Both type I and type II diabetes are related to β-cell defects in the pancreatic islet of Langerhans. Deriving β-cells from stem cells and other mature cell types provides an important cell source for transplantation-based therapy to treat diabetes. Mechanistic studies of β-cell maturation and functional maintenance are crucial in providing novel insights for the generation of glucose-responsive and long-term sustainable β-cells. In this study, I found that two gene/gene families, synaptotagmin IV (Syt4) and Myt factors are essential to promote β-cell maturation and functional maintenance. Mouse studies provided evidence that Syt4 modulates insulin Ca2+ vesicle sensitivity to facilitate β-cell maturation the neonatal stage. Loss of Syt4 in mice resulted in Ca2+ hypersensitivity of insulin vesicles in β-cells and compromised glucose-stimulated insulin secretion. Conversely, Syt4 overexpression reduced insulin Ca2+ vesicle sensitivity and established the mature insulin secretion profile in the newborn β-cells. Moreover, Myt factors are essential to generate functional β-cells. Postnatal β-cells in a Myt knockout mouse model were characterized by functional failure, cell apoptosis and loss of mature β-cell identity. Loss of Myt factors in β-cells disrupted the expression of genes involved in insulin secretion, β-cell survival and identity maintenance. These combined results suggest that Syt4 and Myt factors can be exploited as molecular targets to promote β-cell maturity and long-term functional maintenance for better clinical β-cell regeneration.

Apico-basolateral polarity is a fundamental property of epithelial cells, and its loss is a hallmark of colorectal cancer (CRC). Role(s) for lateral integrins in this polarization process and the consequences of their disruption are incompletely understood. We observed an increase in collagen disorganization and higher prevalence of an integrin β1/EGF receptor-containing complex in human CRC. To better understand the contribution of integrin signaling to epithelial cell morphogenesis and receptor tyrosine kinase (RTK) signal transduction, we used an approach combining 3D type-1 collagen culture and integrin β1 function-altering antibodies. We found that induction of integrin α5β1 clustering at lateral, intercellular surfaces contributes to apico-basolateral polarization in a fibronectin-dependent manner. Preliminary work suggests a role for integrin α5β1 in regulating CRC-derived cell response to RTK ligands EGF, NRG1 and HGF. All together, these data show a novel role for integrin α5β1 in regulating colonic epithelial morphogenesis.

ABSTRACT The Discoidin, CUB and LCCL domain-containing protein 2 (DCBLD2/ESDN/CLCP1) is a type-I, transmembrane receptor that mediates diverse cellular processes such as angiogenesis, vascular remodeling, cellular migration and proliferation. Identification of DCBLD2 in a proteomics screen to identify substrates of Src family tyrosine kinases that bind the Src homology 2 domain of CT10 regulator of kinase-Like (CrkL), a critical scaffolding protein for neuronal development, led to a need for further characterization of the protein. To elucidate the role of this interaction and potential novel function of DCBLD2, an in vivo approach utilizing Danio rerio (zebrafish) was conducted. dcbld2 was found localized in neuronal tissues during development, with strong expression in the retina. Knockdown of the protein led to a deficiency of retinal ganglion cells and the optic tracts, or nerve bundles, they project to innervate the brain. Serial sections revealed malformation of the normally discrete layering of retinal cell types, and smaller eye area overall. These findings suggest a role for dcbld2 in developing nervous tissue, specifically neuronal migration during interkinetic nuclear migration. While it is has been shown that dcbld2 has a role in the developmental patterning of intersegmental vessels in the tail of zebrafish, the protein has not been investigated in the context of neurogenesis. The loss of RGCs and lamination defects observed in the eye, along with its association with the CrkL-SH2 domain, implicate it in processes that allow for the proper differentiation of neurons. This study has brought us further down the path to understanding the multiple functions of the receptor; however, further studies are required to delineate the exact mechanistic function of the dcbld2 receptor.

