Blog Post #4

As I previously mentioned in my last blog post, my primary task thus far this semester has been to look at Immanuel Kant’s seemingly unique interpretation of the Parable of the Unjust Steward (Luke 16:1-13) in Religion within the Boundaries of Mere Reason. This interpretation is distinct within the larger context of Kant’s work, as the steward acts according to self-interest, rather than out of respect for the moral law, yet he still is commended and gains heavenly reward. As such, Kant has to interpret the parable rather unexpectedly in order to make the parable align with his very strict black and white notions of morality. To see if there is any precedent for Kant’s unanticipated interpretation within the Christian exegetical tradition, I have been reviewing numerous texts and series in an attempt to become familiar with the existing interpretations.

This has proven to be a bit more difficult than I previously expected. Since we are attempting to discover if any said precedent in fact exists within the Christian exegetical tradition, it can be a bit discouraging to pour over volumes of books and not really find anything. Most of the instances of citations I have found of the parable have just been passing references, so it would appear, at least for the time being, that no such precedent exists. However, since there is a seemingly limitless amount of literature to review that could perhaps mention the parable, it is a bit difficult to definitively say that no such interpretation exists. Fortunately, the Luke edition of Ancient Christian Commentary on Scripture, which I have been long awaiting from the New York Public Library’s Inter-Library Loan service, has finally arrived at the library. This particular book will be quite helpful in letting me know if there is anything notable with regards to the parable that I may have happened to overlook.

I am quite excited to continue (and eventually complete) my work, and to present to my peers at the showcase. This entire experience has been very productive and I have learned quite a lot, not only in relation to what I’m researching, but how to research (such as research methods and writing an abstract). I think this knowledge will be quite valuable throughout the remainder of my time at Pace.

Blog Post #3

This new semester brings along with it new objectives for our research project. My professor, Dr. Miller, has completed his infinitely helpful manuscript, titled Kant’s Religion Within the Boundaries of Mere Reason: A Reader’s Guide. As such, my responsibilities of reading Kant’s original text and Dr. Miller’s reading guide are completed. Though the finalization of the manuscript was slightly delayed, I believe it was necessary and allowed the manuscript to realize its full potential. I am profoundly proud and happy for my professor for the realization of what was certainly a time-consuming and challenging endeavor and I am exceedingly honored to have had the opportunity to provide him with assistance and feedback.

As the prodigious task of finishing the manuscript is now concluded, we have the opportunity to explore some new possible areas of research. As of now, one idea for this semester that we are currently examining is analyzing Kant’s exceptional interpretation of the Parable of the Unjust Steward, as I mentioned in my previous student blog. Specifically, we will be exploring if there is any precedent for this interpretation in the Christian exegetical tradition. Currently, I am conducting reviews of prior interpretations of the parable that I have been able to locate at locations such as the New York Public Library. Though becoming more familiarized with such an enormous library was quite a daunting task, it has been one of the more “fun” challenges in my research that I’ve encountered thus far. Additionally, we expect to receive word within the next few weeks as to whether or not the paper that we submitted for the North American Kant Society Conference in Philadelphia was accepted. I am excited to continue working this semester, as it seems that our research plans provide quite a bit more leeway than those of last semester.

Blog Post #2

Thus far, our research project has been progressing fairly close to the projection rate. I have completed reading both prefaces and Part I & II of Immanuel Kant’s Religion Within the Boundaries of Mere Reason, and I am currently working on reading Part III. The prefaces primarily establish that although religion is not necessary for morality, religion is a method of reconciling ethical behavior with a rewarding consequence (happiness in proportion to our deservedness). Part I concerns the demonstration of the natural radical evil within the human being, with a fair amount of attention given to resolving the discord between Kant’s conception of morality only being legitimate through free choice and the human being’s natural propensity to evil. In Part II, Kant analyzes the personification of the good principle, its objective reality, and the extent to which a human being could follow such an ideal as an example, if at all. Additionally, I have read the corresponding sections of Dr. Miller’s reading guide for the text, and provided appropriate feedback.

