The use of visual analogue scales to assess motivation to eat in human subjects: a review of their reliability and validity with an evaluation of new hand-held computerized stsytems for temporal tracking of appetite ratings.

This present paper reviews the reliability and validity of visual analogue scales (VAS) in terms of (1) their ability to predict feeding behaviour, (2) their sensitivity to experimental manipulations, and (3) their reproducibility. VAS correlate with, but do not reliably predict, energy intake to the extent that they could be used as a proxy of energy intake. They do predict meal initiation in subjects eating their normal diets in their normal environment. Under laboratory conditions, subjectively rated motivation to eat using VAS is sensitive to experimental manipulations and has been found to be reproducible in relation to those experimental regimens. Other work has found them not to be reproducible in relation to repeated protocols. On balance, it would appear, in as much as it is possible to quantify, that VAS exhibit a good degree of within-subject reliability and validity in that they predict with reasonable certainty, meal initiation and amount eaten, and are sensitive to experimental manipulations. This reliability and validity appears more pronounced under the controlled (but more arti®cial) conditions of the laboratory where the signal : noise ratio in experiments appears to be elevated relative to real life. It appears that VAS are best used in within-subject, repeated-measures designs where the effect of different treatments can be compared under similar circumstances. They are best used in conjunction with other measures (e.g. feeding behaviour, changes in plasma metabolites) rather than as proxies for these variables. New hand-held electronic appetite rating systems (EARS) have been developed to increase reliability of data capture and decrease investigator workload. Recent studies have compared these with traditional pen and paper (P&P) VAS. The EARS have been found to be sensitive to experimental manipulations and reproducible relative to P&P. However, subjects appear to exhibit a signi®cantly more constrained use of the scale when using the EARS relative to the P&P. For this reason it is recommended that the two techniques are not used interchangeably

Selective effects of acute exercise and breakfast interventions on mood and motivation to eat

The effects of exercise and breakfast manipulations on mood and motivation to eat were assessed in 11 healthy females who were regular exercisers and habitual breakfast eaters. The study involved a two by two repeated-measures design, with exercise (or no exercise) and a high-energy breakfast (or low-energy breakfast) as the repeated measures. The exercise or no-exercise session (0800 h) was followed by consumption of the low- or high-energy breakfast (0900 h). An ad libitum lunch test meal was provided 4 hours after the beginning of the exercise session (1200 h). Mood and motivation to eat were continuously tracked from 0800 until 1700 h by an electronic appetite ratings system (EARS). In general, morning subjective mood states (e.g., contentment) were significantly lower in the low-energy breakfast condition, but exercise reversed this effect. Exercise also significantly decreased feelings of lethargy, independent of the breakfast condition. Desire-to-eat and fullness ratings were significantly increased in the low-energy breakfast and high-energy breakfast conditions, respectively. Impairments of mood disappeared in the afternoon after consumption of an ad libitum lunch. In these healthy young adults, the condition inducing the largest energy deficit (exercise and low-energy breakfast) was not associated with the lowest mental states.

Effects of Sweetness and Energy in Drinks on Food Intake Following Exercise

Exercise is known to cause physiological changes that could affect the impact of nutrients on appetite control. This study was designed to assess the effect of drinks containing either sucrose or high-intensity sweeteners on food intake following exercise. Using a repeated-measures design, three drink conditions were employed: plain water (W), a low-energy drink sweetened with artificial sweeteners aspartame and acesulfame-K (L), and a high-energy, sucrose-sweetened drink (H). Following a period of challenging exercise (70% VO2 max for 50 min), subjects consumed freely from a particular drink before being offered a test meal at which energy and nutrient intakes were measured. The degree of pleasantness (palatability) of the drinks was also measured before and after exercise. At the test meal, energy intake following the artificially sweetened (L) drink was significantly greater than after water and the sucrose (H) drinks (p < 0.05). Compared with the artificially sweetened (L) drink, the high-energy (H) drink suppressed intake by approximately the energy contained in the drink itself. However, there was no difference between the water (W) and the sucrose (H) drink on test meal energy intake. When the net effects were compared (i.e., drink + test meal energy intake), total energy intake was significantly lower after the water (W) drink compared with the two sweet (L and H) drinks. The exercise period brought about changes in the perceived pleasantness of the water, but had no effect on either of the sweet drinks. The remarkably precise energy compensation demonstrated after the higher energy sucrose drink suggests that exercise may prime the system to respond sensitively to nutritional manipulations. The results may also have implications for the effect on short-term appetite control of different types of drinks used to quench thirst during and after exercise.

