Exploring the potential role of allostatic load biomarkers in risk assessment of patients presenting with depressive symptoms

Background: Depression is a major health problem worldwide and the majority of patients presenting with depressive symptoms are managed in primary care. Current approaches for assessing depressive symptoms in primary care are not accurate in predicting future clinical outcomes, which may potentially lead to over or under treatment. The Allostatic Load (AL) theory suggests that by measuring multi-system biomarker levels as a proxy of measuring multi-system physiological dysregulation, it is possible to identify individuals at risk of having adverse health outcomes at a prodromal stage. Allostatic Index (AI) score, calculated by applying statistical formulations to different multi-system biomarkers, have been associated with depressive symptoms. Aims and Objectives: To test the hypothesis, that a combination of allostatic load (AL) biomarkers will form a predictive algorithm in defining clinically meaningful outcomes in a population of patients presenting with depressive symptoms. The key objectives were: 1. To explore the relationship between various allostatic load biomarkers and prevalence of depressive symptoms in patients, especially in patients diagnosed with three common cardiometabolic diseases (Coronary Heart Disease (CHD), Diabetes and Stroke). 2 To explore whether allostatic load biomarkers predict clinical outcomes in patients with depressive symptoms, especially in patients with three common cardiometabolic diseases (CHD, Diabetes and Stroke). 3 To develop a predictive tool to identify individuals with depressive symptoms at highest risk of adverse clinical outcomes. Methods: Datasets used: ‘DepChron’ was a dataset of 35,537 patients with existing cardiometabolic disease collected as a part of routine clinical practice. ‘Psobid’ was a research data source containing health related information from 666 participants recruited from the general population. The clinical outcomes for 3 both datasets were studied using electronic data linkage to hospital and mortality health records, undertaken by Information Services Division, Scotland. Cross-sectional associations between allostatic load biomarkers calculated at baseline, with clinical severity of depression assessed by a symptom score, were assessed using logistic and linear regression models in both datasets. Cox’s proportional hazards survival analysis models were used to assess the relationship of allostatic load biomarkers at baseline and the risk of adverse physical health outcomes at follow-up, in patients with depressive symptoms. The possibility of interaction between depressive symptoms and allostatic load biomarkers in risk prediction of adverse clinical outcomes was studied using the analysis of variance (ANOVA) test. Finally, the value of constructing a risk scoring scale using patient demographics and allostatic load biomarkers for predicting adverse outcomes in depressed patients was investigated using clinical risk prediction modelling and Area Under Curve (AUC) statistics. Key Results: Literature Review Findings. The literature review showed that twelve blood based peripheral biomarkers were statistically significant in predicting six different clinical outcomes in participants with depressive symptoms. Outcomes related to both mental health (depressive symptoms) and physical health were statistically associated with pre-treatment levels of peripheral biomarkers; however only two studies investigated outcomes related to physical health. Cross-sectional Analysis Findings: In DepChron, dysregulation of individual allostatic biomarkers (mainly cardiometabolic) were found to have a non-linear association with increased probability of co-morbid depressive symptoms (as assessed by Hospital Anxiety and Depression Score HADS-D≥8). A composite AI score constructed using five biomarkers did not lead to any improvement in the observed strength of the association. In Psobid, BMI was found to have a significant cross-sectional association with the probability of depressive symptoms (assessed by General Health Questionnaire GHQ-28≥5). BMI, triglycerides, highly sensitive C - reactive 4 protein (CRP) and High Density Lipoprotein-HDL cholesterol were found to have a significant cross-sectional relationship with the continuous measure of GHQ-28. A composite AI score constructed using 12 biomarkers did not show a significant association with depressive symptoms among Psobid participants. Longitudinal Analysis Findings: In DepChron, three clinical outcomes were studied over four years: all-cause death, all-cause hospital admissions and composite major adverse cardiovascular outcome-MACE (cardiovascular death or admission due to MI/stroke/HF). Presence of depressive symptoms and composite AI score calculated using mainly peripheral cardiometabolic biomarkers was found to have a significant association with all three clinical outcomes over the following four years in DepChron patients. There was no evidence of an interaction between AI score and presence of depressive symptoms in risk prediction of any of the three clinical outcomes. There was a statistically significant interaction noted between SBP and depressive symptoms in risk prediction of major adverse cardiovascular outcome, and also between HbA1c and depressive symptoms in risk prediction of all-cause mortality for patients with diabetes. In Psobid, depressive symptoms (assessed by GHQ-28≥5) did not have a statistically significant association with any of the four outcomes under study at seven years: all cause death, all cause hospital admission, MACE and incidence of new cancer. A composite AI score at baseline had a significant association with the risk of MACE at seven years, after adjusting for confounders. A continuous measure of IL-6 observed at baseline had a significant association with the risk of three clinical outcomes- all-cause mortality, all-cause hospital admissions and major adverse cardiovascular event. Raised total cholesterol at baseline was associated with lower risk of all-cause death at seven years while raised waist hip ratio- WHR at baseline was associated with higher risk of MACE at seven years among Psobid participants. There was no significant interaction between depressive symptoms and peripheral biomarkers (individual or combined) in risk prediction of any of the four clinical outcomes under consideration. Risk Scoring System Development: In the DepChron cohort, a scoring system was constructed based on eight baseline demographic and clinical variables to predict the risk of MACE over four years. The AUC value for the risk scoring system was modest at 56.7% (95% CI 55.6 to 57.5%). In Psobid, it was not possible to perform this analysis due to the low event rate observed for the clinical outcomes. Conclusion: Individual peripheral biomarkers were found to have a cross-sectional association with depressive symptoms both in patients with cardiometabolic disease and middle-aged participants recruited from the general population. AI score calculated with different statistical formulations was of no greater benefit in predicting concurrent depressive symptoms or clinical outcomes at follow-up, over and above its individual constituent biomarkers, in either patient cohort. SBP had a significant interaction with depressive symptoms in predicting cardiovascular events in patients with cardiometabolic disease; HbA1c had a significant interaction with depressive symptoms in predicting all-cause mortality in patients with diabetes. Peripheral biomarkers may have a role in predicting clinical outcomes in patients with depressive symptoms, especially for those with existing cardiometabolic disease, and this merits further investigation.

