Assalamu alaikum. This is Introduction to Programming and today is lecture number 6. Let again, as usual, let's start with a recap of what we have done in the last lecture. Last time, we looked at the conditional constructs of C language, IF and IF ELSE. There is another conditional construct, but we will cover it in the following lectures.  Now, we can make some decisions. There is another very important construct that we will discuss today, which we will discuss today. That is loop, that is, repetition structures. Many times in our daily life, we have to repeat many things. Let's take a simple, trivial example. We say that every morning and evening, and so on, if you look at a month, you have repeated the same cycle 30 times, if there are 30 days in that month. So, repetition structures occur all the time. If we say that we have to process a payroll of a factory, then payroll is something that we have to calculate from the first person to the last person. Usually, these calculations are of the same type, so we have to repeat them again and again. So, again, a repetition structure. To introduce these repetition structures, let's do some exercises. Let's do some interesting mental exercises. Let's suppose that I give you a problem. Right now, we are talking about integers. I give you a problem that I write a program, in which you print the sum of the first 10 integers from one to ten. So, you can do this program with the tools you have. And the simple approach would be something like, either you say, int sum, and the next line says, sum is equal to 1 plus 2 plus 3 plus 4 plus 5 and so on, plus 10 semicolon, cout, less than, less than sum. So, the program is written, but it doesn't go so easily. I say, it's written, it's gone, there is no syntax error, you have seen the output. So, now you know what the sum of the first 10 integers starting from one is. So, let's do another problem. And the next problem is, find me the sum of the first 100 integers starting from one. So, what will you do? Okay? I say, very, very good. You have now obtained the sum of the first 100 integers. So, please go and now get me the sum of the first 1000. Do you see where we are going? Bad practice, the way to look at this problem is as follows. I say, we know the integer. We haven't defined the problem. We have to sum it from one to 1000. If you have integer number one, do you have any way of finding out what the next integer is? I'm sure you have. You've done it in calculus. It is the previous integer plus one. A generic statement is given that if you're given one, what is the next integer? It is one plus one. Answer two. If you're given two, what is the next integer? It's two plus one. Every time to get the next integer, you gather one in the previous integer. So, if you look at our original problem, find the sum of the first 1000 integers starting from one, how to approach it. We will do this for its analysis. First, we take a variable. Do you remember the concept of the variable? Balty. So, there's a bucket line over there. The sum is written on it. And the balty is empty right now, which means we will declare a variable of type integer int sum. We will initialize that. Again, good programming practice. First, make sure the bucket is empty or it contains what we want it to contain. Because we haven't started the work yet. We say that the sum is zero in the balty. So, it says sum is equal to zero. Now, what do we have to do? The analysis is going on. So, we say, first, pick up the first integer. That, by definition, for our problem is one. You put that in the bucket. Then you find the next integer, which is the previous integer, in this case one. Add one to it. That gives us two. Now, put this in the bucket in a way that it adds to the previous value. You find the next integer plus one. Put it in the bucket in such a way that it adds to the next value and so on. So, see what we're doing is that in the talks, this thing has been cleared that one thing we are repeatedly repeating. So, we should have some programming construct, through which we can do this repetition easily. In C, there are many programming constructs with which we call looping. We call it loop. We can do looping. For that, we will start with one construct today. That is, while statement. While is the word in English, w-h-i-l-e. Here, let me just pause and reiterate. See, whenever I tell you a word for C, in the last lecture, I talked about if and else. Today, we are talking about while. These are the keywords for C. You cannot use these keywords as variable names. So, let's start with while today. What do we do in the while construct? While means, j-b-t-k. As long as a condition is true, keep doing it. These things will be statements. Just like we talked about if, you can also use braces with while, curly brackets, or let them stay. When will you let them stay? If you only need a single statement because of while, then you don't need braces. But again, repeating myself, for good programming practice, just try to make sure that whenever you write while, after the condition, you open the braces. Then you have to write one statement or a thousand. You can write that and indent it. Because this block is coming in the braces and then you have to close the braces. This will not have unwanted side effects or logical errors. So, while loop is the first one that we worry about. It's a very interesting construct. It's a very elegant construct. And its basic syntax is like this. As long as this condition is true, keep doing this. So, in C, the syntax is while. Here, parentheses or parentheses can get a logical expression. That logical expression obviously will contain a logical or a relational operator. And while means that as long as this