Luciano Gonçalves Grácio

Abstract
Regulated exocytosis consists of the fusion between vesicles and the plasma membranes, leading to the formation of a narrow fusion pore through which secretions exit the vesicle lumen into the extracellular space. An increase in the cytosolic concentration of free Ca2+ ([Ca2+](i)) is considered the stimulus of this process. However, whether this mechanism can be preserved in a simplified system of membrane lawns with docked secretory vesicles, devoid of cellular components, is poorly understood. Here, we studied peptide discharge from individual secretory vesicles docked at the plasma membrane, prepared from primary endocrine pituitary cells (the lactotrophs), releasing hormone prolactin. To label secretory vesicles, we transfected lactotrophs to express the fluorescent atrial natriuretic peptide (ANP.emd), previously shown to be expressed in and released from prolactin-containing vesicles. We used stimulating solutions containing different [Ca2+] to evoke vesicle peptide discharge, which appeared similar in membrane lawns and in intact stimulated lactotrophs. All vesicles examined discharged peptides in a subquantal manner, either exhibiting a unitary or sequential time course. In the membrane lawns, the unitary vesicle peptide discharge was predominant and slightly slower than that recorded in intact cells, but with a shorter delay with respect to the stimulation onset. This study revealed directly that Ca2+ triggers peptide discharge from docked single vesicles in the membrane lawns with a half-maximal response of similar to 8 mu M [Ca2+], consistent with previous whole-cell patch-clamp studies in endocrine cells where the rapid component of exocytosis, interpreted to represent docked vesicles, was fully activated at <10 mu M [Ca2+]. Interestingly, the sequential subquantal peptide vesicle discharge indicates that fluctuations between constricted and dilated fusion pore states are preserved in membrane lawns and that fusion pore regulation appears to be an autonomously controlled process.

Abstract
Counting subgraph occurrences in complex networks is an important analytical task with applicability in a multitude of domains such as sociology, biology and medicine. This task is a fundamental primitive for concepts such as motifs and graphlet degree distributions. However, there is a lack of online algorithms for computing and updating subgraph counts in dynamic networks. Some of these networks exist as a streaming of edge additions and deletions that are registered as they occur in the real world. In this paper we introduce StreamFaSE, an efficient online algorithm for keeping track of exact subgraph counts in dynamic networks, and we explain in detail our approach, showcasing its general applicability in different network scenarios. We tested our method on a set of diverse real directed and undirected network streams, showing that we are always faster than the current existing methods for this task, achieving several orders of magnitude speedup when compared to a state-of-art baseline.