In anticipation of next semester’s objectives, I will soon begin reviewing this text for evidence in order to further develop our hypothesis. Specifically, I will be searching for examples that may potentially substantiate the claim that Kant’s Religion is an esoteric text that utilizes subtle textual strategies in order to conceal his intended message from religious censors in eighteenth-century Prussia. In addition, we will begin analyzing Kant’s unique interpretation of the Parable of the Unjust Steward. Our intention is to determine whether or not there is any precedent for said interpretation in the Christian exegetical tradition.

In conclusion, I have learned quite a lot concerning Kant’s philosophical arguments made in Religion Within the Boundaries of Mere Reason this semester, and I look forward to our continued work.

The Small Heat Shock Protein, Hsp42, counteracts Huntington Pathology in S. ceravisiae models.

The Small Heat Shock Protein, Hsp42, counteracts Huntington Pathology in S. ceravisiae models.

Abstract:

Intellectual Merit:

The overall goal is to understand Huntington pathology using yeast models expressing isolated molecular chaperones, particularly the heat shock proteins Hsp42, Hsp26, and the Hsp40/70 complex. Huntington’s Disease is one of several neurodegenerative diseases’ that exhibits pathology through the formation of plaques surrounding neuron cells eventually leading to cell death. The Huntington (Htt) protein, when in its native state, is harmless to cells. Only when the Htt protein becomes mutated does it become toxic to cells, elongating its polyglutamine repeat, and eventually mis-folding the protein into a non-native state leading to the formation of aggregates. One mechanism for cells to counteract the accumulation of mis-folded proteins is by the utilization of molecular chaperones also known as heat shock proteins. Several heat shock proteins have been tested on in yeast Huntington models such as Hsp70, Hsp90, and Hsp104. Each of the three molecular chaperones is thought to counteract non-native proteins by targeting aggregates for re-folding or ubiquitination through the cellular proteasome pathway. The small heat shock protein, Hsp42, is known to counteract non-native protein formation in prion [PSI+] disease models by a similar mechanism to other molecular chaperones, however Hsp42’s role in Huntington disease models has yet to be illuminated upon. Objectives include evaluating the effects of Hsp42 on mutant Htt mis-folded proteins, understanding the mechanisms of Hsp42 expression in comparison to other heat shock protein families, and ultimately to find possible therapeutic means in which to counteract Huntington pathology using Hsp42 as the primary molecular chaperone.

Project Description

Specific Aims

Aim 1: To determine the effects of Hsp42 expression on mutant Huntington proteins displaying three different lengths of polyglutamine (polyQ) repeats: Htt25Q, Htt72Q, and Htt103Q. Htt pathology is determined by the length of its polyQ (CAG) repeats. The longer the glutamine repeats, the more toxicity mutant Htt protein displays. Htt25Q is known to be non-toxic to cells while Htt72Q is known to form insoluble aggregates and Htt103Q is the most toxic of all mutations forming the highest concentration of insoluble cytoplasmic aggregates. We will define the mechanisms in which Hsp42 works on all mutant Htt protein aggregates within S. cerevisiae models and whether localization is determined by the cytoskeleton microtubules.

Aim 2: To differentiate Hsp42 activity compared to the mechanisms of other molecular chaperones such as Hsp40, Hsp70, and Hsp104 when expressed in S. cerevisiae models. Known molecular chaperone activity with Htt expression include the heat shock proteins Hsp40, Hsp70, and Hsp104. In order to fully understand Hsp42’s effects on Huntington, other molecular chaperone mechanisms must be evaluated and understood. We will isolate and define whether Hsp42 is associated with a particular family of molecular chaperones or whether it works uniquely to other heat shock proteins.

Aim 3: To determine whether Hsp42 can work in conjunction with other molecular chaperones to counteract Huntington toxicity. Molecular protein complexes have been shown to work together in counteracting mis-folded protein aggregation such as the Hsp40/Hsp70 complex.  After evaluating the effects of specific molecular chaperone families on Huntington pathology, we will attempt to identify whether Hsp42 can work together with other heat shock proteins in clearing Htt aggregates from cells.

