 Good afternoon, everyone. I'm Epshika Zutshi. I'm from Birla Institute of Technology and Science, which lies in a relatively small village in the north of India. And it basically lies there because the founder of my institute, GD Birla, who is an eminent industrialist, was born there. And so by accident or by luck, the town of Birla got my institute. And it's around 50 years old. And currently, I'm a third year undergraduate student there. I'm pursuing a dual degree program for an MSc honors in biological sciences and a BE honors in computer science. So for the summer, I've been working at Professor Ronald Curry's laboratory. And my work has been on nesting expressive cells in the third ventricle of the adult rat brain. To give you a brief outlook of what my presentation will be involving, I will first be talking about the third ventricle because I realize some people may not be familiar with it and the importance as to why I'm working with the third ventricle in the first place. I'll be talking about the expression of two proteins, nesting and vimentin, in cells of the third ventricle. And I'd also be then focusing on nesting expressing cells in the third ventricle and the possible functions this protein may be playing in these cells. So the third ventricle, firstly, what is it? It's a narrow channel passing through the basal forebrain, which is filled with cerebrospinal fluid to give you a better idea of where the third ventricle exactly is located. So this, which you see over here, is a rat brain. And if we take a corolla section, that is, if you take a section like this, we get a rat brain section similar to this. And the third ventricle is located where you can see the star. So it's a hollow channel passing through the basal forebrain. And similarly, if you take a sagittal section, we can see how the third ventricle encompasses this entire area. And it moves on to form the fourth ventricle and so on. So now that you have a better idea of exactly where the third ventricle is located, I'll talk about nested. So the image you see over here is a normal progression of a stem cell towards a mature differentiated neural cell. And the why nested is so important is because it's been observed to be expressed at the progenitor cell level. It's a tight-six intermediate filament. And so why is this important to us? Because as nested is expressed at the progenitor cell level, it is a marker of multi-linear neural progenitor cells. And so its presence may indicate multi-potentiality. So now in linking nested and the third ventricle together, we see that there are nested and expressing cells present all along the third ventricle. And these cells can be divided into two subclasses. The first being the abandonment cells. So over here, these cells are, these are cell bodies stained with crescent violet. And so these are the abandonment cells. And the hollow you can see over here is the third ventricle. And these abandonment cells are multicellated, as we can see clearly, the cilia projecting outwards into the third ventricle. And this image is stained for acetyl-lated tubulin. And so we can see the cilia coming out of the cells. And the second subgroup of the nested and expressing cells are the tannocytes. Tannocytes are modified abandonment cells. So this image has been stained for nested. And so we can see the black cells are all tannocytes. And what's interesting about them is that they have processes extending out of their cell body. And so these processes extend into the brain parenchyna. And they generally terminate at blood vessels, which I will be talking more about in detail later. So what is known on the, on these nested and expressing cells of the third ventricle? There have been several reports of neurotenesis by different groups on cells of, on these cells of the third ventricle. But till now, all the data that we have is not conclusive because these cells appear to be proliferating at a very low rate. So we're not so sure as to whether it's these nested cells which are proliferating or if there's something else that actually is proliferating. And several groups observe slight proliferation, but with no co-localization with nested. So till date, there is no convincing correlation between nested and proliferation in cells of the third ventricle, which is why I moved on to approach vimentin. So vimentin is another protein which is expressed in most primitive cell types. And it's interesting because vimentin has been linked with nested expression. So it's been observed that vimentin is necessary for nested assembly. And conversely, nested is necessary for vimentin disassembly. So what this complicated mechanism seems to imply is that somehow nested and vimentin link, nested and vimentin expression is linked. And so that is why we started observing for vimentin also in the third episode. Necessary background, I'd like to state out what my objectives were. So my project was a two-fold approach. Firstly, as nested appears to have no major correlation with proliferation, is it possible that other intermediate filaments such as vimentin are associated with cell division? And secondly, if nested expressing cells are not normally proliferating, then what are they doing? And why is nested present in the third ventricle in the first place? So to go about my project, I observed the expression pattern of vimentin. And I also mapped nested and expressing cells along the third ventricle to identify regions around the any seed for nested and expressing cells to suggest nested and functions. So when I first started out in the lab, my PI asked me a question, and I found it awkward then, but I really understand what he meant by it now. So he had asked me whether I know how to cook. And considering I did a whole lot of chromogenic immunohistochemistry, I now understand what he meant by it. So I've seen for acetylated tubulin, for GFAP, for vimentin, and for niston, specifically because as I talked about the second part of my project, which entails mapping the entire nested expression along the third ventricle, I, to do the mapping portion of