User:Mitali Kini - Revision history

Mitali Kini: /* Contact Info */

Mitali Kini — Mon, 06 May 2013 22:59:00 GMT

Contact Info

←Older revision		Revision as of 22:59, 6 May 2013
Line 9:		Line 9:
	*[[Special:Emailuser/Mitali Kini\|Email me through OpenWetWare]]		*[[Special:Emailuser/Mitali Kini\|Email me through OpenWetWare]]

-	I learned about [[OpenWetWare]] from MIT, and I've joined because I would like to join OWW to be able to receive notifications related to the biological engineering course, 20.109 at MIT..	+	I learned about [[OpenWetWare]] from MIT, and I've joined because I would like to join OWW to be able to receive notifications related to the biological engineering course, 20.109 at MIT..

	==Education==		==Education==

Mitali Kini: /* Research interests */

Mitali Kini — Mon, 06 May 2013 22:44:07 GMT

Research interests

←Older revision		Revision as of 22:44, 6 May 2013
Line 19:		Line 19:

	*[[Mitali Kini, Sarah Yu TR Purple Research Proposal]]		*[[Mitali Kini, Sarah Yu TR Purple Research Proposal]]
-
-	~~Research proposal~~
-
-	~~A brief project overview~~
-
-	*General Topic: Detection and Prevention of Protein Aggregation and Misfolding
-
-	Specific Idea: Combining the biophysical study of cellular lipid membrane properties and their influences on protein aggregation and misfolding, with the protein engineering study of GFP or luciferase-based reporters in detecting protein aggregation, we propose the design and creation of a chimeric fluorescent protein reporter that can bind to cellular lipid bilayers and detect aggregate formation in different tissues. With this reporter, we hope to elucidate the mechanism and extent to which lipid membranes play a catalytic role in aggregate formation. We hope to potentially examine the effect of amyloid-forming protein-membrane binding on the viability of cells due to changes induced in membrane permeability, rigidity, and disruption of membrane surface properties. Specific proteins which we expect to study and track with our reporter are AB, htt, and IAPP, which are prominent in many neurodegenerative diseases resulting from aggregation of these proteins in the brain.
-
-	*Sufficient background information for everyone to understand your proposal
-
-	~~Aggregate proteins cause many neurodegenerative diseases such as Alzheimer’s and Parkinson's.~~
-	~~It is important to be able to detect aggregates and, if possible, inhibit their activities. Detection can be done through known fluorescent reporters utilizing GFP or luciferase catalytic activity.~~
-	Furthermore, it has been found that amyloid proteins form aggregates upon interaction with surfaces and lipid bilayers, and in some cases can cause cell death through permeability changes. The aggregate form of these proteins is promoted and stabilized by association with a rigid surface or with peripheral membrane receptors in physiological contexts, and can often integrate and disrupt cell membranes through the presence of transmembrane domains.
-
-	*Project details and methods
-
-	~~-Design and generating chimeric proteins with membrane-binding domain and fluorescence domain~~
-
-	~~- Detection of aggregates without binding membranes (optimizing fluorescence of reporter protein)~~
-
-	~~-Fluorescence (GFP or luciferase) assay and/or aggregate structure analysis~~
-
-	~~- Potential use of flow cytometry~~
-
-	~~- Optimizing model lipids for maintaining surface rigidity and resembling physiological conditions~~
-
-	~~- Detection of cell viability where aggregates are present~~
-
-	~~- Testing cell aggregation with either a) different aggregate-forming cells or b) different surface properties (solid vs. lipid bilayers)~~
-
-	*Predicted outcomes if everything goes according to plan and if nothing does
-
-	~~We hope to see an indication of process of protein misfolding/aggregation by changes in fluorescence. We intend to detect a decrease in cell viability where aggregation is most concentrated.~~