Background

Hsp42 Overview:

The small heat shock protein, Hsp42, has been shown to localize non-native protein aggregates to the peripheral regions (IPODs) within the cell’s cytoplasm. Protein targeting and re-folding are assisted by the expression of Hsp42. Interestingly, Hsp42 is a constitutively active molecular chaperone. One other heat shock protein, which is known to work in conjunction with Hsp42 is Hsp26. The N-terminal domain (NTD) of hsp42 displays high variability in nucleotide sequences as well as varying lengths. The NTD is thought to mediate functional specificity of hsp42 and hsp26/hsp42 complexes. When the NTD sequence of Hsp42 is deleted and transformed into yeast models, aggregates localize to the juxta-nuclear region (JunQ). In strains expressing Hsp26 with the NTD of Hsp42 deleted, IPOD localization of aggregates was restored, however in vivo Hsp26 activation is only induced by heat stress. In prion [PSI+] models, Hsp42 has been shown to target/bind to non-native proteins preventing aggregation and assisting non-native protein folding into native protein conformations reducing cellular toxicity under elevated stress.

Huntington Pathology Overview:

The gene encoding the Htt protein is non-toxic to animal neural cells but can become toxic through mutations in the Htt gene. Pathology in Htt mutants exhibit expanded polyQ residue repeats,  >25Q leading to toxicity.  Through the expansion in the polyQ tail, pathogenicity is accompanied by the mis-folding of the CAG or CAA repeats, producing non-native Htt protein aggregates. Upon the accumulation of mutant Htt proteins, neurons are thought to undergo apoptosis, thus leading to neurodegenerative pathology. The Htt103Q mutation exhibits the most potent toxicity in cell models while the Htt25Q mutation appears to display no detrimental effects. It is also believed that Htt toxicity is partially due to the deletions of flanking Proline-Rich Regions (PRR) along the polyQ repeats, thus the pathology of Huntington is partially masked by the PRR.

Mutant Huntington and Molecular Chaperones Overview:

            The heat shock proteins Hsp70 and Hsp104 have been shown to effect Huntington aggregation. When over-expressed, Hsp70 displays around two intense foci from GFP-tagged mutant Htt proteins as opposed to wild-type cells showing one intense aggresome. Hsp104 displays a similar effect, having more than one less-intense foci and showing diffuse fluorescence indicating soluble Htt protein fragments. Interestingly, it is thought that in Hsp104null strains, pathology of the Huntington protein is absent; indicating that expression of Hsp104 also may facilitate Htt toxicity.

Hypothesis:

Molecular chaperones have a distinct effect on Huntington aggregation. To further elaborate on this particular study, I propose to investigate the role of the small molecular chaperone, Hsp42, on Htt aggregates exhibiting polyQ repeats of 25Q, 72Q, and 103Q. I believe that the over-expression of Hsp42 will result in reduced toxicity, less aggregation, and diffuse Htt fragments in mutant Huntington S. cerevisiae models. The mechanisms by which Hsp42 acts on mutant Htt proteins are to be examined. Where does Hsp42 localize to within the cytoplasm as Htt aggregation occurs? Where do the Huntington proteins localize to in the cell when Hsp42 is over-expressed? Do they localize by utilizing microtubule pathways within the S. cerevisiae cytoskeleton? Does Hsp42 have an effect on the flanking PRR? Will localization of Huntington proteins change when Hsp42 is deleted and thus is not expressed? How long does it take for mutant Htt aggregates to form when Hsp42 is over-expressed? Does Hsp42 hinder Htt protein formation or does it only act on Htt proteins after they have formed? Can Hsp42 be linked with other heat shock proteins when counteracting Htt aggregation? We will investigate each of these questions in the hope of unmasking the exact mechanisms in which Hsp42 acts on Huntington aggregation pathology.

Preliminary Results

Hsp40 on Mutant Huntington aggregates:

            Overexpression of Sis1, a member of the Hsp40 family, leads to formation of two less-intense foci as opposed to one large single aggresome.

Hsp70 on Mutant Huntington aggregates:

Overexpression of Ssa1, a member of the Hsp70 family, leads to a higher background of diffuse fluorescence and multiple less-intense foci.

