried out an experiment in which individuals select actions to reproduce this relationship. 126 participants were informed that the risk of first choice would be an asset average of all standard deviations of the portfolios consist of a single asset, and the same procedure should be used for portfolios composed of two, three and so on, up to 30 actions . They selected the assets they want in their portfolios without the support of a financial analysis. For comparison we also tested a hypothetical simulation of 126 investors who selected shares the same universe, through a random number generator. Thus, each real participant is compensated for random hypothetical investor facing the same opportunity. Patterns were observed in the portfolios of individual participants, characterizing the curves for the components of the samples. Because these groupings are somewhat arbitrary, it was used a more objective measure of behavior: a simple linear regression for each participant, in order to predict the variance of the portfolio depending on the number of assets. In addition, we conducted a pooled regression on all observations by analyzing cross-section. The result of pattern occurs on average but not for most individuals, many of which effectively "de-diversify" when adding seemingly random bonds. Furthermore, the results are slightly worse using a random number generator. This finding challenges the belief that only a small number of titles is necessary for diversification and shows that there is only applicable to a large sample. The implications are important since many individual investors holding few stocks in their portfolios

DNA extraction and RAPD markers to assess the genetic similarity among Gelidium sesquipedale (Rhodophyta) populations

A simple method developed for genomic DNA isolation from fungus was tested on the red alga, Gelidium sesquipedale (Clem.) Born et Thur., which is commercially exploited for its high sulfated polysaccharide (agar) content. This method is faster, cheaper, and less toxic than conventional phenol/chloroform methods. Random amplified polymorphic DNA (RAPD) amplifications were performed successfully without the necessity of purifying the DNA. RAPD markers were used to investigate the genetic similarity among three natural populations of G. sesquipedale from southern Portugal. Bulked-genomic DNA samples of 15 different individuals were made in each population. These can be conceived of as a sample of the population DNA. Of the 62 primers screened, 41 produced bands and 22 revealed polymorphisms. Genetic similarities among populations were high. Populations that are further away from each other have the lowest similarity coefficients, whereas the intermediate Ingrina population, located on the south coast, showed higher genetic similarity with the Odeceixe population located on the southwest coast, than with the Sao Rafael southern population. This suggests a higher genetic flow between Odeceixe and Ingrina or the result may be a founder effect in the sense that the species has propagated from the east coast to the south coast of Portugal. We conclude that the use of this isolation method with RAPD analysis is appropriate to characterize the genetic variability of this commercial species along its geographical distribution. Large sample sizes can be screened at a relatively low cost. Finding genetic markers for commercial populations of C. sesquipedale may be of industrial interest.

DNA extraction and RAPD markers to assess the genetic similarity among Gelidium sesquipedale (Rhodophyta) populations

A simple method developed for genomic DNA isolation from fungus was tested on the red alga, Gelidium sesquipedale (Clem.) Born et Thur., which is commercially exploited for its high sulfated polysaccharide (agar) content. This method is faster, cheaper, and less toxic than conventional phenol/chloroform methods. Random amplified polymorphic DNA (RAPD) amplifications were performed successfully without the necessity of purifying the DNA. RAPD markers were used to investigate the genetic similarity among three natural populations of G. sesquipedale from southern Portugal. Bulked-genomic DNA samples of 15 different individuals were made in each population. These can be conceived of as a sample of the population DNA. Of the 62 primers screened, 41 produced bands and 22 revealed polymorphisms. Genetic similarities among populations were high. Populations that are further away from each other have the lowest similarity coefficients, whereas the intermediate Ingrina population, located on the south coast, showed higher genetic similarity with the Odeceixe population located on the southwest coast, than with the Sao Rafael southern population. This suggests a higher genetic flow between Odeceixe and Ingrina or the result may be a founder effect in the sense that the species has propagated from the east coast to the south coast of Portugal. We conclude that the use of this isolation method with RAPD analysis is appropriate to characterize the genetic variability of this commercial species along its geographical distribution. Large sample sizes can be screened at a relatively low cost. Finding genetic markers for commercial populations of C. sesquipedale may be of industrial interest.

Cosmic Explosions: Observations Of Infant Hydrogen-Free Supernovae Towards An Understanding Of Their Parent Systems

Radiation in the first days of supernova explosions contains rich information about physical properties of the exploding stars. In the past three years, I used the intermediate Palomar Transient Factory to conduct one-day cadence surveys, in order to systematically search for infant supernovae. I show that the one-day cadences in these surveys were strictly controlled, that the realtime image subtraction pipeline managed to deliver transient candidates within ten minutes of images being taken, and that we were able to undertake follow-up observations with a variety of telescopes within hours of transients being discovered. So far iPTF has discovered over a hundred supernovae within a few days of explosions, forty-nine of which were spectroscopically classified within twenty-four hours of discovery.