Molecular markers of obesity and diabetes

Recently it has been shown that the consumption of a diet high in saturated fat is associated with impaired insulin sensitivity and increased incidence of type 2 diabetes. In contrast, diets that are high in monounsaturated fatty acids (MUFAs) or polyunsaturated fatty acids (PUFAs), especially very long chain n-3 fatty acids (FAs), are protective against disease. However, the molecular mechanisms by which saturated FAs induce the insulin resistance and hyperglycaemia associated with metabolic syndrome and type 2 diabetes are not clearly defined. It is possible that saturated FAs may act through alternative mechanisms compared to MUFA and PUFA to regulate of hepatic gene expression and metabolism. It is proposed that, like MUFA and PUFA, saturated FAs regulate the transcription of target genes. To test this hypothesis, hepatic gene expression analysis was undertaken in a human hepatoma cell line, Huh-7, after exposure to the saturated FA, palmitate. These experiments showed that palmitate is an effective regulator of gene expression for a wide variety of genes. A total of 162 genes were differentially expressed in response to palmitate. These changes not only affected the expression of genes related to nutrient transport and metabolism, they also extend to other cellular functions including, cytoskeletal architecture, cell growth, protein synthesis and oxidative stress response. In addition, this thesis has shown that palmitate exposure altered the expression patterns of several genes that have previously been identified in the literature as markers of risk of disease development, including CVD, hypertension, obesity and type 2 diabetes. The altered gene expression patterns associated with an increased risk of disease include apolipoprotein-B100 (apo-B100), apo-CIII, plasminogen activator inhibitor 1, insulin-like growth factor-I and insulin-like growth factor binding protein 3. This thesis reports the first observation that palmitate directly signals in cultured human hepatocytes to regulate expression of genes involved in energy metabolism as well as other important genes. Prolonged exposure to long-chain saturated FAs reduces glucose phosphorylation and glycogen synthesis in the liver. Decreased glucose metabolism leads to elevated rates of lipolysis, resulting in increased release of free FAs. Free FAs have a negative effect on insulin action on the liver, which in turn results in increased gluconeogenesis and systemic dyslipidaemia. It has been postulated that disruption of glucose transport and insulin secretion by prolonged excessive FA availability might be a non-genetic factor that has contributed to the staggering rise in prevalence of type 2 diabetes. As glucokinase (GK) is a key regulatory enzyme of hepatic glucose metabolism, changes in its activity may alter flux through the glycolytic and de novo lipogenic pathways and result in hyperglycaemia and ultimately insulin resistance. This thesis investigated the effects of saturated FA on the promoter activity of the glycolytic enzyme, GK, and various transcription factors that may influence the regulation of GK gene expression. These experiments have shown that the saturated FA, palmitate, is capable of decreasing GK promoter activity. In addition, quantitative real-time PCR has shown that palmitate incubation may also regulate GK gene expression through a known FA sensitive transcription factor, sterol regulatory element binding protein-1c (SREBP-1c), which upregulates GK transcription. To parallel the investigations into the mechanisms of FA molecular signalling, further studies of the effect of FAs on metabolic pathway flux were performed. Although certain FAs reduce SREBP-1c transcription in vitro, it is unclear whether this will result in decreased GK activity in vivo where positive effectors of SREBP-1c such as insulin are also present. Under these conditions, it is uncertain if the inhibitory effects of FAs would be overcome by insulin. The effects of a combination of FAs, insulin and glucose on glucose phosphorylation and metabolism in cultured primary rat hepatocytes at concentrations that mimic those in the portal circulation after a meal was examined. It was found that total GK activity was unaffected by an increased concentration of insulin, but palmitate and eicosapentaenoic acid significantly lowered total GK activity in the presence of insulin. Despite the fact that total GK enzyme activity was reduced in response to FA incubation, GK enzyme translocation from the inactive, nuclear bound, to active, cytoplasmic state was unaffected. Interestingly, none of the FAs tested inhibited glucose phosphorylation or the rate of glycolysis when insulin is present. These results suggest that in the presence of insulin the levels of the active, unbound cytoplasmic GK are sufficient to buffer a slight decrease in GK enzyme activity and decreased promoter activity caused by FA exposure. Although a high fat diet has been associated with impaired hepatic glucose metabolism, there is no evidence from this thesis that FAs themselves directly modulate flux through the glycolytic pathway in isolated primary hepatocytes when insulin is also present. Therefore, although FA affected expression of a wide range of genes, including GK, this did not affect glycolytic flux in the presence of insulin. However, it may be possible that a saturated FA-induced decrease in GK enzyme activity when combined with the onset of insulin resistance may promote the dys-regulation of glucose homeostasis and the subsequent development of hyperglycaemia, metabolic syndrome and type 2 diabetes.

