title: Work integrated learning

authors: Ferns, Sonia

abstract: In the coming decades, environmental, cultural, economic and social changes will have a profound global impact (Hajkowicz, Cook & Littleboy, 2012). The higher education sector is under pressure to transform the way it operates in response to these forces (Ernst & Young, 2012). The emerging knowledge economy, progressing technological capabilities, increasing global mobility, and growing demands for economic productivity, require a proficient, innovative and competitive work force. Education is perceived as a key mechanism for preparing the population to meet the global demands of the 21st century. Work integrated learning (WIL) is internationally recognized and nationally endorsed as a strategy for ensuring students are exposed to authentic learning experiences with the opportunity to apply theoretical concepts to practice-based tasks, ultimately enhancing graduate employability (Knight & Yorke, 2004; Peach & Matthews, 2011).

text: Work integrated learning In the coming decades, environmental, cultural, economic and social changes will have a profound global impact (Hajkowicz, Cook & Littleboy, 2012). The higher education sector is under pressure to transform the way it operates in response to these forces (Ernst & Young, 2012). The emerging knowledge economy, progressing technological capabilities, increasing global mobility, and growing demands for economic productivity, require a proficient, innovative and competitive work force. Education is perceived as a key mechanism for preparing the population to meet the global demands of the 21st century. Work integrated learning (WIL) is internationally recognized and nationally endorsed as a strategy for ensuring students are exposed to authentic learning experiences with the opportunity to apply theoretical concepts to practice-based tasks, ultimately enhancing graduate employability (Knight & Yorke, 2004; Peach & Matthews, 2011).

year: 2014

title: The effects of honey on the inflammatory response of cells with respect to wound healing

