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Gene therapy targeting the spinal cord is an important tool for analyzing mechanisms of nervous system diseases and the development of gene therapies. Analogous to a lumbar puncture in humans, the rodent spinal cord can be accessed through an efficient, noninvasive injection. Here we describe a method for AAV-mediated gene transfer to cells of the spinal cord by intrathecal injection of small quantities of AAV vector.

Gene therapy targeting the spinal cord is an important tool for analyzing mechanisms of nervous system diseases and the development of gene therapies. Analogous to a lumbar puncture in humans, the rodent spinal cord can be accessed through an efficient, noninvasive injection. Here we describe a method for AAV-mediated gene transfer to cells of the spinal cord by intrathecal injection of small quantities of AAV vector.

Neuroplasticity in the dorsal horn following peripheral nerve damage contributes critically to the establishment of chronic pain. The neurosecretory protein VGF (non-acronymic) is rapidly and robustly upregulated following nerve injury, and therefore peptides generated from it are positioned to serve as signals for peripheral damage. The goal of this project was to understand the spinal modulatory effects of the C-terminal VGF derived peptide TLQP-62 at the cellular level and gain insight into the function of the peptide in the development of neuropathic pain. In a rodent model of neuropathic pain, we demonstrate that endogenous levels of TLQP-62 increased in spinal cord, and its immunoneutralization led to prolonged attenuation of the development of nerve injury-induced hypersensitivity. Using multiphoton imaging of submaximal glutamate-induced Ca(2+) responses in spinal cord slices, we demonstrate the ability of TLQP-62 to potentiate glutamatergic responses in the dorsal horn. We further demonstrate that the peptide selectively potentiates responses of high threshold spinal neurons to mechanical stimuli in singe-unit in vivo recordings. These findings are consistent with a function of TLQP-62 in spinal plasticity that may contribute to central sensitization following nerve damage.

A 70-year-old man was admitted to our hospital for a penile circumcision due to phimosis using continuous dose spinal anaesthesia. On postoperative day 10 he came to the emergency department with a superficial abscess localised at the injection site of the spinal catheter. He was treated with intravenous antibiotics for 10 days, and the superficial abscess was incised and drained. Ten days later, the patient was readmitted to the emergency department with complaints of back pain and fever. A repeat MRI scan of his lumbar sacral area was done and showed epidural abscesses without any compression of the medulla or the myelum. The patient did not have any signs of spinal cord or nerve root compression at that time. He was treated with intravenous antibiotics with resolution of symptoms.

Food-borne infection with enterohemorrhagicEscherichia coli(EHEC) is a major cause of diarrheal illness in humans, and can lead to severe complications such as hemolytic uremic syndrome. Cattle and other ruminants are the main reservoir of EHEC, which enters the food-chain through contaminated meat, dairy, or vegetables. However, how EHEC transitions from the transmission vector to colonizing the intestinal tract, and how virulence-specific genes are regulated during this transition, is not well understood. Here, we describe the establishment of a vertebrate model for food-borne EHEC infection, using the protozoanParamecium caudatumas a vector and the zebrafish (Danio rerio) as a host. At 4 days post fertilization, zebrafish have a fully developed intestinal tract, yet are fully transparent. This allows us to follow intestinal colonization, microbe-host cell interactions, and microbial gene induction within the live host and in real time throughout the infection. Additionally, this model can be adapted to compare food- and water-borne infections, under gnotobiotic conditions or against the backdrop of an endogenous (and variable) host microbiota. Finally, the zebrafish allows for investigation of factors affecting shedding and transmission of bacteria to naïve hosts. High-resolution analysis of EHEC gene expression within the zebrafish host emphasizes the need for tight transcriptional regulation of virulence factors for within-host fitness.IMPORTANCEEnterohemorrhagicEscherichia coli(EHEC) is a food-borne pathogen which can cause diarrhea, vomiting and in some cases, severe complications such as kidney problems in humans. Up to 30% of cattle are colonized with EHEC, which can enter the food-chain through contaminated meat, dairy and vegetables. In order to control infections and stop transmission, it is important to understand what factors allow EHEC to colonize its hosts, cause virulence and aid transmission. Since this cannot be systematically studied in humans, it is important to develop animal models of infection and transmission. We developed a model which allows us to study food-borne infection in zebrafish, a vertebrate host that is transparent and genetically tractable. Using the zebrafish host, we can follow the bacterial infection cycle in real time, and gain important information regarding bacterial physiology and microbe-host interactions. This will allow us to identify potential new targets for infection control and prevention.