A collaborative approach to home care delivery for older clients: Perspectives of home care providers

A collaborative approach to home care (HC) delivery for older clients has taken centre stage (Nies, 2006). In Finland, public home help and home health care services have been combined to form the home care unit, whose goal is to provide a collaborative approach to care delivery through cooperation and sharing of responsibilities. In this model, the general practitioner (GP), home care nurses (HCN) and home help workers (HHW) care for shared clients. GPs and HCNs provide health care, such as monitoring of clients’ health status, and HHWs assist with personal care tasks such as dressing, washing and meal preparation. As the needs of older clients are multiple, collaboration is needed as one professional group cannot take sole responsibility (Nies, 2006). This paper reports on a study undertaken to examine home care unit care providers’ perspectives of the collaborative approach to HC delivery for older clients.

Acute exercise improves postprandial cardiovascular risk factors in overweight and obese individuals

Objectives The effects of 30 min of exercise on postprandial lipaemia in the overweight and obese are unknown as previous studies have only investigated bouts of at least 60 min in lean, healthy individuals. The aim of this study was to investigate whether a single 30-min bout of resistance, aerobic or combined exercise at moderate-intensity would decrease postprandial lipaemia, glucose and insulin levels as well as increase resting energy expenditure and increase fat oxidation following a high fat meal consumed 14 h after the exercise bout, in overweight and obese individuals compared to no exercise. We also compared the effects of the different exercise modalities. Methods This study was a randomized cross-over design which examined the postprandial effects of 30 min of different types of exercise in the evening prior to a breakfast meal in overweight and obese men and women. Participants were randomized on four occasions, each one-week apart, to each condition; either no exercise, aerobic exercise, resistance exercise or a combination of aerobic exercise and resistance exercise. Results An acute bout of combination training did not have any significant effect on postprandial measurements compared to no exercise. However, aerobic exercise significantly reduced postprandial triglyceride levels by 8% compared to no exercise (p = 0.02) and resistance exercise decreased postprandial insulin levels by 30% compared to aerobic exercise (p = 0.01). Conclusion These results indicate that a single moderate-intensity 30 min bout of aerobic or resistance exercise improves risk factors associated with cardiovascular disease in overweight and obese individuals.

