Magnetic Strength Compatibilization of Soft Magnetic Composites using a Magnetic Force Sensor

In this thesis, Fe-based Soft Magnetic Composites (SMC) varying in thermoset resins are prepared using a variety of metallurgical techniques. Their magnetic properties are then studied, tested, then compared with each other. Three various resins were used for specimen preparation: Biresin CR131, G/Flex, and Biothan. The magnetic pull strength between the SMC samples and a permanent magnet (PM) was measured, with respect to each other's distances, using a device I built entirely with limited resources available. This device is called a magnetic force sensor (MFS). The details and methodology used to construct this device will be clearly described within this paper. A finite element method software is used to compare and validate the credibility of the MFS. The outcome proves to corroborate my device's capabilities as a credible tool for research. Of the SMC experiments taken with the device, resin base CR131 showed the strongest magnetic response to the PM while Biothan showed the weakest response. The quality of SMC due to preparation will be a key topic of my results and discussion

Partial Regulatory T Cell Depletion Prior to Acute Feline Immunodeficiency Virus Infection Does Not Alter Disease Pathogenesis

Feline immunodeficiency virus (FIV) infection in cats follows a disease course similar to HIV-1, including a short acute phase characterized by high viremia, and a prolonged asymptomatic phase characterized by low viremia and generalized immune dysfunction. CD4+CD25hiFoxP3+ immunosuppressive regulatory T (Treg) cells have been implicated as a possible cause of immune dysfunction during FIV and HIV-1 infection, as they are capable of modulating virus-specific and inflammatory immune responses. Additionally, the immunosuppressive capacity of feline Treg cells has been shown to be increased during FIV infection. We have previously shown that transient in vivo Treg cell depletion during asymptomatic FIV infection reveals FIV-specific immune responses suppressed by Treg cells. In this study, we sought to determine the immunological influence of Treg cells during acute FIV infection. We asked whether Treg cell depletion prior to infection with the highly pathogenic molecular clone FIV-C36 in cats could alter FIV pathogenesis. We report here that partial Treg cell depletion prior to FIV infection does not significantly change provirus, viremia, or CD4+ T cell levels in blood and lymphoid tissues during the acute phase of disease. The effects of anti-CD25 mAb treatment are truncated in cats acutely infected with FIV-C36 as compared to chronically infected cats or FIV-naïve cats, as Treg cell levels were heightened in all treatment groups included in the study within two weeks post-FIV infection. Our findings suggest that the influence of Treg cell suppression during FIV pathogenesis is most prominent after Treg cells are activated in the environment of established FIV infection

Multivariate Statistical Analyses Demonstrate Unique Host Immune Responses to Single and Dual Lentiviral Infection

Feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV) are recently identified lentiviruses that cause progressive immune decline and ultimately death in infected cats and humans. It is of great interest to understand how to prevent immune system collapse caused by these lentiviruses. We recently described that disease caused by a virulent FIV strain in cats can be attenuated if animals are first infected with a feline immunodeficiency virus derived from a wild cougar. The detailed temporal tracking of cat immunological parameters in response to two viral infections resulted in high-dimensional datasets containing variables that exhibit strong co-variation. Initial analyses of these complex data using univariate statistical techniques did not account for interactions among immunological response variables and therefore potentially obscured significant effects between infection state and immunological parameters.Here, we apply a suite of multivariate statistical tools, including Principal Component Analysis, MANOVA and Linear Discriminant Analysis, to temporal immunological data resulting from FIV superinfection in domestic cats. We investigated the co-variation among immunological responses, the differences in immune parameters among four groups of five cats each (uninfected, single and dual infected animals), and the "immune profiles" that discriminate among them over the first four weeks following superinfection. Dual infected cats mount an immune response by 24 days post superinfection that is characterized by elevated levels of CD8 and CD25 cells and increased expression of IL4 and IFNgamma, and FAS. This profile discriminates dual infected cats from cats infected with FIV alone, which show high IL-10 and lower numbers of CD8 and CD25 cells.Multivariate statistical analyses demonstrate both the dynamic nature of the immune response to FIV single and dual infection and the development of a unique immunological profile in dual infected cats, which are protected from immune decline

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Functional activation of p53 induces apoptosis in the absence of MDM2

