PhD

thesis1. LD50 values and 95% confidence limits were determined for the ip route with one strain each o EAT and S-37 tumors. The values obtained were: for EAT 2,000 ± 560; for S-37 45,000 ± 11,866. 2. DNP and DNA were prepared from EAT and S-37 tumors and the prepared material was used for immunization. 3. Mice immunized with DNP prepared from EAT and S-37 and subsequently challenged with viable EAT and S-37 tumor cells showed a considerable degree of resistance as compared to the unimmunized control animals. 4. Cross immunity was demonstrated between mice immunized with DNP from one tumor and challenged with tumor cell other than the type used for immunization. 5. Mice immunized with DNA prepared from EAT and S-37 cells and challenged with viable EAT and S-37 tumor cells showed increased resistance as compared to the unimmunized control animals. The degree of resistance induce using material without the protein moiety was less that that induced by using DNP. 6. Cross immunity was also demonstrated between mice immunized with DNA from one type of tumor cell and then challenged with tumor cells other than the type from which the nucleic acid for immunization was obtained. However, the immunity in this latter circumstance was considerably less than when the immunization carried out with the corresponding DNP. 7. DNP prepared from normal mouse tissue and used for immunization showed no measurable induction of resistance when the mice were tested by challenge with viable tumor cells. 8. Microscopic observation of peritoneal washing from immunized and normal mice that had been challenged with viable tumor cells showed a significantly different cellular and humoral response over a 72-hour period. This response was indicated by lymphocytic and histiocytic infiltration and by lysis of tumor cell

Novel Epigenetic Biomarkers for the Barrett’s – Adenocarcinoma Sequence in Oesophageal Cancer

Introduction Oesophageal adenocarcinoma presents an ever increasing challenge to the NHS, with rising incidence and poor overall survival. There are few robust biomarkers available for this disease – either for detection or stratification. Barrett’s Oesophagus, a precursor to adenocarcinoma, is common. In the non-dysplastic setting, few patients will progress to cancer. There are no current biomarkers to aid in this stratification process. Aims To assess the role of methylation biomarkers in the context of diagnosis of adenocarcinoma and their role in tumour biology. To provide a methylation stratification tool in the identification of high risk non-dysplastic Barrett’s Oesophagus. Methods Genome wide methylation assessment was performed with validation using bisulphite pyrosequencing on carefully selected tissues to reveal novel methylation biomarkers Results Methylation of TRIM15, has been shown to be a robust biomarker in disease identification and has a role in tumour biology. OR3A4, a long none coding RNA, has been identified as a way to reliably risk stratify the non-dysplastic Barrett’s patient and forms the first biomarker of this kind. Conclusions Methylation biomarkers play a key role in disease identification and risk of cancer development. They also appear to play a role in tumour biology

Ex Vivo Modeling of Chemical Synergy in Prenatal Kidney Cystogenesis

Cyclic adenosine monophosphate (cAMP) drives genetic polycystic kidney disease (PKD) cystogenesis. Yet within certain PKD families, striking differences in disease severity exist between affected individuals, and genomic and/or environmental modifying factors have been evoked to explain these observations. We hypothesized that PKD cystogenesis is accentuated by an aberrant fetal milieu, specifically by glucocorticoids. The extent and nature of cystogenesis was assessed in explanted wild-type mouse embryonic metanephroi, using 8-Br-cAMP as a chemical to mimic genetic PKD and the glucocorticoid dexamethasone as the environmental modulator. Cysts and glomeruli were quantified by an observer blinded to culture conditions, and tubules were phenotyped using specific markers. Dexamethasone or 8-Br-cAMP applied on their own produced cysts predominantly arising in proximal tubules and descending limbs of loops of Henle. When applied together, however, dexamethasone over a wide concentration range synergized with 8-Br-cAMP to generate a more severe, glomerulocystic, phenotype; we note that prominent glomerular cysts have been reported in autosomal dominant PKD fetal kidneys. Our data support the idea that an adverse antenatal environment exacerbates renal cystogenesis

Mechanisms underpinning adaptations in placental calcium transport in normal mice and those with fetal growth restriction

Fetal delivery of calcium, via the placenta, is crucial for appropriate skeletal mineralization. We have previously demonstrated that maternofetal calcium transport, per gram placenta, is increased in the placental specific insulin-like growth factor 2 knockout mouse (P0) model of fetal growth restriction (FGR) compared to wild type littermates (WTL). This effect was mirrored in wild-type (WT) mice comparing lightest vs. heaviest (LvH) placentas in a litter. In both models increased placental calcium transport was associated with normalization of fetal calcium content. Despite this adaptation being observed in small normal (WT), and small dysfunctional (P0) placentas, mechanisms underpinning these changes remain unknown. Parathyroid hormone-related protein (PTHrP), elevated in cord blood in FGR and known to stimulate plasma membrane calcium ATPase, might be important. We hypothesized that PTHrP expression would be increased in LvH WT placentas, and in P0 vs. WTL. We used calcium pathway-focused PCR arrays to assess whether mechanisms underpinning these adaptations in LvH WT placentas, and in P0 vs. WTL, were similar. PTHrP protein expression was not different between LvH WT placentas at E18.5 but trended toward increased expression (139%; P = 0.06) in P0 vs. WTL. PCR arrays demonstrated that four genes were differentially expressed in LvH WT placentas including increased expression of the calcium-binding protein calmodulin 1 (1.6-fold; P < 0.05). Twenty-four genes were differentially expressed in placentas of P0 vs. WTL; significant reductions were observed in expression of S100 calcium binding protein G (2-fold; P < 0.01), parathyroid hormone 1 receptor (1.7-fold; P < 0.01) and PTHrP (2-fold; P < 0.05), whilst serum/glucocorticoid-regulated kinase 1 (SGK1), a regulator of nutrient transporters, was increased (1.4 fold; P < 0.05). Tartrate resistant acid phosphatase 5 (TRAP5 encoded by Acp5) was reduced in placentas of both LvH WT and P0 vs. WTL (1.6- and 1.7-fold, respectively; P < 0.05). Signaling events underpinning adaptations in calcium transport are distinct between LvH placentas of WT mice and those in P0 vs. WTL. Calcium binding proteins appear important in functional adaptations in the former whilst PTHrP and SGK1 are also implicated in the latter. These data facilitate understanding of mechanisms underpinning placental calcium transport adaptation in normal and growth restricted fetuses

The atrial natriuretic peptide (ANP) knockout mouse does not exhibit the phenotypic features of pre-eclampsia or demonstrate fetal growth restriction

The ANP knockout mouse is reported to exhibit pregnancy-associated hypertension, proteinuria and impaired placental trophoblast invasion and spiral artery remodeling, key features of pre-eclampsia (PE). We hypothesized that these mice may provide a relevant model of human PE with associated fetal growth restriction (FGR). Here, we investigated pregnancies of ANP wild type (ANP+/+), heterozygous (ANP+/-) and knockout (ANP−/-) mice. Maternal blood pressure did not differ between genotypes (E12.5, E17.5), and fetal weight (E18.5) was unaffected. Placental weight was greater in ANP−/− versus ANP+/+ mice. Therefore, in our hands, the ANP model does not express phenotypic features of PE with FGR

Colección José Arencibia [Material gráfico]

Copia digital. Madrid : Ministerio de Educación, Cultura y Deporte. Subdirección General de Coordinación Bibliotecaria, 201