CELL AND DEVELOPMENTAL BIOLOGY Structural Analysis of the Helicobacter pylori toxin VacA Tasia Marie Pyburn Dissertation under the direction of Associate Professor Melanie Ohi Helicobacter pylori is a Gram-negative bacterium that colonizes the human stomach and contributes to peptic ulceration and gastric adenocarcinoma. One of the most important H. pylori virulence determinants is a secreted pore-forming toxin known as vacuolating cytotoxin A (VacA). Secreted as an 88 kDa protein, VacA is composed of an N-terminal p33 domain and a C-terminal p55 domain which assemble into multiple types of water-soluble oligomers including hexamers, heptamer, dodecamers, and tetradecamers. We have determined three-dimensional (3D) structures of VacA s1/i1/m1 oligomeric conformations at ~15 Ã resolution as well as three mutant forms of VacA. At this resolution, differences between the mutants and VacA s1/i1/m1 could not be discerned. Therefore, cryo-EM has been performed on VacA s1/i1/m1 and a structure has been determined of a VacA dodecamer to the highest resolution to date, ~10Ã resolution. The structural organization of membrane-bound VacA has not been characterized in any detail and the role(s) of specific VacA domains in membrane binding and insertion are unclear. Our goal is to understand how VacA transitions from a soluble protein to a membrane inserted protein and how it organizes on membrane. Using a combination of in vitro liposome binding, biochemical assays, and single particle electron microscopy (EM), we show membrane-bound VacA organizes into hexameric oligomers. Comparison of the two-dimensional averages of membrane-bound and soluble VacA hexamers generated using single particle EM reveals structural differences within the central pore-forming region of the oligomers indicating that membrane interactions induce a structural change within the p33 domain. Analyses of VacA variants demonstrate that while the p55 domain can bind membranes, the p33 domain is required for membrane insertion. Surprisingly, neither VacA oligomerization nor the presence of putative transmembrane GXXXG repeats in the p33 domain is required for membrane insertion. These findings provide new insights into the process by which VacA binds and inserts into the lipid bilayer to form membrane channels. Approved _______________________________________________ Date __________________ Melanie D. Ohi, Ph.D.

Reprogramming of pancreatic cells into new beta-like cells is a potential therapy for Type 1 diabetes. Pancreatic acinar cells are an appealing target for cellular reprogramming since they are abundant, derived from a common progenitor cell during pancreatic organogenesis, and exhibit significant transcriptional plasticity. Towards this end, it has been reported that adenoviral-mediated expression of three pancreas-specific transcription factors MafA, Pdx1 and Neurog3 (3TF) in immunocompromised Rag1-/- mice resulted in the conversion of pancreatic acinar cells into new, insulin-secreting, beta-like cells. Using a transgenic mouse model to express 3TF in a pancreatic acinar cell- and doxycycline-dependent manner, we discovered that the outcome of transcription factor-mediated acinar to beta-like cellular reprogramming is dependent on both the magnitude of 3TF expression and on reprogramming-induced inflammation. Overly robust 3TF expression causes acinar cell necrosis resulting in marked inflammation and acinar-to-ductal metaplasia. Generation of new beta-like cells requires limiting reprogramming-induced inflammation, either by reducing 3TF expression or by eliminating macrophages. The new beta-like cells were able to reverse streptozotocin-induced diabetes 6 days after inducing 3TF expression but failed to sustain their function after removal of the reprogramming factors.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

A theory is presented of the development of pattern recognition and looking behaviour in infancy. It is proposed that scanning habits are acquired and patterns recognized with the reproduction of fixations and eye movements in the order in which they originally occurred. Recognition is achieved by correctly predicting the current input for each fixation. Evidence supporting this proposal is discussed, and the limitations of other theories are examined. A case is made for the storage of two kinds of visual information, originating from central and peripheral vision respectively. Infants indicate recognition of familiar patterns by looking less at them than patterns which are new. This can be explained by the discrepancy principle which proposes a curvilinear relation between the amount of looking and degree of discrepancy between a pattern and its representation in memory. This principle is incorporated in the theory to account for the control of the length of sequences of fixations. A computer model of the theory is described. This contains a simulation of the cortical processing of visual input, a number of oculomotor reflexes, learning mechanisms, and the means of controlling the length of a fixation sequence by assessing its discrepancy with the contents of memory. The model was run on a computer and learned to recognize patterns by scanning them and reproducing the original sequences of fixations. The ability of the model to mimic infant looking behaviour is shown in three simulations of different infant experiments. Recognition was demonstrated by a decline in looking at familiar relative to new patterns, and this ability was retained after a delay. Such behaviour took time to develop, and the model required a certain level of visual experience before it appeared. Individual differences in the performance of the model resembling tempo differences in infants were also produced.