The role of context in image annotation and recommendation

With the rise of smart phones, lifelogging devices (e.g. Google Glass) and popularity of image sharing websites (e.g. Flickr), users are capturing and sharing every aspect of their life online producing a wealth of visual content. Of these uploaded images, the majority are poorly annotated or exist in complete semantic isolation making the process of building retrieval systems difficult as one must firstly understand the meaning of an image in order to retrieve it. To alleviate this problem, many image sharing websites offer manual annotation tools which allow the user to “tag” their photos, however, these techniques are laborious and as a result have been poorly adopted; Sigurbjörnsson and van Zwol (2008) showed that 64% of images uploaded to Flickr are annotated with < 4 tags. Due to this, an entire body of research has focused on the automatic annotation of images (Hanbury, 2008; Smeulders et al., 2000; Zhang et al., 2012a) where one attempts to bridge the semantic gap between an image’s appearance and meaning e.g. the objects present. Despite two decades of research the semantic gap still largely exists and as a result automatic annotation models often offer unsatisfactory performance for industrial implementation. Further, these techniques can only annotate what they see, thus ignoring the “bigger picture” surrounding an image (e.g. its location, the event, the people present etc). Much work has therefore focused on building photo tag recommendation (PTR) methods which aid the user in the annotation process by suggesting tags related to those already present. These works have mainly focused on computing relationships between tags based on historical images e.g. that NY and timessquare co-exist in many images and are therefore highly correlated. However, tags are inherently noisy, sparse and ill-defined often resulting in poor PTR accuracy e.g. does NY refer to New York or New Year? This thesis proposes the exploitation of an image’s context which, unlike textual evidences, is always present, in order to alleviate this ambiguity in the tag recommendation process. Specifically we exploit the “what, who, where, when and how” of the image capture process in order to complement textual evidences in various photo tag recommendation and retrieval scenarios. In part II, we combine text, content-based (e.g. # of faces present) and contextual (e.g. day-of-the-week taken) signals for tag recommendation purposes, achieving up to a 75% improvement to precision@5 in comparison to a text-only TF-IDF baseline. We then consider external knowledge sources (i.e. Wikipedia & Twitter) as an alternative to (slower moving) Flickr in order to build recommendation models on, showing that similar accuracy could be achieved on these faster moving, yet entirely textual, datasets. In part II, we also highlight the merits of diversifying tag recommendation lists before discussing at length various problems with existing automatic image annotation and photo tag recommendation evaluation collections. In part III, we propose three new image retrieval scenarios, namely “visual event summarisation”, “image popularity prediction” and “lifelog summarisation”. In the first scenario, we attempt to produce a rank of relevant and diverse images for various news events by (i) removing irrelevant images such memes and visual duplicates (ii) before semantically clustering images based on the tweets in which they were originally posted. Using this approach, we were able to achieve over 50% precision for images in the top 5 ranks. In the second retrieval scenario, we show that by combining contextual and content-based features from images, we are able to predict if it will become “popular” (or not) with 74% accuracy, using an SVM classifier. Finally, in chapter 9 we employ blur detection and perceptual-hash clustering in order to remove noisy images from lifelogs, before combining visual and geo-temporal signals in order to capture a user’s “key moments” within their day. We believe that the results of this thesis show an important step towards building effective image retrieval models when there lacks sufficient textual content (i.e. a cold start).