Hsp104 on Mutant Huntington aggregates:

Overexpression of Hsp104 increased the number of less-intense foci as well as the fluorescent saturation of the background. Overexpression of hsp104 increased the number of foci as well as the fluorescent saturation of the background.

          

The Proline-Rich Region in Huntington pathology:

The number of glutamine repeats as well as the inclusion or deletion of the flanking proline-rich regions determines Huntington toxicity. In Huntington, two proline residues, which are separated by a proline rich region, are located at the carboxyl terminus of the polyglutamine repeat.  Mutant plasmids with deletions in the proline rich region are believed to increase toxicity and reduce solubility of Huntington aggregates. In vitro, oligoprolines decrease the rate of synthetic polyglutamine aggregation. It has been speculated the proline-rich region may act to recruit protein interactors, which protect the cell from toxicity or, alternatively, the proline-rich region may act to change Huntington polypeptide conformations.

Research Design and Methods:

Aim 1: To determine the effects of Hsp42 expression on mutant Huntington proteins displaying three different lengths of polyglutamine (polyQ) repeats: Htt25Q, Htt72Q, and Htt103Q.

The haploid wild type S. cerevisiae strain BY4742 – MATalpha his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0, will be used for all transformations. The nucleotide sequence for Hsp42 will be used for designing primers for amplification via Polymerase Chain Reaction (PCR) with Topo TA over-hangs on the 5’ and 3’ end of PCR product. The PCR product will then be inserted into a Galactose inducible pYES 2.1/V5-His-TOPO plasmid allowing for ampicillin selection after bacterial transformation.

The bacteria will then be incubated, harvested, and mini-prepped. Plasmids extracted will then be transformed with lithium acetate into WT and Hsp42null strains. The strains will then be selected for using the Ura3 marker. Huntington Plasmids used for PCR are 1181 htt p426 25Q GPD, 15576 p426 htt25QDPro GFP, 1188 htt p426 103Q GPD, 15833 htt p303 FLAG 72QDPro GFP (may have sequencing problems), and 15582 htt p416 GAL 72Q+ProGFP. Sequences isolated for PCR are as follows:

 All primers will include a 5’ XbaI over-hang and a 3’ SalI over-hang for insertion into a p415 template with a constitutive GPD promoter.

Restriction enzymes that we are using: (for 25Q+P, 25QDP, 72Q+P, 103Q+P, and 103QDP)

 

  • Xba1 5’

 

 

  • Sal1 3’

 

 

Double Digest recommendations for Xba1 and Sal1 from www.neb.com

 

  • NEBuffer 3.1 is the best supplied NEBuffer
  • Xba1 75% and Sal1 100% activity

 

 

Forward primer design for Xba1/htt/Sal1

TTAA-TCTAGA-ATGGCGACCCTGGAAAAGCTG

Xba1                htt gene

IDT DNA oligo analyzer melting temperature: 61.50C

31 bp

 

 

 

Reverse primer design

CATGGACGAGCTGTACAAG-      TTA         -GTCGAC-TTAA

GFP                                          stop codon      Sal1

IDT DNA oligo analyzer melting temperature: 60.70C

32bp

 

                 

            Cloned Htt p415 plasmids will then be transformed into bacteria and selected for using ampicillin. Bacteria will be harvested and mini-prepped. All Htt plasmids, 25Q, 25QDP, 72Q, 72QDP, 103Q, and 103QDP will be will then be transformed into WT, WT+Hsp42, Hsp42null+Hsp42, and Hsp42null strains and selected for using URA3 and LEU2 markers. All strains will then be grown to mid-log phase and examined using confocal fluorescence microscopy. Hsp42 expression in Htt models will be induced by galactose and aggregates will be monitored in WT, WT + over-expression, Hsp42null, and Hsp42null + expression. Comparisons in imaging will be compared, contrasted, and analyzed using Velocity software in all cloned strains.

Aim 2: To differentiate Hsp42 activity compared to the mechanisms of other molecular chaperones such as Hsp40, Hsp70, and Hsp104 when expressed in S. cerevisiae models.