This thesis considers infant social development from the viewpoint of the perceptual and memory capacities necessary for particular social abilities. Some social abilities, e.g. facial or voice discrimination, require visual or auditory integrity, thus the development of visual and auditory capacities are reviewed. Recognition of familiar faces and/or voices requires memory. Hence the development of memory abilities is considered. Subsequently the development of social behaviour is reviewed. After these literature reviews, three experimental studies are described. The first of these investigates the recognition of mother's voice and reports evidence of that such recognition develops during the first month of life. The second experiment considers visual recognition of the mother and differential responsivity to face-to-face and averted gaze and to different tones of voice. One month old infants did not reveal any conclusive evidence on these points. However, post-hoc analysis suggested the importance of the physical characteristics of faces in eliciting infant visual attention. Experience in these studies suggested the need for the study of more naturalistic encounters and hence a methodology for the study and analysis of naturalistic social interactions was developed. This methodology was then applied to a study of interactions between mothers and strangers with infants seen from one to eight months of age. This study revealed a surprising developmental pattern of differentiation between mother and stranger, with an unexpected period of positive responsiveness to strangers occurring at five months of age. The sequential analysis of interactions revealed evidence of a progressive development of infant receptivity to gaze, and also an exploratory analysis of receptivity to adult smiles and vocalizations suggested infants may respond to these adult behaviours. Subsequently the results of these studies are linked to other recent research.

This investigation consisted of field-descriptive and experimental analysis of young children's verbal behaviour, aiming at the identification and description of parental verbal stimulation and assessment of reinforcement variables. Five 21-month-old children and their respective mothers participated in the field-descriptive study. Observational sessions were carried out at the subjects' home and in a playroom; the situation was one of free-play. Verbal behaviour was taped; non-verbal behaviour was recorded according to selected categories. The audio-tapes were submitted to a technique designed to record the kind and frequency of utterances and the temporal interval between them. The interactive sequences of mother-child utterances were analysed with regard to these three aspects. Indices were computed to describe the characteristics of the patterns of interaction with regard to maternal verbal behaviour and to the child's verbal performance. The results indicated relationships among the categories aid descriptive indices of maternal behaviour and the child's speech: 1) the frequency of the child's verbalisations did not relate to the total amount of maternal verbal output in itself but to the mother's utterances which consisted of a direct response, within 4 sec, to the child's previous utterance; 2) the child's usage of speech correlated with the degree in which the mother responded selectively to the child's utterances; 3) the mother presented different verbal responses as consequences to the child's utterances, which had significant differential effects on the child's verbal performance as related to initiation, maintenance and ending of verbal chains of interaction. Two out of these five children participated in the experimental study which tested the effectiveness of 'repetition' (plus praise and/or the subject's name) as compared with the effectiveness of a material reinforcer (a small toy) on the emission of "correct utterances" as opposed to "incorrect utterances". The verbal reinforcer was delivered by a 'talking clown' and the material reinforcer by feeder. The results indicated that the verbal reinforcer was relatively more effective in controlling the subjects' rate of 'correct' verbal responses. When reinforcement was delayed the main effect observed was the decrease of rate of responses during the verbal periods to a level similar to that observed during the periods of contingent material reinforcement. The results were discussed within a reinforcement theory framework, and suggestions concerning certain methodological requirements to analyse parental stimulation in relation to children's language development were presented.

The regulation of protein biosynthesis at fertilisation was studied in North Sea Plaice (Pleuronectus platessa). RNA and protein synthesis were studied in fertilised and unfertilised plaice eggs. There was no detectable RNA synthesis in the egg in early embryonic stages, but protein synthesis at all stages of development. However, using RITA synthesis inhibitors (actinomycin D and ethidium bromide), it was demonstrated that certain groups of proteins were synthesised independently of any newly synthesised messenger RNA. Different rates of protein synthesis in various subcellular fractions from both fertilised and unfertilised eggs were demonstrated. Proteins precipitated by vinblastine were shown to exhibit the most changes between the egg and fertilised egg. These proteins were further characterized on several polyacrylamide-gel electrophoresis systems. RNA was isolated from the polysomes and the supernatant fractions of eggs and fertilised eggs. This RITA was tested for messenger RITA activity in a rabbit reticulocyte cell-free system and also in a wheat germ in vitro system. Messenger RNA activity was demonstrated in both the polysomal and supernatant fractions from the egg and also from the fertilised egg, and was purified by affinity chromatography and sucrose gradient centrifugation. Regulatory mechanisms have been demonstrated at the translational level of protein synthesis in early plaice embryogenesis.