Tomato Shade Avoidance: Unraveling Internode Elongation and Hormonal Harmony

In a quest to unravel the intricate dance of plants with light, our journey begins with the pivotal role of light and plant growth general introduction. The competition among neighboring vegetation sparks the phenomenon of shade avoidance syndrome (SAS), driven by far-red light enrichment. Armed with a foundation of molecular insights from prior research on shade avoidance syndrome, our thesis sets sail into uncharted territory. Chapter 1 delves into the shade avoidance syndrome (SAs) in tomato cultivars M82 and Moneymaker, dissecting cellular developmental plasticity under white light and far-red supplemented conditions. Microscopy-based quantification zooms into the first internode, revealing significant cellular anatomy responses, setting the stage for deeper exploration in subsequent chapters. Chapter 2 explores the molecular mechanism with tissue specific signally in SAS. A time series RNAseq sheds light on auxin’s early role in internode elongation and identification of transcription factors, paving the way for Chapter 4’s exploration of hormones, identifying potential key regulators. The hormonal dynamics take center stage in Chapter 4, as we probe into auxin, gibberellins, and brassinosteroids during SAS. The study unveils complex interactions governing stem elongation, highlighting the nuanced influence of auxin, while GA and BR emerge as potent players. The intricate interplay of these hormones shapes plant responses to shade, emphasizing their crucial roles. Chapter 5 encapsulates a multidimensional exploration, examining cell expansion, the regulatory roles of transcription factors, and the dynamics of shade avoidance responses across 8 dicots. Pith elongation patterns, transcriptional regulation, and motif distribution offer a nuanced understanding of conserved regions, showcasing the interdisciplinary nature of plant biology.Our journey concludes with a spotlight on the distribution of dissimilar transcription factors across plant families, paving the way for future investigations. In the final chapter, we cast a broad perspective on the constraints of RNAseq analysis, hormone analysis, and evolutionary considerations. The discovery of a general conservation pattern between pith-specific expression in far-red across diverse species and transcription factor conservation opens avenues for deeper research. While our proposed linkage model awaits further confirmation, this ongoing exploration promises to unveil the molecular intricacies of plant responses to light, enriching our understanding of adaptation strategies

Molecular characterization of animal microRNAs : sequence, expression, and stability

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2004.Includes bibliographical references.(cont.) miRNAs. These cells lines may also be useful for other functional studies, such as validation of putative mRNA target genes.Multicellular organisms possess natural gene-regulatory pathways that employ small RNAs to negatively regulate gene expression. In nematodes, the small temporal RNAs (stRNAs), lin-4 and let-7, negatively regulate genes important in specifying developmental timing. A gene-silencing pathway present in plants, fungi and animals called RNA interference, involves the conversion of long double-stranded RNA into short interfering RNAs, which can serve to negatively regulate endogenous genes or suppress the replication of viruses and transposons. To investigate how wide a role small RNAs play in regulating gene expression in animals, we developed a RNA cloning procedure and first applied it to the cloning of small RNAs from the nematode, Caenorhabditis elegans. In addition to cloning lin-4 and let-7 sequences, our study revealed a large number of conserved and highly expressed small RNAs with features reminiscent of stRNAs. Because not all of these small RNAs were expressed in temporal fashion, we and others have referred to this novel class of tiny RNAs as microRNAs. We completed an extensive census of microRNA (miRNA) genes in C.elegans by cloning and bioinformatics searches to lay the groundwork for future functional studies. Our census marked the detection of nearly 90 C.elegans miRNAs, estimated an upper-bound of about 120 miRNA genes in C.elegans, and detailed the conservation and clustering of miRNA sequences. We also determined the high molecular abundance of several miRNAs in C.elegans and Hela cells. In an effort to understand the reason for the high molecular abundance of miRNAs, we constructed an inducible miRNA-expressing cell line to measure the stability of animal miRNAs. Time course measurements suggested a long (>24 hours) half-life for twoby Nelson C. Lau.Ph.D

