Thelohania solenopsae are intracellular pathogens found in the red imported fire ant, Solenopsis invicta. These pathogens cause detrimental effects to their fire ant hosts. The present study revealed that the midgut and the meconium materials from pupating fourth instar larvae were possible vehicles for the horizontal transmission of the disease. The pathogen was further found to cause a reduction of humeral proteins. In SDS-PAGE stained with silver, several proteins were observed only in controls but not in infected fire ant queens. Different queens were found to have variable proteins reduced due to infection of this pathogen. Furthermore, vitellogenin titers were found to be significantly reduced in infected fire ant queens, although the infection rates of the fat body cells was found to be less than 20%. Finally, although the pathogens did not directly induce apoptosis in infected cells, there were more infected cells undergoing apoptosis than uninfected cells. There was no evidence to support the idea that infected fat body cells became more resistant to apoptosis inducers.

Stroke is the fifth leading cause of death in the United States and a major cause of long-term disability in industrialized countries. The core region of an ischemic stroke dies within minutes due to activation of necrotic pathways. Outside of this core region is the penumbral zone, where some perfusion is maintained via collateral arteries. Delayed cell death occurs in this area due to the triggering of apoptotic mechanisms, which expands the ischemic injury over time. The cellular and molecular events that produce the expansion of the ischemic core continue to be poorly understood. The increases in the amyloid precursor protein and pathogenic secretases lead to the increase in amyloid-β (Aβ) production. The relatively small amount of research in this area has hampered development of stroke therapy designed to prevent neuronal and glial cell degeneration in the penumbra. Currently, there is a significant lack of therapeutic options for acute ischemic stroke, and no drug has been approved for treating patients at delayed time points (≥ 4.5 hr post-stroke). Afobazole, an anxiolytic currently used clinically in Russia, has been shown to reduce neuronal and glial cell injury in vitro following ischemia, both of which have been shown to play important roles following an ischemic stroke. Treatment with afobazole decreased microglial activation in response to ATP and Aβ, as indicated by reduced membrane ruffling and cell migration. Prolonged exposure of microglia to ischemia or Aβ conditions resulted in glial cell death that was associated with increased expression of the pro-apoptotic protein, Bax, the death protease, caspase-3 and a reduced expression in Bcl-2. Co-application of afobazole decreased the number of cells expressing both Bax and caspase-3, while increasing the cells expressing Bcl-2 resulting in a concomitant enhancement in cell survival. While afobazole inhibited activation of microglia cells by Aβ25-35, it preserved normal functional responses in these cells following exposure to the amyloid peptide. Intracellular calcium increases induced by ATP were depressed in microglia after 24 hr exposure to Aβ25-35. However, co-incubation with afobazole returned these responses to near control levels. Therefore, stimulation of sigma-1 and sigma-2 receptors by afobazole prevents Aβ25-35 activation of microglia and inhibits Aβ25-35-associated cytotoxicity. Examining the molecular mechanisms involved in the increased neuronal survival demonstrates that ischemia or Aβ results in an increased expression of the pro-apoptotic protein Bax and the death protease caspase-3, while at the same time decreasing expression of the anti-apoptotic protein, Bcl-2. However, unlike observations made with microglia, afobazole was unable to modulate this ischemia-induced expression, but was able to modulate Aβ-induced expression of apoptotic proteins while still rescuing neurons from death. Additional experiments were carried out to understand this disparity between the failures of afobazole to prevent the up-regulation of pro-apoptotic genes while retaining the ability to mitigate neuronal death. Although the neurons were still alive they were in a senescent state and were unresponsive to depolarization by high K+. However, these findings are still positive due to the ability of afobazole to delay neuron death, thus minimalizing the toxic environment of the penumbra. These comorbidities of ischemia and Aβ toxicity may lead to potentiated responses and increase the risk for various vascular dementias. It was of particular interest to study how the convergence of ischemia, acidosis and Aβ influence cellular activity and survival within core and penumbral regions. Application of Aβ increased the [Ca2+]i overload produced by concurrent ischemia + acidosis application in isolated cortical neurons. We found that the acid-sensing ion channels 1a (ASIC1a) are involved in the potentiation of [Ca2+]i overload induced by Aβ. Furthermore, afobazole (100 uM) abolished Aβ potentiation of ischemia + acidosis evoked [Ca2+]i overload, which may represent a therapeutic strategy for mitigating injury produced by Aβ and stroke.

