title: In vitro cell culture to study microglial inflammation

authors: Newland, Julia

subjects: CNs

abstract: Inflammation of the central nervous system (CNS) characterised by release of cytokines such as tumour necrosis factor-alpha (TNF-a) and interleukin-1 (IL-1) is an event that is often detrimental to neuronal cells and is thought to contribute to the neuronal loss observed during neurodegeneration.
One objective of this study was to investigate the potential anti-inflammatory properties of kawakawa leaf extracts using in vitro model cell systems. Kawakawa leaf extracts anti-inflammatory properties have anecdotally been documented in traditional Maori medicinal practices (Rangoa). However, before this aspect could be carried out, experiments were undertaken to investigate the effects the kawakawa leaf extracts had on the growth rates of two mammalian cell lines (HeLa and THP-1 cells).
Interestingly, the growth assays showed that the extracts to have both a stimulatory and inhibitory effect on cell growth. Toxicity of cells can be categorised as either necrotic or apoptotic cell death. In this study, HeLa cells and THP-1 cells were incubated under standard growth conditions with various concentrations (10 to 100ug/ml) of Kawakawa leave extracts prepared at room temperature or 60OC.
Growth data as seen in figures 2 and 3 typically have a long lag phase before they start to reach exponential growth between days 4 and 5. The extract having the largest stimulatory effect on HeLa cells as shown by figure 6 was 25 ug/ml at 60OC and the largest inhibitory effect seen on HeLa cells was seen at 50 ug/ml RT shown by figure 6. The extract which has the largest stimulatory effect on THP-1 cells is 25 ug/ml 60OC as shown by figure 11 whereas the extract producing the largest inhibitory effect is 25 ug/ml RT.

Apoptotic and necrotic cell death mechanisms were examined by using in vitro biochemistry techniques. Assessments were largely made based on biochemical changes of HeLa cells and THP-1 cells post treatment of Kawakawa leave extracts.
Measurement of lactate dehydrogenase (LDH assays) in the growth media of cells that were shown to be growth inhibited during exposure to Kawakawa extracts indicated that the growth inhibition resulting from exposure to the extracts was not due to necrotic cell death processes. (ie that cells maintained their membrane integrity) When compared to the control, a lower amount of cytosolic LDH was seen in THP-1 cell lines. However, HeLa cell lines produced a high level of cytosolic LDH which may be due to mycoplasma contamination. On average, Kawakawa concentrations at 12.5ug/ml and 25ug/ml yielded less lysed cells percentage across both cell lines.
The demonstrated cell growth inhibition was unlikely to be due to necrosis and because of this it was decided to see if cell death was caused by mitochondrial processes that would further support apoptotic cell death. The biochemical assay MTT indirectly measures mitochondrial dehydrogenase activity in the presence of kawakawa leave extracts.
The MTT data illustrated mitochondrial impairment where HeLa cells exposed to Kawakawa 60OC concentrations 6.25 ug/ml and 12.5 ug/ml had the lowest average mitochondrial activity. THP-1 cells exposed to Kawakawa 60OC concentrations 12.5 ug/ml and 50 ug/ml had the lowest average mitochondrial activity.
The main results of this study include, the treatment of HeLa and THP-1 cells with kawakawa extracts demonstrated that cytotoxicity was not due to necrosis. The most interesting finding of this study is when kawakawa extracts inhibit cell growth there is no increase in LDH. This is suggestive that the cellular membrane remains intact when exposed to the extract.

text: In vitro cell culture to study microglial inflammation Inflammation of the central nervous system (CNS) characterised by release of cytokines such as tumour necrosis factor-alpha (TNF-a) and interleukin-1 (IL-1) is an event that is often detrimental to neuronal cells and is thought to contribute to the neuronal loss observed during neurodegeneration.
One objective of this study was to investigate the potential anti-inflammatory properties of kawakawa leaf extracts using in vitro model cell systems. Kawakawa leaf extracts anti-inflammatory properties have anecdotally been documented in traditional Maori medicinal practices (Rangoa). However, before this aspect could be carried out, experiments were undertaken to investigate the effects the kawakawa leaf extracts had on the growth rates of two mammalian cell lines (HeLa and THP-1 cells).
Interestingly, the growth assays showed that the extracts to have both a stimulatory and inhibitory effect on cell growth. Toxicity of cells can be categorised as either necrotic or apoptotic cell death. In this study, HeLa cells and THP-1 cells were incubated under standard growth conditions with various concentrations (10 to 100ug/ml) of Kawakawa leave extracts prepared at room temperature or 60OC.
Growth data as seen in figures 2 and 3 typically have a long lag phase before they start to reach exponential growth between days 4 and 5. The extract having the largest stimulatory effect on HeLa cells as shown by figure 6 was 25 ug/ml at 60OC and the largest inhibitory effect seen on HeLa cells was seen at 50 ug/ml RT shown by figure 6. The extract which has the largest stimulatory effect on THP-1 cells is 25 ug/ml 60OC as shown by figure 11 whereas the extract producing the largest inhibitory effect is 25 ug/ml RT.