relational expression is true, it keeps giving an answer. As long as this expression is true, the rest of the statements will continue to be executed. Now, let's go back to our addition problem and see if we can do it in a nice, simple, elegant manner. Now, what was our problem? Let me reiterate. It said, find the sum of the first 1,000 integers starting from 1. So, in terms of C programming. Now, I am not talking about the IOS stream. I am not talking about the main key. I am talking about the body of the program. First, we declare a variable in which the sum will go. Let's name it as sumricks. Again, programming is self-explanatory. It explains itself. If you are reading the program, you will know what will come in this variable. So, we say int sum, semicolon. Again, good programming practice. When you declare a variable, please initialize it. So, we say sum equals 0, semicolon. So, we made sure that we have got a bucket in which we will keep an answer. And that bucket is empty right now. For this, there is a shorthand in C. When you declare any variable, you can initialize it on the same line. It is a matter of style. So, you could have said int sum equals 0, semicolon. And that statement would have done two things. It would have declared a variable called sum of type integer. And it would have initialized it to 0. Which means now we have written one statement instead of two statements. It is completely optional. You can adopt any style for yourself. Personally, I write int sum, and sum equals to 0. I write that initialization or the value assigned to it is seen on a single line. So, we have initialized it up to here. Can we now collect the number in it? Well, first, we need a variable for a number. So, where we wrote int sum, we also write comma with it. So now we have int sum comma number, semicolon. What will happen? The number is the variable in which we will keep integers. At this time, what integer is being worked with? So, the first few lines of the code will be complete. Int sum comma number, semicolon. Then we initialized sum. We said sum equals 0, semicolon. After that, we said take the first number. We said integers. So, take the first integer. And going back to the problem statement, we wrote that the first integer will be 1. So, we said number equals 1, semicolon. I'll keep repeating things. These are both assignment statements. What did they do? They did the calculations, the memory locations, which we labeled 1 as sum and 1 as number. We kept 0 in the sum, and we kept 1 in the number location. Remember, this is assignment, not any quality. Now, let's move on. Now, what is our problem? The problem is that we got a place, the number was also given, now start collecting them. How far do we have to collect them? This is the condition of the while statement. We said that we need a thousand numbers first. We need to take 1 to 1,000 integers. So, our ending condition is 1,000. When the value of the number becomes 1,000, we will stop at that time. So, what do we do? What we do is, we say initialized over, number is equal to 1. So, we say while parentheses open, number is less than or equal to 1,000. Parenthesis is closed. Relational operator, last time we talked, less than equal to sign, will be written together. There is no space between them. This means less than or equal to. So, now what condition have we made? We said that the number of the variable is less than or equal to 1,000. This is the condition, so this is the half statement.  What do we do until this? So, we say while parentheses, number less than or equal to 1,000, parentheses closed, braces open. Now, I am putting it in the bracket, again a stylistic issue. How will we get the sum in it? So, what do we have to do now? First, we have to analyze, think a little about the action. The action is, the current value of our number, pick it up and put it in the sum bracket. After that, what should we do? Generate the next number. So, the first line is very simple. Sum is equal to sum plus number. Why did I write this way? Why did I not write sum equal to number? Simple reason. If we had written sum equal to number, then it would have been an assignment statement. And the sum bracket would have been replaced by the value of number. We do not want to replace it by number. What we want to do is, add the number to the original contents. So, what we have used is, sum is equal to sum plus number, which means, first, take the original contents of sum, add the number to it, and store the result in the sum again. I was just repeating the good old statement that I had told you in the first or second lecture, that it is so common, x equal to x plus 1. We are using the same variant here. Sum is equal to sum plus number. Next, what do we have to do? Generate the next number. So, given any integer, what is the next integer? It is integer plus 1. So, we put in the next statement, number equals number plus 1. After that, we close the brace, and the wild structure is over. Just think about it. It is so elegant. You have neither typed the number 10, nor typed the number 100, nor typed the number 1000. We have put a small wild loop. We have put the three lines of the four lines. While number is less than equal to 1000, sum is equal to sum plus number, number equal to number plus 1. This loop will work. What is happening? The value of the number is increasing in every loop. 1, then 2, then 3, then 4, and so on. The condition that is applied to the top of the wild, it says, while number is less than equal to 1000, this is going on. When number 1000 happened, then this condition is still true. You will enter the loop. You will say, sum is equal to sum plus number. The last number is here. 1000 will be added to it. The next line