authors: Harcourt, Nichola Robyn

abstract: The primary aim of this thesis was to test the effect of two types of honey, a manuka honey and an Otago pasture honey, on a range of key cell types involved in the inflammatory response of wound healing. A range of in vitro assays were used to test the effect of honey on various cells with a view to the implications of the results for wound healing, focusing on the mechanisms by which honey has been observed to have beneficial effects on both scar formation and inflammation. 
To investigate whether honey could stimulate bovine T cells to proliferate in vitro, both MTT and BrDU assays of proliferation and flow cytometry analysis were used. It was found that low concentrations of honey stimulated resting T cells to proliferate and express the IL-2 receptor in a dose-dependent manner with progressive dilution. This suggests that honey contains lymphomitogenic factors. Manuka honey was stimulatory at higher dilutions than pasture honey. Low concentrations of honey induced cell division profiles similar to those obtained with Con A-stimulated cells. The stimulatory activity of honey was found to be in a high molecular weight fraction. Sugars alone had no effect on T cell proliferation, as demonstrated by use of artificial honey (a syrup of sugars as in honey). 
The ability for honey to induce messenger RNA expression for key cytokines involved in wound healing was investigated. Conventional reverse transcriptase-PCR was used to detect the production of mRNA for honey at 0.25% concentrations for various times (0-24 h). The more sensitive molecular technique, quantitative real-time RT-PCR was then used to quantify the abundance of cytokine mRNA transcripts expressed in bovine blood exposed to 0.25% manuka honey as compared with Con A or control cultures. Transcriptional activity of ten genes, IL-1, IL-5, IL-12, IL-18, IFN-𝛾, HSP70, HSP90, iNOS, TNF-α, and TGF-β were studied at the mRNA level during a 0-24 h exposure of whole blood to honey. To test for any modulatory effects of honey on gene expression in an inflammatory model, whole blood was exposed to honey at the same time as LPS and the mRNA expression for the genes was measured. The results show that honey upregulates a wide range of mediators, including TNF-α, IL-1β, and TGF-β, and this supports the hypothesis that honey induces cytokine release. Honey gave a transient and moderate induction of cytokine mRNA compared with a massive and prolonged induction by the mitogens, Con A and LPS. The inclusion of honey with LPS led to a reduced expression of mRNA for key inflammatory mediators, including iNOS and TGF-β, compared with LPS alone. This supports the hypothesis that honey modulates inflammation. 
To investigate whether honey could induce THP-1 monocytes to release TNF-α, bioassays using WEHI cells were carried out to measure TNF-α production after the monocytes were exposed to honey. Honey was found to stimulate release of TNF-α by the monocytes when at a range of concentrations between 0.000025-0.1%, with no differences between the levels produced at the various concentrations of honey. At concentrations of honey from 0.25-1% the TNF-α production decreased as the concentration of honey increased. This may indicate that an anti-inflammatory action overrides the stimulatory effect at concentrations of honey greater than 0.25%. Sugar content had no effect upon TNF-α release, as demonstrated by the artificial honey control. There were no differences between honey types (manuka honey and pasture honey) in induction of TNF-α release. Time-course analysis confirmed that a 4-6 h incubation period of cells with 0.25% honey gave maximal TNF-α production. A 2 h minimum exposure period of cells to honey was critical for TNF-α production. Incubation of LPS-stimulated monocytes with honey had no effect on their subsequent TNF-α production. A good correlation was found between the TNF measurements detected by ELISA and the WEHI Bioassay. 
To test whether honey could modulate LPS-stimulated NO production by THP-1 monocytes and bovine peripheral blood mononuclear cells, Greiss assays were performed. Both manuka honey and pasture honey at 0.5% and 1% concentrations suppressed LPS-induced nitrite release in a dose-wise manner, indicating modulation of nitric oxide production. Manuka honey had a more potent modulating effect on LPS driven nitrite production than pasture honey, and maintained activity at 0.25%. Sugars alone had no effect. High molecular weight dialysis fractions of either honey contained the activity, but some of the activity was lost by fractionation. An ether extract of manuka honey led to the greatest modulation of nitrite production by LPS-stimulated monocytes.  
To investigate whether honey has an effect on phagocytosis, whole blood was incubated with honey and the ability of neutrophils to take up fluorescent-labelled bacteria was measured using the Phagotest® assay. The artificial honey control provided clear evidence that low concentrations of honey (optimal at 0.25%) induce phagocytosis by neutrophils due to the supply of sugars. Manuka honey had an additional opsonizing effect on bacteria, which enhances the phagocytic response beyond that seen with sugars alone. 
The effects of honey on tight junction (TJ) resistance were assessed for MDCK cell monolayers subjected to an EGTA challenge. It was found that manuka honey and pasture honey have protective effects on TJ following the challenge, and enhance postchallenge recovery of transepithelial resistance. Manuka honey had greater modulatory activity on TJ with increased concentration from 0.1-1%, and 1% concentrations of both honeys gave the greatest protective effects. Manuka honey appeared to have greater protective effects than pasture honey. Application of manuka honey (at 1% concentrations) to both the apical and basolateral sides of the MDCK cell monolayer significantly enhanced TJ tightness beyond the control. Dialysis of the honey confirmed that the high molecular weight fraction contained the active component. Diffusate fractions from either honey type had no effect on TJ. Artificial honey had no effect. 
The effect of various honey concentrations on the proliferation of the 3T3-Ll fibroblast cell-line in vitro was investigated using MTT proliferation assays. Both manuka honey and pasture honey (0.25%) increased fibroblast proliferation. Artificial honey had no effect on fibroblast proliferation, indicating sugars have no role in mitogenic activity. This suggests that honey contains factors which directly promote cell division in fibroblasts. 
An in vitro model was used to test whether honey-induced factors produced by peripheral blood mononuclear cells (PBMC) could activate fibroblast proliferation. These assays were performed to examine whether honey could have an indirect stimulatory effect on fibroblasts. Incubating fibroblasts with supernatants derived from honey-stimulated PBMCs (at 0.025% concentrations of honey) led to significant fibroblast proliferation. Low concentrations of honey (less than 0.1%) do not directly stimulate fibroblast proliferation, therefore factors produced by honey-stimulated PBMCs must promote fibroblast proliferation. A high molecular weight fraction of manuka honey obtained from dialysis contained the active component. The diffusate obtained by dialysis (containing sugars) had no activity. 
To investigate whether honey could modulate the response of fibroblasts to an inflammatory agent, fibroblasts were exposed to honey for various times prior to and at the same time as IL-1β. Honey did not augment fibroblast proliferation when added at the same time as IL-1β. Prior incubation of fibroblasts with honey (0.25-1%) for 2 h before IL-1β-stimulation decreased the cell response to IL-1β, and this anti-inflammatory active component was of a high molecular weight.
It is proposed, on the basis of this in vitro study, that honey provides a neatly controlled therapy for optimising tissue repair, with potential for use in inflammatory disorders. It is the central thesis of this study that the stimulatory agent in honey induces cytokine production necessary for healing to occur, but that the oxidant species produced by these cells are effectively regulated by a second agent, thereby creating a feedback-regulated delivery system. The results presented in the current study suggest that honey can both stimulate and modulate cell activity, and show that honey interferes with a large number of regulatory steps in the inflammatory pathway, e.g. cell division, transcription, tight junction integrity, production of oxidant species. While the stimulatory activity was observed at lower concentrations, the modulatory effects required higher concentrations of honey. The dual ability for honey to stimulate moderate cellular activation in the absence of an immune stimulus, yet not augment mitogenic stimulation, and in some cases to modulate cell response to a mitogen, indicates that it will promote healing without setting up harmful inflammation. 
If further experiments confirm this to be the case, honey will have potential for therapeutic application. The work also identifies some new areas of research, which if completed would further enhance the understanding of the role honey plays in tissue healing.