Depression and physical disability in chronic pain: The mediation role of emotional intelligence and acceptance

Emotional intelligence (EI) and acceptance have previously been identified as potential factors in the adjustment to chronic pain (CP). This study examined the associations between CP experiences, depression, and physical disability. It further investigated the mediating effect of EI and acceptance in the relationship between CP experiences, depression, and physical disability and how this changes with the duration of the CP. Method: A cross-sectional design, employing validated questionnaires, was used to measure pain experience, physical disability, depression, EI, and acceptance in 133 CP patients. Results: All variables were found to be significantly associated in theoretically predicted ways. The relationship between CP experiences and depression was mediated by both factors, as high EI and acceptance promoted a decreased influence of pain on depression. By contrast, the relationship between CP experiences and physical disability was mediated by acceptance, but not by EI. Further, the temporal stability analysis of this mediation model showed that long-term CP patients are better able to make use of these factors. Conclusions: The relationship between the experience of pain and depression or physical disability seems to be significantly mediated by factors such as EI and acceptance. This study lends further support to the development of more encompassing models that take both control and non-control variables into account when conceptualising the adjustment to CP. Theoretical and clinical implications are discussed.

The Role of Attachment in a Social Cognitive Model of Social Domain Satisfaction in College Students

The study examined a modified social cognitive model of domain satisfaction (Lent, 2004). In addition to social cognitive variables and trait positive affect, the model included two aspects of adult attachment, attachment anxiety and avoidance. The study extended recent research on well-being and satisfaction in academic, work, and social domains. The adjusted model was tested in a sample of 454 college students, in order to determine the role of adult attachment variables in explaining social satisfaction, above and beyond the direct and indirect effects of trait positive affect. Confirmatory factor analysis found support for 8 correlated factors in the modified model: social domain satisfaction, positive affect, attachment avoidance, attachment anxiety, social support, social self-efficacy, social outcome expectations, and social goal progress. Three alternative structural models were tested to account for the ways in which attachment anxiety and attachment avoidance might relate to social satisfaction. Results of model testing provided support for a model in which attachment avoidance produced only an indirect path to social satisfaction via self-efficacy and social support. Positive affect, avoidance, social support, social self-efficacy, and goal progress each produced significant direct or indirect paths to social domain satisfaction, though attachment anxiety and social outcome expectations did not contribute to the predictive model. Implications of the findings regarding the modified social cognitive model of social domain satisfaction were discussed.