says, number is equal to number plus 1. So, the value of the number will be 1001. Now, when the wild loop will test it again, it will say, while number is less than equal to 1000, so the answer to this will be false. When this is false, the loop is over. So, now your control of the program, it will not execute the statements written in the wild, it will go ahead with the end brace. There, we can put in the simple line saying, see out less than quotation marks, again to be self-explanatory and be polite. The sum of the first 1000 integers starting from 1 is, put all these things in quotation marks, then less than less than sign and then sum, semicolon. So, see out that you remember the property, it knows how to handle character strings, it knows how to handle integers. So, it wrote a character string, after that, the value of sum was printed on the screen, your problem was solved. Now, look at this, a little on this program, a little on this program, and let's see if there is any improvement in this or not. First thing, now if I come back to the same frame of mind, that I have to trouble the students today, I say that I have taken the sum of 1000, so now you go and take the sum of 20,000 first integers starting from 1. What will you do? When I talked about this for the first time, it was about heart attack, that after 10 numbers you have to type 100, then you have to type 1000, then we put the loop, life became very easy. Now, what is it? You open the program, where we have written 1000, whatever the near target value is, let's say 2000, if you do 2000 there, then the loop you have, while, number less than equal to 2000, and first the sum of 2000 integers will give you the program, 3000, 4000, 5000, you may be wanting to try it out, you try it out for yourself, in fact it might be interesting to find out that how far can you take the sum? If you say that first the sum of 100,000 integers, then what is the right answer? There is no correct answer. Let's see how far you can reach, take it to the discussion board, talk about it. The limit I am talking about will give you a hint, that we are working with integer variables, for integer variables, there is a special place reserved for a specific size in memory, if the number becomes so big that it does not fit in that place, then there is an overflow condition, now there will be two things, either your program will give you an error while walking, it will not give you an error on compile time, because it does not know what value will be given in that place, we are talking about the sum of variables, the number is correct, excuse me, after that, if we talk about the overflow, then either there is a run time error, or there will be some other value written there, how will the value be written, the value that the computer wants to write in the memory location, the extra bits that do not come in that place, will disappear somewhere, they will go into space, therefore the answer that is now written in some variable, that is not the answer to the question, this is some other number, so you might want to play with it and discover the limits of your computer, and your compiler, how many numbers are there that we can take the sum, let's go on with the same problem, and see how we develop it further, see when I said that the 1,000 problem has been solved, now take it to 2,000, take it to 5,000, what were you doing every time, you were doing that you opened the program in your editor, remember editor, you have to open the program in your integrated development environment, and there you did some modification, then you compiled the program, and prayed that while modifying, there should be no syntax error, that there should be no extra comma, and then you executed it, then you took the answer, I came and gave you another target, then you edited the program, so now you are in this loop, edit, compile, execute, change, meaning edit again, compile, execute, so can you do something better, now I am going towards reusability, can you write the program, where I come and tell you, if you give me the sum of integers, then without changing the program, you can tell me the sum of integers, think what approach will be, approach is quite simple, and I am sure that many of you would already have thought of it, suppose in the wild statement we have written, number less than equal to 1000, we change that, we say wild number is less than or equal to upper limit, so where did this upper limit come from, you declare a variable, then tell the user that while running the program, you should tell them how many numbers you need, so again good practice, good style, thinking of reuse, what would we do, we had already put two variables, sum and number, we put one more variable together, so int sum, number, upper limit, semicolon, now initialize sum and number to 0 and 1 respectively, and then tell the user what is the upper limit, now its interesting side effects will come, you said see out, let's give the user a prompt, please enter the upper limit for which you want the sum, and after that with see in, greater than greater than upper limit, semicolon, the number type the user, I will do it 1, 2, 3, 4, now the program will interpret it, first 1,234 integers need to be summed, after doing this when you will come to the while, so now the upper limit value has come, you said while number less than or equal to upper limit, that same thing sum equal to sum plus number, number is equal to number plus 1, now this program, at least till the time to gather integers, has become generic, from this we have done enough, this program did not take any more time, but see, now if I go to the upper limit when the program asks me, while walking, there I will put 0, then what will