Analysis of cell allocation in GFP chimeric blastocysts by confocal microscopy

This study combined confocal microscopy with the use of a tau-GFP (green fluorescent protein) transgenic mouse strain to study cell fate in two main types of chimeric blastocysts. In each case one component of the chimera was known to contribute poorly to the fetal lineage at later stages. The experiments were designed to test whether this was due to non-random allocation to different tissues at the blastocyst stage.The initial part of this thesis involved the establishment of confocal microscopy techniques and the characterisation of two novel tau-GFP transgenic mouse strains. A method of culturing embryos on the confocal microscope was established for use in further studies. Two tau-GFP transgenic mouse lines, TgTP6.3 and TgTP6.4, were evaluated for their use in following chimera studies by assessing the timing of the onset of GFP expression during preimplantation development and the viability of heterozygote and homozygote mice.The remaining studies involved the use of tau-GFP chimeric embryos. Mouse tetraploid«-*diploid chimeras have previously been used as a model of confined placental mosaicism (CPM). Approximately 2% of human conceptuses investigated by chorionic villus sampling contain chromosomally abnormal cells that are confined to the placenta. This condition, known as human CPM, can lead to incorrect prenatal diagnosis. Animal models would be useful for investigating the mechanisms responsible for the exclusion of abnormal cells from the fetus. As spontaneous chromosomal mosaicism is rare in mouse embryos, mouse aggregation chimeras have been used as a model. Previous results have shown that tetraploid cells are excluded from the epiblast derivatives, including the fetus, of mid gestation tetraploid*-* diploid chimeras. Tetraploid cells have been shown to be preferentially allocated to the trophectoderm, in particular the mural trophectoderm, of mouse tetraploid-^diploid blastocysts. However, tetraploid cells are present within the inner cell mass region of the blastocyst. Therefore, the current study used tau-GFP tetraploid*-*diploid aggregation chimeras to determine if tetraploid cells are present within the epiblast and lost later or are excluded from the epiblast region by preferential allocation to the hypoblast. Tetraploid<-*diploid chimeras were produced using TgTP6.3 embryos. Analysis of these chimeras at E3.5 and E4.5 has confirmed that tetraploid cells are preferentially allocated to the mural trophectoderm. However, tetraploid cells were present within the region of the blastocyst that forms the epiblast. Analysis of expanded chimeric blastocysts at E5.5 and E7.5, produced by transferring them to delayed implantation females, also showed that tetraploid cells were present within the epiblast region. This suggests that tetraploid cells are initially present within the epiblast region but lost from the epiblast later by some mechanism of cell selection against tetraploid cells.Embryos from some inbred strains, such as BALB/c, also tend to contribute poorly to chimeras, so producing 'unbalanced chimeras'. Therefore unbalanced BALB/c chimeras could be a possible model of CPM. BALB/c^GFP aggregation chimeras were analysed using the established time-lapse technique. This was to determine if BALB/c cells are underrepresented in mid-gestation BALB/c chimeras by preferential allocation of BALB/c cells to the mural trophectoderm. These results showed that BALB/c cells were not preferentially allocated to the mural trophectoderm and indicate that a general cell selection mechanism takes place

