title: Quiescence induces epigenetic changes in bovine fibroblasts and improves their reprogramming into cloned embryos

authors: Kallingappa, Prasanna Kumar

subjects: Quiescence, Epigenetics, Reprogramming, Somatic cell nuclear transfer (SCNT), Histone Methylation, Histone Acetylation, Histone varients, DNA methylation, Polycomb group (PcG) proteins

abstract: Cloning by somatic cell nuclear transfer (SCNT) forces cells to lose their lineage-specific epigenetic marks and become totipotent again. This reprogramming process often results in epigenetic and transcriptional aberrations that compromise development. Development rates after SCNT can thus serve as a functional assay for genome-wide epigenetic reprogramming. Dolly the sheep, the first mammalian SCNT clone, was derived from a donor cell that was induced into quiescence by serum starvation. We hypothesized that quiescence alters the epigenetic status of donor cells and elevates their reprogrammability. In order to test this idea, we compared chromatin composition and cloning efficiency of serum-starved, quiescent (G₀), bovine fibroblasts vs non-starved, diploid G₁ controls. Mechanically synchronized G₁ cells were generated by mitotic shake-off and harvested within 3 h post-mitosis. Based on morphological assessment and EdU incorporation during continuous labeling, >93% of cells were captured in G₁.
Using quantitative confocal immunofluorescence microscopy and fluorometric ELISA, we show that G₀ fibroblasts were significantly hypomethylated at lysines (K) of histone 3 (H3), specifically H3K4me3, H3K9me2, H3K9me3 and H3K27me3, but not H3K9me1. Histone acetylation was reduced at H3K9 and H4K5, increased at H3K12 and remained unchanged at H3K16. G₀ cells also significantly reduced DNAme. In addition, they significantly down-regulated the nuclear abundance of RNA polymerase II, histone variant H2A.Z, as well as Polycomb group (PcG) proteins EED, SUZ12, PHC1 and RING2. Histone variant H3.3, PcG proteins EZH2 and histone deacetylase HDAC1 did not change compared to the G₁ controls. Following NT into metaphase-arrested oocytes, G₀ DNA condensed slower than that of G₁ cells, indicating a more relaxed chromatin configuration. After seven days of in vitro culture, H3K9me3, but not H3K4me3, H3K27me3, SUZ12 and RING2, remained hypomethylated in G₀- vs G₁-derived NT blastocysts, both in the inner cell mass and trophectoderm. Furthermore, G₀ donors significantly improved development into cloned blastocysts. In conclusion, quiescence induced long-term epigenetic changes, specifically H3K9me3 hypomethylation, that correlated with increased donor cell reprogrammability.

text: Quiescence induces epigenetic changes in bovine fibroblasts and improves their reprogramming into cloned embryos Cloning by somatic cell nuclear transfer (SCNT) forces cells to lose their lineage-specific epigenetic marks and become totipotent again. This reprogramming process often results in epigenetic and transcriptional aberrations that compromise development. Development rates after SCNT can thus serve as a functional assay for genome-wide epigenetic reprogramming. Dolly the sheep, the first mammalian SCNT clone, was derived from a donor cell that was induced into quiescence by serum starvation. We hypothesized that quiescence alters the epigenetic status of donor cells and elevates their reprogrammability. In order to test this idea, we compared chromatin composition and cloning efficiency of serum-starved, quiescent (G₀), bovine fibroblasts vs non-starved, diploid G₁ controls. Mechanically synchronized G₁ cells were generated by mitotic shake-off and harvested within 3 h post-mitosis. Based on morphological assessment and EdU incorporation during continuous labeling, >93% of cells were captured in G₁.
Using quantitative confocal immunofluorescence microscopy and fluorometric ELISA, we show that G₀ fibroblasts were significantly hypomethylated at lysines (K) of histone 3 (H3), specifically H3K4me3, H3K9me2, H3K9me3 and H3K27me3, but not H3K9me1. Histone acetylation was reduced at H3K9 and H4K5, increased at H3K12 and remained unchanged at H3K16. G₀ cells also significantly reduced DNAme. In addition, they significantly down-regulated the nuclear abundance of RNA polymerase II, histone variant H2A.Z, as well as Polycomb group (PcG) proteins EED, SUZ12, PHC1 and RING2. Histone variant H3.3, PcG proteins EZH2 and histone deacetylase HDAC1 did not change compared to the G₁ controls. Following NT into metaphase-arrested oocytes, G₀ DNA condensed slower than that of G₁ cells, indicating a more relaxed chromatin configuration. After seven days of in vitro culture, H3K9me3, but not H3K4me3, H3K27me3, SUZ12 and RING2, remained hypomethylated in G₀- vs G₁-derived NT blastocysts, both in the inner cell mass and trophectoderm. Furthermore, G₀ donors significantly improved development into cloned blastocysts. In conclusion, quiescence induced long-term epigenetic changes, specifically H3K9me3 hypomethylation, that correlated with increased donor cell reprogrammability.