happen, if the upper limit value is 0, then when you will come to the while statement, there it will be written while number is less than or equal to 0, and the number at that time for the first time, what was its value, above you have written number equals 1, and then you have come to the while and said, while number is less than or equal to 0, is this condition true, answer is obvious, condition is false, if the condition is false, the loop will not run, not even one time, so while statement will fail for the first time, will come down, and your written will come down, the sum of the first, here also put the upper limit, in the output statement, so you would say see out less than quotation marks, the sum of the first, space leave the end quotation marks, then less than upper limit, then continue the statement, less than less than, then open quotation marks, then leave space, and the integers is, space leave the end quotation marks, less than less than, sum, semicolon, here when this program will run, and you put the upper limit 0, then written will come, the sum of the first, 0 integers is 0, very correct, so it is interesting that, there is a boundary condition, an abnormal condition, that the sum of the first 0 numbers, so the sum of the first 0 numbers, should be 0, its answer is correct, so you can see that just by playing with one simple program, you can get it to become more and more and more interesting, how about if we make it even more interesting, let us take the same problem, but in its statement, let us change a small change, I tell you that, that the first n integers, 1000 let us say, starting from 1, the even integers in it, I need to explain, now see, we are doing 2-3 things, one is, as I said earlier, you should think loop, loop will go here, but I have also qualified it, that I need even integer, so when even integer is required, then this is a condition, so now as soon as you think, or hear the word condition, you should start thinking if, so what will we do to do for this, our program becomes a little more modified, we let the first part be the same, upper limit one, the wild statement will also be the same, but in the wild, we are saying, what do we need to connect, if the current number is even, then connect it in front, otherwise don't connect, in other words do nothing, so it's a simple if, it doesn't have an else, in the wild it will come, if, and then in the bracket we need the condition, that condition is, if number is even, this is what I said in English, the question is, how do we write it in C, number is even, what do you think, now you have some arithmetic operators, you had seen, plus, minus, multiplied, divide, modulo, if any of them has such an operator, through which we can know, if an integer is even or not, if it's not even then it will be odd, remember, think about it, if we use mod operator, the percentage sign we used, and we say, number, modulo, two, what is the test of even number, that it is divided exactly from two, so, if we say number modulo two, and its answer is zero, so that means the number is even, now formulate the C statement, how it will be written, this test we said, if number is even, so the way we write it is, if, parentheses, number, percentage, two, equal, equal, zero, parentheses is closed, again I am recapping, that when we have to test equality, so single equal to, no, that is assignment, double equal to sign is used, so we say if, number, modulo two, equals, equals zero, parentheses is closed, and then the same style issue, put the brace, however the statement is the same, curly bracket open, indentation by one unit, and there we will write, sum equals sum plus number, next line brace is closed, now what is happening, this we have written in the if statement, in the while statement, there is no need, in the while it can be if, in the while it can be another while, it becomes nested while loop, now let's see our program, simple, initialized, sum is zero, number is one, we got the upper limit from the user, let's say he put 1234, now we are in the while, in the while it is written, while number is less than or equal to, upper limit, while goes in, there we tested first, the number which we are handling, is even or odd, we said if, number, percentage two, i.e. number, mod two, equals zero, with braces we gave the if statement, sum is equal to sum plus number, if statement is over, and after that we said, number is equal to number plus one, here we have to increase the number, regardless of whether it is even or odd, so first one will be odd, sum will not be added, number equal to number plus one will go, one will be two, return to loop, sum will be two, because number, percentage two will be zero, and the loop runs, and now when it will come out, so obviously you should change the C out, you should say, the sum of all even integers, between one and upper limit, which is its value is, and then next sum, obviously that is an exercise for you, but you already have this program in your handouts, so you should be able to see, the syntax of the C out statement, what is being made, so interesting we have found, how to determine even numbers, let us exercise a little, what if the percentage operator in the C is not there, all other things are present, all other rules are the same, but the modulo operator is not there, can you think of a trick, in which you can determine even numbers, think about it, I can tell you the answer, but give it a serious thought, how would you do that, look much earlier on, some lectures we talked about, one thing is integer division, and at that time I told you, the integer division truncates the answer, meaning 5 slash 2, when it is executed in C, as an integer division, its answer will be 