Smoking and Second Hand Smoking in Adolescents with Chronic Kidney Disease: A Report from the Chronic Kidney Disease in Children (CKiD) Cohort Study

The goal of this study was to determine the prevalence of smoking and second hand smoking [SHS] in adolescents with CKD and their relationship to baseline parameters at enrollment in the CKiD, observational cohort study of 600 children (aged 1-16 yrs) with Schwartz estimated GFR of 30-90 ml/min/1.73m2. 239 adolescents had self-report survey data on smoking and SHS exposure: 21 [9%] subjects had “ever” smoked a cigarette. Among them, 4 were current and 17 were former smokers. Hypertension was more prevalent in those that had “ever” smoked a cigarette (42%) compared to non-smokers (9%), p\u3c0.01. Among 218 non-smokers, 130 (59%) were male, 142 (65%) were Caucasian; 60 (28%) reported SHS exposure compared to 158 (72%) with no exposure. Non-smoker adolescents with SHS exposure were compared to those without SHS exposure. There was no racial, age, or gender differences between both groups. Baseline creatinine, diastolic hypertension, C reactive protein, lipid profile, GFR and hemoglobin were not statistically different. Significantly higher protein to creatinine ratio (0.90 vs. 0.53, p\u3c0.01) was observed in those exposed to SHS compared to those not exposed. Exposed adolescents were heavier than non-exposed adolescents (85th percentile vs. 55th percentile for BMI, p\u3c 0.01). Uncontrolled casual systolic hypertension was twice as prevalent among those exposed to SHS (16%) compared to those not exposed to SHS (7%), though the difference was not statistically significant (p= 0.07). Adjusted multivariate regression analysis [OR (95% CI)] showed that increased protein to creatinine ratio [1.34 (1.03, 1.75)] and higher BMI [1.14 (1.02, 1.29)] were independently associated with exposure to SHS among non-smoker adolescents. These results reveal that among adolescents with CKD, cigarette use is low and SHS is highly prevalent. The association of smoking with hypertension and SHS with increased proteinuria suggests a possible role of these factors in CKD progression and cardiovascular outcomes

Plasma membrane profiling of multiple myeloma and the identification of novel monoclonal antibody targets

Supported by a Cancer Research UK (CRUK) studentship (C48525/A18347

Paediatric inflammatory bowel disease - bench to bedside and nationwide : a detailed analysis of Scottish children with IBD