year: 2013

title: Clay mineralogy of tephras and associated paleosols and soils, and hydrothermal deposits, North Island [New Zealand]

authors: Lowe, David J.

subjects: clay minerals

abstract: Tour themes and itinerary
The tour centres on the occurrence and genesis of clay minerals, especially allophane, halloysite, and ferrihydrite, associated with both Quaternary rhyolitic airfall tephra (volcanic ash) deposits and volcanogenic alluvium, and on mineralisation and thermal activity in hydrothermal fields. After a brief overview of the basaltic volcanoes of Auckland City, our route essentially traverses the Central Volcanic Region by way of a large loop with overnight stops at Rotorua (2 nights), Tokaanu, and Auckland (Fig. 0.1). We have around five stops planned for each day (including lunch), three of these being scientific stops except on Day 4 when we have only one scientific stop because of the need to travel greater distances.
Our route takes us progressively towards the locus of the most recently active volcanic centres of the Central Volcanic Region, and so the surficial tephra deposits and buried paleosols become successively younger and generally less weathered: tephras at the Mangawara section (Day 1) span c. 1 Ma; at Tapapa (Day 2), c. 140 ka; at Te Ngae (Day 2), c. 20 ka; and at De Bretts, c. 10 ka, and Wairakei, c. 2 ka (Day 3). Interspersed with these tephra-paleosol sections are stops to examine an allophane-halloysite soil drainage (leaching) sequence on volcanogenic alluvium (Day 1), hydrothermal activity and mineral deposits at Whakarewarewa (Day 2) and Waiotapu (Day 3), and pure ferrihydrite seepage deposits in Hamilton (Day 4). Following introductory and detailed background review material, the tour guide has been arranged on a day-by-day basis and includes an outline of the route and stops, and several pages describing the stratigraphy, mineralogy, chemistry, and pedology of the deposits or features at each of the main stops. We will attempt to point out and describe geological and other features as appropriate during travel periods.
Other activities
Examples of New Zealand's distinctive fauna and flora, including kiwis and tuataras, will be seen at close quarters at Rainbow Springs (Day 2), where we will also enjoy an agricultural farm show. In Rotorua we will partake in a Maori hangi (steam-cooked feast) and concert including traditional dance forms (hakas) and songs (Day 2). In Tokaanu, hot pools will be available to relax in near the slopes of Mt Tongariro (Day 3). At Waitomo, we will visit the Waitomo Cave and in Hamilton spend a short time at the Waikato Museum of Art and History (Day 4). Finally, the tour will conclude with a farewell dinner in Auckland.

text: Clay mineralogy of tephras and associated paleosols and soils, and hydrothermal deposits, North Island [New Zealand] Tour themes and itinerary
The tour centres on the occurrence and genesis of clay minerals, especially allophane, halloysite, and ferrihydrite, associated with both Quaternary rhyolitic airfall tephra (volcanic ash) deposits and volcanogenic alluvium, and on mineralisation and thermal activity in hydrothermal fields. After a brief overview of the basaltic volcanoes of Auckland City, our route essentially traverses the Central Volcanic Region by way of a large loop with overnight stops at Rotorua (2 nights), Tokaanu, and Auckland (Fig. 0.1). We have around five stops planned for each day (including lunch), three of these being scientific stops except on Day 4 when we have only one scientific stop because of the need to travel greater distances.
Our route takes us progressively towards the locus of the most recently active volcanic centres of the Central Volcanic Region, and so the surficial tephra deposits and buried paleosols become successively younger and generally less weathered: tephras at the Mangawara section (Day 1) span c. 1 Ma; at Tapapa (Day 2), c. 140 ka; at Te Ngae (Day 2), c. 20 ka; and at De Bretts, c. 10 ka, and Wairakei, c. 2 ka (Day 3). Interspersed with these tephra-paleosol sections are stops to examine an allophane-halloysite soil drainage (leaching) sequence on volcanogenic alluvium (Day 1), hydrothermal activity and mineral deposits at Whakarewarewa (Day 2) and Waiotapu (Day 3), and pure ferrihydrite seepage deposits in Hamilton (Day 4). Following introductory and detailed background review material, the tour guide has been arranged on a day-by-day basis and includes an outline of the route and stops, and several pages describing the stratigraphy, mineralogy, chemistry, and pedology of the deposits or features at each of the main stops. We will attempt to point out and describe geological and other features as appropriate during travel periods.
Other activities
Examples of New Zealand's distinctive fauna and flora, including kiwis and tuataras, will be seen at close quarters at Rainbow Springs (Day 2), where we will also enjoy an agricultural farm show. In Rotorua we will partake in a Maori hangi (steam-cooked feast) and concert including traditional dance forms (hakas) and songs (Day 2). In Tokaanu, hot pools will be available to relax in near the slopes of Mt Tongariro (Day 3). At Waitomo, we will visit the Waitomo Cave and in Hamilton spend a short time at the Waikato Museum of Art and History (Day 4). Finally, the tour will conclude with a farewell dinner in Auckland.