2, answer will also be an integer, fractional part will be thrown away truncated, so we have got, 5 slash 2 is equal to 2, now how are we doing the test for even numbers, this is a programming trick, so I am telling you that, the more tricks you have in your good old tool bag, the more equip you will be to handle more complex problems, we did that, we took a number, and the number is an integer, we divided it from 2, integer division, whatever the answer is, we multiplied it from 2, again integer multiplication, so what will be the answer, so we have got 2 star parenthesis, number slash 2 parenthesis is closed, what is the result, see what will happen, if the number is even, then the division will be number divided by 2, it will give the exact answer, it will give an accurate answer, you will multiply it from 2, then you will get the original number again, if the value of the number is not even, then the integer division by 2, the answer will be truncated, and you will get the next lower integer as an answer, when you multiply it from 2, then you will not get the original number, so you can think of formulating another test, for finding whether a number is even or not, without using the modular operator, this was an aside just to add a little trick to your toolkit, because who knows, when you become a proficient programmer, what language are you working in, it is possible that the mod operator is not in that language, and if it is not, then what does it mean, we can not even determine the numbers, we can, keep that in mind, now look at the loops, what happened, we have seen 2-3 small programs, the first thing I will tell you, we got a very clean and efficient and elegant way of programming, a long program in which we had to do 1 plus 2 or whatever, in a small concise language, abbreviated and programmed in 3 lines, it works, and what are those 3-4 lines doing, calculations of thousands, how many times do you want to make a loop, you determine, the loops that we have placed, they are going to the upper limit, until this happens, keep doing this, and normally, the loop like this, the test of that, changes the condition of the body of the loop, in this case, we were testing the value of the number on the top, and the value of the number in the loop changes, this is almost necessary, in the structure, when you change the value of it, then it changes the condition of the original while, otherwise, there can be a problem too, let's take a simple example, if we talk about, we put another variable on the top, and give another name of the variable, it is called junk, it is also an inch junk, and you called junk, junk is equal to 1, we write loop while junk is less than or equal to upper limit, and we don't do anything in the loop, we say sum is equal to sum plus number, number is equal to number plus 1, what will happen, it will be a bad thing, this is what is called an infinite loop, we are not changing the junk, the condition of the while will always be true, the computer will be going crazy, until there is no overflow condition, or there is no electricity, or there is no smoke coming out of the computer, it will keep going, your program will be stuck for infinity, watch out for that, make sure that when you are putting any loop test, in this case number less than or equal to upper limit, that loop test keeps an adequate exit, some condition is changing, in which it gets an exit point, be very careful with that, we don't want infinite loops, while loop gives us a way to handle complex problems, combine it with an if statement, like we just did, you can do many examples for yourself, for example, I will pose you, now you take odd numbers, between 1 and let's say 1000, or whatever number is keyed in at the keyboard, take out the odd numbers in between, you take them out separately, even numbers are this, odd numbers are this, and use only one loop, think what we will do, hint, if, else, you have either an even number or odd number, if we are talking about integers, you can use that too, there are many things, so here we will introduce you to the loop flow chart, its structure is a little different, you will have to understand it a little, but it is not difficult, it is simple, see what is the first thing in while loop, condition, for condition we have a diamond symbol, with condition you have to put an exit, or how to get out of the while loop, and later the body of the while loop comes, what is the statement, so let's look at this construct, that you entered from above, made a rectangle, in which you wrote while, and from the right hand side of the rectangle, you took out the line, and on top of that you made a diamond, and in the diamond, you wrote the condition of the while, now you will make the line from the diamond, if the condition is true then what will happen, that is, what will happen if the while loop goes, below you make a rectangle, make one, make five, those are the processes that will go inside the loop, the line coming from below these rectangles, you bring it up, as if you have given it in the diagram, and it becomes a loop structure, and the rectangle of the while, we took out the line from below, and it became the exit of the while, that is, the closing brace will be here, so if you see the structure of the while, then from the top to the bottom, there is a straight line in between, there is a rectangle in which the while is written, so the rest of the things will be visible to the right, this is the thing that we were using in the code, that when we wrote the while loop, we indented the body of it, so again this flow charting construct, this also helps us with the code, in one to one correspondence, now we are doing the opposite, we write the code first, then we