Francisella tularensis is a zoonotic bacterium that must exist in diverse environments ranging from arthropod vectors to mammalian hosts. To better understand how genes are regulated in these different environments, a transcriptional response- regulator gene (genome locus FTL0552) was deleted in F. tularensis live vaccine strain (LVS). The FTL0552 deletion mutant exhibited slightly reduced rates of extracellular growth but was unable to replicate or survive in mouse macrophages and was avirulent in the mouse model using either BALB/c or C57BL/6 mice. Mice infected with the FTL0552 mutant produced reduced levels of inflammatory cytokines, exhibited reduced histopathology and cleared the bacteria quicker than mice infected with LVS. Mice that survived infection with the FTL0552 mutant were afforded partial protection when challenged with a lethal dose of the virulent Schu S4 strain (4 of 10 survivors, day 21 post infection) when compared to naïve mice (0 of 10 survivors by day 7 post infection). Microarray experiments indicate that 148 genes are regulated in the FTL0552 mutant. Most of the genes are down regulated, indicating that FTL0552 controls transcription of genes in a positive manner. The list of down regulated genes includes genes located within the Francisella Pathogenicity Island (FPI) that are essential for intracellular survival and virulence of Francisella tularensis. Furthermore, a mutant in FTL0552 or the comparable locus in Schu S4 (FTT1557c) may be an alternative candidate vaccine for tularemia.

<p>Auditory sensations can arise from objects in our environment or from our own actions, such as when we speak or make music. We must able to distinguish such sources of sounds, as well as form new associations between our actions and the sounds they produce. The brain is thought to accomplish this by conveying copies of the motor command, termed corollary discharge signals, to auditory processing brain regions, where they can suppress the auditory consequences of our own actions. Despite the importance of such transformations in health and disease, little is known about the mechanisms underlying corollary discharge in the mammalian auditory system. Using a range of techniques to identify, monitor, and manipulate neuronal circuits, I characterized a synaptic and circuit basis for corollary discharge in the mouse auditory cortex. The major contribution of my studies was to identify and characterize a long-range projection from motor cortex that is responsible for suppressing auditory cortical output during movements by activating local inhibitory interneurons. I used similar techniques to understand how this circuit is embedded within a broader neuromodulatory brain network important for learning and plasticity. These findings characterize the synaptic and circuit mechanisms underlying corollary discharge in mammalian auditory cortex, as well as uncover a broad network interaction potentially used to pattern neural associations between our actions and the sounds they produce.</p> / Dissertation

The protein Hedgehog (Hh) is a highly conserved, secreted ligand (and morphogen) capable of patterning many different tissues during development. Recently, Sonic Hedgehog (SHH) a human homolog of Drosophila Hh was found to be a causative agent in certain cancers. While several drugs are being developed to combat the binding of SHH to its receptor Patched or the Patched-target Smoothened, very little is known about how SHH is secreted from the producing cell, another site for therapeutic targeting. We report here the characterization of a novel proteolytic event and genetic pathway that controls Hh intracellular sorting and axon transport using the Drosophila eye imaginal disc as our model system. In fly larval photoreceptor neurons the developmental signal Hh is guided to the apical (retina) and basal (growth cone, GC) ends where secretion of the morphogen is an inductive factor in photoreceptor differentiation and establishment of eye/brain neural connections. The Hh secreted from the basal side induces lamina development while Hh secreted at the retina induces ommatidial development. Hedgehog processing consists of autocleavage from its 46 kDa form (HhU) to become a lipid-modified N-terminal signaling molecule (HhN; 19kDa) and a C-terminal molecule (HhC24; 24 kDa). Following autocleavage, a fraction of the C-terminal auto-cleavage product then undergoes a second cleavage event leading to 16 kDa (HhC16) and 9 kDa products. Nothing is known about the significance of the C-terminal “2nd cleavage” other than its occurrence in both fly and human tissue. In an effort to identify regulators of Hh sorting, we discovered that the HhC “2nd cleavage” is a determining factor in the sorting of the HhN signaling domain. That is, if a cell induces more cleavage (more HhC16) we observe more HhN in the apical domain. Likewise, if a cell inhibits 2nd cleavage (less HhC16) we see more basal HhN. Creation of a “2nd cleavage mutant” shows that this process has developmental significance. Further, biochemical characterization of the 2nd cleavage suggests it occurs in the ER after autocleavage and that HhC24 can exit the cell in a Golgi independent manner (via lipid droplets) while HhC16 remains intracellular. The ER exit of HhC24 appears to be controlled by a conserved PP2A (Mts) /PKB (Akt) kinase pathway which potentially regulates the size and number of lipid droplets produced. These findings are an important first step in understanding the intracellular sorting and transport of Hh and highlight new targets for the treatment of SHH-related cancers. The discovery of divergent modes of Hh secretion and the “2nd cleavage” open novel avenues for Hh research by offering an alternative, and very direct, line of attack in the treatment of Hh-related cancer.