The p53 tumor suppressor gene product is negatively regulated by the product of its downstream target, mdm2. The mdm2 oncogene abrogates p53 transactivation function. Amplification of mdm2 occurs in 36% of human sarcomas, which often retain p53 in wild type form, suggesting that overexpression of mdm2 in tumors results in p53 inactivation. Thus, the relationship of p53 to mdm2 is important in tumorigenesis. The deletion of mdm2 in the mouse results in embryonic lethality by 5.5 days post coitum. Embryonic lethality of the mdm2 null embryos was overcome by simultaneous loss of the p53 tumor suppressor, which substantiates the importance of the negative regulatory function of MDM2 on p53 function in vivo. These data suggest that the loss of MDM2 function allowed the constitutively active p53 protein to induce either a complete G1 arrest or the p53-dependent apoptotic pathway, resulting in the death of the mdm2−/− embryos. The present study examines the hypothesis that the absence of mdm2 induces apoptosis due to p53 activation. Viability of the p53−/−mdm2−/− mice has allowed establishment of mouse embryo fibroblasts (MEFs) and a detailed examination of the properties of these cells. To introduce p53 into this system, and essentially recreate a mdm2 null cell, a temperature sensitive p53 (tsp53) point mutant (A135V) was used, which exhibits a nonfunctional, mutant conformation at 39°C and wild type, functional conformation at 32°C. Infected pools of p53−/− and p53−/−mdm2−/− MEFs with the tsp53 gene were established and single-cell clonal populations expressing tsp53 were selected. Shifting the cells from 39°C to 32°C caused p53−/−mdm2 −/− lines expressing tsp53 to undergo up to 80% apoptosis, which did not occur in the p53−/− lines expressing tsp53 nor the parental lines lacking p53 expression. Furthermore, the amount of p53 present in the clonal population determined the extent of apoptosis. Tsp53 is transcriptionally active in this system, however, it discriminates among different target promoters and does not induce the apoptosis effector targets bax or Fas/Apo1. In summary, this study indicates that the presence or absence of mdm2 is the determining factor for the ability of p53 to trigger apoptosis in this system. The loss of mdm2 promotes p53-dependent apoptosis in MEFs in a cell cycle and dose-dependent manner. p53 is differentially phosphorylated in the presence and absence of mdm2, but does not induce the apoptosis effectors, bax or Fas/ Apo1

Role of PABP1 on Influenza A virus mRNA Translation Initiation

The Influenza A virus (IAV) is a pathogen with a long-standing history of seasonal spread across human communities. Infection by IAV can cause upper respiratory distress in humans that can be fatal especially for those with weakened immune systems. The reservoirs of IAV in animals as well as the rapid mutation rate makes IAV challenging to prevent and eradicate, therefore more understanding of its mechanisms of hijacking host cellular machinery is required to find new solutions for treating this disease. Results from the field suggests that Influenza RNA are acted upon by translation factors in the same manner as host mRNA. Capped and poly (A) tailed viral mRNA are recognized by eIF4E at the 5’ end and PABP1 at the 3’ end to recruit the subsequent initiation factors until the ribosome is assembled and can commence translation. This mechanism of translation initiation however, fails to explain why viral proteins are synthesized to such a high degree. We set out to study the role PABP1 plays on IAV RNA due to it being targeted by the IAV protein NS1. Using qualitative and quantitative binding assays we have further characterized the PABP1-NS1 interaction by discovering that it occurs in the absence of Poly(A). This suggests that NS1 acts upon PABP1 independent of PABP1 binding to a poly(A) tail. Furthermore, we discovered a novel interaction that PABP1 has to the 5’ UTR regions of viral mRNAs. The differences in binding affinity correlate with the protein production measured for those segments. We thus tested the validity of these observations by studying the benefits of IAV 5’ UTRs in a cell free in vitro translation system where cap dependent translation was inhibited. We found that RNAs driven with the viral 5’ UTRs are more resistant to suppression of cap-dependent translation than RNAs driven by a eukaryotic 5’ UTR. To follow up on our in vitro results, we pulled down PABP1 from IAV infected cells and found via RT-qPCR that the 5’ UTRs of IAV RNAs are enriched post pulldown. This suggests that IAV mRNA translation can be initiated by PABP1 acting on the 5’ end

Probing VCMA in MTJs with in-plane magnetization

Voltage controlled magnetic anisotropy (VCMA) is a novel method to switch magnetizations in low-power and ultra-fast applications based on magnetic tunnel junctions (MTJs). Here we explore the ferromagnetic resonance (FMR) technique to probe VCMA in situations where other methods cannot be applied. We quantify VCMA in CoFeB/MgO/CoFeB MTJ nanopillars with in-plane magnetizations where our FMR method is unique in providing direct information about VCMA. We observe a quadratic shift of the FMR resonance field when a voltage bias is applied across the MTJ. The VCMA energy corresponding to the quadratic shift varies with an energy factor of 8.2μJ/m2 for 1 V2/nm2. These results are important for understanding magnetodynamics in MTJ-based applications with in-plane magnetizations