Keeping House : a novel and novel companion

The creative work of this study is a novel-length work of literary fiction called Keeping House (published as Grace's Table, by University of Queensland Press, April 2014). Grace has not had twelve people at her table for a long time. Hers isn't the kind of family who share regular Sunday meals. As Grace prepares the feast, she reflects on her life, her marriage and her friendships. When the three generations of her family come together, simmering tensions from the past threaten to boil over. The one thing that no one can talk about is the one thing that no one can forget. Grace's Table is a moving and often funny novel using food as a language to explore the power of memory and the family rituals that define us. The exegetical component of this study does not adhere to traditional research pedagogies. Instead, it follows the model of what the literature describes as fictocriticism. It is the intention that the exegesis be read as a hybrid genre; one that combines creative practice and theory and blurs the boundaries between philosophy and fiction. In offering itself as an alternative to the exegetical canon it provides a model for the multiplicity of knowledge production suited to the discipline of practice-led research. The exegesis mirrors structural elements of the creative work by inviting twelve guests into the domestic space of the novel to share a meal. The guests, chosen for their diverse thinking, enable examination of the various agents of power involved in the delivery of food. Their ideas cross genders, ages and time periods; their motivations and opinions often collide. Some are more concerned with the spatial politics of where food is consumed, others with its actual preparation and consumption. Each, however, provides a series of creative reflective conversations throughout the meal which help to answer the research question: How can disempowered women take authority within their domestic space? Michel de Certeau must defend his "operational tactics" or "art of the weak" 1 as a means by which women can subvert the colonisation of their domestic space against Michel Foucault's ideas about the functions of a "disciplinary apparatus". 2 Erving Goffman argues that the success of de Certeau's "tactics" depends upon his theories of "performance" and "masquerade" 3; a claim de Certeau refutes. Doreen Massey and the author combine forces in arguing for space, time and politics to be seen as interconnected, non-static and often contested. The author calls for identity, or sense of self, to be considered a further dimension which impacts on the function of spatial models. Yu-Fi Tuan speaks of the intimacy of kitchens; Gaston Bachelard the power of daydreams; and Jean Anthelme Brillat-Savarin gives the reader a taste of the nourishing arts. Roland Barthes forces the author to reconsider her function as a writer and her understanding of the reader's relationship with a text. Fictional characters from two texts have a place at the table – Marian from The Edible Woman by Margaret Atwood 4 and Lilian from Lilian's Story by Kate Grenville. 5 Each explores how they successfully subverted expectations of their gender. The author interprets and applies elements of the conversations to support Grace's tactics in the novel as well as those related to her own creative research practice. Grace serves her guests, reflecting on what is said and how it relates to her story. Over coffee, the two come together to examine what each has learned.

Natural and engineered kallikrein inhibitors: an emerging pharmacopoeia

The kallikreins and kallikrein-related peptidases are serine proteases that control a plethora of developmental and homeostatic phenomena, ranging from semen liquefaction to skin desquamation and blood pressure. The diversity of roles played by kallikreins has stimulated considerable interest in these enzymes from the perspective of diagnostics and drug design. Kallikreins already have well-established credentials as targets for therapeutic intervention and there is increasing appreciation of their potential both as biomarkers and as targets for inhibitor design. Here, we explore the current status of naturally occurring kallikrein protease-inhibitor complexes and illustrate how this knowledge can interface with strategies for rational re-engineering of bioscaffolds and design of small-molecule inhibitors.

A systematic review of environmental factors and obesogenic dietary intakes among adults : are we getting closer to understanding obesogenic environments?

This study examined whether physical, social, cultural and economical environmental factors are associated with obesogenic dietary behaviours and overweight/obesity among adults. Literature searches of databases (i.e. PubMed, CSA Illumina, Web of Science, PsychInfo) identified studies examining environmental factors and the consumption of energy, fat, fibre, fruit, vegetables, sugar-sweetened drinks, meal patterns and weight status. Twenty-eight studies were in-scope, the majority (n= 16) were conducted in the USA. Weight status was consistently associated with the food environment; greater accessibility to supermarkets or less access to takeaway outlets were associated with a lower BMI or prevalence of overweight/obesity. However, obesogenic dietary behaviours did not mirror these associations; mixed associations were found between the environment and obesogenic dietary behaviours. Living in a socioeconomically-deprived area was the only environmental factor consistently associated with a number of obesogenic dietary behaviours. Associations between the environment and weight status are more consistent than that seen between the environment and dietary behaviours. The environment may play an important role in the development of overweight/obesity, however the dietary mechanisms that contribute to this remain unclear and the physical activity environment may also play an important role in weight gain, overweight and obesity.