Apoptotic and necrotic cell death mechanisms were examined by using in vitro biochemistry techniques. Assessments were largely made based on biochemical changes of HeLa cells and THP-1 cells post treatment of Kawakawa leave extracts.
Measurement of lactate dehydrogenase (LDH assays) in the growth media of cells that were shown to be growth inhibited during exposure to Kawakawa extracts indicated that the growth inhibition resulting from exposure to the extracts was not due to necrotic cell death processes. (ie that cells maintained their membrane integrity) When compared to the control, a lower amount of cytosolic LDH was seen in THP-1 cell lines. However, HeLa cell lines produced a high level of cytosolic LDH which may be due to mycoplasma contamination. On average, Kawakawa concentrations at 12.5ug/ml and 25ug/ml yielded less lysed cells percentage across both cell lines.
The demonstrated cell growth inhibition was unlikely to be due to necrosis and because of this it was decided to see if cell death was caused by mitochondrial processes that would further support apoptotic cell death. The biochemical assay MTT indirectly measures mitochondrial dehydrogenase activity in the presence of kawakawa leave extracts.
The MTT data illustrated mitochondrial impairment where HeLa cells exposed to Kawakawa 60OC concentrations 6.25 ug/ml and 12.5 ug/ml had the lowest average mitochondrial activity. THP-1 cells exposed to Kawakawa 60OC concentrations 12.5 ug/ml and 50 ug/ml had the lowest average mitochondrial activity.
The main results of this study include, the treatment of HeLa and THP-1 cells with kawakawa extracts demonstrated that cytotoxicity was not due to necrosis. The most interesting finding of this study is when kawakawa extracts inhibit cell growth there is no increase in LDH. This is suggestive that the cellular membrane remains intact when exposed to the extract.

year: 2016

title: Food insecurity in urban New Zealand. The case of the Kopa family

authors: Graham, Rebekah Sarah

abstract: Poverty brings insecurity and chaos into the home, forcing families to develop strategies for navigating the difficulties of food insecurity. We document how one household, the Kopa family, responds to poverty in ad hoc and agentive ways that assist and hinder their attempts to keep themselves fed. This study is based on a series of eight qualitative interviews with family members, including photo-elicitation and go-along techniques, as well as extensive field notes from more casual research interactions such as shared meals. Our analysis demonstrates how food insecurity shapes this family’s culturally patterned social practices surrounding the procurement, consumption, and sharing of food.

text: Food insecurity in urban New Zealand. The case of the Kopa family Poverty brings insecurity and chaos into the home, forcing families to develop strategies for navigating the difficulties of food insecurity. We document how one household, the Kopa family, responds to poverty in ad hoc and agentive ways that assist and hinder their attempts to keep themselves fed. This study is based on a series of eight qualitative interviews with family members, including photo-elicitation and go-along techniques, as well as extensive field notes from more casual research interactions such as shared meals. Our analysis demonstrates how food insecurity shapes this family’s culturally patterned social practices surrounding the procurement, consumption, and sharing of food.

year: 2018

title: Molecular Characterisation of Sex Differentiation Genes in Yellowtail Kingfish (Seriola lalandi)

authors: Elliott, Nicholas B.

subjects: seriola

abstract: Species diversification is crucial for the successful establishment and growth of a finfish aquaculture industry in New Zealand. The yellowtail kingfish Seriola lalandi is a promising candidate species with a fast growth rate, high market value, and is successfully farmed overseas, along with several other Seriola species. To truly domesticate any farmed species, understanding reproduction is vital. Sexual differentiation is a key part of early reproductive development and is characterised by the migration of primordial germ cells through the body to the site of the presumptive gonad, where they proliferate and differentiate into spermatogonia and oogonia. A number of key genes are expressed during sex differentiation, but currently very little molecular research has been conducted on S. lalandi. 

