Using Lyve1-Cre+ Mice to Visualize Lymphatics for Laser Capture Microdissection

Background: Lymphedema is characterized by limb swelling secondary to lymphatic dysfunction. Lymphedema most frequently develops following breast cancer treatment due to iatrogenic damage of the lymphatics from surgery and radiation. Lymphedema affects 20-40% of breast cancer survivors. There is no cure for this disease. For determination of successful delivery of gene-based therapies, target cells are often isolated and analyzed via real-time PCR. One method to isolate a region of cells within a tissue section is laser capture microdissection (LCM). This process involves outlining the desired regions, which are cut on membrane slides and captured using a laser. Using LCM requires visualization and identification of the target tissue. In the case of lymphedema therapies, the target tissue is lymphatic vasculature. Rationale of Project: While lymphatics can be visualized using immunohistochemistry antibodies specific to lymphatic markers, the process is time consuming and can interfere with RNA levels in the tissue. Another option to visualize lymphatics is to use Lyve1-Cre+ mice. These mice express enhanced green fluorescent protein on lymphatic cells. The purpose of this project is to assess the utility of Lyve1-Cre+ and develop the methodology to capture the lymphatics in the murine tail to enable utilization of this methodology in murine tail model of lymphedema. Methodology Development: Samples of Lyve1-Cre+ mouse tails were harvested. Sections (10µm) were captured on LCM slides and dehydrated. The slides were visualized on the LCM microscope system, lymphatics vessels fluoresced green. They were captured via laser dissection. The captured samples were analyzed for the lymphatic specific genes (Prox1 and Lyve1) via quantitative real-time PCR to determine the purity of capture.

Methodology development for the synthesis of iminosugars

<p>This thesis investigated the development and application of methodology for the synthesis of iminosugars. The first portion of this thesis (Chapters 2 and 3) explored the scope of previously established protecting-group-free Vasella-reductive-amination and I2-mediated carbamate annulation methodology initially developed within the Stocker-Timmer group for the synthesis of pyrrolidines and piperidines from aldose sugars. In this thesis, the Vasella-reductive-amination methodology was extended to include the use of ketose sugars as starting materials, thereby allowing for the synthesis of primary amines directly from in situ formed ketones under protecting-group-free conditions. The scope of the carbamate annulation was then explored, whereby it was determined that both steric and electronic effects appear to affect transition state energies during the annulation reaction. Here, formation of pyrrolidines with the 2,5-trans and 3,4-cis relationships are favoured, however, in circumstances were conflicting electronic- and steric-effects are present, steric-effects dominate thereby favouring the formation of the 2,5-trans product. Using a combination of this Vasella-reductive-amination and carbamate annulation methodology, 2,5-dideoxy-2,5-imino-L-iditol was thus synthesised in 6 steps and 18% overall yield from D-fructose. Next, the same methodology was applied to the synthesis of the promising molecular chaperone 2,5-dideoxy-2,5-imino-D-altritol. Thus, 2,5-dideoxy-2,5-imino-D-altritol was synthesised over 7 steps and in 22% yield from D-tagatose, which is the most efficient synthesis of this iminosugar to date. The second part of this thesis (Chapters 4 and 5) focused on the optimisation and development of synthetic methodology that would allow for the highly efficient synthesis of a variety of iminosugars including piperidines and azepanes. To this end, modifications to existing synthetic methodology allowed for the rapid synthesis of a variety of iodoglycosides, which are important synthons. Next, reductive amination/cyclisation methodology that allowed for the direct transformation of methyl iodoglycosides or isopropylidene-protected iodoglycosides into iminosugars was developed. As such, the piperidines 1-Deoxynojirimycin, 1-Deoxymannojirimycin (DMJ), L-1-Deoxygalactojirimycin (L-DGJ), and (3R,4r,5S)-piperidine-3,4,5-triol were prepared in 4 steps and good overall yields (44%, 62%, 67%, and 53%, respectively). In the case of DMJ and (3R,4r,5S)-piperidine-3,4,5-triol, these are the most efficient syntheses of these materials to date. Factors influencing the stereochemical outcome of the reductive amination reaction were also explored, and evidence suggests that the reduction occurs from the least sterically hindered face of an intermediate cyclic imine, whereby the preferred conformation of the imine is the one which places the largest number of substituents in the pseudo-equatorial position. Using analogous methodology, the azepane (3S,4R,5S,6R)-azepane-3,4,5,6-tetraol was also prepared in 4 steps and good yield (53%).</p>