A systematic review of studies on socioeconomic inequalities in dietary intakes associated with weight gain and overweight/obesity conducted among European adults

This Review examined socioeconomic inequalities in intakes of dietary factors associated with weight gain, overweight/obesity among adults in Europe. Literature searches of studies published between 1990 and 2007 examining socioeconomic position (SEP) and the consumption of energy, fat, fibre, fruit, vegetables, energy-rich drinks and meal patterns were conducted. Forty-seven articles met the inclusion criteria. The direction of associations between SEP and energy intakes were inconsistent. Approximately half the associations examined between SEP and fat intakes showed higher total fat intakes among socioeconomically disadvantaged groups. There was some evidence that these groups consume a diet lower in fibre. The most consistent evidence of dietary inequalities was for fruit and vegetable consumption; lower socioeconomic groups were less likely to consume fruit and vegetables. Differences in energy, fat and fibre intakes (when found) were small-to-moderate in magnitude; however, differences were moderate-to-large for fruit and vegetable intakes. Socioeconomic inequalities in the consumption of energy-rich drinks and meal patterns were relatively under-studied compared with other dietary factors. There were no regional or gender differences in the direction and magnitude of the inequalities in the dietary factors examined. The findings suggest that dietary behaviours may contribute to socioeconomic inequalities in overweight/obesity in Europe. However, there is only consistent evidence that fruit and vegetables may make an important contribution to inequalities in weight status across European regions.

Adolescent experiences of 'family meals' in Australia

Popular discourse laments the decline of the ‘family meal’, leading to family fragmentation and nutritional compromise. This article reports findings of a study investigating beliefs and practices surrounding the ‘family meal’, using data drawn from an on-line survey completed by 625 adolescents in Perth, Western Australia. The results challenge current concerns about the loss of the ‘family meal’, demonstrating that, for a majority, meals are eaten together rather than in isolation; are home-made rather than store bought or fast food; and are sites of conversation regardless of the presence of a television. Adolescents are divided, however, on the value of the ‘family meal’, with half seeing it as a positive experience of family togetherness and half regarding it negatively or as unimportant. The findings go some way to dispelling the notion that the ‘family meal’ no longer exists in Australia.

Product labelling can confer sustained increases in expected and actual satiety

Expected satiety has been shown to play a key role in decisions around meal size. Recently it has become clear that these expectations can also influence the satiety that is experienced after a food has been consumed. As such, increasing the expected and actual satiety a food product confers without increasing its caloric content is of importance. In this study we sought to determine whether this could be achieved via product labelling. Female participants (N=75) were given a 223-kcal yoghurt smoothie for lunch. In separate conditions the smoothie was labelled as a diet brand, a highly-satiating brand, or an ‘own brand’ control. Expected satiety was assessed using rating scales and a computer-based ‘method of adjustment’, both prior to consuming the smoothie and 24 hours later. Hunger and fullness were assessed at baseline, immediately after consuming the smoothie, and for a further three hours. Despite the fact that all participants consumed the same food, the smoothie branded as highly-satiating was consistently expected to deliver more satiety than the other ‘brands’; this difference was sustained 24 hours after consumption. Furthermore, post-consumption and over three hours, participants consuming this smoothie reported significantly less hunger and significantly greater fullness. These findings demonstrate that the satiety that a product confers depends in part on information that is present around the time of consumption. We suspect that this process is mediated by changes to expected satiety. These effects may potentially be utilised in the development of successful weight-management products.

Comparing the medium-term effects of exercise or dietary restriction on appetite regulation and compensatory responses