text: The effects of honey on the inflammatory response of cells with respect to wound healing The primary aim of this thesis was to test the effect of two types of honey, a manuka honey and an Otago pasture honey, on a range of key cell types involved in the inflammatory response of wound healing. A range of in vitro assays were used to test the effect of honey on various cells with a view to the implications of the results for wound healing, focusing on the mechanisms by which honey has been observed to have beneficial effects on both scar formation and inflammation. 
To investigate whether honey could stimulate bovine T cells to proliferate in vitro, both MTT and BrDU assays of proliferation and flow cytometry analysis were used. It was found that low concentrations of honey stimulated resting T cells to proliferate and express the IL-2 receptor in a dose-dependent manner with progressive dilution. This suggests that honey contains lymphomitogenic factors. Manuka honey was stimulatory at higher dilutions than pasture honey. Low concentrations of honey induced cell division profiles similar to those obtained with Con A-stimulated cells. The stimulatory activity of honey was found to be in a high molecular weight fraction. Sugars alone had no effect on T cell proliferation, as demonstrated by use of artificial honey (a syrup of sugars as in honey). 
The ability for honey to induce messenger RNA expression for key cytokines involved in wound healing was investigated. Conventional reverse transcriptase-PCR was used to detect the production of mRNA for honey at 0.25% concentrations for various times (0-24 h). The more sensitive molecular technique, quantitative real-time RT-PCR was then used to quantify the abundance of cytokine mRNA transcripts expressed in bovine blood exposed to 0.25% manuka honey as compared with Con A or control cultures. Transcriptional activity of ten genes, IL-1, IL-5, IL-12, IL-18, IFN-𝛾, HSP70, HSP90, iNOS, TNF-α, and TGF-β were studied at the mRNA level during a 0-24 h exposure of whole blood to honey. To test for any modulatory effects of honey on gene expression in an inflammatory model, whole blood was exposed to honey at the same time as LPS and the mRNA expression for the genes was measured. The results show that honey upregulates a wide range of mediators, including TNF-α, IL-1β, and TGF-β, and this supports the hypothesis that honey induces cytokine release. Honey gave a transient and moderate induction of cytokine mRNA compared with a massive and prolonged induction by the mitogens, Con A and LPS. The inclusion of honey with LPS led to a reduced expression of mRNA for key inflammatory mediators, including iNOS and TGF-β, compared with LPS alone. This supports the hypothesis that honey modulates inflammation. 
To investigate whether honey could induce THP-1 monocytes to release TNF-α, bioassays using WEHI cells were carried out to measure TNF-α production after the monocytes were exposed to honey. Honey was found to stimulate release of TNF-α by the monocytes when at a range of concentrations between 0.000025-0.1%, with no differences between the levels produced at the various concentrations of honey. At concentrations of honey from 0.25-1% the TNF-α production decreased as the concentration of honey increased. This may indicate that an anti-inflammatory action overrides the stimulatory effect at concentrations of honey greater than 0.25%. Sugar content had no effect upon TNF-α release, as demonstrated by the artificial honey control. There were no differences between honey types (manuka honey and pasture honey) in induction of TNF-α release. Time-course analysis confirmed that a 4-6 h incubation period of cells with 0.25% honey gave maximal TNF-α production. A 2 h minimum exposure period of cells to honey was critical for TNF-α production. Incubation of LPS-stimulated monocytes with honey had no effect on their subsequent TNF-α production. A good correlation was found between the TNF measurements detected by ELISA and the WEHI Bioassay. 
To test whether honey could modulate LPS-stimulated NO production by THP-1 monocytes and bovine peripheral blood mononuclear cells, Greiss assays were performed. Both manuka honey and pasture honey at 0.5% and 1% concentrations suppressed LPS-induced nitrite release in a dose-wise manner, indicating modulation of nitric oxide production. Manuka honey had a more potent modulating effect on LPS driven nitrite production than pasture honey, and maintained activity at 0.25%. Sugars alone had no effect. High molecular weight dialysis fractions of either honey contained the activity, but some of the activity was lost by fractionation. An ether extract of manuka honey led to the greatest modulation of nitrite production by LPS-stimulated monocytes.  