-	If nothing goes according to plan, we expect to see no signals with our reporters, or increased cell death due to binding of our reporter to membranes, or incorrect signaling even in the absence of aggregates.
-
-	*Needed resources to complete the work
-
-	~~We will need access to model bilayers, solid surfaces, and cell culture constructs to test in vitro binding of aggregates to membranes at physiological conditions.~~
-	~~We will also require access to plate-readers for fluorescence assays, sequencing and PCR equipment to induce mutational changes/create chimeric proteins.~~
-	~~Any additional technology-intensive experiments will require money to acquire equipment and time to learn use of the equipment.~~
-
-	*References (Summary)
-
-	1. Burke KA, Yates EA, Legileiter J. Biophysical insights into how surfaces, including lipid membranes, modulate protein aggregation related to neurodegeneration. Fronteirs in Neurology 2013. doi: 10.3389/fneur.2013.00017
-
-	In many cases of protein aggregate formation, a key component is the interaction of amyloid proteins and cellular surfaces, especially lipid bilayer membranes. Bilayer-protein interactions have been shown in this paper to modulate protein misfolding and play a somewhat catalytic role in protein aggregate propagation. Furthermore, in cases such as the common AB amyloid-forming proteins as well as IAPP and htt, the binding to lipid bilayers can cause conformational changes in the permeability and stability of the membrane, leading to cell death. Furthermore, some bilayer-amyloid interactions can stabilize the aggregate form of the protein and promote propagation of the aggregation process. These interactions occur through transmembrane domains present in AB or chemical reactions that alter permeability and surface tension of the membranes. These cases allow us to understand the importance of direct cell membrane interactions with aggregate-forming proteins and the need to detect/identify specific proteins involved in these interactions in order to elucidate cell-to-cell signaling that are responsible for aggregate formation in neurodegenerative diseases.
-
-	~~2. Gregoire S, Kwon I.~~
-
-	~~A revisited folding reporter for quantitative assay of protein misfolding and aggregation in mammalian cells. Biotechnology Journal 2012, 7, 1297-1307. doi: 10.1002/biot.201200103~~
-	In this paper, a simple non-invasive assay for protein misfolding and aggregation in mammalian cells was performed by fusing the GFP fluorescent protein to the C terminus of human copper/zinc superoxide dismutase mutants proteins. The results from this chimeric protein show that the extent of misfolding and aggregation of a target protein can be quantitatively estimated by the fluorescence intensity of the cells by GFP. Hence GFP-tagging of aggregated proteins can be used to determine residues causing protein aggregation and understand the aggregation process of SOD1 variants. These technique of fusing the fluorescent protein to the protein aggregate of interest will allow us to detect protein misfolding/aggregation in our potential in vitro experiment.
-
-	~~3. Gregoire S, Irwin J, Kwon I. Techniques for Monitoring Protein Misfolding and Aggregation in Vitro and in Living Cells. Korean J Chem Eng 2012. 29(6): 693-702. doi: 10.1007/s11814-012-0060-x.~~
-
-	This paper outlines some key techniques enabling monitoring protein misfolding and aggregation in vitro. These techniques include molecular probes for aggregate characterization (binding and fluorescence assays), methods for obtaining quaternary structure of aggregate species (electron microscopy), methods for obtaining secondary structure (CD and FTIR), and most importantly, the use of fluorescent protein reporters by fusion to the C-terminus of target protein in detecting aggregation, as well as enzyme-based reporters. These techniques will be useful in our own design of a fluorescent protein reporter, as well as understanding associated techniques to characterizing protein aggregation in specific membrane-bound species.