Our observations of infant Type Ia supernovae provide evidence for both the single-degenerate and double-degenerate progenitor channels. On the one hand, a low-velocity Type Ia supernova iPTF14atg revealed a strong ultraviolet pulse within four days of its explosion. I show that the pulse is consistent with the expected emission produced by collision between the supernova ejecta and a companion star, providing direct evidence for the single degenerate channel. By comparing the distinct early-phase light curves of iPTF14atg to an otherwise similar event iPTF14dpk, I show that the viewing angle dependence of the supernova-companion collision signature is probably responsible to the difference of the early light curves. I also show evidence for a dark period between the supernova explosion and the first light of the radioactively-powered light curve. On the other hand, a peculiar Type Ia supernova iPTF13asv revealed strong near-UV emission and absence of iron in the spectra within the first two weeks of explosion, suggesting a stratified ejecta structure with iron group elements confined to the slow-moving part of the ejecta. With its total ejecta mass estimated to exceed the Chandrasekhar limit, I show that the stratification and large mass of the ejecta favor the double-degenerate channel.

In a separate approach, iPTF found the first progenitor system of a Type Ib supernova iPTF13bvn in the pre-explosion HST archival mages. Independently, I used the early-phase optical observations of this supernova to constrain its progenitor radius to be no larger than several solar radii. I also used its early radio detections to derive a mass loss rate of 3e-5 solar mass per year for the progenitor right before the supernova explosion. These constraints on the physical properties of the iPTF13bvn progenitor provide a comprehensive data set to test Type Ib supernova theories. A recent HST revisit to the iPTF13bvn site two years after the supernova explosion has confirmed the progenitor system.

Moving forward, the next frontier in this area is to extend these single-object analyses to a large sample of infant supernovae. The upcoming Zwicky Transient Facility with its fast survey speed, which is expected to find one infant supernova every night, is well positioned to carry out this task.

Prevalence and risk factors for mild and high-frequency bilateral sensorineural hearing loss at age 11 years old:a UK prospective cohort study

OBJECTIVE: This study aimed to assess prevalence and risk factors for mild/high-frequency bilateral sensorineural hearing loss within a UK population of children at age 11 years. DESIGN: Prospective birth cohort study. STUDY SAMPLE: Repeat hearing thresholds were measured in 5032 children, as part of the Avon Longitudinal Study of Parents and Children (ALSPAC) at age 7, 9, and 11 years. Pregnancy, birth, and early medical history were obtained prospectively through parental questionnaires and medical records. RESULTS: Twenty children had mild and seven had high-frequency bilateral sensorineural hearing loss, giving a combined prevalence of 0.5% (95% CI 0.4-0.8%). These children were more likely than the rest of the study sample to have been admitted to hospital at 6-18 months (OR 2.7, 95% CI 1.00-7.30). Parents of these children were more likely to have suspected a hearing problem when the children were 3 years old (OR 2.4, 95% CI 1.05-5.60). CONCLUSIONS: This is the first UK prospective cohort study to investigate the prevalence of mild and high-frequency hearing loss. This study, which has the advantage of a large sample size and repeat hearing measures over a four year period, reports lower prevalence values than US cross-sectional studies.

Development of molecular approaches to assess the diversity and abundance of marine nematodes assemblages for assessment of Good Environmental Status of the estuarine and marine ecosystems

In Europe, the concerns with the status of marine ecosystems have increased, and the Marine Directive has as main goal the achievement of Good Environmental Status (GES) of EU marine waters by 2020. Molecular tools are seen as promising and emerging approaches to improve ecosystem monitoring, and have led ecology into a new era, representing perhaps the most source of innovation in marine monitoring techniques. Benthic nematodes are considered ideal organisms to be used as biological indicator of natural and anthropogenic disturbances in aquatic ecosystems underpinning monitoring programmes on the ecological quality of marine ecosystems, very useful to assess the GES of the marine environment. dT-RFLP (directed Terminal-Restriction Fragment Length Polymorphism) allows to assess the diversity of nematode communities, but also allows studying the functioning of the ecosystem, and combined with relative real-time PCR (qPCR), provides a high-throughput semi-quantitative characterization of nematode communities. These characteristics make the two molecular tools good descriptors for the good environmental status assessment. The main aim of this study is to develop and optimize the dT-RFLP and qPCR in Mira estuary (SW coast, Portugal). A molecular phylogenetic analysis of marine and estuarine nematodes is being performed combining morphological and molecular analysis to evaluate the diversity of free-living marine nematodes in Mira estuary. After morphological identification, barcoding of 18S rDNA and COI genes are being determined for each nematode species morphologically identified. So far we generated 40 new sequences belonging to 32 different genus and 17 families, and the study has shown a good degree of concordance between traditional morphology-based identification and DNA sequences. These results will improve the assessment of marine nematode diversity and contribute to a more robust nematode taxonomy. The DNA sequences are being used to develop the dT-RFLP with the ability to easily process large sample numbers (hundreds and thousands), rather than typical of classical taxonomic or low throughput molecular analyses. A preliminary study showed that the digest enzymes used in dT-RFLP for terrestrial assemblages separated poorly the marine nematodes at taxonomic level for functional group analysis. A new digest combination was designed using the software tool DRAT (Directed Terminal Restriction Analysis Tool) to distinguished marine nematode taxa. Several solutions were provided by DRAT and tested empirically to select the solution that cuts most efficiently. A combination of three enzymes and a single digest showed to be the best solution to separate the different clusters. Parallel to this, another tool is being developed to estimate the population size (qPCR). An improvement in qPCR estimation of gene copy number using an artificial reference is being performed for marine nematodes communities to quantify the abundance. Once developed, it is proposed to validate both methodologies by determining the spatial and temporal variability of benthic nematodes assemblages across different environments. The application of these high-throughput molecular approaches for benthic nematodes will improve sample throughput and their implementation more efficient and faster as indicator of ecological status of marine ecosystems.