     Monitoring heat shock protein activity will be achieved by tagging molecular chaperones with a mCherry FP. Integrating the mCherry nucleotide sequence into the hsp42 strains will be done directly by homologous recombination of the mCherry PCR product into the hsp42 chromosome.  Protocol is the same as the lithium acetate transformation protocol, only using the PCR product for transformation rather than a plasmid for fusing.

Forward primer sequence:

 

Hsp42mCherryF:

 

ATTGGAGTTTGAAGAAAATCCCAACCCTACGGTAGAAAATggtgacggtgctggttta

 

Reverse primer sequence:

 

Hsp42mCherryR:

 

ATAAATATAAATGTATGTGTGTATAAACAGATACGATatcgatgaattcgagctcg

 

 

After mCherry fusion, Hsp42 will be monitored for localization and concentration when expressed or over-expressed in cloned Htt-GFP strains. We will then analyze the degree of Htt aggregation, diffusion, and localization in conjunction to Hsp42 activity, thus illuminating Hsp42’s mechanisms on clearing Htt aggregates from the cellular cytoplasm. We will then repeat all cloning procedures with the molecular chaperones Hsp40/Hsp70 complex and Hsp104. Analysis of the activity of these heat shock proteins will then be compared to the analysis of Hsp42 activity.

Aim 3: To determine whether Hsp42 can work in conjunction with other molecular chaperones to counteract Huntington toxicity.

WT+ Hsp42 + Hsp40/70 or Hsp104 over-expression, Hsp42null + Hsp40/70 or Hsp104 expression, and Hsp42null+Hsp40/70null or Hsp104null strains will be sub-cloned and analyzed to determine whether Hsp42 can act in conjunction other molecular chaperones in clearing Huntington aggregates. Possible FP tagging on Hsp40/70 and Hsp104 will be Citrine. This will allow us to visually distinguish between the Htt-GFP, the Hsp42-mCherry, and Hsp40/70 or Hsp104-Citrine.

Conclusions:

            Further investigations into the mechanisms of molecular chaperone activity on clearing Huntington aggregates in cells is needed in order understand methods of counteracting Huntington pathology. As of today, Huntington as well as other neurodegenerative diseases displaying mis-folded protein activity (Alzheimer’s, spongiform encephalopathy, etc.) has no definite cure.  If molecular chaperones function to clear non-native protein aggregation, perhaps this is one route researchers can proceed with in finding therapeutic means in clearing Huntington toxicity in mammalian neurons. The molecular chaperone, Hsp42, currently has very little literature surrounding it within the context of mutant Huntington cell models. I believe Hsp42 will have a drastic effect on clearing Huntington mis-folded proteins and that Hsp42 will be able to work with other molecular chaperone families in reducing Huntington aggregation, fragmenting Huntington proteins into a soluble state, and clearing non-native proteins, thus reducing Huntington pathology.

Kant’s Religion within the Boundaries of Mere Reason: A Reader’s Guide (Blog Post #1)

 

Our research project, titled Kant’s Religion within the Boundaries of Mere Reason: A Reader’s Guide, does exactly what the title suggests: provides readers with a guide to understanding Immanuel Kant’s Religion within the Boundaries of Mere Reason. The goal is to present advanced undergraduate as well as beginner graduate students a comprehensive explanation of the text, in addition to commentary. While my professor is writing the reading guide, my job is to examine his work in order to provide him with helpful feedback. As a member of his target audience, I can help him accurately determine which areas of the text a student is most likely to find particularly challenging. Furthermore, I am aiding in the compilation of a bibliography, mostly by locating and reviewing scholarly articles to be included.

Through this research project, I hope to gain an in-depth understanding of Kant’s religious philosophy.  This will hopefully allow us to work on additional articles throughout the second semester. Moreover, on a personal level, I am hoping to further refine my reading skills. Kant’s writing is notoriously troublesome; by first reading Kant’s text independently, I am afforded the opportunity to cultivate my abilities to comprehend challenging literature. I often find myself realizing why such a reading guide is necessary throughout the process of reading such a frustrating text. Overall, I hope to aid in the development of published secondary literature for the purpose of helping fellow students.