year: 1993-07-01

title: Te Awa Atua, Te Awa Tapu, Te Awa Wahine: An examination of stories, ceremonies and practices regarding menstruation in the pre-colonial Māori world.

authors: Murphy, Ngahuia Aroha Grace

subjects: mana wahine

abstract: This thesis examines Māori cosmological stories, ceremonies, and traditional practices regarding menstruation in pre-colonial Māori society. I use kaupapa Māori and mana wahine as a theoretical and methodological framework, contextualising these stories within Māori cultural paradigms.This is important because menstruation has been framed within deeply misogynist, colonial ideologies in some ethnographic accounts, distorting menstrual rites and practices beyond recognition. These interpretations have been used to inform colonialist narratives of female inferiority in traditional Māori society, attempting to change Native constructs of womanhood. Such narratives have been perpetuated in contemporary literature, reinforcing powerful discourses of menstrual pollution and female inferiority. This thesis is a challenge to such representations. By examining menstrual stories located in Māori cosmologies, and investigating tribal histories, oral literatures, ceremonies and rites, I argue that menstruation was seen as a medium of whakapapa (genealogy) that connected Māori women to our pantheon of atua (supernatural beings). A study of ancient menstrual rites, recorded in tribal songs and chants, reveal that menstrual blood was used for psychic and spiritual protection. These examples unveil striking Indigenous constructs of womanhood that transform colonialist interpretations and radically challenge notions of female inferiority and menstrual pollution. I maintain in this thesis that presenting menstruation and menstrual blood as putrid is a politically motivated act of colonial violence that specifically targets the source of our continuity as Indigenous People, the whare tangata (house of humanity – womb of women). I pose the question ‘if menstrual blood symbolises whakapapa, what does it mean to present it as ‘unclean’ and how do such representations cut across the politics of tino rangatiratanga (autonomy)?’ Through in-depth semi-structured interviews, kōrero (dialogue), and wānanga (series of conversations) with Māori women, including cultural experts, scholars, artists, and mana wahine exponents, I gather a collection of ceremonies, stories, and wisdoms that reclaim Māori spiritualities which celebrate menstruation as divine. Within the context of a colonial history of marginalisation, this work is an activist site of political resistance which takes a step towards re-threading the feminine strands in the spiritual fabric of our world, torn asunder by the ideological imposition of a colonial, Christian male god. I argue, that menstruation is a potent site of decolonisation, cultural reclamation, and resistance toward the perpetuation of colonial hegemony.

text: Te Awa Atua, Te Awa Tapu, Te Awa Wahine: An examination of stories, ceremonies and practices regarding menstruation in the pre-colonial Māori world. This thesis examines Māori cosmological stories, ceremonies, and traditional practices regarding menstruation in pre-colonial Māori society. I use kaupapa Māori and mana wahine as a theoretical and methodological framework, contextualising these stories within Māori cultural paradigms.This is important because menstruation has been framed within deeply misogynist, colonial ideologies in some ethnographic accounts, distorting menstrual rites and practices beyond recognition. These interpretations have been used to inform colonialist narratives of female inferiority in traditional Māori society, attempting to change Native constructs of womanhood. Such narratives have been perpetuated in contemporary literature, reinforcing powerful discourses of menstrual pollution and female inferiority. This thesis is a challenge to such representations. By examining menstrual stories located in Māori cosmologies, and investigating tribal histories, oral literatures, ceremonies and rites, I argue that menstruation was seen as a medium of whakapapa (genealogy) that connected Māori women to our pantheon of atua (supernatural beings). A study of ancient menstrual rites, recorded in tribal songs and chants, reveal that menstrual blood was used for psychic and spiritual protection. These examples unveil striking Indigenous constructs of womanhood that transform colonialist interpretations and radically challenge notions of female inferiority and menstrual pollution. I maintain in this thesis that presenting menstruation and menstrual blood as putrid is a politically motivated act of colonial violence that specifically targets the source of our continuity as Indigenous People, the whare tangata (house of humanity – womb of women). I pose the question ‘if menstrual blood symbolises whakapapa, what does it mean to present it as ‘unclean’ and how do such representations cut across the politics of tino rangatiratanga (autonomy)?’ Through in-depth semi-structured interviews, kōrero (dialogue), and wānanga (series of conversations) with Māori women, including cultural experts, scholars, artists, and mana wahine exponents, I gather a collection of ceremonies, stories, and wisdoms that reclaim Māori spiritualities which celebrate menstruation as divine. Within the context of a colonial history of marginalisation, this work is an activist site of political resistance which takes a step towards re-threading the feminine strands in the spiritual fabric of our world, torn asunder by the ideological imposition of a colonial, Christian male god. I argue, that menstruation is a potent site of decolonisation, cultural reclamation, and resistance toward the perpetuation of colonial hegemony.

year: 2011