Critical core mass for enriched envelopes: the role of H2O condensation

Context. Within the core accretion scenario of planetary formation, most simulations performed so far always assume the accreting envelope to have a solar composition. From the study of meteorite showers on Earth and numerical simulations, we know that planetesimals must undergo thermal ablation and disruption when crossing a protoplanetary envelope. Thus, once the protoplanet has acquired an atmosphere, not all planetesimals reach the core intact, i.e. the primordial envelope (mainly H and He) gets enriched in volatiles and silicates from the planetesimals. This change of envelope composition during the formation can have a significant effect on the final atmospheric composition and on the formation timescale of giant planets. Aims. We investigate the physical implications of considering the envelope enrichment of protoplanets due to the disruption of icy planetesimals during their way to the core. Particular focus is placed on the effect on the critical core mass for envelopes where condensation of water can occur. Methods. Internal structure models are numerically solved with the implementation of updated opacities for all ranges of metallicities and the software Chemical Equilibrium with Applications to compute the equation of state. This package computes the chemical equilibrium for an arbitrary mixture of gases and allows the condensation of some species, including water. This means that the latent heat of phase transitions is consistently incorporated in the total energy budget. Results. The critical core mass is found to decrease significantly when an enriched envelope composition is considered in the internal structure equations. A particularly strong reduction of the critical core mass is obtained for planets whose envelope metallicity is larger than Z approximate to 0.45 when the outer boundary conditions are suitable for condensation of water to occur in the top layers of the atmosphere. We show that this effect is qualitatively preserved even when the atmosphere is out of chemical equilibrium. Conclusions. Our results indicate that the effect of water condensation in the envelope of protoplanets can severely affect the critical core mass, and should be considered in future studies.

The role of perceived housing quality and perceived choice to recovery: An ecological perspective on a housing first program in Lisbon

This study explores, from an ecological perspective, the relationship between perceived housing quality and the perception of choice, and between perceived choice and recovery of 45 Housing First Lisbon participants. For this purpose, we used a quantitative method and applied three instruments that report perceived housing quality, perceived choice and severe mental illness recovery. The findings reveal a significant and positive association between perceived housing quality and perceived choice, and between perceived choice and recovery, with choice being predicted by housing quality and recovery predicted by choice. These results reinforce the scientific evidence regarding the success of housing first models as a consumer choice-driven intervention, addressing pertinent environmental factors that contribute to housing stability. The study demonstrates that recovery processes can be maximized through services that empower their consumers by allowing them to choose and control the priority and order of the support services received.

COLLEGE STUDENTS’ ATTITUDES TOWARD SEEKING PROFESSIONAL HELP: THE ROLE OF SOCIAL CLASS, CLASSISM, AND STIGMA

Research on attitudes toward seeking professional help among college students has examined the influence of social class and stigma. This study tested 4 theoretically and empirically derived structural equation models of college students’ attitudes toward seeking counseling with a sample of 2230 incoming university students. The models represented competing hypotheses regarding the manners in which objective social class, subjective social class, classism, public stigma, stigma by close others, and self-stigma related to attitudes toward seeking professional help. Findings supported the social class direct and indirect effects model, as well as the notion that classism and stigma domains could explain the indirect relationships between social class and attitudes. Study limitations, future directions for research, and implications for counseling are discussed.

Corporate governance in european transition economies: Emerging models

The purpose of this paper is to contribute to the debate on corporate governance models in European transition economies. The paper consists of four parts. After a historic overview of the evolution of corporate governance, the introduction presents various understandings of the corporate governance function and describes current issues in corporate governance. Part two deals with governance systems in the (mainly domestically) privatized former state-owned companies in Central European transition countries, with the main types of company ownership structures, relationships between governing and management functions, and deficiencies in existing governance systems. Part three is dedicated to the analysis of factors that determine the efficiency of the relationship between the corporate governance and management functions in Central European transition economies. It deals with the issue of why the German (continental European) governance model is usually the preferred choice and why the chosen models underperform. In the conclusion the author offers his suggestions on how the Central European transition countries should improve their corporate governance in the future.

The role of the Indian Ocean sector and sea surface salinity for prediction of the coupled Indo-Pacific system