The inflammatory bowel diseases (IBDs) are a group of chronic conditions affecting the gastrointestinal tract, often presenting with non-specific clinical features such as abdominal pain, weight loss and diarrhoea. Approximately 25% of patients are diagnosed with IBD in childhood. For epidemiological studies, previously collected (1990-1995) and original (2003-2008) Scottish incidence data were used to determine national trends in newly diagnosed paediatric IBD (PIBD). A smaller, geographically defined, prospective 14-year cohort (1997- 2011) in South-East Scotland (SES) was used to assess regional trends in incidence, point prevalence, disease extent, medication use and PIBD surgery rates in 326 children. For the detailed analysis of the role of ICOSLG and CRP in Scottish children with PIBD, haplotype-tagging of both genes in 448 children (and their parents) registered on the Paediatric Inflammatory bowel disease Cohort and Treatment Study (PICTS) database was performed. Further clinical information from this database and previously gathered adult mRNA microarray data were also used to inform the analysis. For the faecal calprotectin (FC) case-control study, all PIBD patients diagnosed in SES between 01.01.05 and 31.12.10 (aged 1- 17yrs) with a FC performed during initial workup were identified; controls were matched non- IBD patients who had similarly undergone endoscopy with a referral FC level available. The systematic review and meta-analysis of FC case-control studies was performed with keywords relating to IBD and calprotectin in electronic resources from 1946 to May 2012. Inclusion criteria were studies that reported FC levels prior to the endoscopic investigation of IBD in children less than 18 years old. Laboratory work used newly derived HEK293 and HCT116 cell lines stably expressing wild-type NOD2 and the CD-associated NOD2 frameshift mutant, as well as utilising previously derived HEK293 and HCT116 cells stably expressing green fluorescent-labelled protein LC3 during the assessment of autophagy. Western blot, immunofluorescent microscopy and flow cytometry were used for analysis. There was a significant rise in PIBD incidence in Scotland since the early 1990s, with 260 new cases between 1990-1995 (4.45/100,000/year) and 436 in the 2003-2008 epoch (7.82/100,000/year) (p<0.001). A five-fold increase in Crohn's disease (CD) in the last 40 years was also demonstrated. SES was shown to have the highest recorded PIBD incidence rate in the UK for the six-year epoch from 2006-2011 (9.50/100,000/year) with a significant rise in ulcerative colitis (UC) to 2.67/100,000/year (p=0.010). Point prevalence rates for PIBD in SES had also risen significantly to 41.2/100,000 between the 2000-2005 and 2006-2011 epochs (p=0.016). With a follow up of 1577 patient years, the severe phenotype in children with PIBD was confirmed; 34% of children with CD presented with pan-enteric disease (44% at follow up), and 76% of children with UC had pancolonic disease at diagnosis (81% at follow up). 26% of patients required methotrexate and 18% were exposed to infliximab/adalimumab, with the time to first exposure of both significantly lower in children diagnosed between 2006-2011 (p=0.001 and p<0.001 respectively). A total of 70% of children were exposed to azathioprine and 20% underwent IBD-related surgery. Using a haplotype-tagging approach and transmission disequilibrium testing (TDT) in 230 PIBD case-parent trios there was significant overtransmission of the rs8126734-A single nucleotide polymorphism (SNP) in ICOSLG following correction (p=0.0467). In the CD TDT analysis the same SNP was overtransmitted (p=0.0084). The strongest susceptibility signal was evident across the two marker haplotype rs762421-A / rs8126734-G (p=0.0072), suggesting that the 3-prime untranslated region in ICOSLG may be targeted for deep sequencing. mRNA microarray data from adult patients showed downregulation of ICOSLG expression in the ascending colon (p=0.023) and upregulation in the descending colon (p=0.0351) in uninflamed biopsies from CD patients and non-IBD controls; no difference in gene expression was shown in UC patients. Using a similar approach, the A allele of two SNPs tagging CRP showed significant over-transmission to affected IBD patients after correction (rs1417938, p=0.006; rs1130864, p=0.015). The six-marker haplotype (ACACAC) showed significant distortion of transmission to affected individuals (p=8x10-4). CD and UC patients demonstrated differences in rs1205 genotype (p=0.0085) and CRP haplotype (p=0.0024), with the influence of the rs1205 SNP on response to anti-tumour necrosis factor-alpha therapy also shown (p=0.021). During the FC case-control study significantly elevated FC levels at diagnosis were demonstrated compared to controls (1265 μg/g vs 65 μg/g; p<0.001). FC also outperformed commonly used blood parameters (e.g. CRP, ESR, platelets), with an area under the curve of 0.93 (95% CI 0.89-0.97) and good sensitivity (0.93 [95% CI 0.86-0.98]) and specificity (0.74 [95% CI 0.64-0.82]) when values above 200μg/g were used. FC levels were not influenced by disease location in CD or UC. The systematic review and meta-analysis highlighted the often poor methodological quality of previous studies and concluded that across all studies FC had a pooled sensitivity of 0.98 (95% CI 0.95-1.00) and pooled specificity of 0.68 (95% CI 0.50-0.86) for PIBD at diagnosis. Characterisation of cells stably-expressing wild-type NOD2 or the CD-associated NOD2 frameshift mutation demonstrated increased cell proliferation compared to empty vector, and an accentuated apoptotic response to serum starvation. The NOD2 frameshift protein had a shorter half-life (at 11 hours) than the wild-type protein, with degradation of the NOD2 protein shown to be mediated through a proteasome-dependent pathway, possibly through lysine residues on the CARD domain. Following the establishment of a robust method of assessing autophagy in a cell culture system, experimental work showed that muramyl dipeptide-induced autophagy is unlikely to signal through the mammalian target of rapamycin, with the intermediate filament vimentin shown to be intimately involved in this pathway; the vimentin gene (Vim) was also shown to be a candidate susceptibility gene for CD. Using a panel of PIBD drugs azathioprine was shown to induce autophagy in a dose-dependent manner through an mTOR-dependent, ERK-independent pathway. It can be seen that with the increasing incidence and prevalence of PIBD in Scotland that a greater understanding of epidemiological trends, the role of genetic susceptibility, the optimal use of biomarkers and translational functional biology are all needed to understand further the aetiopathogenesis of PIBD. This future work will undoubtedly help to inform service design and the clinical care pathways utilised to provide the best care for children in addition to targeting pathways for potential drug development, with these measures helping to prepare for the increasing disease burden generated by PIBD

