An immune modulatory role for the fes proto-oncogene

The Fes protein tyrosine kinase is abundantly expressed in phagocytic immune cells, including tumor associated macrophages. Fes knockout mice (fes-/-) display enhanced sensitivity to LPS, and this was shown to be associated with increased NF-κB signaling and TNFα production from fes-/- macrophages. Interestingly, tumor onset in the mouse mammary tumor virus (MMTV-Neu) transgenic mouse model of breast cancer is significantly delayed in fes-/- mice, and this was associated with increased frequency of CD11b+ myeloid and CD3+ T cells in the premalignant mammary glands. Recent studies have also implicated Fes in cross-talk between MHC-I and the NF-κB and IRF-3 pathways in macrophages. Signal 3, the production of inflammatory cytokines and Type I interferons downstream of NF-κB and IRF-3 pathways in antigen presenting cells, is considered an important component of T-cell activation, after engagement of T cell receptor by MHC presented antigen (Signal 1) and co-receptors by their ligands (Signal 2). Using a lymphocytic choriomeningitis virus (LCMV) model of immune activation, I show that LPS stimulated fes-/- macrophages promote more robust activation of LCMV antigenspecific CD8+ T cells than wild type macrophages (fes+/+). Furthermore, LPS stimulated fes-/- macrophages showed increased phosphorylation of NF-B and IRF-3. I also showed that Fes colocalizes with MHC-I in dynamic vesicular structures within macrophages. These observations are consistent with a model where Fes regulates Signal 3 in antigen presenting cells through roles in cross-talk between MHC-I and the NF-kB and IRF-3 signaling pathways. This suggests that Fes plays an immune checkpoint role at the level of Signal 3, and that Fes inhibition could promote tumor immunity through increased Signal 3 driven T cell activation.

A Minor Change?: A Case Study of Carleton Place's Integration of Minor Variance Procedures into Ontario's Development Permit System

The Development Permit System has been introduce with minimal directives for establishing a decision making process. This is in opposition to the long established process for minor variances and suggests that the Development Permit System does not necessarily incorporate all of Ontario’s fundamental planning principles. From this concept, the study aimed to identify how minor variances are incorporated into the Development Permit System. In order to examine this topic, the research was based around the following research questions: • How are ‘minor variance’ applications processed within the DPS? • To what extent do the four tests of a minor variance influence the outcomes of lower level applications in the DPS approval process? A case study approach was used for this research. The single-case design employed both qualitative and quantitative research methods including a review of academic literature, court cases, and official documents, as well as a content analysis of Class 1, 1A, and 2 Development Permit application files from the Town of Carleton Place that were decided between 2011 and 2015. Upon the completion of the content analysis, it was found that minor variance issues were most commonly assigned to Class 1 applications. Planning staff generally met approval timelines and embraced their delegated approval authority, readily attaching conditions to applications in order to mitigate off-site impacts. While staff met the regulatory requirements of the DPS, ‘minor variance’ applications were largely decided on impact alone, demonstrating that the principles established by the four tests, the defining quality of the minor variance approval process, had not transferred to the Development Permit System. Alternatively, there was some evidence that the development community has not fully adjusted to the requirements of the new approvals process, as some applications were supported using a rationale containing the four tests. Subsequently, a set of four recommendations were offered which reflect the main themes established by the findings. The first two recommendations are directed towards the Province, the third to municipalities and the fourth to developers and planning consultants: 1) Amend Ontario Regulation 608/06 so that provisions under Section 4(3)(e) fall under Section 4(2). 2) Change the rhetoric from “combining elements of minor variances” to “replacing minor variances”. 3) Establish clear evaluation criteria. 4) Understand the evaluative criteria of the municipality in which you are working.

Unraveling the function of bacterial-type phosphoenolpyruvate carboxylase in vascular plants

Two distinct phosphoenolpyruvate carboxylase (PEPC) isozymes occur in vascular plants and green algae: plant-type PEPC (PTPC) and bacterial-type PEPC (BTPC). PTPC polypeptides typically form a tightly regulated cytosolic Class-1 PEPC homotetramer. BTPCs, however, appear to be less widely expressed and to exist only as catalytic and regulatory subunits that physically interact with co-expressed PTPC subunits to form hetero-octameric Class-2 PEPC complexes that are highly desensitized to Class-1 PEPC allosteric effectors. Yeast two-hybrid studies indicated that castor plant BTPC (RcPPC4) interacts with all three Arabidopsis thaliana PTPC isozymes, and that it forms stronger interactions with AtPPC2 and AtPPC3, suggesting that specific PTPCs are preferred for Class-2 PEPC formation. In contrast, Arabidopsis BTPC (AtPPC4) appeared to interact very weakly with AtPPC2 and AtPPC3, suggesting that BTPCs from different species may have different physical properties, hypothesized to be due to sequence dissimilarities within their ~10 kDa intrinsically disordered region. Recent RNA-seq and microarray data were analyzed to obtain a better understanding of BTPC expression patterns in different tissues of various monocot and dicot species. High levels of BTPC transcripts, polypeptides and Class-2 PEPC complexes were originally discovered in developing castor seeds, but the analysis revealed a broad range of diverse tissues where abundant BTPC transcripts are also expressed, such as the developing fruits of cucumber, grape, and tomato. Marked BTPC expression correlated well with the presence of ~116 kDa immunoreactive BTPC polypeptides, as well as Class-2 PEPC complexes in the immature fruit of cucumbers and tomatoes. It is therefore hypothesized that in vascular plants BTPC and thus Class-2 PEPC complexes maintain anaplerotic PEP flux in tissues with elevated malate levels that would potently inhibit ‘housekeeping’ Class-1 PEPCs. Elevated levels of malate can be used by biosynthetically active sink tissues such as immature tomatoes and cucumbers for rapid cell expansion, drought or salt stressed roots for osmoregulation, and developing seeds and pollen as a precursor for storage lipid and protein biosynthesis.