Kynurenines in neurological disorders

The kynurenine pathway is thought to be involved in neurological disorders but its precise role and the mechanisms involved have yet to be established. Tryptophan can be metabolised via this pathway to produce the neurotoxic N-methyl-D-aspartate (NMDA) receptor agonist, quinolinic acid (QUIN), and the direct generators of reactive oxygen species, 3-hydroxykynurenine (3HKYN) and 3-hydroxyanthranilic acid (3HANA), as well as the neuroprotective NMDA receptor antagonist, kynurenic acid (KYNA). High performance liquid chromatography (HPLC) methods were successfully developed and validated for measuring tryptophan, kynurenine, KYNA, 3HANA and anthranilic acid (ANA) in blood samples, using absorbance and fluorescence detection. The method for determining 3HKYN using electrochemical detection was more problematic and was only used for tryptophan loaded samples and their respective baseline samples. Using HPLC, the concentrations of tryptophan, kynurenine, KYNA, 3HKYN and 3HANA were measured in the blood of Huntington's disease (HD) patients and patients with chronic brain injury, where the injury had occurred at least one year previously. QUIN was also determined for these patients using gas chromatography-mass spectrometry (GC-MS). In addition, the dynamics of the kynurenine pathway were investigated following oral tryptophan depletion and loading. In contrast to these chronic conditions, patients with acute stroke were also studied. The concentrations of tryptophan, kynurenine, KYNA, ANA and 3HANA were determined in the blood of the stroke patients, examining any changes in these concentrations during the two weeks after the stroke. The extent of inflammation and oxidative stress were also assessed for all patients, by measuring the levels of neopterin and the lipid peroxidation products, malondialdehyde and 4-hydroxynonenal. Patients with late stage HD showed abnormal tryptophan metabolism via the kynurenine pathway, together with increased inflammation and oxidative stress. Increased levels of kynurenine together with increased kynurenine: tryptophan (K:T) ratios, indicating greater indoleamine 2,3-dioxygenase (IDO) activity, were observed in blood samples from HD patients in comparison with healthy control subjects. In conjunction with this increased IDO activity, there was a decrease in the ratios of KYNA: kynurenine, suggesting decreased kynurenine aminotransferase (KAT) activity. Inflammation, which may be stimulating IDO activity, could also be decreasing KAT activity, suggested by negativecorrelations between the KYNA: kynurenine ratios and the inflammatory marker, neopterin. The inactivity of KAT suggests a small deficiency in KYNA over a long period of time which could cause a reduction in NMDA receptor antagonism, resulting in slow progressive excitotoxicity contributing to the neurodegeneration in HD. Low KYNA: kynurenine ratios were observed in baseline and tryptophan depleted samples, but after tryptophan loading, HD patients showed similar ratios compared with control subjects. This suggests that loading may be beneficial for HD patients, as more of the neuroprotectant, KYNA can potentially be produced. However, the results suggest that concentrations of the neurotoxin, QUIN, may also be increasing after tryptophan loading. Low concentrations of 3HKYN and 3HANA, with no change in QUIN levels, were also observed in the blood of HD patients. 3HANA levels continued to be decreased for the HD patients after loading. This may suggest degradation of 3HKYN and 3HANA by autoxidation producing reactive oxygen species which could contribute to the high levels of oxidative stress found in these patients. Tryptophan loading in healthy control subjects showed significant increases in the inflammatory marker, neopterin, and in the lipid peroxidation products. These results should be considered when tryptophan loading is used in psychiatric practice and in diets high in tryptophan, such as the Atkins diet. Patients with severe chronic brain injury showed similar alterations in kynurenine pathway metabolism as HD patients, at baseline and throughout the loading and depletion protocols. Although the brain injury had occurred at least one year previously, these patients showed persistent inflammation and oxidative stress, demonstrated by their increased levels of neopterin and lipid peroxidation products compared with healthy controls. In baseline blood samples, there were increased K:T ratios indicating greater IDO activity in the patients. Patients with chronic brain injury showed decreased concentrations of the neuroprotectant, KYNA, as well as low KAT activity, indicated by the decreased KYNA: kynurenine ratios. After tryptophan loading, K:T ratios decreased and the KYNA: kynurenine ratios increased in patients in comparison with controls, suggesting a reversal in the activities of the enzymes IDO and KAT. Similar levels of the inflammatory marker, neopterin, were observed in patients and controls after tryptophan loading. This suggests that these changes in IDO and KAT activities may be related to inflammation. As for the HD patients, patients with chronic brain injury showed lower levels of 3HKYN and 3HANA in their blood, with no change in QUIN levels. These metabolites may be undergoing autoxidation, producing reactive oxygen species which contribute to the ongoing oxidative stress in these patients.The kynurenine pathway was activated following an acute stroke, as indicated by the increased K:T ratios, suggesting greater IDO activity. Stroke patients also had raised levels of neopterin and lipid peroxidation products, indicating inflammation and oxidative stress. There were no changes in the blood concentrations of kynurenines, neopterin or lipid peroxidation products during the fourteen days after a stroke. Stroke patients had reduced levels of 3HANA in their blood, as observed for the HD and chronic brain injury patients. There were negative correlations between the concentration of 3HANA and the volume of the brain lesion, measured by computed tomography (CT) scan, demonstrating the importance of the decreased concentrations of 3HANA. In addition, there were increased levels of ANA in the blood of the stroke patients and the ratios of 3HANA: ANA also correlated with brain lesion volume. Another measurement which correlated with lesion volume was lipid peroxidation, suggesting that oxidative stress contributes to the extent of the brain damage after a stroke. This may suggest that the role of 3HANA in stroke is related to its autoxidation and the generation of reactive oxygen species. Increased concentrations of KYNA were observed in patients who died within three weeks of having a stroke. These high levels of KYNA may have been produced following excitotoxicity and the generation of free radicals, and may cause excessive NMDA receptor blockade or reduced mitochondrial adenosine triphosphate (ATP) synthesis, thus contributing to cell death. The kynurenine pathway was activated and showed abnormal metabolism in all the patient groups, suggesting a potential role for these metabolites in neuronal dysfunction in HD, chronic brain injury and acute stroke. Further work is required to elucidate the role of tryptophan metabolites and whether they may have a direct contribution to neuronal damage in neurological disorders