tell it what is the flow chart, that is, we are introducing these things now, later when the problems will get more complex, then normally in their analysis, you will first make the flow chart, and then you will write the code, so keep these constructs with you, keep them in your tool bag, then we put an if statement in the while, if we look at the same construct, that the line from the dimension of the while, where we are making processes, you will put an if statement there, remember that I told you about the if statement, that you put a point at the top and bottom, and whatever the if construct is made with a point, there are the starting and ending points of the if statement, it is very simple, wherever there is a rectangle in the body of the while, you remove the rectangle and connect those points there, so what you will see in the picture, the picture is the flow chart, if the while loop starts, then the body of the while is indented, if the if statement comes in the body of the while, then another indentation comes, so second level indentation, so in the picture the same thing will be seen, which you will see in the code, very easy to identify, so I think you should practice drawing some loops in terms of flow charts, use the while loop, and then with that, put some nested ifs, put a nested while of Bisha, see what happens, because we will cover the nested loops as well, let's look at another problem for a second, simple problem, but something that we use, see you know the factorial, you must have heard the factorial, you must have used it, factorial of a number, its definition is, if you have a number n, then n factorial is defined as, n multiplied by n minus 1, multiplied by n minus 2, multiplied by n minus 3, and so on up to, multiplied by 3, multiplied by 2, multiplied by 1, so it's a series of multiplications, can we get a factorial of a number, by writing a program, and if we can get a factorial, then we must get it out of the loop, we must write it straight out, so now I won't be washing the whole adding of 10 numbers, now you must have so many skills, that you must think about it, you have to do the same thing again and again, so for this, let's jump to the factorial program, using a while loop, again, let us keep the concept of generic programming, let us work easily, first think that, if we get a factorial of 6 today, then maybe in the evening, let's calculate the factorial of 5 or 7, or calculate the factorial of 8, then let's use that as a variable, so factorial program, let's first look at the code, let's jump right there, I have a stream, main, opening press, what do we need, in the variables, we need the factorial of which number, so we say int number, by definition, these are integers, after that, next will come the answer to this, that variable called factorial, let's go ahead, you should see out and prompt, see the value of the number, so we have a queue, in terms of, which number factorial is being drawn, get it from the user, now let's come to the definition of factorial, so now the number we get, that int number, the value, so this will come in the while loop, now the while loop will be, while number is greater than or equal to 1, because in factorial, we have to count down, n, n minus 1, n minus 2, n minus 3, until it reaches 1, so our while condition is, while number is greater than or equal to 1, I have told you the while condition, but before that, another very subtle thing, we have done some programs, okay, some is equal to some plus number, etc., and we initialize some to zero, the value used to be empty, and then we used to collect the number, this problem that we are tackling right now, that is factorial, in factorial, our result will come, we have kept that variable, its name is factorial, that result is the result of multiplication, every time, whatever number we have, we have to multiply something, so now the question is, which value do we have to initialize factorial? think, we will keep zero, if we initialize factorial with zero, then when we multiply n by n minus 1, then we will say factorial is equal to factorial times number, factorial's value is zero, times number will be zero, whatever is fun, factorial's value will be zero, so this also needs to be taken into consideration in programming, whenever you are taking something's products, or repetitive product, then usually we initialize it from one, in this case also it makes sense, that the variable has factorial, its initialization has to be from one, the number you have got from the user, and the factorial is initialized by one, now the loop is shown, while number is greater than or equal to one, the loop's body is here, factorial is equal to factorial multiplied by number, so the first time it was one, second time the number will come in it, next line, now the equation we are solving is, n multiplied by n minus one, i.e. which number has to be multiplied, the original value of the number is decremented by one, so the next line will come in the loop, number is equal to number minus one, and remember there was a condition above, it was greater than or equal to the condition, so now the number has started to go down, and eventually it will reduce from one, so the loop will end, now if you want to run this program manually, I think it's a good exercise, you put the conditions, make a small table, what will be the value of the number, what will be the value of the factorial, and then run the loop from the hand, first this line came, then this line came, that the value of factorial will continue to build up, do it from a small number, don't do it with a very large number, otherwise you will have problems, but nonetheless, this is also an interesting exercise, simulation by hand, so