my project, I used a microscope projector, and I traced out the outline of the third ventricle on which I then located the nested positive cells, scanned it into a computer, and with the series of images that I got for each section, I stacked all of them using a software by MLJ, and generated a 3D projection using V3D. So coming to my results, this shows the staining pattern of vimentin. And when we stained for vimentin, we observed that several cells in the dorsal end of the first 0.8 millimeters. So if you remember the image of the third ventricle that I showed you, there's the rostrum side, which is the front portion, and there's the cordon side, which is the back portion. And so in the first 0.8 millimeters and the last 2.5 millimeters, we could see several cells which were vimentin negative. Now to explain what I mean by vimentin negative, so this is the third ventricle, again. These are cell bodies stained for crescent violet. So the purple cells that you see are vimentin negative, whereas the brown cells that you see are all vimentin positive. And so we can see a clear pocket of cells which are vimentin negative. And we also observed that most tanninside stained for nesting. As again, we can see the third ventricle here and the tanninside processes extending outwards from the third ventricle. So what does this tell us? This told us that there may be a leak between vimentin and nesting, we're not sure. But there may be, because they both do stain for the tanninsides. And so currently, this research is preliminary, we haven't got convincing reports yet, but we did a double stain for vimentin and nesting. And so in green, you can see the vimentin over here. And for example, this would be a clear have vimentin positive cell. And as you can see several more all along this ventricle wall. And so when the same image was scanned using a cornfocus microscope in red, where we can see the nesting, we again observed a few more cells positive for nesting, much lesser than vimentin, but they were there. And so when we performed a merge of the green and the red, overlap of any green and red would result in a yellow image. Which interestingly in the merge, we can see several yellow cells all along the third ventricle wall. Now, what we can infer from this is that while nesting seems to be co-localizing with vimentin, there are several cells which seem to be only vimentin positive. And right now it's too preliminary to state that there is a clear link in the expressions. And but it is likely that there are several other proteins which are acting as factors promoting the expression of nesting and vimentin. But it also states that vimentin possibly could be involved in the proliferation. We don't know yet, but it's worth looking at. So coming to the second part of my project, which is the staining pattern of nesting expressing cells. The images you can see here are for nesting. So the dark images are for nesting. And along the 3.5 millimeter extent of the third ventricle, in the first 0.8 millimeters, in figures A and C, we can see the nesting positive and nesting cells and the tannicytes. In, we saw rare expression all along the middle 0.6 millimeters. And again, in the last 2.1 millimeters, there was dense tannicytes staining. And there was a bit of a problem that we faced over here because due to the dense tannicytes staining, the most of the ventricular wall was obscured due to which we couldn't quite make out whether there are any appendable cells lying underneath. So that might be one shortcoming. So given the sections, I then prepared the 3D projection of the third ventricle. So each of these are single sections of the brain all compiled together. So in yellow is the appendable cell, in white are the tannicytes, all these are nesting positive and nesting cells. And I just like to play this for you. It shows you the same thing that it looks fancy. So for a better image, I let just, so there's one very clear that observation that we can make from this image. And that is that the nesting expression is clearly localized to two regions along the third ventricle and it seems to not be occurring in the remaining areas. Which suggests that nesting does have a function, which is why it's being so specifically expressed in certain areas. So we took a look around at the neighboring regions along the third ventricle. And we observed that these regions include the median pre-optic nucleus, the sub-formical organ, the median annulance. Basically there is something very common linking these areas together. And that is that all these regions are circumventricular organs. Now what are circumventricular organs? These organs have an incomplete blood-brill barrier, which makes them instrumental in the transport of hormones from the brain to the blood. So what this tells you is that you have nesting, you have nesting expressing cells and somehow they always seem to co-localize in severe regions. So is there a link? Because this seems to suggest there is some sort of underlining factor. And so, and this is not surprising apparently because tannocytes and CVOs do have an inherent link. Tannocytes are involved in hormone transport from the cerebrospinal fluid to the blood. And they're also speculated to secrete gene knowledge. So tannocytes being localized to severe regions for that matter is not surprising at all. What is surprising is that all tannocytes express nested. And that means that nesting, which is an intermediate filament, could possibly be involved in the specialized transport functions of tannocytes. And it's also possible that as tannocytes secrete gonadotropin-releasing hormone, and the amount of hormones secreted into the body has to keep varying according to different conditions due to puberty, due to different stages of development. Nesting may be involved in such plasticity and modeling also required to regulate the amount of hormones that are released. So to summarize all that I found out, the role of bi-mention and its possible link with nesting and proliferation, we still do early in the studies to make a clear cut assumption.