Sample-to-sample fluctuations of the overlap distributions in the three-dimensional Edwards-Anderson spin glass

We study the sample-to-sample fluctuations of the overlap probability densities from large-scale equilibrium simulations of the three-dimensional Edwards-Anderson spin glass below the critical temperature. Ultrametricity, stochastic stability, and overlap equivalence impose constraints on the moments of the overlap probability densities that can be tested against numerical data. We found small deviations from the Ghirlanda Guerra predictions, which get smaller as system size increases. We also focus on the shape of the overlap distribution, comparing the numerical data to a mean-field-like prediction in which finite-size effects are taken into account by substituting delta functions with broad peaks.

Large-volume methacrylate monolith for plasmid purification : process engineering approach to synthesis and application

The extent of exothermicity associated with the construction of large-volume methacrylate monolithic columns has somewhat obstructed the realisation of large-scale rapid biomolecule purification especially for plasmid-based products which have proven to herald future trends in biotechnology. A novel synthesis technique via a heat expulsion mechanism was employed to prepare a 40 mL methacrylate monolith with a homogeneous radial pore structure along its thickness. Radial temperature gradient was recorded to be only 1.8 °C. Maximum radial temperature recorded at the centre of the monolith was 62.3 °C, which was only 2.3 °C higher than the actual polymerisation temperature. Pore characterisation of the monolithic polymer showed unimodal pore size distributions at different radial positions with an identical modal pore size of 400 nm. Chromatographic characterisation of the polymer after functionalisation with amino groups displayed a persistent dynamic binding capacity of 15.5 mg of plasmid DNA/mL. The maximum pressure drop recorded was only 0.12 MPa at a flow rate of 10 mL/min. The polymer demonstrated rapid separation ability by fractionating Escherichia coli DH5α-pUC19 clarified lysate in only 3 min after loading. The plasmid sample collected after the fast purification process was tested to be a homogeneous supercoiled plasmid with DNA electrophoresis and restriction analysis.

Optimal Data Distributions in Machine Learning

In the first part of the thesis we explore three fundamental questions that arise naturally when we conceive a machine learning scenario where the training and test distributions can differ. Contrary to conventional wisdom, we show that in fact mismatched training and test distribution can yield better out-of-sample performance. This optimal performance can be obtained by training with the dual distribution. This optimal training distribution depends on the test distribution set by the problem, but not on the target function that we want to learn. We show how to obtain this distribution in both discrete and continuous input spaces, as well as how to approximate it in a practical scenario. Benefits of using this distribution are exemplified in both synthetic and real data sets.

In order to apply the dual distribution in the supervised learning scenario where the training data set is fixed, it is necessary to use weights to make the sample appear as if it came from the dual distribution. We explore the negative effect that weighting a sample can have. The theoretical decomposition of the use of weights regarding its effect on the out-of-sample error is easy to understand but not actionable in practice, as the quantities involved cannot be computed. Hence, we propose the Targeted Weighting algorithm that determines if, for a given set of weights, the out-of-sample performance will improve or not in a practical setting. This is necessary as the setting assumes there are no labeled points distributed according to the test distribution, only unlabeled samples.

Finally, we propose a new class of matching algorithms that can be used to match the training set to a desired distribution, such as the dual distribution (or the test distribution). These algorithms can be applied to very large datasets, and we show how they lead to improved performance in a large real dataset such as the Netflix dataset. Their computational complexity is the main reason for their advantage over previous algorithms proposed in the covariate shift literature.

In the second part of the thesis we apply Machine Learning to the problem of behavior recognition. We develop a specific behavior classifier to study fly aggression, and we develop a system that allows analyzing behavior in videos of animals, with minimal supervision. The system, which we call CUBA (Caltech Unsupervised Behavior Analysis), allows detecting movemes, actions, and stories from time series describing the position of animals in videos. The method summarizes the data, as well as it provides biologists with a mathematical tool to test new hypotheses. Other benefits of CUBA include finding classifiers for specific behaviors without the need for annotation, as well as providing means to discriminate groups of animals, for example, according to their genetic line.