	==Publications==		==Publications==

Mitali Kini: /* Research interests */

Mitali Kini — Mon, 06 May 2013 22:35:22 GMT

Research interests

←Older revision		Revision as of 22:35, 6 May 2013
Line 18:		Line 18:
	<!-- Feel free to add brief descriptions to your research interests as well -->		<!-- Feel free to add brief descriptions to your research interests as well -->

-	*[[Research ~~proposal~~]]	+	*[[Mitali Kini, Sarah Yu TR Purple Research Proposal]]

	Research proposal		Research proposal

Mitali Kini: /* Research interests */

Mitali Kini — Mon, 06 May 2013 22:34:27 GMT

Research interests

←Older revision		Revision as of 22:34, 6 May 2013
Line 18:		Line 18:
	<!-- Feel free to add brief descriptions to your research interests as well -->		<!-- Feel free to add brief descriptions to your research interests as well -->

-	*[[Research proposal]	+	*[[Research proposal]]

	Research proposal		Research proposal

Mitali Kini: /* Research interests */

Mitali Kini — Mon, 06 May 2013 22:33:53 GMT

Research interests

←Older revision		Revision as of 22:33, 6 May 2013
Line 17:		Line 17:
	==Research interests==		==Research interests==
	<!-- Feel free to add brief descriptions to your research interests as well -->		<!-- Feel free to add brief descriptions to your research interests as well -->
		+
		+	*[[Research proposal]

	Research proposal		Research proposal

Mitali Kini: /* Contact Info */

Mitali Kini — Mon, 06 May 2013 22:02:46 GMT

Contact Info

←Older revision		Revision as of 22:02, 6 May 2013
Line 9:		Line 9:
	*[[Special:Emailuser/Mitali Kini\|Email me through OpenWetWare]]		*[[Special:Emailuser/Mitali Kini\|Email me through OpenWetWare]]

-	~~I work in the Vander Heiden lab in the Koch Institute.~~ I learned about [[OpenWetWare]] from MIT, and I've joined because I would like to join OWW to be able to receive notifications related to the biological engineering course, 20.109 at MIT..	+	I learned about [[OpenWetWare]] from MIT, and I've joined because I would like to join OWW to be able to receive notifications related to the biological engineering course, 20.109 at MIT..

	==Education==		==Education==

Mitali Kini: /* Research interests */

Mitali Kini — Mon, 06 May 2013 20:58:51 GMT

Research interests

Mitali Kini: /* Research interests */

Mitali Kini — Mon, 06 May 2013 20:57:48 GMT

Research interests

←Older revision		Revision as of 20:57, 6 May 2013
Line 20:		Line 20:
	Research proposal		Research proposal

-	A brief project overview	+	A brief project overview

	General Topic: Detection and Prevention of Protein Aggregation and Misfolding		General Topic: Detection and Prevention of Protein Aggregation and Misfolding