The purpose of this dissertation is to evaluate the potential downstream influence of the Indian Ocean (IO) on El Niño/Southern Oscillation (ENSO) forecasts through the oceanic pathway of the Indonesian Throughflow (ITF), atmospheric teleconnections between the IO and Pacific, and assimilation of IO observations. Also the impact of sea surface salinity (SSS) in the Indo-Pacific region is assessed to try to address known problems with operational coupled model precipitation forecasts. The ITF normally drains warm fresh water from the Pacific reducing the mixed layer depths (MLD). A shallower MLD amplifies large-scale oceanic Kelvin/Rossby waves thus giving ~10% larger response and more realistic ENSO sea surface temperature (SST) variability compared to observed when the ITF is open. In order to isolate the impact of the IO sector atmospheric teleconnections to ENSO, experiments are contrasted that selectively couple/decouple the interannual forcing in the IO. The interannual variability of IO SST forcing is responsible for 3 month lagged widespread downwelling in the Pacific, assisted by off-equatorial curl, leading to warmer NINO3 SST anomaly and improved ENSO validation (significant from 3-9 months). Isolating the impact of observations in the IO sector using regional assimilation identifies large-scale warming in the IO that acts to intensify the easterlies of the Walker circulation and increases pervasive upwelling across the Pacific, cooling the eastern Pacific, and improving ENSO validation (r ~ 0.05, RMS~0.08C). Lastly, the positive impact of more accurate fresh water forcing is demonstrated to address inadequate precipitation forecasts in operational coupled models. Aquarius SSS assimilation improves the mixed layer density and enhances mixing, setting off upwelling that eventually cools the eastern Pacific after 6 months, counteracting the pervasive warming of most coupled models and significantly improving ENSO validation from 5-11 months. In summary, the ITF oceanic pathway, the atmospheric teleconnection, the impact of observations in the IO, and improved Indo-Pacific SSS are all responsible for ENSO forecast improvements, and so each aspect of this study contributes to a better overall understanding of ENSO. Therefore, the upstream influence of the IO should be thought of as integral to the functioning of ENSO phenomenon.

Investigating the Role of O-GlcNAc Glycosylation in Neurodegeneration

O-GlcNAc glycosylation of nuclear and cytosolic proteins is an essential post-translational modification implicated in many diseases, from cancer to diabetes. Importantly, many important neuronal proteins are also O-GlcNAc modified, and aberrant O-GlcNAcylation of these proteins may contribute to the pathology of neurodegenerative diseases although these mechanisms have not been well defined. Here we investigated the role of O-GlcNAc glycosylation in the brain, utilizing both chemistry and molecular biology to study O-GlcNAc transferase (OGT), the enzyme that adds the sugar modification. To evaluate the role of OGT in adult neurons, we generated a forebrain-specific conditional knockout of OGT (OGT cKO) in mice. Although indistinguishable from wild-type littermates at birth, after three weeks we observe progressive neurodegeneration in OGT cKO mice. Hallmarks of Alzheimer’s disease, including neuronal loss, neuroinflammation, behavioral deficits, hyperphosphorylated tau, and amyloid beta peptide accumulation, are observed. Furthermore, decreases in OGT protein levels were found in human AD brain tissue, suggesting that altered O-GlcNAcylation likely contributes to neurodegenerative diseases in humans. This model is one of a few mouse models that recapitulate AD phenotypes without mutating and overexpressing human tau, amyloid precursor protein, or presenilin, highlighting the essential role of OGT in neurodegenerative pathways.

Given the importance of OGT in the brain, we further investigated the regulation of the OGT enzyme by phosphorylation. We found that phosphorylation of OGT near its C-terminus reduces its activity in cancer cells, and have developed phosphorylation-specific antibodies to aid mechanistic studies. Furthermore, mutation of this phosphorylation site on OGT, followed by overexpression in neurons was shown to enhance neurite outgrowth, demonstrating a functional consequence for this site. Thus phosphorylation of OGT inhibits its activity and enhances neurite outgrowth, and current studies aim to characterize the signaling pathway that regulates OGT phosphorylation in neurons.

Role of Ccbe1 during cardiac differentiation of mouse ESCs

Tese de Doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016

Changing family models in Spain: the impact of the sociodemographic factors

Abstract During the last few decades, there has been an increasing international recognition of the studies related to the analysis of the family models change, the focus being the determinants of the female employment and the problems related to the work family balance (Lewis, 2001; Petit & Hook, 2005Saraceno, Crompton & Lyonette, 20062008; Pfau-Effinger, 2012). The majority of these studies have been focused on the analysis of the work-family balance problems as well as the effectiveness of the family and gender policies in order to encourage female employment (Korpi et al., 2013). In Spain, special attention has been given to the family policies implemented, the employability of women and on the role of the father in the family (Flaquer et al., 2015; Meil, 2015); however, there has been far less emphasis on the analysis of the family cultural models (González and Jurado, 2012; Crespi and Moreno, 2016). The purpose of this paper is to present some of the first results on the influence of the socio-demographic factors on the expectations and attitudes about the family models. This study offers an analytical reflection upon the foundation of the determinants of the family ambivalence in Spain from the cultural and the institutional dimension. This study shows the Spanish family models of preferences following the Pfau-Effinger (2004) classification of the famiy living arrangements. The reason for this study is twofold; on the one hand, there is confirmed the scarcity of studies that have focused their attention on this objective in Spain; on the other hand, the studies carried out in the international context have confirmed the analytical effectiveness of researching on the attitude and value changes to explain the meaning and trends of the family changes. There is also presented some preliminary results that have been obtained from the multinomial analysis related to the influence of the socio-demographic factors on the family model chosen by the individuals in Spain (father and mother working full time; mother part-time father full-time; mother not at work father full-time; mother and father part-time). 3 The database used has been the International Social Survey Programme: Family and Changing Gender Roles IV- ISSP 2012-. Spain is the only country of South Europe that has participated in the survey. For this reason it has been considered as a representative case study.