you might want to try it out, and see that you can run the program with your hand, and then you can always come back, because factorial, when numbers pass a little, the answer becomes so big, that it will be very difficult for you to verify so try this out, write the factorial program, run it for various values, again good point for discussion board, what is the value, whose factorial becomes so big, that it cannot be stored in the memory of the computer, as an int variable, try finding that limit, small while loop program, make a flow chart, practice all of these things, practice makes perfect, it has to be done, program a lot, practice a lot, draw your flow charts, it's important for you to do that, one, while finishing, I will tell you a property of the while statement, in the next lecture, there will be a lot of discussion on this, see in the middle, we reached a place in this lecture, when I said, if we find the sum of the first zero integers, we discovered that the while loop is not running, the first condition failed, the statements were not executed, and we came out, so a property of the while loop is, that it executes zero or more times, think about it, it can run at a zero level, depending on the condition, or it can run more than that, we will see its variants, we will see other types of loops, where at least one loop will run, this is not a property of the while loop, it can run at a zero level, so please keep that in mind, today's lecture, was on repetition structures, that is, looping structures, we discussed one loop of C, which is the while loop, the good thing is, the meaning of the while loop of C, is used in C, similarly, as we said, if and else are English words, and the same meaning is used for decision making, in the C language, so it's very easy to learn and understand, again, if you have a list of C keywords, I would strongly recommend that you keep looking at those keywords, so that you know which words are used as variable names, we made a flow chart of the while structure, and we looked at its construct, again, I would strongly recommend that you draw yourself many, many, many flow charts for the while statement, and you have two important programming constructs, one condition, if else, or if simple, and one loop, while loop, along with that, you can write 90% of all programs, moving forward, we will talk about structured programming, we haven't mentioned it yet, in that, I will tell you that you need these two things, to write any and all programs, until then, excuse me, goodbye.


Lagos Transport Fest by EventThive.ng caught out to shape the future mobility for Africa, had lots of speakers that have invested a lot in the transport system and the business opportunities this brings in terms of development. They have seen the prospects in this industry ranging from road, rail, ports and logistics. Positively Africa's correspondent Destiny has more. Transport business is no doubt a lucrative venture that continues to attract investors into the Nigerian economy due to our ever-rising population. There is an increase in demand in the transportation industry with a growing population of over 200,000 people in Nigeria. This requires much supply, hence the need for people to venture into various land transportation businesses. EventThive CEO spoke about vital deliberations across the mobility industry and the reason for this setup. Basically we believe it is important for us to constantly provide platforms for policy makers and of course private sector stakeholders to learn new developments within Nigeria's transport sector. That's where we thought we should kickstart the idea of Lagos Transport Fest. It was launched in 2019 and this is currently the third edition of the event where we provide mobility solutions provided with an opportunity for them to showcase their solutions at platforms like this and of course we bring together the guys from the public sector for them to learn new developments from the guys that are developing this solution in the private sector. So that's basically what we can do at Lagos Transport Fest every year. Youth gaze with full attention in these whole field to capacity as key speakers inform on the way forward investing in the transportation industry. We have people who come around to talk about terminals. They want to operate terminal to move continents. We have people who actually want to participate that become in. We talk about granting of an access. Lagos State for example is running Islamata on our line. All he's asking for now is access just like another state will come in. We have another state coming in. And the state government has also been visiting. We have been discussing and we have individuals who want to bring their wagons. They want to bring locomotive wagons to run. All we need to do is just to sit down the MOU and discuss it in modality or how to defray. Because I'm really descent for you to move around with. It provides you with security such that you're able to commute from place to place in a very safe manner. What we've been able to do in the last two years is build that up into having over half a million about 560,000 people that have used that trips up to commute within bus stops in Lagos across cities in Nigeria and not just doing that in Nigeria but doing that in Nigeria. We currently have operations in Ghana and we currently have operations in Uganda. With the availability of e-hailing cabs there is indeed a beat of a transition in the transport industry, perhaps only for the elite. There is a growing expectation that the real sector will function fully like in other climes so people can start keeping their cars at home and thereby reducing traffic. Destiny Mama for Plus TV Africa. you