Using bioinformatics approaches, the available gene databases and the implementation of a transcriptomic library prepared from the gonad, ovary and pituitary tissue of S. lalandi, a number of key sex differentiation genes were identified, which included, Vasa, Amh and Cyp19a1a. Primers were designed to each gene, to enable confirmation of the gene sequences, by PCR and RACE-PCR. Using this approach, no confirmed sequence was obtained for Cyp19a1a, however sequences were obtained for part of the Vasa gene and a large part of the Amh gene, including the 3’end. 

The availability of these sex differentiation gene sequences allowed expression of Vasa, Amh and Cyp19a1a to be investigated. Primers were designed using the confirmed sequences for Vasa and Amh, as well as the unconfirmed sequence for Cyp19a1a. These were used in real-time PCR to examine the expression of each gene during development, within larvae at hatching, 3 days post hatch (dph), 12 dph and 18 dph. Expression of each gene was found to increase by 12 dph, but then decrease at 18dph, however, no meaningful significant difference in expression was seen statistically between any of the time points.

Lastly, a protocol was developed for embedding and sectioning of larvae from a range of different ages. A number of attempts were made to optimise an approach, with fish from 3 dph to 60 dph eventually being paraffin embedded, sectioned and stained with either toluidine blue or hematoxylin and eosin (H&E). A number of regions of interest were described, with one showing good similarity to early gonad formation, detailed in other studies. However, due to technical issues and time constraints, no PGCs or presumptive gonad tissues could be positively identified. Future work will use prepared sections to stain for the sex differentiation genes, to gain a better understanding of their role during development. 

This study has established a platform for further work on S. lalandi by creating a framework of molecular tools for studying sex differentiation genes and their expression in this species. It has also worked towards refining the histological techniques required to successfully analyse structural development in larvae. This will be valuable for future studies with techniques such as in-situ hybridisation, assisting in the development of tools for the improvement of S. lalandi aquaculture, for applications such as monosex culture and surrogate broodstock technology.

text: Molecular Characterisation of Sex Differentiation Genes in Yellowtail Kingfish (Seriola lalandi) Species diversification is crucial for the successful establishment and growth of a finfish aquaculture industry in New Zealand. The yellowtail kingfish Seriola lalandi is a promising candidate species with a fast growth rate, high market value, and is successfully farmed overseas, along with several other Seriola species. To truly domesticate any farmed species, understanding reproduction is vital. Sexual differentiation is a key part of early reproductive development and is characterised by the migration of primordial germ cells through the body to the site of the presumptive gonad, where they proliferate and differentiate into spermatogonia and oogonia. A number of key genes are expressed during sex differentiation, but currently very little molecular research has been conducted on S. lalandi. 

Using bioinformatics approaches, the available gene databases and the implementation of a transcriptomic library prepared from the gonad, ovary and pituitary tissue of S. lalandi, a number of key sex differentiation genes were identified, which included, Vasa, Amh and Cyp19a1a. Primers were designed to each gene, to enable confirmation of the gene sequences, by PCR and RACE-PCR. Using this approach, no confirmed sequence was obtained for Cyp19a1a, however sequences were obtained for part of the Vasa gene and a large part of the Amh gene, including the 3’end. 

The availability of these sex differentiation gene sequences allowed expression of Vasa, Amh and Cyp19a1a to be investigated. Primers were designed using the confirmed sequences for Vasa and Amh, as well as the unconfirmed sequence for Cyp19a1a. These were used in real-time PCR to examine the expression of each gene during development, within larvae at hatching, 3 days post hatch (dph), 12 dph and 18 dph. Expression of each gene was found to increase by 12 dph, but then decrease at 18dph, however, no meaningful significant difference in expression was seen statistically between any of the time points.

Lastly, a protocol was developed for embedding and sectioning of larvae from a range of different ages. A number of attempts were made to optimise an approach, with fish from 3 dph to 60 dph eventually being paraffin embedded, sectioned and stained with either toluidine blue or hematoxylin and eosin (H&E). A number of regions of interest were described, with one showing good similarity to early gonad formation, detailed in other studies. However, due to technical issues and time constraints, no PGCs or presumptive gonad tissues could be positively identified. Future work will use prepared sections to stain for the sex differentiation genes, to gain a better understanding of their role during development. 

This study has established a platform for further work on S. lalandi by creating a framework of molecular tools for studying sex differentiation genes and their expression in this species. It has also worked towards refining the histological techniques required to successfully analyse structural development in larvae. This will be valuable for future studies with techniques such as in-situ hybridisation, assisting in the development of tools for the improvement of S. lalandi aquaculture, for applications such as monosex culture and surrogate broodstock technology.

year: 2015