Previous studies have shown that exercise (Ex) interventions create a stronger coupling between energy intake (EI) and energy expenditure (EE) leading to increased homeostasis of the energy-balance (EB) regulatory system compared to a diet intervention where an un-coupling between EI and EE occurs. The benefits of weight loss from Ex and diet interventions greatly depend on compensatory responses. The present study investigated an 8-week medium-term Ex and diet intervention program (Ex intervention comprised of 500kcal EE five days per week over four weeks at 65-75% maximal heart rate, whereas the diet intervention comprised of a 500kcal decrease in EI five days per week over four weeks) and its effects on compensatory responses and appetite regulation among healthy individuals using a between- and within-subjects design. Effects of an acute dietary manipulation on appetite and compensatory behaviours and whether a diet and/or Ex intervention pre-disposes individuals to disturbances in EB homeostasis were tested. Energy intake at an ad libitum lunch test meal after a breakfast high- and low-energy pre-load (the high energy pre-load contained 556kcal and the low energy pre-load contained 239kcal) were measured at the Baseline (Weeks -4 to 0) and Intervention (Weeks 0 to 4) phases in 13 healthy volunteers (three males and ten females; mean age 35 years [sd + 9] and mean BMI 25 kg/m2 [sd + 3.8]) [participants in each group included Ex=7, diet=5 (one female in the diet group dropped out midway), thus, 12 participants completed the study]. At Weeks -4, 0 and 4, visual analogue scales (VAS) were used to assess hunger and satiety and liking and wanting (L&W) for nutrient and taste preferences using a computer-based system (E-Prime v1.1.4). Ad libitum test meal EI was consistently lower after the HE pre-load compared to the LE pre-load. However, this was not consistent during the diet intervention however. A pre-load x group interaction on ad libitum test meal EI revealed that during the intervention phase the Ex group showed an improved sensitivity to detect the energy content between the two pre-loads and improved compensation for the ad libitum test meal whereas the diet group’s ability to differentiate between the two pre-loads decreased and showed poorer compensation (F[1,10]=2.88, p-value not significant). This study supports previous findings of the effect Ex and diet interventions have on appetite and compensatory responses; Ex increases and diet decreases energy balance sensitivity.

The social orders of family mealtime

This study examined the everyday practices of families within the context of family mealtime to investigate how members accomplished mealtime interactions. Using an ethnomethodological approach, conversation analysis and membership categorization analysis, the study investigated the interactional resources that family members used to assemble their social orders moment by moment during family mealtimes. While there is interest in mealtimes within educational policy, health research and the media, there remain few studies that provide fine-grained detail about how members produce the social activity of having a family meal. Findings from this study contribute empirical understandings about families and family mealtime. Two families with children aged 2 to 10 years were observed as they accomplished their everyday mealtime activities. Data collection took place in the family homes where family members video recorded their naturally occurring mealtimes. Each family was provided with a video camera for a one-month period and they decided which mealtimes they recorded, a method that afforded participants greater agency in the data collection process and made available to the analyst a window into the unfolding of the everyday lives of the families. A total of 14 mealtimes across the two families were recorded, capturing 347 minutes of mealtime interactions. Selected episodes from the data corpus, which includes centralised breakfast and dinnertime episodes, were transcribed using the Jeffersonian system. Three data chapters examine extended sequences of family talk at mealtimes, to show the interactional resources used by members during mealtime interactions. The first data chapter explores multiparty talk to show how the uniqueness of the occasion of having a meal influences turn design. It investigates the ways in which members accomplish two-party talk within a multiparty setting, showing how one child "tells" a funny story to accomplish the drawing together of his brothers as an audience. As well, this chapter identifies the interactional resources used by the mother to cohort her children to accomplish the choralling of grace. The second data chapter draws on sequential and categorical analysis to show how members are mapped to a locally produced membership category. The chapter shows how the mapping of members into particular categories is consequential for social order; for example, aligning members who belong to the membership category "had haircuts" and keeping out those who "did not have haircuts". Additional interactional resources such as echoing, used here to refer to the use of exactly the same words, similar prosody and physical action, and increasing physical closeness, are identified as important to the unfolding talk particularly as a way of accomplishing alignment between the grandmother and grand-daughter. The third and final data analysis chapter examines topical talk during family mealtimes. It explicates how members introduce topics of talk with an orientation to their co-participant and the way in which the take up of a topic is influenced both by the sequential environment in which it is introduced and the sensitivity of the topic. Together, these three data chapters show aspects of how family members participated in family mealtimes. The study contributes four substantive themes that emerged during the analytic process and, as such, the themes reflect what the members were observed to be doing. The first theme identified how family knowledge was relevant and consequential for initiating and sustaining interaction during mealtime with, for example, members buying into the talk of other members or being requested to help out with knowledge about a shared experience. Knowledge about members and their activities was evident with the design of questions evidencing an orientation to coparticipant’s knowledge. The second theme found how members used topic as a resource for social interaction. The third theme concerned the way in which members utilised membership categories for producing and making sense of social action. The fourth theme, evident across all episodes selected for analysis, showed how children’s competence is an ongoing interactional accomplishment as they manipulated interactional resources to manage their participation in family mealtime. The way in which children initiated interactions challenges previous understandings about children’s restricted rights as conversationalists. As well as making a theoretical contribution, the study offers methodological insight by working with families as research participants. The study shows the procedures involved as the study moved from one where the researcher undertook the decisions about what to videorecord to offering this decision making to the families, who chose when and what to videorecord of their mealtime practices. Evident also are the ways in which participants orient both to the video-camera and to the absent researcher. For the duration of the mealtime the video-camera was positioned by the adults as out of bounds to the children; however, it was offered as a "treat" to view after the mealtime was recorded. While situated within family mealtimes and reporting on the experiences of two families, this study illuminates how mealtimes are not just about food and eating; they are social. The study showed the constant and complex work of establishing and maintaining social orders and the rich array of interactional resources that members draw on during family mealtimes. The family’s interactions involved members contributing to building the social orders of family mealtime. With mealtimes occurring in institutional settings involving young children, such as long day care centres and kindergartens, the findings of this study may help educators working with young children to see the rich interactional opportunities mealtimes afford children, the interactional competence that children demonstrate during mealtimes, and the important role/s that adults may assume as co-participants in interactions with children within institutional settings.

