No description available.

No description available.

No description available.

No description available.

No description available.

The cytoskeleton is a dynamic network of microtubules constantly being reorganized to meet the spatiotemporal demands of the cell. Microtubules are organized into subcellular highways to control cell processes such as cell division, cargo transport, and neuronal development and maintenance. Reorganization of this intricate network is tightly regulated by various stabilizing and destabilizing microtubule-associated proteins that decorate the network. Katanin p60 is a microtubule destabilizing enzyme from the ATPases Associated with various Activities (AAA+) family. It can both sever and depolymerize microtubules. In order to sever microtubules, katanin recognizes the tubulin carboxy-terminal tails (CTTs) and hydrolyzes ATP. Using super-resolution microscopy and image analysis, we find that the tubulin CTTs are not required for katanin to depolymerize microtubules. We also characterize the regulation of microtubule severing and depolymerization by katanin in various nucleotide states. A better understanding of how CTTs and nucleotides regulate microtubule severing and depolymerization by katanin will help future research aimed to correct katanin activity when these processes goes awry as in improper chromosome segregation during mitosis or loss of microtubule integrity in neuronal diseases.

Mitosis, or cell division, is the mechanism by which cells divide and is an intricate process requiring the action and control of numerous proteins. Such proteins serve either as structural entities within the mitotic spindle, or perform the "work" within the apparatus. In particular, Kinesin-5 motor proteins, a subset within the kinesin motor protein superfamily, are primarily responsible for organization of microtubules (MTs) within the mitotic apparatus, and are consequently vital for efficient mitosis. These proteins utilize energy from ATP hydrolysis in order to "walk" along antiparallel MTs, positioning them into the bipolar mitotic spindle. Loss of Kinesin-5 activity results in formation of a monoastral spindle and subsequent cell cycle arrest. Recently, a wide variety of small molecules have been identified that possess the ability to inhibit certain Kinesin-5 motors. Such compounds, including monastrol (the first Kinesin-5 inhibitor identified), have been employed to study Kinesin-5 activity. A thorough understanding of Kinesin-5 function, combined with the ability to specifically target these proteins with small molecules, may provide the capability to control cell division and may therefore have significant implications in anti-cancer therapies. The following dissertation describes research that utilizes small molecules to probe the function (ATPase activity and MT interactions) of various Kinesin-5 proteins and provides information that will lead to a better understanding of exactly how such proteins function in vivo. Further, a greater knowledge of Kinesin-5 protein activity as well as specific interactions with small-molecule compounds, may lead to the development of more potent, less toxic anti-cancer drugs. / Ph. D.

Mosquitoes affect human health worldwide as a result of their ability to vector multiple diseases. Mosquitocide resistance is a serious public health challenge that warrants the development of improved chemical control strategies for mosquitoes. Previous studies demonstrate the mosquito blood-brain barrier (BBB) to interfere with the target-site delivery and action of anticholinesterase chemistries. The ATP-binding cassette (ABC) transporters are efflux proteins that assist in maintaining the BBB interface and serve as a first line of defense to mosquitocide exposures. To date, there are three subfamilies (ABC -B, -C, -G) of ABC transporters; however, knowledge of these chemistries interacting with mosquito ABC transporter(s) is limited. Here, I report that tacrine and bis(7)-tacrine are relative non-toxic anticholinesterases at solubility limits; however, the addition of verapamil enhances toxicity of both tacrine and bis(7)-tacrine to mosquitoes. Verapamil significantly increases the mortality of mosquitoes exposed to tacrine and bis(7)-tacrine compared to the tacrine- and bis(7)- tacrine-only treatments. Tacrine and bis(7)-tacrine reduce acetylcholinesterase activity in mosquito head preparations compared to the untreated mosquitoes; however, the addition of verapamil significantly increases the anticholinesterase activity of tacrine and bis(7)-tacrine compared to the tacrine-and bis(7)-tacrine-only treatments. Tacrine and bis(7)-tacrine increase ATPase activity in Aedes aegypti at lower concentrations compared to that of verapamil (Fig. 3). The differential increase in ATPase activity suggests that tacrine and bis(7)-tacrine are more suitable substrates for ABC transporter(s) compared to verapamil and, thus, provides putative evidence that ABC transporter(s) is a pharmacological obstacle to the delivery of these anticholinesterases to their intended target site. / Master of Science in Life Sciences