The hedgehog in the coal mine: Exploring hedgehog extinction accounting in the agrochemical sector

The ongoing 6th mass extinction has alerted the accounting community to the need to go beyond accounting for biodiversity. With over 1 million species currently threatened by extinction, the extinction accounting framework has been getting traction with the financial and investment sectors, alerting companies more than ever of the need to account for species as a double material risk. Following current literature in extinction accounting on bee extinction, the thesis focusing on the agrochemical industry in relation to hedgehogs extinction in the UK, the population of which has diminished in over 30% in urban areas and 50% in rural areas since 2000. To examine the implementation of the extinction accounting framework in the agrochemical sector and the hedgehog protection arena, the following research questions were posed: 1. Are the discourses identified in the texts working to ensure the survival of all living beings or is there a need to search for new stories? Which discourses are destructive, predominantly working against the ecosophy? Which discourses are ambivalent can beneficial discourses be found to resonate with the ecosophy? 2. How is the natural world represented and constructed by the agrochemical corporations via multimodal semiosis such as images and videos? 3. What discourses do other stakeholders and organisations such as NGOs, local authorities, hedgehog carers in the hedgehog arena use? 4. How can the extinction accounting framework improve agrochemical accountability in the UK context, in relation to disappearing hedgehogs? To answer the research questions, the methodology, anchored in social constructionism, theorises that agrochemical companies construct a shadow reality, using Beck’s (1992) application of Plato’s allegory of the cave. The methodology positions accounting practices and reporting as a social construct that is discursively constructed. Therefore, through applying an ecolinguistic analysis of textual, multimodal of two agrochemical corporations and spoken discourse of 32 interviewees spanning a wide range of stakeholders within the hedgehog and agrochemical arena, the thesis examines the discourses against the researcher’s ecosophy. A political theory of animals rights is applied as the ecosophy to argue that for disclosures to be truly emancipatory, they must be anchored in positive political rights awarded to animals. The findings from the four empirical chapters are compared and contrasted to reveal that agrochemical companies reject the adoption of the extinction accounting framework as they deny the 6th mass extinction and biodiversity loss and do not view hedgehog extinction, or any other species, as a material risk. The findings demonstrate that companies de-legitimise NGOs in the hedgehog and environmental arena. In turn, the findings suggest NGOs do not acknowledge hedgehog rescuers’ knowledge and expertise. In fact, beyond the economic and financial restricting factors faced by NGOs and local councils, their lack of coordination and accountability, coupled with pressures to appear ‘metric’ and ‘scientific’ presents an obstacle to halting hedgehog extinction. Finally, the thesis reveals that hedgehog rescuers, although disparate, are the ones who transmit the plight of hedgehogs