Rational design of serine protease inhibitors

Proteases regulate a spectrum of diverse physiological processes, and dysregulation of proteolytic activity drives a plethora of pathological conditions. Understanding protease function is essential to appreciating many aspects of normal physiology and progression of disease. Consequently, development of potent and specific inhibitors of proteolytic enzymes is vital to provide tools for the dissection of protease function in biological systems and for the treatment of diseases linked to aberrant proteolytic activity. The studies in this thesis describe the rational design of potent inhibitors of three proteases that are implicated in disease development. Additionally, key features of the interaction of proteases and their cognate inhibitors or substrates are analysed and a series of rational inhibitor design principles are expounded and tested. Rational design of protease inhibitors relies on a comprehensive understanding of protease structure and biochemistry. Analysis of known protease cleavage sites in proteins and peptides is a commonly used source of such information. However, model peptide substrate and protein sequences have widely differing levels of backbone constraint and hence can adopt highly divergent structures when binding to a protease’s active site. This may result in identical sequences in peptides and proteins having different conformations and diverse spatial distribution of amino acid functionalities. Regardless of this, protein and peptide cleavage sites are often regarded as being equivalent. One of the key findings in the following studies is a definitive demonstration of the lack of equivalence between these two classes of substrate and invalidation of the common practice of using the sequences of model peptide substrates to predict cleavage of proteins in vivo. Another important feature for protease substrate recognition is subsite cooperativity. This type of cooperativity is commonly referred to as protease or substrate binding subsite cooperativity and is distinct from allosteric cooperativity, where binding of a molecule distant from the protease active site affects the binding affinity of a substrate. Subsite cooperativity may be intramolecular where neighbouring residues in substrates are interacting, affecting the scissile bond’s susceptibility to protease cleavage. Subsite cooperativity can also be intermolecular where a particular residue’s contribution to binding affinity changes depending on the identity of neighbouring amino acids. Although numerous studies have identified subsite cooperativity effects, these findings are frequently ignored in investigations probing subsite selectivity by screening against diverse combinatorial libraries of peptides (positional scanning synthetic combinatorial library; PS-SCL). This strategy for determining cleavage specificity relies on the averaged rates of hydrolysis for an uncharacterised ensemble of peptide sequences, as opposed to the defined rate of hydrolysis of a known specific substrate. Further, since PS-SCL screens probe the preference of the various protease subsites independently, this method is inherently unable to detect subsite cooperativity. However, mean hydrolysis rates from PS-SCL screens are often interpreted as being comparable to those produced by single peptide cleavages. Before this study no large systematic evaluation had been made to determine the level of correlation between protease selectivity as predicted by screening against a library of combinatorial peptides and cleavage of individual peptides. This subject is specifically explored in the studies described here. In order to establish whether PS-SCL screens could accurately determine the substrate preferences of proteases, a systematic comparison of data from PS-SCLs with libraries containing individually synthesised peptides (sparse matrix library; SML) was carried out. These SML libraries were designed to include all possible sequence combinations of the residues that were suggested to be preferred by a protease using the PS-SCL method. SML screening against the three serine proteases kallikrein 4 (KLK4), kallikrein 14 (KLK14) and plasmin revealed highly preferred peptide substrates that could not have been deduced by PS-SCL screening alone. Comparing protease subsite preference profiles from screens of