Mitali Kini: /* Research interests */

Mitali Kini — Mon, 06 May 2013 20:57:06 GMT

Research interests

Mitali Kini: /* Research interests */

Mitali Kini — Mon, 06 May 2013 20:56:21 GMT

Research interests

←Older revision		Revision as of 20:56, 6 May 2013
Line 17:		Line 17:
	==Research interests==		==Research interests==
	<!-- Feel free to add brief descriptions to your research interests as well -->		<!-- Feel free to add brief descriptions to your research interests as well -->
-	~~Biochemistry~~, ~~Oncology~~ (~~Cancer Metabolism~~), ~~want~~ to learn ~~about Computational Bio~~/ ~~Tissue Engineering~~	+
		+	Research proposal
		+
		+	A brief project overview
		+	General Topic: Detection and Prevention of Protein Aggregation and Misfolding
		+	Specific Idea: Combining the biophysical study of cellular lipid membrane properties and their influences on protein aggregation and misfolding, with the protein engineering study of GFP or luciferase-based reporters in detecting protein aggregation, we propose the design and creation of a chimeric fluorescent protein reporter that can bind to cellular lipid bilayers and detect aggregate formation in different tissues. With this reporter, we hope to elucidate the mechanism and extent to which lipid membranes play a catalytic role in aggregate formation. We hope to potentially examine the effect of amyloid-forming protein-membrane binding on the viability of cells due to changes induced in membrane permeability, rigidity, and disruption of membrane surface properties. Specific proteins which we expect to study and track with our reporter are AB, htt, and IAPP, which are prominent in many neurodegenerative diseases resulting from aggregation of these proteins in the brain.
		+
		+	Sufficient background information for everyone to understand your proposal
		+	Aggregate proteins cause many neurodegenerative diseases such as Alzheimer’s and Parkinson's.
		+	It is important to be able to detect aggregates and, if possible, inhibit their activities. Detection can be done through known fluorescent reporters utilizing GFP or luciferase catalytic activity.
		+	Furthermore, it has been found that amyloid proteins form aggregates upon interaction with surfaces and lipid bilayers, and in some cases can cause cell death through permeability changes. The aggregate form of these proteins is promoted and stabilized by association with a rigid surface or with peripheral membrane receptors in physiological contexts, and can often integrate and disrupt cell membranes through the presence of transmembrane domains.
		+
		+	Project details and methods
		+	-Design and generating chimeric proteins with membrane-binding domain and fluorescence domain
		+	- Detection of aggregates without binding membranes (optimizing fluorescence of reporter protein)
		+	-Fluorescence (GFP or luciferase) assay and/or aggregate structure analysis
		+	- Potential use of flow cytometry
		+	- Optimizing model lipids for maintaining surface rigidity and resembling physiological conditions
		+	- Detection of cell viability where aggregates are present
		+	- Testing cell aggregation with either a) different aggregate-forming cells or b) different surface properties (solid vs. lipid bilayers)
		+
		+	Predicted outcomes if everything goes according to plan and if nothing does
		+	We hope to see an indication of process of protein misfolding/aggregation by changes in fluorescence. We intend to detect a decrease in cell viability where aggregation is most concentrated.
		+	If nothing goes according to plan, we expect to see no signals with our reporters, or increased cell death due to binding of our reporter to membranes, or incorrect signaling even in the absence of aggregates.
		+
		+	Needed resources to complete the work
		+	We will need access to model bilayers, solid surfaces, and cell culture constructs to test in vitro binding of aggregates to membranes at physiological conditions.
		+	We will also require access to plate-readers for fluorescence assays, sequencing and PCR equipment to induce mutational changes/create chimeric proteins.
		+	Any additional technology-intensive experiments will require money to acquire equipment and time to learn use of the equipment.
		+
		+	References (Summary)
		+	1. Burke KA, Yates EA, Legileiter J. Biophysical insights into how surfaces, including lipid membranes, modulate protein aggregation related to neurodegeneration. Fronteirs in Neurology 2013. doi: 10.3389/fneur.2013.00017
		+
		+	In many cases of protein aggregate formation, a key component is the interaction of amyloid proteins and cellular surfaces, especially lipid bilayer membranes. Bilayer-protein interactions have been shown in this paper to modulate protein misfolding and play a somewhat catalytic role in protein aggregate propagation. Furthermore, in cases such as the common AB amyloid-forming proteins as well as IAPP and htt, the binding to lipid bilayers can cause conformational changes in the permeability and stability of the membrane, leading to cell death. Furthermore, some bilayer-amyloid interactions can stabilize the aggregate form of the protein and promote propagation of the aggregation process. These interactions occur through transmembrane domains present in AB or chemical reactions that alter permeability and surface tension of the membranes. These cases allow us to understand the importance of direct cell membrane interactions with aggregate-forming proteins and the need to detect/identify specific proteins involved in these interactions in order to elucidate cell-to-cell signaling that are responsible for aggregate formation in neurodegenerative diseases.
		+
		+	2. Gregoire S, Kwon I. A revisited folding reporter for quantitative assay of protein misfolding and aggregation in mammalian cells. Biotechnology Journal 2012, 7, 1297-1307. doi: 10.1002/biot.201200103
		+	In this paper, a simple non-invasive assay for protein misfolding and aggregation in mammalian cells was performed by fusing the GFP fluorescent protein to the C terminus of human copper/zinc superoxide dismutase mutants proteins. The results from this chimeric protein show that the extent of misfolding and aggregation of a target protein can be quantitatively estimated by the fluorescence intensity of the cells by GFP. Hence GFP-tagging of aggregated proteins can be used to determine residues causing protein aggregation and understand the aggregation process of SOD1 variants. These technique of fusing the fluorescent protein to the protein aggregate of interest will allow us to detect protein misfolding/aggregation in our potential in vitro experiment.
		+
		+	3. Gregoire S, Irwin J, Kwon I. Techniques for Monitoring Protein Misfolding and Aggregation in Vitro and in Living Cells. Korean J Chem Eng 2012. 29(6): 693-702. doi: 10.1007/s11814-012-0060-x.
		+
		+	This paper outlines some key techniques enabling monitoring protein misfolding and aggregation in vitro. These techniques include molecular probes for aggregate characterization (binding and fluorescence assays), methods for obtaining quaternary structure of aggregate species (electron microscopy), methods for obtaining secondary structure (CD and FTIR), and most importantly, the use of fluorescent protein reporters by fusion to the C-terminus of target protein in detecting aggregation, as well as enzyme-based reporters. These techniques will be useful in our own design of a fluorescent protein reporter, as well as understanding associated techniques to characterizing protein aggregation in specific membrane-bound species.

