Synthesis and Biological Investigation into the Metabolism of Retinoids

Retinoids have a wide variety of applications. They can act as potent inducers of stem cell differentiation, as chemopreventative and chemotherapeutic agents, and have been utilised for the treatment of dermatological conditions. However, natural retinoids readily undergo photo-isomerisation in the presence of natural light and / or in physiological conditions. This propensity for isomerisation is believed to be the cause of many unwanted problems / side effects. Synthetic retinoids have been made by replacing key functional groups with more robust pharmacophores which retain the key functionalities of natural retinoids but are resistant to isomerisation. Previous studies have shown that synthetic retinoids, such as EC23 and EC19 have very high stability and are more potent than their natural analogues ATRA and 13cisRA. The high potency of synthetic retinoids such as EC23 implies that they do not undergo metabolism. An in vivo investigation into the metabolism of retinoids was initiated. AG1 X2 polymer beads were used to deliver various concentrations of AT retinol solutions in DMSO to the anterior margin of a stage 20 chick limb bud which was then observed at stage 36 for signs of abnormal development such as digit duplication. Occurrence of digit duplication would suggest that AT retinol had been metabolised to ATRA, a known inducer of teratogenic effects, within the limb bud; however, only normal development was observed. This suggests that an alternative route for delivering uncharged molecules to limb buds is required. Sonogashira cross coupling reactions were employed to synthesise ether acetate analogues of EC23 and EC19, (EC23 OAc and EC19 OAc) from TMNA with acetic acid 4-bromo-benzyl ester and acetic acid 3-iodo-benzyl ester respectively, with the intention of hydrolysing them into EC23 OH and EC19 OH, i.e. synthetic analogues of AT retinol, to be investigated for teratogenic effects in the chick limb bud as above. If successful delivery of AT retinol produced limb defects, while EC23-OH did not, this would be consistent with the idea that EC23 and its analogues are refractory to normal retinoid metabolism. A variety of useful retinoid precursors were also synthesised from cross coupling reactions and borylation techniques. Synthetic retinoids methyl 5',6',7',8'-tetrahydro-2,2'-binaphthyl-6-carboxylate and (E)-methyl-3-(4-(5,6,7,8-tetrahydronaphthalen-2-yl)phenyl)acrylate were made by Suzuki Miyaura Cross coupling reactions, also to be tested in vivo

The role of microvascular endothelial cells in the pathogenesis of emphysema

PhD ThesisCOPD comprising small airways disease and emphysema is a chronic, debilitating often fatal lung condition that approximately 20% of smokers develop. Current therapies mostly target inflammation and airflow obstruction caused by small airways disease however there are no current therapies which treat emphysema, the pathogenesis of which remains poorly understood. The microvascular hypothesis of COPD is a credible alternative to the classical hypothesis of inflammation and protease driven lung destruction, whereby an initial insult to the microvasculature leads to loss of alveolar structure which typifies emphysema. I planned to investigate the role of the microvasculature in the pathogenesis of COPD by isolating susceptible lung microvascular endothelial cells (LMVECs) from individuals with emphysema in an attempt to mimic in vivo conditions more closely. LMVECs were isolated from explanted emphysematous lungs removed at transplantation. Following successful isolation (71%) and characterisation of emphysema LMVECs, I sought to study cellular responses to cigarette smoke injury, namely apoptosis and endothelial to mesenchymal transition. Apoptosis was investigated on tissue blocks via caspase 3 immunohistochemistry and by ex vivo methods including flow cytometry (annexin V), TUNEL and live cell imaging for activated caspase 3. Unfortunately cigarette smoke extract caused autofluorescence of cells and as all of these techniques employed the use of fluorescence for detection, any conclusions that can be made as to whether cells underwent apoptosis are limited. Endothelial to mesenchymal transition was investigated in response to TGFβ1 and cigarette smoke extract. While there was evidence of down regulation of endothelial markers in response to cigarette smoke on confocal imaging there was no convincing evidence of upregulation of mesenchymal markers with no corresponding change in protein expression via western blotting. One explanation may be that such changes in cell structure and endothelial cell expression may be more in keeping with endothelial activation rather than a true phenotypic switch. In summary, this study presents a new model of emphysema, with attempts to gain insight into endothelial injury in the pathogenesis of COPD, highlighting the challenges and limitations of working with primary diseased cells in response to cigarette smoke injury

Aging, Emotion, Attention, and Binding in the Taboo Stroop Task: Data and Theories.

