Cancer is a set of varied and diverse diseases that share common characteristics, such as active proliferation, increased replicative potential, and tissue invasion or metastasis. One protein, 14-3-3ζ, is shown to be upregulated in a number of different cancers and also correlates with poor patient prognosis, recurrence, and mortality. This protein comes from a family of adapter proteins known for their scaffolding ability, pro- and anti-oncogenic capabilities, and affinity for phosphorylated substrates. It has been shown previously to participate in cancer progression, subversion of apoptosis, and to increase chemoresistance. Herein we will discuss the ability of 14-3-3ζ to promote distant-site metastasis and we propose that it does so through a variety of different mechanisms including the MAPK signaling cascade, HER2/ErbB2 pathway, and by mediation of cell adhesion through regulation of LAR. Liprin-β was identified as a novel 14-3-3ζ interactor in a mass spectrometry-based interactomics analysis. 14-3-3ζ was found to co-immunoprecipitate with both Liprin-α and Liprin-β. We will discuss the identification and mutation of putative 14-3-3ζ binding sites on both Liprins, the effect these have on the binding of both Liprins to 14-3-3ζ and of Liprin-α to LAR, and the possible downstream consequences of these interactions. The results described herein are inconclusive due in part to our inability to obtain a reliable Liprin-β pulldown and in part our inability to identify the 14-3-3ζ-binding sites on Liprin-α and Liprin-β. The concluding chapter contains a discussion of the possible future directions, including the creation of further Liprin mutants as well as fluorescent imaging of LAR localization and focal adhesion turnover.

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Synaptic plasticity describes efficacy changes in synaptic transmission and ranges in duration from tens to hundreds of milliseconds (short-term), to hours and days (long-term). Short-term plasticity plays crucial roles in synaptic computation, information processing, learning, working and short-term memory as well as its dysfunction in psychiatric and neurodegenerative diseases. The main aim of my PhD thesis was to determine the molecular mechanisms of different forms of presynaptic plasticity. Short-term facilitation increases neurotransmitter release in response to a high-frequency pair (paired-pulse facilitation; PPF) or train (train facilitation; TF) of presynaptic stimuli. Synaptotagmin 7 (Syt7) has been shown to act as residual calcium (Ca$_{res}$) sensor for PPF and TF at various synapses. Syt7 also seems to be involved in recovery from depression, whereas its role in neurotransmission remains controversial. My aim was to express Syt7 in a synapse where it is not normally found and determine how it affects short-term synaptic plasticity. Immunohistochemistry indicated that Syt7 is not localized to cerebellar climbing fibers (CFs). Wild-type (WT) and Syt7 knockout (KO) recordings at CF to Purkinje cell (CF-PC) synapses established that at near-physiological external calcium (Ca$_{ext}$) levels both genotypes displayed similar recovery from paired-pulse depression. In low Ca$_{ext}$,WT CF-PC synapses showed robust PPF, which turned out to be independent of Syt7. All my experiments strongly suggested that WT CFs do not express native Syt7, but display low Ca$_{ext}$ CF-PC PPF and TF. Thus, channelrhodopsin-2 and Syt7 were bicistronically expressed via AAV9 virus in CFs. This ectopic Syt7 expression in CFs led to big increases in low-Ca$_{ext}$ CF-PC facilitation, more than doubling PPF and more than tripling TF. While overexpression of Syt7 might turn out to have an effect on the initial release probability (pr), the observed CF-PC facilitation increase still critically depended on presynaptic Syt7 expression. And when comparing only cells in a defined EPSC1 amplitude range, the Syt7-induced increase in low-Ca$_{ext}$ PPF could not be accounted for by changes in initial pr, suggesting a general role for Syt7 as calcium sensor for facilitation. Another form of short-term plasticity, post-tetanic potentiation (PTP), is believed to be mediated presynaptically by calcium-dependent protein kinase C (PKC) isoforms that phosphorylate Munc18-1 proteins. It is unknown how generally applicable this mechanism is throughout the brain and if other proteins might be able to modulate PTP. Combining genetic (PKCαβy triple knockout [TKO] and Munc18-1SA knock-in [Munc18 KI] mice, in which Munc18- 1 cannot get phosphorylated) with pharmacological tools (PKC inhibitor GF109203), helped us show that PTP at the cerebellar parallel fiber to Purkinje cell (PF-PC) synapse seems to depend on PKCs but seems mostly independent of Munc18-1 phosphorylation. In addition, compared to WT animals, genetic elimination of presynaptic active zone protein Liprin-α3 led to similar PF-PC PTP and paired-pulse ratios (PPRs). At the hippocampal CA3-CA1 synapse previous pharmacological studies suggested that PKC mediates PTP. A genetic approach helped to show that calcium dependent PKCs do not seem to be required for CA3-CA1 PTP. Pharmacologically inhibiting protein kinase A as well as genetically eliminating Syt7 also had no effect on CA3-CA1 PTP. In addition, Ca IM-AA mutant mice, in which Ca$_{v}$2.1 channels have a mutated IQ-like motif (IM) so that it cannot get bound by calcium sensor proteins any more, not only displayed regular PTP, but also normal PPF and TF at CA3-CA1 synapses. In conclusion, my PhD thesis helped further characterize different forms of presynaptic plasticity, underlined that short-term synaptic plasticity can be achieved through diverse mechanisms across the Mammalian brain and supported a potentially general role for synaptotagmin 7 acting as residual calcium sensor for facilitation.