Personality and Performance: Assessing the Mediating Role of Mental Model Formation in the Personality-Performance Relationship

Personality has long been linked to performance. Evolutions in this relationship have brought forward new questions regarding the true nature of how personality impacts performance. Both direct and indirect relationships have been proven significant. This study further investigated potential indirect relationships by including a mediating variable, mental model formation, in the personality-performance relationship. Undergraduate students were assessed in a 6-week period, Time 1 - Time 2 experiment. Conceptualizations of personality included measures of the Big 5 model and Self-efficacy, with performance measured by content quiz and overall course scores. Findings showed that the Big 5 personality traits, extraversion and agreeableness, positively and significantly impacted commonality with the instructor’s mental model. However, commonality with the instructor’s mental model did not impact performance. In comparison, commonality with an expert mental model positively and significantly impacted performance for both the content quiz and overall course score. Furthermore, similarity with an expert mental model positively and significantly impacted overall course performance. Hypothesized full mediation of mental model formation for the personality-performance relationship was not supported due to a lack of direct effect relationships required for mediation. However, a revised conceptualization of results emerged. Findings from the current study point to the novel and unique role mental models play in the personality-performance relationship. While personality traits do impact mental model formation, accuracy in the mental models formed is critical to performance.

The Role of Existential Beliefs Within the Relation of Centenarians’ Health and Well-Being

The purpose of this study was to explore the role of existential beliefs in mediating the influence of health on centenarians' well-being. A total of 80 centenarians (mean age 101.1; SD = 1.3; 81.3 % women) with no/minor cognitive impairment were included. The OARS questionnaire for diseases and functional capacity (ADL, IADL), the Satisfaction with Life Scale, and the existential beliefs subscale were used for data collection. The findings suggest that existential resources are a crucial element for mitigating the impact of health constraints in subjective well-being in this population. Appropriate models of intervention for very old age that recognize the importance of religion, spirituality, and meaning of life are to be considered.

The role of area-level deprivation and gender in participation in population-based faecal immunochemical test (FIT) colorectal cancer screening

This study aimed to investigate the effects of sex and deprivation on participation in a population-based faecal immunochemical test (FIT) colorectal cancer screening programme. The study population included 9785 individuals invited to participate in two rounds of a population-based biennial FIT-based screening programme, in a relatively deprived area of Dublin, Ireland. Explanatory variables included in the analysis were sex, deprivation category of area of residence and age (at end of screening). The primary outcome variable modelled was participation status in both rounds combined (with “participation” defined as having taken part in either or both rounds of screening). Poisson regression with a log link and robust error variance was used to estimate relative risks (RR) for participation. As a sensitivity analysis, data were stratified by screening round. In both the univariable and multivariable models deprivation was strongly associated with participation. Increasing affluence was associated with higher participation; participation was 26% higher in people resident in the most affluent compared to the most deprived areas (multivariable RR = 1.26: 95% CI 1.21–1.30). Participation was significantly lower in males (multivariable RR = 0.96: 95%CI 0.95–0.97) and generally increased with increasing age (trend per age group, multivariable RR = 1.02: 95%CI, 1.01–1.02). No significant interactions between the explanatory variables were found. The effects of deprivation and sex were similar by screening round. Deprivation and male gender are independently associated with lower uptake of population-based FIT colorectal cancer screening, even in a relatively deprived setting. Development of evidence-based interventions to increase uptake in these disadvantaged groups is urgently required.