How does aging impact relations between emotion, memory, and attention? To address this question, young and older adults named the font colors of taboo and neutral words, some of which recurred in the same font color or screen location throughout two color-naming experiments. The results indicated longer color-naming response times (RTs) for taboo than neutral base-words (taboo Stroop interference); better incidental recognition of colors and locations consistently associated with taboo versus neutral words (taboo context-memory enhancement); and greater speed-up in color-naming RTs with repetition of color-consistent than color-inconsistent taboo words, but no analogous speed-up with repetition of location-consistent or location-inconsistent taboo words (the consistency type by repetition interaction for taboo words). All three phenomena remained constant with aging, consistent with the transmission deficit hypothesis and binding theory, where familiar emotional words trigger age-invariant reactions for prioritizing the binding of contextual features to the source of emotion. Binding theory also accurately predicted the interaction between consistency type and repetition for taboo words. However, one or more aspects of these phenomena failed to support the inhibition deficit hypothesis, resource capacity theory, or socio-emotional selectivity theory. We conclude that binding theory warrants further test in a range of paradigms, and that relations between aging and emotion, memory, and attention may depend on whether the task and stimuli trigger fast-reaction, involuntary binding processes, as in the taboo Stroop paradigm

Barriers to infection of human cells by feline leukemia virus: insights into resistance to zoonosis

The human genome displays a rich fossil record of past gamma-retrovirus infections, yet no current epidemic is evident, despite environmental exposure to viruses that infect human cells in vitro. Feline leukemia viruses (FeLVs) rank high on this list, but domestic or workplace exposure has not been associated with detectable serological responses. Non-specific inactivation of gamma-retroviruses by serum factors appears insufficient to explain these observations. To investigate further we explored the susceptibility of primary and established human cell lines to FeLV-B, the most likely zoonotic variant. Fully permissive infection was common in cancer-derived cell lines, but was also a feature of non-transformed keratinocytes and lung fibroblasts. Cells of haematopoietic origin were less generally permissive and formed discrete groups on the basis of high or low intracellular protein expression and virion release. Potent repression was observed in primary human blood mononuclear cells and a subset of leukemia cell lines. However, the early steps of reverse transcription and integration appear to be unimpaired in non-permissive cells. FeLV-B was subject to G->A hypermutation with a predominant APOBEC3G signature in partially permissive cells but was not mutated in permissive cells or in non-permissive cells that block secondary viral spread. Distinct cellular barriers that protect primary human blood cells are likely to be important in protection against zoonotic infection with FeLV

Jumping Off the treadmill: transforming NRM to systemic governing with systemic co-inquiry

While there is continued interest in Deliberative Policy Analysis (DPA) its practice element appears to have been underappreciated. We reflect on our experience of using a systemic co-inquiry to provide new insights into operationalising DPA that may assist it to speak more immediately to issues related to governing in the Anthropocene. Natural resource management (NRM) in Australia embraced the global turn to governance, but demonstrated how difficult it is to achieve systemic, collaborative approaches to management policy. The treadmill of our title symbolises the experience of community and organizational stakeholders in the case area, who were constantly in motion but achieving no forward movement in collaborative governance. A systemic coinquiry into how decision making and action taking in NRM could be improved began in 2015. Systemic co-inquiry is a facilitated process that enables emergence of ideas and opportunities for transforming a situation. We describe this process, present how it was used in the case area, then critically reflect on its contributions for governance and practice, and its theoretical and political implications. Describing and critiquing our use of systemic co-inquiry provides new insights to address challenges for future DPA

Expression of CD44 molecules and CD44 ligands during human thymic fetal development: expression of CD44 isoforms is developmentally regulated