The type IV pilus (T4P) is a dynamic long thin fiber found on the surface of many bacterial groups. T4P is a versatile nanomachine; it plays many important roles such as for surface attachment, virulence factor, and surface motility apparatus. This research focuses on understanding the kinetics of PilB, the T4P assembly ATPase. PilB crystal structure exhibits an elongated hexamer with 2-fold symmetry indicating a symmetric rotary mechanism model. Except for its structure, the symmetric rotary mechanism of PilB has not been demonstrated experimentally. Its conformation and relatively low activity constrained previous in vitro studies of PilB. This study identified PilB from thermophilic organism Chloracidobacterium thermophilum (Ct) to be a model for in vitro studies. An active CtPilB was successfully expressed and purified as a hexamer. Malachite green phosphate assay was used to examine CtPilB ATPase activity. The examination indicated that CtPilB is a robust ATPase with a complex kinetics profile. The profile has a stepwise incline in ATPase activity as a function of [ATP] that led to a decline in higher [ATP]. The decline was confirmed to be a substrate inhibition by the enzyme-coupled assay. As for the incline, the detailed mechanism is still less clear to explain the multiphasic profile. The overall incline did not conform with classical Michaelis-Menten kinetic but the first part of the incline was shown to conform with Michaelis-Menten kinetics. The complex kinetics profile of PilB is consistent with the symmetric rotary mechanism of catalysis. / Master of Science / This research was conducted to understand type IV pilus (T4P), a hair-like structure found on the surface of many bacteria groups. T4P is a versatile structure; it plays many vital roles in bacterial life such as in surface motility, surface attachment, gene transfer, and virulence factor. Pilus is a dynamic polymer composed of many small pilin proteins that can be assembled or disassembled. Structurally, pilus is supported by machinery that helps to extend and retract pilus by adding or removing pilin proteins. At the core of the machinery, two different proteins are responsible to power the assemble and disassemble process by converting the chemical energy in ATP into mechanical energy. This study focuses on the protein that powers pilus assembly, PilB. Understanding PilB will be very beneficial in elucidating how the strongest biological motor work in action. The structure of PilB was determined to be a hexamer consist of six identical copies of the same protein forming a ring structure with 2-fold symmetry. This structure suggests that PilB works using symmetric rotary mechanism. Previous studies of PilB have not been productive because the purified PilB did not behave well during the assay. In this study, PilB from Chloracidobacterium thermophilum (CtPilB) was determined to be a reasonable model for the study. CtPilB was successfully purified and it was identified to have a robust activity outside the cell allowing for further biochemistry studies. The profile of CtPilB kinetics was unique and it did not conform with the classical kinetic profile. The analysis of the profile suggests that CtPilB exhibit a complex mechanism in hydrolyzing ATP.

O Trypanosoma cruzi expressa um grupo de glicoprotcinas de superfície, denominadas Tc-85, que pertencem à superfumília gêmca das gp85/traus-sialidases. Nosso laboratório clonou e caracterizou um membro da fumília Tc85 (Tc85-11), cuja região carboxila tenninal (clone Tc85-1) adere em laminina e em células de mamífero. Usando peptídeos sintéticos, correspondendo em seqüência à Tc85-1, caracterizou-se o motivo mais conservado da superfamilia gênica das gp85/trans-sialidases (VTVxNVFLYNR), o qual não adere em laminina. Esse motivo foi chamado peptídeo J. Por cromatografia de extratos de membrana de cardiomiócitos em coluna de afmidade contendo peptídeo J, foi isolada uma molécula de 30kDa identificada como sendo a subunidade β3 da Na+, K+ ATPase. A porção extracelular da subunidade β3 da Na+, K+ ATPase foi clonada e a interação in vitro desta proteína com peptídeo J foi observada. Deste modo, é sugerido aqui que a subunidade β3 da Na+, K+ ATPase pode ter um papel importante na interação do parasita com a célula hospedeira. / Abstract not available.