the two types of peptide libraries showed that the most preferred substrates were not detected by PS SCL screening as a consequence of intermolecular cooperativity being negated by the very nature of PS SCL screening. Sequences that are highly favoured as result of intermolecular cooperativity achieve optimal protease subsite occupancy, and thereby interact with very specific determinants of the protease. Identifying these substrate sequences is important since they may be used to produce potent and selective inhibitors of protolytic enzymes. This study found that highly favoured substrate sequences that relied on intermolecular cooperativity allowed for the production of potent inhibitors of KLK4, KLK14 and plasmin. Peptide aldehydes based on preferred plasmin sequences produced high affinity transition state analogue inhibitors for this protease. The most potent of these maintained specificity over plasma kallikrein (known to have a very similar substrate preference to plasmin). Furthermore, the efficiency of this inhibitor in blocking fibrinolysis in vitro was comparable to aprotinin, which previously saw clinical use to reduce perioperative bleeding. One substrate sequence particularly favoured by KLK4 was substituted into the 14 amino acid, circular sunflower trypsin inhibitor (SFTI). This resulted in a highly potent and selective inhibitor (SFTI-FCQR) which attenuated protease activated receptor signalling by KLK4 in vitro. Moreover, SFTI-FCQR and paclitaxel synergistically reduced growth of ovarian cancer cells in vitro, making this inhibitor a lead compound for further therapeutic development. Similar incorporation of a preferred KLK14 amino acid sequence into the SFTI scaffold produced a potent inhibitor for this protease. However, the conformationally constrained SFTI backbone enforced a different intramolecular cooperativity, which masked a KLK14 specific determinant. As a consequence, the level of selectivity achievable was lower than that found for the KLK4 inhibitor. Standard mechanism inhibitors such as SFTI rely on a stable acyl-enzyme intermediate for high affinity binding. This is achieved by a conformationally constrained canonical binding loop that allows for reformation of the scissile peptide bond after cleavage. Amino acid substitutions within the inhibitor to target a particular protease may compromise structural determinants that support the rigidity of the binding loop and thereby prevent the engineered inhibitor reaching its full potential. An in silico analysis was carried out to examine the potential for further improvements to the potency and selectivity of the SFTI-based KLK4 and KLK14 inhibitors. Molecular dynamics simulations suggested that the substitutions within SFTI required to target KLK4 and KLK14 had compromised the intramolecular hydrogen bond network of the inhibitor and caused a concomitant loss of binding loop stability. Furthermore in silico amino acid substitution revealed a consistent correlation between a higher frequency of formation and the number of internal hydrogen bonds of SFTI-variants and lower inhibition constants. These predictions allowed for the production of second generation inhibitors with enhanced binding affinity toward both targets and highlight the importance of considering intramolecular cooperativity effects when engineering proteins or circular peptides to target proteases. The findings from this study show that although PS-SCLs are a useful tool for high throughput screening of approximate protease preference, later refinement by SML screening is needed to reveal optimal subsite occupancy due to cooperativity in substrate recognition. This investigation has also demonstrated the importance of maintaining structural determinants of backbone constraint and conformation when engineering standard mechanism inhibitors for new targets. Combined these results show that backbone conformation and amino acid cooperativity have more prominent roles than previously appreciated in determining substrate/inhibitor specificity and binding affinity. The three key inhibitors designed during this investigation are now being developed as lead compounds for cancer chemotherapy, control of fibrinolysis and cosmeceutical applications. These compounds form the basis of a portfolio of intellectual property which will be further developed in the coming years.