Understanding microscale biological processes as cells develop into tissues is one of the most important, yet most difficult, problems in modern biology. Cells encounter a dynamic chemical and physical environment and delineating the myriad of variables proves daunting with even the most sophisticated experiments. This dissertation focuses on the development and application of unique enabling technologies designed to sample and control biological microenvironments. By developing two approaches – one aimed at intracellular biochemistry and another for extracellular targets – based on photochemistry and optical force generation, research presented here will allow new areas of subcellular dynamics to be addressed. On the intracellular side, enzyme-immobilized polymeric microspheres or enzyme microstructures are placed into the cell cytosol via optical tweezers for sustained and localized chemical modification of the intracellular environment. This approach is complemented by the use of extracellular guidance barriers formed from photo-induced crosslinking of proteins. Through the use of minimally toxic photosensitizers and femtosecond (fs) near infrared (NIR) light, it is possible to fabricate three-dimensional protein structures in a living cell’s environment. Moreover, this work explores the ability to form protein structures with enzymatic activity as well as with high aspect-ratio features at micron resolution. Finally, the photochemical transformation of serotonin into a highly fluorescent visible photoproduct is investigated as a means to overcome problems associated with sample size in neurotransmitter detection during synaptic chemical signaling. Optimization of this multiphoton process entails understanding the mechanism by which the photoproduct is created and experiments towards this goal are presented here. Ultimately, the precision and flexibility of these technologies will allow access to new areas of the biosciences. / text

We demonstrate intracellular delivery of various molecules by inducing controlled and reversible cell damage through pulsed laser irradiation of carbon black (CB) nanoparticles. We then characterized and optimized the system for maximal uptake and minimal loss of viability. At our optimal condition 88% of cells exhibited uptake with almost no loss of viability. In other more intense cases it was shown that cell death could be prevented through addition of poloxamer. The underlying mechanism of action is also studied and our hypothesis is that the laser heats the CB leading to thermal expansion, vapor formation and/or chemical reaction leading to generation of acoustic waves and then there is energy transduction to the cell causing poration of the cell membrane. We also delivered anti-EGFR siRNA to ovarian cancer cells. Cells exposed to a laser at 18.75 mJ/cm2 for 7 minutes resulted in a 49% knockdown of EGFR compared to negative control. We established an alternative way to deliver siRNA to knockdown proteins, for the first time using laser CB interaction.