Introduction Isolated limb infusion (ILI) is a minimally invasive alternative to isolated limb perfusion (ILP) for delivering high-dose regional chemotherapy to treat locally advanced limb melanoma. The current study aimed to evaluate the applicability of ILI in four Australian tertiary referral centers outside of its originating institution, the Sydney Melanoma Unit (SMU; currently known as the Melanoma Institute Australia). Methods Data of 131 patients, treated between 1992 and 2008 were collectively analyzed. The ILI procedures were based on the Sydney Melanoma Unit protocol using melphalan. Response was determined using the WHO criteria and toxicity was assessed using the Wieberdink scale. Results The median patient age was 74 years (range 28–100). Fifty-six percent were female. Overall response (OR) rate to ILI was 63% (CR 27%; PR 36%). Wieberdink toxicity grade III or higher was seen in 13%. No toxicity-related amputations occurred. Median follow-up was 24 months; median survival was 58 months. In patients with a complete response (CR), median survival was 101 months; in patients with a partial response (PR) this was 41 months (P = 0.026). On univariate analysis a younger age, lower-limb procedures and a lower Breslow thickness of the primary melanoma were associated with a favorable response. On multivariate analysis Breslow thickness and lower-limb ILI remained significant predictors for response. Conclusion In this, to date, largest multi-center study of ILI for melanoma the results are comparable to other reports and demonstrate that ILI can be widely implemented and safely applied across tertiary referral centers. J. Surg. Oncol. 2014 109:780–785. © 2014 Wiley Periodicals, Inc.

The neurosecretory protein VGF (non-acronymic) is rapidly and robustly upregulated by primary somatosensory afferent neurons following peripheral nerve damage. VGF is proteolytically processed to yield a number of bioactive peptides, including the C-terminal peptide TLQP-62. Our work demonstrates that spinal TLQP-62 increases after nerve injury and that immunoneutralization of C-terminal VGF peptides attenuates the development of mechanical allodynia. This peptide has been shown to induce BDNF-dependent neuronal plasticity in the hippocampus. We have shown that TLQP-62 induces potentiation of glutamatergic responses in a subset of dorsal horn neurons and exposure of spinal cord slices to TLQP-62 increases TrkB phosphorylation, suggesting that TLQP-62 may facilitate BDNF release. Since primary afferent neurons are a potential source of spinal BDNF, we examined the effects of TLQP-62 on these neurons. Using mice that express a genetically encoded calcium indicator restricted to primary afferent neurons (Pirt-GCamp3 mice), we observed that TLQP-62 induced calcium transients in a subset of primary afferent fibers in the dorsal horn of spinal cord slices. The responses occurred immediately after TLQP-62 exposure and were sustained during prolonged exposure. TLQP-62 also induced calcium transients in DRG cultures, both in capsaicin-responsive neurites, as well as in neurites that did not respond to capsaicin. Ongoing work is investigating the functional consequences of these TLQP-62 effects and their relationship to BDNF via patch-clamp and BDNF-pHluorin imaging, respectively. Our observations of TLQP-62-induced calcium responses in the processes of primary afferent neurons are consistent with the idea that the peptide may modulate neurotransmitter release.