	==Publications==		==Publications==

←Older revision		Revision as of 20:58, 6 May 2013
Line 20:		Line 20:
	Research proposal		Research proposal

-	A brief project overview	+	A brief project overview

-	General Topic: Detection and Prevention of Protein Aggregation and Misfolding	+	*General Topic: Detection and Prevention of Protein Aggregation and Misfolding

	Specific Idea: Combining the biophysical study of cellular lipid membrane properties and their influences on protein aggregation and misfolding, with the protein engineering study of GFP or luciferase-based reporters in detecting protein aggregation, we propose the design and creation of a chimeric fluorescent protein reporter that can bind to cellular lipid bilayers and detect aggregate formation in different tissues. With this reporter, we hope to elucidate the mechanism and extent to which lipid membranes play a catalytic role in aggregate formation. We hope to potentially examine the effect of amyloid-forming protein-membrane binding on the viability of cells due to changes induced in membrane permeability, rigidity, and disruption of membrane surface properties. Specific proteins which we expect to study and track with our reporter are AB, htt, and IAPP, which are prominent in many neurodegenerative diseases resulting from aggregation of these proteins in the brain.		Specific Idea: Combining the biophysical study of cellular lipid membrane properties and their influences on protein aggregation and misfolding, with the protein engineering study of GFP or luciferase-based reporters in detecting protein aggregation, we propose the design and creation of a chimeric fluorescent protein reporter that can bind to cellular lipid bilayers and detect aggregate formation in different tissues. With this reporter, we hope to elucidate the mechanism and extent to which lipid membranes play a catalytic role in aggregate formation. We hope to potentially examine the effect of amyloid-forming protein-membrane binding on the viability of cells due to changes induced in membrane permeability, rigidity, and disruption of membrane surface properties. Specific proteins which we expect to study and track with our reporter are AB, htt, and IAPP, which are prominent in many neurodegenerative diseases resulting from aggregation of these proteins in the brain.

-	Sufficient background information for everyone to understand your proposal	+	*Sufficient background information for everyone to understand your proposal

	Aggregate proteins cause many neurodegenerative diseases such as Alzheimer’s and Parkinson's.		Aggregate proteins cause many neurodegenerative diseases such as Alzheimer’s and Parkinson's.
Line 32:		Line 32:
	Furthermore, it has been found that amyloid proteins form aggregates upon interaction with surfaces and lipid bilayers, and in some cases can cause cell death through permeability changes. The aggregate form of these proteins is promoted and stabilized by association with a rigid surface or with peripheral membrane receptors in physiological contexts, and can often integrate and disrupt cell membranes through the presence of transmembrane domains.		Furthermore, it has been found that amyloid proteins form aggregates upon interaction with surfaces and lipid bilayers, and in some cases can cause cell death through permeability changes. The aggregate form of these proteins is promoted and stabilized by association with a rigid surface or with peripheral membrane receptors in physiological contexts, and can often integrate and disrupt cell membranes through the presence of transmembrane domains.