Cystic fibrosis (CF) is an inborn, hereditary disease, due to mutations in the gene for a cAMP-activated chloride (Cl-) channel, the cystic fibrosis transmembrane conductance regulator (CFTR). As a result of impaired ion and water transport,the airway mucus is abnormally viscous, which leads to bacterial colonization.Recurrent infections and inflammation result in obstructive pulmonary disease.Similar changes in the pancreas lead to pancreatic insufficiency.Several compounds have been tested to improve transepithelial ion transport in CF patients, either via activation of the mutant CFTR, or via stimulation of alternative chloride channels. The main purpose of this thesis was to find substances that might correct the defective ion transport in epithelial cells in CFand could be useful for the pharmacological treatment of CF patients. Long-term treatment with the macrolide antibiotic azithromycin (AZM)improved clinical parameters and lung function in CF patients and increased Cl- transport in CF bronchial epithelial cells (CFBE) (Paper I); although mRNA expression of the CFTR gene remained unchanged.In contrast, pre-exposure to the mucolytic antioxidant N-acetylcysteine (NAC) increased CFTR protein expression and was associated with increased Cl- efflux from CFBE cells (Paper II). Clinical trials of this substance might be warranted. Duramycin has been the subject of clinical trials that finished in June2009. Up till now, no results from this study are available. The effect of this substance on Cl- efflux from three CF and three non-CF cell lines (Paper III) was disappointing. An effect was found only in CFBE cells, the effect was minimal, occurred in a narrow concentration range, and was not associated with an increase in the intracellular calcium concentration [Ca2+]i. The fact that NO-donors stimulated Cl- efflux from CFBE cells (but did notchange [Ca2+]i) after several hours of preincubation suggests that these substances may be a potentially interesting group of compounds for the treatment of CF (Paper IV). A model for the effect of NO-donors on Cl- efflux is presented. / Cystisk fibros (CF) är en medfödd, ärftlig, sjukdom, som förorsakas av en mutation i en gen som innehåller koden för en kloridkanal som aktiveras av cykliskt AMP (cystic fibrosis transmembrane conductance regulator, CFTR). Som en följd av otillräcklig transport av joner och vatten är slemmet i luftvägarna onormalt segt, vilket leder till att det koloniseras av bakterier. Upprepade infektioneroch inflammation av luftvägarna leder slutligen till obstruktiv lungsjukdom.Liknande förändringar i bukspottkörteln leder till att också detta organ inte fungerar. Flera kemiska ämnen har testats för sin förmåga att förbättra jontransporten över epitelet hos CF-patienter. Detta skulle kunna göras antingen genom aktivering av det muterade CFTR-proteinet, eller genom stimulering av alternativa kloridkanaler. Huvudsyftet med den forskning som beskrivs i denna avhandling var att hitta kemiska substanser som skulle kunna korrigera den defekta jontransporten i epitelceller hos CF-patienter, och därför vara nyttiga för behandlingen av patienterna. Behandling under längre tid med azithromycin (AZM), ett makrolidantibiotikum,förbättrade CF-patienternas kliniska status och lungfunktion,samt ökade kloridutflödet från CF bronkialepitelceller (CFBE-celler) (Arbete I).Däremot ändrades inte uttrycket av mRNA för CFTR-genen. I kontrast till detta ökade uttrycket av CFTR-proteinet om CFBE-cellerna utsattes för den slemlösande anti-oxidanten N-acetylcystein (NAC), vilket ledde till ökat kloridutflöde från denna cellinje (Arbete II). Det vore rimligt att utföra kliniska prövningar av detta ämne. Duramycin har testats i kliniska prov som slutade i juni 2009, men några resultatfrån dessa prov har inte offentliggjorts än. Effekten av detta ämne på kloridutflödet från tre CF-cellinjer och tre icke-CF cellinjer (Arbete III) var en besvikelse. Duramycin hade endast effekt på CFBE-celler, effekten var mycket liten, förekom endast i ett litet koncentrationsområde av duramycin, och var inte kopplad till en ökning av den intracellulära kalciumkoncentrationen [Ca2+]i. Att ämnen som avger kväveoxid (NO) stimulerade kloridutflödet från CFceller (men inte påverkade [Ca2+]i) efter några timmar, visar att denna grupp av ämnen kan vara potentiellt intressant för behandlingen av CF (arbete IV). En modell för effekten av NO på kloridtransporten i CF-celler presenteras.

No description available.

This thesis uses methods and models from non-equilibrium statistical physics to describe intracellular processes. Bidirectional microtubule-based transport within axons is modeled as a quasi-one-dimensional stochastic lattice gas with two particle species moving in opposite directions under mutual exclusion interaction. Generically occurring clusters of particles in current models for intracellular transport can be dissolved by additionally considering the dynamics of the transport lattice, i.e., the microtubule. An idealized model for the lattice dynamics is used to create a phase transition toward a homogenous state with efficient transport in both directions. In the thermodynamic limit, a steady state property of the dynamic lattice limits the maximal size of clusters. Lane formation mechanisms which are due to specific particle-particle interactions turn out to be very sensitive to the model assumptions. Furthermore, even if some particle-particle interaction is considered, taking the lattice dynamics into account almost always improves transport. Thus the lattice dynamics seems to be the key aspect in understanding how nature regulates intracellular traffic. The last part introduces a model for the dynamics of a microtubule which is limited in its growth by the cell boundary. The action of a rescue-enhancing protein which is added to the growing tip of a microtubule and then slowly dissociates leads to interesting aging effects which should be experimentally observable.