To investigate whether honey has an effect on phagocytosis, whole blood was incubated with honey and the ability of neutrophils to take up fluorescent-labelled bacteria was measured using the Phagotest® assay. The artificial honey control provided clear evidence that low concentrations of honey (optimal at 0.25%) induce phagocytosis by neutrophils due to the supply of sugars. Manuka honey had an additional opsonizing effect on bacteria, which enhances the phagocytic response beyond that seen with sugars alone. 
The effects of honey on tight junction (TJ) resistance were assessed for MDCK cell monolayers subjected to an EGTA challenge. It was found that manuka honey and pasture honey have protective effects on TJ following the challenge, and enhance postchallenge recovery of transepithelial resistance. Manuka honey had greater modulatory activity on TJ with increased concentration from 0.1-1%, and 1% concentrations of both honeys gave the greatest protective effects. Manuka honey appeared to have greater protective effects than pasture honey. Application of manuka honey (at 1% concentrations) to both the apical and basolateral sides of the MDCK cell monolayer significantly enhanced TJ tightness beyond the control. Dialysis of the honey confirmed that the high molecular weight fraction contained the active component. Diffusate fractions from either honey type had no effect on TJ. Artificial honey had no effect. 
The effect of various honey concentrations on the proliferation of the 3T3-Ll fibroblast cell-line in vitro was investigated using MTT proliferation assays. Both manuka honey and pasture honey (0.25%) increased fibroblast proliferation. Artificial honey had no effect on fibroblast proliferation, indicating sugars have no role in mitogenic activity. This suggests that honey contains factors which directly promote cell division in fibroblasts. 
An in vitro model was used to test whether honey-induced factors produced by peripheral blood mononuclear cells (PBMC) could activate fibroblast proliferation. These assays were performed to examine whether honey could have an indirect stimulatory effect on fibroblasts. Incubating fibroblasts with supernatants derived from honey-stimulated PBMCs (at 0.025% concentrations of honey) led to significant fibroblast proliferation. Low concentrations of honey (less than 0.1%) do not directly stimulate fibroblast proliferation, therefore factors produced by honey-stimulated PBMCs must promote fibroblast proliferation. A high molecular weight fraction of manuka honey obtained from dialysis contained the active component. The diffusate obtained by dialysis (containing sugars) had no activity. 
To investigate whether honey could modulate the response of fibroblasts to an inflammatory agent, fibroblasts were exposed to honey for various times prior to and at the same time as IL-1β. Honey did not augment fibroblast proliferation when added at the same time as IL-1β. Prior incubation of fibroblasts with honey (0.25-1%) for 2 h before IL-1β-stimulation decreased the cell response to IL-1β, and this anti-inflammatory active component was of a high molecular weight.
It is proposed, on the basis of this in vitro study, that honey provides a neatly controlled therapy for optimising tissue repair, with potential for use in inflammatory disorders. It is the central thesis of this study that the stimulatory agent in honey induces cytokine production necessary for healing to occur, but that the oxidant species produced by these cells are effectively regulated by a second agent, thereby creating a feedback-regulated delivery system. The results presented in the current study suggest that honey can both stimulate and modulate cell activity, and show that honey interferes with a large number of regulatory steps in the inflammatory pathway, e.g. cell division, transcription, tight junction integrity, production of oxidant species. While the stimulatory activity was observed at lower concentrations, the modulatory effects required higher concentrations of honey. The dual ability for honey to stimulate moderate cellular activation in the absence of an immune stimulus, yet not augment mitogenic stimulation, and in some cases to modulate cell response to a mitogen, indicates that it will promote healing without setting up harmful inflammation. 
If further experiments confirm this to be the case, honey will have potential for therapeutic application. The work also identifies some new areas of research, which if completed would further enhance the understanding of the role honey plays in tissue healing.