It has recently been recognized that CD44 comprises a large family of alternatively spliced forms.In the thymus, CD44 has been postulated to play an important role in immature T cell migration and maturation. In this paper, we have studied the expression of CD44 molecules and two CD44 ligands, hyaluronan (HA) and fibronectin (FN), during human thymic fetal development. We found that mAbs against all CD44 isoforms (A3D8 or A1G3) reacted with both thymic epithelial (TE) cells and thymocytes beginning at the time of initial colonization of the human thymus by hematopoietic stem cells at 8.2 weeks of fetal gestation. However, mAbs specific for splice variants of CD44 containing membrane-proximal inserts (11.24, 11.10 and 11.9) reacted only with terminally differentiated TE cells in and around Hassall's bodies beginning at 16-19 weeks of fetal gestation. Studies of differentiated versus undifferentiated TE cells in vitro confirmed the selective expression of CD44 variant isoforms on terminally differentiated TE cells. Expression of HA and FN was determined by fluorescence microscopy using either biotlnylated-HA binding protein or an anti-FN mAb. We found that whereas FN was present throughout the human fetal thymus beginning at 8.2 weeks, HA was not present until 16 weeks of gestational age. These data demonstrate the differential expression of standard versus variant CD44 isoforms during thymic ontogeny and implicate CD44 interactions with ligands other than HA as important in the earlier stages of humanthymus developmen

Comparative analysis reveals a role for TGF-β in shaping the residency-related transcriptional signature in tissue-resident memory CD8+ T cells.

Tissue-resident CD8+ memory T (TRM) cells are immune cells that permanently reside at tissue sites where they play an important role in providing rapid protection against reinfection. They are not only phenotypically and functionally distinct from their circulating memory counterparts, but also exhibit a unique transcriptional profile. To date, the local tissue signals required for their development and long-term residency are not well understood. So far, the best-characterised tissue-derived signal is transforming growth factor-β (TGF-β), which has been shown to promote the development of these cells within tissues. In this study, we aimed to determine to what extent the transcriptional signatures of TRM cells from multiple tissues reflects TGF-β imprinting. We activated murine CD8+ T cells, stimulated them in vitro by TGF-β, and profiled their transcriptomes using RNA-seq. Upon comparison, we identified a TGF-β-induced signature of differentially expressed genes between TGF-β-stimulated and -unstimulated cells. Next, we linked this in vitro TGF-β-induced signature to a previously identified in vivo TRM-specific gene set and found considerable (>50%) overlap between the two gene sets, thus showing that a substantial part of the TRM signature can be attributed to TGF-β signalling. Finally, gene set enrichment analysis further revealed that the altered gene signature following TGF-β exposure reflected transcriptional signatures found in TRM cells from both epithelial and non-epithelial tissues. In summary, these findings show that TGF-β has a broad footprint in establishing the residency-specific transcriptional profile of TRM cells, which is detectable in TRM cells from diverse tissues. They further suggest that constitutive TGF-β signaling might be involved for their long-term persistence at tissue sites

Unilateral Disruptions in the Default Network with Aging in Native Space

BACKGROUND: Disruption of the default-mode network (DMN) in healthy elders has been reported in many studies. METHODS: In a group of 51 participants (25 young, 26 elder) we examined DMN connectivity in subjects' native space. In the native space method, subject-specific regional masks (obtained independently for each subject) are used to extract regional fMRI times series. This approach substitutes the spatial normalization and subsequent smoothing used in prevailing methods, affords more accurate spatial localization, and provides the power to examine connectivity separately in the two hemispheres instead of averaging regions across hemispheres. RESULTS: The native space method yielded new findings which were not detectable by the prevailing methods. The most reliable and robust disruption in elders' DMN connectivity were found between supramarginal gyrus and superior-frontal cortex in the right hemisphere only. The mean correlation between these two regions in young participants was about 0.5, and dropped significantly to 0.04 in elders (P = 2.1 x 10(-5)). In addition, the magnitude of functional connectivity between these regions in the right hemisphere correlated with memory (P = 0.05) and general fluid ability (P = 0.01) in elder participants and with speed of processing in young participants (P = 0.008). These relationships were not observed in the left hemisphere. CONCLUSION: These findings suggest that analysis of DMN connectivity in subjects' native space can improve localization and power and that it is important to examine connectivity separately in each hemisphere