A Thesis Submitted to the Faculty of Graduate Studies and Research In Partial Fulfillment of the Requirements for the Degree of Master of *Arts in Religious Studies, University of Regina. v , 142 p. The goals of this thesis are to showcase the creation of a method for scraping and cataloging data on smartphone applications that were accumulated under a set criteria of individual religion search parameters. It is through the analysis of this data in tandem with establishing demographics for smartphone use that this thesis explores the relationship between religious community and space and internet communication technologies. To establish this connection this thesis begins by dedicating the first chapter to outlining current scholarship on religious space and community in context of the internet. By creating a baseline for researching religion in the digital sphere this thesis builds an approach to studying the relationship between religion, smartphones, and smartphone applications. In addition, this thesis continues to build on notions of religious community and space and how these notions function on smartphones and with smartphone users. This thesis continues by dedicating the second chapter to creating a historical outline for smartphones and smartphone platforms as well as establishing user demographics and adoption rates based on age, ethnicity, gender, socio-economic position, and education. This is accomplished first by tracing the evolution of the internet from the telegraph to the World Wide Web and secondly by establishing the technological evolution of the smartphone and the smartphone application. By using research data from sources such as Pew Research Center the author illustrates how not all smartphone platforms are created equally and that there is a socio-economic element to these divisions. These statistics demonstrate how as a society our social behaviorisms are becoming increasingly digital in nature. That it is not unreasonable to hypothesize that this same digitization is happening to institutionalized religious beliefs and practices in relation to religious space and community. The proposed method for data collection and the author’s initial findings are outlined in chapter three of this thesis. By organizing and sanitizing the findings for the pre-established search criteria in Microsoft Excel, the author categorizes comparable data from one platform against another and by one religion against another. Using this approach allowed the author to compare findings in regards to operating system, mainreligion, sub-religion, category, and maturity rating. Chapter three concludes by discussing five different forms of religious smartphone applications which are based on what these applications are facilitating for users. This allows the author to establish what kind of impact religious smartphone applications and the dispersal rates of those applications may have on religious community and space. This thesis concludes by speculating that there is a connection between one’s choices of mobile technology and what institutionalized religion a user may be participant. The author hypothesizes that the role religious community plays in one’s digital religious belief and practice may contribute to electronic colonialism in terms of smartphone platforms within those communities. Although the proposed method is a great entry point to studying this phenomenon, further refining of the data procurement process and how the results are displayed would need to be accomplished for further analysis to occur. Student yes

Obligate intracellular chlamydial bacteria of the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) superphylum are important pathogens of terrestrial and marine vertebrates, yet many features of their pathogenesis and host specificity are still unknown. This is particularly true for families such as the which, in addition to epithelia, cellular targets for nearly all , can infect and replicate in macrophages, an important arm of the innate immune system or in their free-living amoebal counterparts. An ideal pathogen model system should include both host and pathogen, which led us to develop the first larval zebrafish model for chlamydial infections with . By varying the means and sites of application, epithelial cells of the swim bladder, endothelial cells of the vasculature and phagocytosing cells of the innate immune system became preferred targets for infection in zebrafish larvae. Through the use of transgenic zebrafish, we could observe recruitment of neutrophils to the infection site and demonstrate for the first time that is taken up and replicates in these phagocytic cells and not only in macrophages. Furthermore, we present evidence that myeloid differentiation factor 88 (MyD88) mediated signaling plays a role in the innate immune reaction to , eventually by Toll-like receptor (TLRs) recognition. Infected larvae with depleted levels of MyD88 showed a higher infection load and a lower survival rate compared to control fish. This work presents a new and potentially powerful non-mammalian experimental model to study the pathology of chlamydial virulence and opens up new possibilities for investigation of other members of the PVC superphylum.

Background Exposure to ethanol (EtOH) in utero alters the disposition of tangentially migrating GABAergic interneurons in the fetal brain. The medial ganglionic eminence (MGE) gives rise to a large portion of cortical GABAergic interneurons, including the parvalbumin-expressing interneurons that shape and contribute to inhibitory/excitatory (I/E) balance of the intracortical circuit. Here, we investigated in the mouse medial prefrontal cortex (mPFC) the hypothesis that low levels of maternal EtOH consumption from closure of the neural tube embryonic day (E) 9.5 until birth result in an enduring interneuronopathy. Methods Pregnant mice were subjected to a 2% w/w EtOH consumption regimen starting at neural tube closure and ending at parturition. Neurogenesis in the MGE was assessed by 5-bromo-2-deoxyuridine (BrdU) immunofluorescence at E12.5. The count and distribution of parvalbumin-expressing interneurons were determined in adult animals, and patch clamp electrophysiology was performed to determine GABAergic function and I/E balance. Open-field behavior in adult mice was assessed to determine whether the EtOH-exposed cohort displayed a lasting alteration in exploratory behavior. Results In embryos exposed to EtOH in utero, we found increased BrdU labeling in the MGE, pointing to increased neurogenesis. Adult mice prenatally exposed to EtOH were hyperactive, and this was associated with an increase in parvalbumin-expressing GABAergic interneurons in the mPFC. In addition, prenatal EtOH exposure altered the balance between spontaneous inhibitory and excitatory synaptic input and attenuated GABAergic tone in layer V mPFC pyramidal neurons in juvenile mice. Conclusions These findings underscore that altered migration of GABAergic interneurons contributes to the EtOH-induced aberration of cortical development and that these effects persist into adulthood as altered cortical form and function.