Methodology development for the synthesis of iminosugars

<p>This thesis investigated the development and application of methodology for the synthesis of iminosugars. The first portion of this thesis (Chapters 2 and 3) explored the scope of previously established protecting-group-free Vasella-reductive-amination and I2-mediated carbamate annulation methodology initially developed within the Stocker-Timmer group for the synthesis of pyrrolidines and piperidines from aldose sugars. In this thesis, the Vasella-reductive-amination methodology was extended to include the use of ketose sugars as starting materials, thereby allowing for the synthesis of primary amines directly from in situ formed ketones under protecting-group-free conditions. The scope of the carbamate annulation was then explored, whereby it was determined that both steric and electronic effects appear to affect transition state energies during the annulation reaction. Here, formation of pyrrolidines with the 2,5-trans and 3,4-cis relationships are favoured, however, in circumstances were conflicting electronic- and steric-effects are present, steric-effects dominate thereby favouring the formation of the 2,5-trans product. Using a combination of this Vasella-reductive-amination and carbamate annulation methodology, 2,5-dideoxy-2,5-imino-L-iditol was thus synthesised in 6 steps and 18% overall yield from D-fructose. Next, the same methodology was applied to the synthesis of the promising molecular chaperone 2,5-dideoxy-2,5-imino-D-altritol. Thus, 2,5-dideoxy-2,5-imino-D-altritol was synthesised over 7 steps and in 22% yield from D-tagatose, which is the most efficient synthesis of this iminosugar to date. The second part of this thesis (Chapters 4 and 5) focused on the optimisation and development of synthetic methodology that would allow for the highly efficient synthesis of a variety of iminosugars including piperidines and azepanes. To this end, modifications to existing synthetic methodology allowed for the rapid synthesis of a variety of iodoglycosides, which are important synthons. Next, reductive amination/cyclisation methodology that allowed for the direct transformation of methyl iodoglycosides or isopropylidene-protected iodoglycosides into iminosugars was developed. As such, the piperidines 1-Deoxynojirimycin, 1-Deoxymannojirimycin (DMJ), L-1-Deoxygalactojirimycin (L-DGJ), and (3R,4r,5S)-piperidine-3,4,5-triol were prepared in 4 steps and good overall yields (44%, 62%, 67%, and 53%, respectively). In the case of DMJ and (3R,4r,5S)-piperidine-3,4,5-triol, these are the most efficient syntheses of these materials to date. Factors influencing the stereochemical outcome of the reductive amination reaction were also explored, and evidence suggests that the reduction occurs from the least sterically hindered face of an intermediate cyclic imine, whereby the preferred conformation of the imine is the one which places the largest number of substituents in the pseudo-equatorial position. Using analogous methodology, the azepane (3S,4R,5S,6R)-azepane-3,4,5,6-tetraol was also prepared in 4 steps and good yield (53%).</p>

Features of using qualitative interviews with injection drug users

This article is dedicated to some features of using qualitative interviews when interviewing injection drug users. The main purpose of this article is to give young researchers and all social scientists some important hints and guidelines that would help them when using qualitative interviews for studying injection drug users. In the first part of the article authors make a literature review concerning specifics and advantages of using qualitative methods for studying specific groups or categories in sociology. However, there are many qualitative methods besides interview, that can be used for studying these groups (focus-groups, observation, content analysis, etc.), authors focus their attention on qualitative interviews as one of the most universal ones, considering its wide-spread use and advantages. In the second part of the article authors reveal and stress some major points on every stage of qualitative interview: preparation, literature review, methodology development, respondent recruiting, interviewing and analysis. Some of the major points are the understanding of the social and cultural context in which the studied group lives, always paying attention to the senses and meanings that respondent give to concepts that are important to the research, the importance of trust for the injection drug users due to the illegality of their actions that is crucial on both recruitment and interviewing stage, ethical issues concerning confidentiality and anonymity, etc. Also researches in this area include such components as practical recommendations and the ways or steps that could be taken to solve problems related to research problematic. Despite concentrating on the use of one particular method for studying people who use injection drugs, hints and guidelines given in this article can be applied to a wider list of specific respondent’s categories and can even be useful when choosing similar methods.