The Effect of Aquifer/Caprock Interface on Geological Storage of CO2

AbstractThe migration of CO2 stored in deep saline aquifers depends on the morphology of the top of the aquifer. Topographical highs, such as anticlines, may trap CO2 and limit the distance migrated, or elevated ridges may provide pathways enabling CO2 to migrate further from the injector. For example, seismic data of the Utsira formation at the Sleipner storage site indicates that a branch of the CO2 plume is moving to the north [1]. It is therefore important to study the interface between the aquifer and the caprock when assessing risk as CO2 storage sites.Undulations in the top surface of an aquifer may either be caused by sedimentary structures [2], or by folding. In addition, irregularities may be generated by faulting [2]. Large-scale features are detected using seismic data (i.e. structures with amplitudes greater than 10 m), and such structures will generally be included in reservoir or aquifer models. However, smaller- scale features could also have an effect on a CO2 plume migration, and this is the topic of our study. We have conducted simulations in models with a range of top-surface morphology, and have examined the distance migrated and the amount of dissolution.The results from this study suggest that the effects of sub-seismic variations in the topography of the aquifer/caprock interface are unlikely to have a significant impact on the migration and dissolution of CO2 in a saline aquifer, compared with tilt or permeability anisotropy. The results were most sensitive to the kv/kh ratio during the injection period

Serine, but not glycine, supports one-carbon metabolism and proliferation of cancer cells

Previous work has shown that some cancer cells are highly dependent on serine/glycine uptake for proliferation. Although serine and glycine can be interconverted and either might be used for nucleotide synthesis and one-carbon metabolism, we show that exogenous glycine cannot replace serine to support cancer cell proliferation. Cancer cells selectively consumed exogenous serine, which was converted to intracellular glycine and one-carbon units for building nucleotides. Restriction of exogenous glycine or depletion of the glycine cleavage system did not impede proliferation. In the absence of serine, uptake of exogenous glycine was unable to support nucleotide synthesis. Indeed, higher concentrations of glycine inhibited proliferation. Under these conditions, glycine was converted to serine, a reaction that would deplete the one-carbon pool. Providing one-carbon units by adding formate rescued nucleotide synthesis and growth of glycine-fed cells. We conclude that nucleotide synthesis and cancer cell proliferation are supported by serine—rather than glycine—consumption

RUNX-mediated growth arrest and senescence are attenuated by diverse mechanisms in cells expressing RUNX1 fusion oncoproteins

RUNX gene over-expression inhibits growth of primary cells but transforms cells with tumor suppressor defects, consistent with reported associations with tumor progression. In contrast, chromosomal translocations involving RUNX1 are detectable in utero, suggesting an initiating role in leukemias. How do cells expressing RUNX1 fusion oncoproteins evade RUNX-mediated growth suppression? Previous studies showed that the TEL-RUNX1 fusion from t(12;21) B-ALLs is unable to induce senescence-like growth arrest (SLGA) in primary fibroblasts while potent activity is displayed by the RUNX1-ETO fusion found in t(8;21) AMLs. We now show that SLGA potential is suppressed in TEL-RUNX1 but reactivated by deletion of the TEL HLH domain or mutation of a key residue (K99R). Attenuation of SLGA activity is also a feature of RUNX1-ETO9a, a minor product of t(8;21) translocations with increased leukemogenicity. Finally, while RUNX1-ETO induces SLGA it also drives a potent senescence-associated secretory phenotype (SASP), and promotes the immortalisation of rare cells that escape SLGA. Moreover, the RUNX1-ETO SASP is not strictly linked to growth arrest as it is largely suppressed by RUNX1 and partially activated by RUNX1-ETO9a. These findings underline the heterogeneous nature of premature senescence and the multiple mechanisms by which this failsafe process is subverted in cells expressing RUNX1 oncoproteins

Serine one-carbon catabolism with formate overflow

Serine catabolism to glycine and a one-carbon unit has been linked to the anabolic requirements of proliferating mammalian cells. However, genome-scale modeling predicts a catabolic role with one-carbon release as formate. We experimentally prove that in cultured cancer cells and nontransformed fibroblasts, most of the serine-derived one-carbon units are released from cells as formate, and that formate release is dependent on mitochondrial reverse 10-CHO-THF synthetase activity. We also show that in cancer cells, formate release is coupled to mitochondrial complex I activity, whereas in nontransformed fibroblasts, it is partially insensitive to inhibition of complex I activity. We demonstrate that in mice, about 50% of plasma formate is derived from serine and that serine starvation or complex I inhibition reduces formate synthesis in vivo. These observations transform our understanding of one-carbon metabolism and have implications for the treatment of diabetes and cancer with complex I inhibitors