year: 2005

title: The recruitment dynamics of post-settlement juvenile toheroa and the potential applications for aquaculture

authors: van der Lee, Marieka

subjects: Toheroa

abstract: Managing the recovery of threatened species due to anthropogenic disturbance is one of the most challenging problems in ecological monitoring. Researching species ecology is vital to help us improve ecosystem health and subsequently develop better management strategies in the environment. Humanity is entirely reliant on ecosystem services for food security. With the world population expected to reach ten billion by 2050, there is a growing need for more sustainable food sources. With the exploitation of many environmental resources worldwide, aquaculture has been championed as a sustainable option for future seafood production. In New Zealand, the toheroa (Paphies ventricosa) has been identified as a potentially valuable asset to the aquaculture industry and for possible conservation strategies via the implementation of reseeding populations with hatchery-reared spat.

Toheroa faced inadequate resource management and exploitation in the 1900s which ultimately led to the closure of an important recreational, cultural and commercial fishery. However, toheroa populations have failed to recover despite over 40 years of protection. It has been proposed that the continued decline of toheroa populations is not due to a lack of juvenile recruitment. Very little is known about juvenile toheroa recruitment, and there is no available information that could facilitate restoration management or determine the appropriate action to take for monitoring juvenile populations. From both a conservation and an aquaculture perspective, understanding the underlying ecology will be pivotal for future endeavours.

My thesis aimed to investigate the distribution dynamics of post-settlement juvenile toheroa on Ripiro Beach and the potential applications for aquaculture. Population surveys were conducted on Ripiro Beach in February and May to establish a sampling methodology that accurately captures the juvenile population both spatially and temporally. Surveys on vertical substrate stratification significantly affected both density and shell size with increasing sediment depth. Additionally, in contrast to the hypothesis formed on the basis of traditional knowledge that juvenile distribution is limited to the dunes and upper intertidal, spat appeared to have far more varied littoral distributions than previously theorised. The data from these surveys were used to make estimates of the total juvenile population on Ripiro Beach with attention to changes spatially (habitat type), temporally (seasonally), and by size cohorts (mm). My results indicated that toheroa populations are not recruit-limited. Estimates for the total February population ranged between 580 (±190) million and 650 (±200) million. By May, large abundance losses (> 90%) dropped the estimated population to between 30 (±15) million and 37 (±17) million, indicating significant bottleneck mortality during the early growth stages. However, the high abundances of small spat (< 5 mm) indicate that toheroa aquaculture is potentially viable. In aid of future aquaculture applications, I investigated how handling, harvesting, and different transportation methods could impact toheroa health, survival, or performance. This experiment aimed to mimic real-world applications wherein juvenile spat would be harvested and ongrown in hatcheries. To do this, I utilised the burrowing behaviour of juvenile toheroa as a quantitative indicator of stress. I found that if appropriate storage methods were implemented, initial handling and harvesting had a greater impact than actual transportation.