-	Project details and methods	+	*Project details and methods

	-Design and generating chimeric proteins with membrane-binding domain and fluorescence domain		-Design and generating chimeric proteins with membrane-binding domain and fluorescence domain
		+
	- Detection of aggregates without binding membranes (optimizing fluorescence of reporter protein)		- Detection of aggregates without binding membranes (optimizing fluorescence of reporter protein)
		+
	-Fluorescence (GFP or luciferase) assay and/or aggregate structure analysis		-Fluorescence (GFP or luciferase) assay and/or aggregate structure analysis
		+
	- Potential use of flow cytometry		- Potential use of flow cytometry
		+
	- Optimizing model lipids for maintaining surface rigidity and resembling physiological conditions		- Optimizing model lipids for maintaining surface rigidity and resembling physiological conditions
		+
	- Detection of cell viability where aggregates are present		- Detection of cell viability where aggregates are present
		+
	- Testing cell aggregation with either a) different aggregate-forming cells or b) different surface properties (solid vs. lipid bilayers)		- Testing cell aggregation with either a) different aggregate-forming cells or b) different surface properties (solid vs. lipid bilayers)

-	Predicted outcomes if everything goes according to plan and if nothing does	+	*Predicted outcomes if everything goes according to plan and if nothing does

	We hope to see an indication of process of protein misfolding/aggregation by changes in fluorescence. We intend to detect a decrease in cell viability where aggregation is most concentrated.		We hope to see an indication of process of protein misfolding/aggregation by changes in fluorescence. We intend to detect a decrease in cell viability where aggregation is most concentrated.
	If nothing goes according to plan, we expect to see no signals with our reporters, or increased cell death due to binding of our reporter to membranes, or incorrect signaling even in the absence of aggregates.		If nothing goes according to plan, we expect to see no signals with our reporters, or increased cell death due to binding of our reporter to membranes, or incorrect signaling even in the absence of aggregates.

-	Needed resources to complete the work	+	*Needed resources to complete the work

	We will need access to model bilayers, solid surfaces, and cell culture constructs to test in vitro binding of aggregates to membranes at physiological conditions.		We will need access to model bilayers, solid surfaces, and cell culture constructs to test in vitro binding of aggregates to membranes at physiological conditions.
Line 53:		Line 59:
	Any additional technology-intensive experiments will require money to acquire equipment and time to learn use of the equipment.		Any additional technology-intensive experiments will require money to acquire equipment and time to learn use of the equipment.

-	References (Summary)	+	*References (Summary)

	1. Burke KA, Yates EA, Legileiter J. Biophysical insights into how surfaces, including lipid membranes, modulate protein aggregation related to neurodegeneration. Fronteirs in Neurology 2013. doi: 10.3389/fneur.2013.00017		1. Burke KA, Yates EA, Legileiter J. Biophysical insights into how surfaces, including lipid membranes, modulate protein aggregation related to neurodegeneration. Fronteirs in Neurology 2013. doi: 10.3389/fneur.2013.00017

←Older revision		Revision as of 20:57, 6 May 2013
Line 21:		Line 21:

	A brief project overview		A brief project overview
		+
	General Topic: Detection and Prevention of Protein Aggregation and Misfolding		General Topic: Detection and Prevention of Protein Aggregation and Misfolding
		+
	Specific Idea: Combining the biophysical study of cellular lipid membrane properties and their influences on protein aggregation and misfolding, with the protein engineering study of GFP or luciferase-based reporters in detecting protein aggregation, we propose the design and creation of a chimeric fluorescent protein reporter that can bind to cellular lipid bilayers and detect aggregate formation in different tissues. With this reporter, we hope to elucidate the mechanism and extent to which lipid membranes play a catalytic role in aggregate formation. We hope to potentially examine the effect of amyloid-forming protein-membrane binding on the viability of cells due to changes induced in membrane permeability, rigidity, and disruption of membrane surface properties. Specific proteins which we expect to study and track with our reporter are AB, htt, and IAPP, which are prominent in many neurodegenerative diseases resulting from aggregation of these proteins in the brain.		Specific Idea: Combining the biophysical study of cellular lipid membrane properties and their influences on protein aggregation and misfolding, with the protein engineering study of GFP or luciferase-based reporters in detecting protein aggregation, we propose the design and creation of a chimeric fluorescent protein reporter that can bind to cellular lipid bilayers and detect aggregate formation in different tissues. With this reporter, we hope to elucidate the mechanism and extent to which lipid membranes play a catalytic role in aggregate formation. We hope to potentially examine the effect of amyloid-forming protein-membrane binding on the viability of cells due to changes induced in membrane permeability, rigidity, and disruption of membrane surface properties. Specific proteins which we expect to study and track with our reporter are AB, htt, and IAPP, which are prominent in many neurodegenerative diseases resulting from aggregation of these proteins in the brain.

	Sufficient background information for everyone to understand your proposal		Sufficient background information for everyone to understand your proposal
		+
	Aggregate proteins cause many neurodegenerative diseases such as Alzheimer’s and Parkinson's.		Aggregate proteins cause many neurodegenerative diseases such as Alzheimer’s and Parkinson's.
	It is important to be able to detect aggregates and, if possible, inhibit their activities. Detection can be done through known fluorescent reporters utilizing GFP or luciferase catalytic activity.		It is important to be able to detect aggregates and, if possible, inhibit their activities. Detection can be done through known fluorescent reporters utilizing GFP or luciferase catalytic activity.
Line 30:		Line 33:

	Project details and methods		Project details and methods
		+
	-Design and generating chimeric proteins with membrane-binding domain and fluorescence domain		-Design and generating chimeric proteins with membrane-binding domain and fluorescence domain
	- Detection of aggregates without binding membranes (optimizing fluorescence of reporter protein)		- Detection of aggregates without binding membranes (optimizing fluorescence of reporter protein)
Line 39:		Line 43:

	Predicted outcomes if everything goes according to plan and if nothing does		Predicted outcomes if everything goes according to plan and if nothing does
		+
	We hope to see an indication of process of protein misfolding/aggregation by changes in fluorescence. We intend to detect a decrease in cell viability where aggregation is most concentrated.		We hope to see an indication of process of protein misfolding/aggregation by changes in fluorescence. We intend to detect a decrease in cell viability where aggregation is most concentrated.
	If nothing goes according to plan, we expect to see no signals with our reporters, or increased cell death due to binding of our reporter to membranes, or incorrect signaling even in the absence of aggregates.		If nothing goes according to plan, we expect to see no signals with our reporters, or increased cell death due to binding of our reporter to membranes, or incorrect signaling even in the absence of aggregates.

	Needed resources to complete the work		Needed resources to complete the work
		+
	We will need access to model bilayers, solid surfaces, and cell culture constructs to test in vitro binding of aggregates to membranes at physiological conditions.		We will need access to model bilayers, solid surfaces, and cell culture constructs to test in vitro binding of aggregates to membranes at physiological conditions.
	We will also require access to plate-readers for fluorescence assays, sequencing and PCR equipment to induce mutational changes/create chimeric proteins.		We will also require access to plate-readers for fluorescence assays, sequencing and PCR equipment to induce mutational changes/create chimeric proteins.
Line 48:		Line 54:

	References (Summary)		References (Summary)
		+
	1. Burke KA, Yates EA, Legileiter J. Biophysical insights into how surfaces, including lipid membranes, modulate protein aggregation related to neurodegeneration. Fronteirs in Neurology 2013. doi: 10.3389/fneur.2013.00017		1. Burke KA, Yates EA, Legileiter J. Biophysical insights into how surfaces, including lipid membranes, modulate protein aggregation related to neurodegeneration. Fronteirs in Neurology 2013. doi: 10.3389/fneur.2013.00017

	In many cases of protein aggregate formation, a key component is the interaction of amyloid proteins and cellular surfaces, especially lipid bilayer membranes. Bilayer-protein interactions have been shown in this paper to modulate protein misfolding and play a somewhat catalytic role in protein aggregate propagation. Furthermore, in cases such as the common AB amyloid-forming proteins as well as IAPP and htt, the binding to lipid bilayers can cause conformational changes in the permeability and stability of the membrane, leading to cell death. Furthermore, some bilayer-amyloid interactions can stabilize the aggregate form of the protein and promote propagation of the aggregation process. These interactions occur through transmembrane domains present in AB or chemical reactions that alter permeability and surface tension of the membranes. These cases allow us to understand the importance of direct cell membrane interactions with aggregate-forming proteins and the need to detect/identify specific proteins involved in these interactions in order to elucidate cell-to-cell signaling that are responsible for aggregate formation in neurodegenerative diseases.		In many cases of protein aggregate formation, a key component is the interaction of amyloid proteins and cellular surfaces, especially lipid bilayer membranes. Bilayer-protein interactions have been shown in this paper to modulate protein misfolding and play a somewhat catalytic role in protein aggregate propagation. Furthermore, in cases such as the common AB amyloid-forming proteins as well as IAPP and htt, the binding to lipid bilayers can cause conformational changes in the permeability and stability of the membrane, leading to cell death. Furthermore, some bilayer-amyloid interactions can stabilize the aggregate form of the protein and promote propagation of the aggregation process. These interactions occur through transmembrane domains present in AB or chemical reactions that alter permeability and surface tension of the membranes. These cases allow us to understand the importance of direct cell membrane interactions with aggregate-forming proteins and the need to detect/identify specific proteins involved in these interactions in order to elucidate cell-to-cell signaling that are responsible for aggregate formation in neurodegenerative diseases.

-	2. Gregoire S, Kwon I. A revisited folding reporter for quantitative assay of protein misfolding and aggregation in mammalian cells. Biotechnology Journal 2012, 7, 1297-1307. doi: 10.1002/biot.201200103	+	2. Gregoire S, Kwon I.
		+
		+	A revisited folding reporter for quantitative assay of protein misfolding and aggregation in mammalian cells. Biotechnology Journal 2012, 7, 1297-1307. doi: 10.1002/biot.201200103
	In this paper, a simple non-invasive assay for protein misfolding and aggregation in mammalian cells was performed by fusing the GFP fluorescent protein to the C terminus of human copper/zinc superoxide dismutase mutants proteins. The results from this chimeric protein show that the extent of misfolding and aggregation of a target protein can be quantitatively estimated by the fluorescence intensity of the cells by GFP. Hence GFP-tagging of aggregated proteins can be used to determine residues causing protein aggregation and understand the aggregation process of SOD1 variants. These technique of fusing the fluorescent protein to the protein aggregate of interest will allow us to detect protein misfolding/aggregation in our potential in vitro experiment.		In this paper, a simple non-invasive assay for protein misfolding and aggregation in mammalian cells was performed by fusing the GFP fluorescent protein to the C terminus of human copper/zinc superoxide dismutase mutants proteins. The results from this chimeric protein show that the extent of misfolding and aggregation of a target protein can be quantitatively estimated by the fluorescence intensity of the cells by GFP. Hence GFP-tagging of aggregated proteins can be used to determine residues causing protein aggregation and understand the aggregation process of SOD1 variants. These technique of fusing the fluorescent protein to the protein aggregate of interest will allow us to detect protein misfolding/aggregation in our potential in vitro experiment.