Exposure to ethanol in utero may result in fetal alcohol spectrum disorder (FASD), hallmarked by varying degrees of cognitive deficits, many of which are associated with abnormalities in processing within the cortical circuitry (Sadrian et al., 2013; Shawa et al., 2013). Developmentally, the formation of cortical circuits is a tightly regulated sequel of events involving genesis, migration, and integration of inhibitory and excitatory neurons. Cajal–Retzius cells, as the transient and first-born neurons of the embryonic rodent cortex, arise from pallial and subpallial origins and migrate tangentially below the pial surface to cover the entire neocortex (Bielle et al., 2005; Garcia-Moreno et al., 2007). They release the glycoprotein reelin (D'Arcangelo et al., 1995; Hirotsune et al., 1995; Ogawa et al., 1995), implicated in regulating neuronal migration, layering, and neurogenesis in the developing cortex (Franco et al., 2011; Frotscher, 1998; Gil-Sanz et al., 2013; Gupta et al., 2003; Jossin and Cooper, 2011; Lakoma et al., 2011; Olson et al., 2006; Sekine et al., 2011). Thus, Cajal–Retzius cells are in a favorable position to orchestrate early aspects of corticogenesis, cortical patterning, and the establishment of nascent cortical circuits (Schwartz et al., 1998; Soriano and Del Rio, 2005). While the importance of Cajal–Retzius cells in corticogenesis is well-documented (Super et al., 2000), whether they are susceptible to ethanol exposure in utero is unexplored. In this study, pregnant mice were administered ethanol in a liquid diet that yielded moderate levels of blood alcohol (x = 20 mg/dl). The adolescent offspring exposed in utero to ethanol displayed altered open-field activity, indicating a relevant neurobehavioral consequence of our in utero ethanol exposure paradigm. We started the maternal consumption regimen on embryonic day (E)9, the time of closure of the neural tube, to span the entire period of cortical development. In this light, beyond regulating migration and neuronal integration during corticogenesis, Cajal–Retzius cells have also been postulated to play a functional role within the developing cortical circuit (Aguilo et al., 1999; Radnikow et al., 2002; Soda et al., 2003). We show for the first time that they receive GABAergic synaptic input as early as E13.5 in the mouse neocortex. In addition, we report that ethanol exposure in utero early on in cortical development alters the genesis, distribution, and GABAergic synaptic input of Cajal–Retzius cells.

Isolated limb infusion (ILI) is a minimally invasive alternative to isolated limb perfusion (ILP) for delivering high-dose regional chemotherapy to treat locally advanced limb melanoma. The current study aimed to evaluate the applicability of ILI in four Australian tertiary referral centers outside of its originating institution, the Sydney Melanoma Unit (SMU; currently known as the Melanoma Institute Australia).Data of 131 patients, treated between 1992 and 2008 were collectively analyzed. The ILI procedures were based on the Sydney Melanoma Unit protocol using melphalan. Response was determined using the WHO criteria and toxicity was assessed using the Wieberdink scale.The median patient age was 74 years (range 28-100). Fifty-six percent were female. Overall response (OR) rate to ILI was 63% (CR 27%; PR 36%). Wieberdink toxicity grade III or higher was seen in 13%. No toxicity-related amputations occurred. Median follow-up was 24 months; median survival was 58 months. In patients with a complete response (CR), median survival was 101 months; in patients with a partial response (PR) this was 41 months (P = 0.026). On univariate analysis a younger age, lower-limb procedures and a lower Breslow thickness of the primary melanoma were associated with a favorable response. On multivariate analysis Breslow thickness and lower-limb ILI remained significant predictors for response.In this, to date, largest multi-center study of ILI for melanoma the results are comparable to other reports and demonstrate that ILI can be widely implemented and safely applied across tertiary referral centers.