The research in this thesis demonstrated that populations do not appear to be recruit-limited. Furthermore, the findings indicate that aquaculture built on the foundation of wild-harvested juveniles is potentially viable. However, further research is required in order to quantify potential annual recruitment variation both spatially and temporally. Additionally, in order to establish a conservation approach, we must first define the niche requirements of toheroa to determine the obstacle (or obstacles) inhibiting recovery.

text: The recruitment dynamics of post-settlement juvenile toheroa and the potential applications for aquaculture Managing the recovery of threatened species due to anthropogenic disturbance is one of the most challenging problems in ecological monitoring. Researching species ecology is vital to help us improve ecosystem health and subsequently develop better management strategies in the environment. Humanity is entirely reliant on ecosystem services for food security. With the world population expected to reach ten billion by 2050, there is a growing need for more sustainable food sources. With the exploitation of many environmental resources worldwide, aquaculture has been championed as a sustainable option for future seafood production. In New Zealand, the toheroa (Paphies ventricosa) has been identified as a potentially valuable asset to the aquaculture industry and for possible conservation strategies via the implementation of reseeding populations with hatchery-reared spat.

Toheroa faced inadequate resource management and exploitation in the 1900s which ultimately led to the closure of an important recreational, cultural and commercial fishery. However, toheroa populations have failed to recover despite over 40 years of protection. It has been proposed that the continued decline of toheroa populations is not due to a lack of juvenile recruitment. Very little is known about juvenile toheroa recruitment, and there is no available information that could facilitate restoration management or determine the appropriate action to take for monitoring juvenile populations. From both a conservation and an aquaculture perspective, understanding the underlying ecology will be pivotal for future endeavours.

My thesis aimed to investigate the distribution dynamics of post-settlement juvenile toheroa on Ripiro Beach and the potential applications for aquaculture. Population surveys were conducted on Ripiro Beach in February and May to establish a sampling methodology that accurately captures the juvenile population both spatially and temporally. Surveys on vertical substrate stratification significantly affected both density and shell size with increasing sediment depth. Additionally, in contrast to the hypothesis formed on the basis of traditional knowledge that juvenile distribution is limited to the dunes and upper intertidal, spat appeared to have far more varied littoral distributions than previously theorised. The data from these surveys were used to make estimates of the total juvenile population on Ripiro Beach with attention to changes spatially (habitat type), temporally (seasonally), and by size cohorts (mm). My results indicated that toheroa populations are not recruit-limited. Estimates for the total February population ranged between 580 (±190) million and 650 (±200) million. By May, large abundance losses (> 90%) dropped the estimated population to between 30 (±15) million and 37 (±17) million, indicating significant bottleneck mortality during the early growth stages. However, the high abundances of small spat (< 5 mm) indicate that toheroa aquaculture is potentially viable. In aid of future aquaculture applications, I investigated how handling, harvesting, and different transportation methods could impact toheroa health, survival, or performance. This experiment aimed to mimic real-world applications wherein juvenile spat would be harvested and ongrown in hatcheries. To do this, I utilised the burrowing behaviour of juvenile toheroa as a quantitative indicator of stress. I found that if appropriate storage methods were implemented, initial handling and harvesting had a greater impact than actual transportation.

The research in this thesis demonstrated that populations do not appear to be recruit-limited. Furthermore, the findings indicate that aquaculture built on the foundation of wild-harvested juveniles is potentially viable. However, further research is required in order to quantify potential annual recruitment variation both spatially and temporally. Additionally, in order to establish a conservation approach, we must first define the niche requirements of toheroa to determine the obstacle (or obstacles) inhibiting recovery.

year: 2022