It's Wednesday afternoon, and we know what time that is. It's Hawaii, the state of clean energy time. And I'm your host, Mitch Yuan. And I'm very pleased to welcome back a multiple or a serial interviewer, interviewee, Toby Kincaid. And we're gonna carry on with our series about hydrogen, the decarbonizer. This is actually the fifth part of our series. And I hope we're gonna have a lot more. Toby's got a lot of good information today, which I will guarantee we're gonna be back many times with Toby on this show. So Toby, welcome to the show. Thank you, aloha, Commander. Good to be with you. So you have a lot of news. I mean, we're talking before the show started. Wow, it was all I could do was to quiet you up so we could start the show. You're so excited and enthusiastic about this. So tell us about your news. You've been all the way into Singapore, I understand. It was a hard flight. And I went two and a half days, three days in Singapore. Tell us about what's going on in Singapore. Well, it was a virtual conference. So I didn't fly very far, but I am astounded. The world is organizing around green hydrogen. I've never seen this before. This is extraordinary. And I remember we had a conversation some months ago when you mentioned, you know, you sensed something happening in the hydrogen industry. Well, my goodness, are you right? This is a sea change. You know, we've been talking about green hydrogen and the way of informing people, but I had no real concept of how advanced the world has become just within the last few years. And how I know this now is I've been attending conferences. There was one in Europe a few weeks ago put out by Mission Hydrogen out of Stuttgart. Very well done. It was a 24-hour marathon session. I look a little older now, probably for that session. And even last week in America, we had a conference put on by the Green Hydrogen Coalition out of Berkeley. Absolutely fantastic panel. I was astounded at the caliber and quality of the venture capitalist of that one of my favorite speakers was the director of the LADWP, the Los Angeles Department of Water and Power. Now, it doesn't sound like that's a big thing, but they control billions of dollars of service area. And they were absolutely on the money. So I was astounded. Now, we're just finishing up actually right now the Singapore Conference. Now, this is the first annual Asia Pacific hydrogen summit. And again, I am floored at the absolute caliber and quality of the perspective of the speakers. Now, this was put on by Mitsubishi Heavy Industries. So that tells you that they are looking very seriously in that they're not beginning to look. They have looked, they are beginning to conquer. And what's happening is that we see these emerging markets in Japan, South Korea and Singapore and they intend to be the green hydrogen hub of the Asia Pacific basin. And what they're doing, which I didn't really realize and you had mentioned before is that the Australians are really positioning themselves to be the supplier of green hydrogen for the entire basin. And in this conference, I find it interesting, they didn't mention the United States. Nobody talked about us. They even didn't mention Hawaii. They are looking at the world as though we're gonna do this and we're gonna lead everybody in doing it. And I was astounded, the venture capitalists, we'll get to some of the slides later, but the amount of capital that is being mobilized is this is a sea change. Right, so let's go to the next slide because you wanna tell us about something. Oh, please. Oh, well, this is your great slide that talks about the water cycle. This is what it's all about. Have you ever heard the phrase from dust to dust? Yeah, okay, well, this is from water to water. And that's what's so amazing is that we're talking about industrial power. We're talking about safe power. We're talking about power that's available to everyone in the world because it's sourced from water. And what your slide points out is once you make the water and you store it as hydrogen, it can store forever. We're not talking about lithium ion which degrades in four hours. No, no, hydrogen is so stable and so safe. Safe, you can store it for weeks, months, years, decades, possibly even centuries. That's how well we know how to store it and how safe it is because there's only hydrogen in the tank. And now in your slide, we're talking about how do you use this hydrogen? Well, the fuel cell is a marvelous device and it gives you the energy, the power density that you need to run not only cars, but pickup trucks, construction vehicles, farm equipment, and as we'll get into later, maritime shipping now that the incentives and the initiatives being born in Europe and in Asia Pacific. Oh my goodness, the world is moving. I was astounded.   You know, and then it shakes, you know, how do you do that? Well, you were in, you're in tune. It's really that momentum. And so well done, called that one right. Yeah, right. Well, thanks. So let's throw up to a slide too. Oh, okay. Let's take a slide. So I'm not sure they're coming on, so. I think I see something on, but I'll give it a preface. I've lamented for a long time, this industry needs a trade journal, a trade magazine. So I've been contemplating how I might do that. And so it reminded me of something my mother said to me when I was a little kid. She saw me one day and I was kind of forlorn and I had this big project and you could tell, I just didn't know how to go at it. And she said, how do you eat an elephant? And I thought for a second, I eat an elephant. I don't know, how do I? She goes, one bite at a time. Right, oh, thanks, mom. That made me understand. So when I look at this elephant of the green hydrogen industry, as you know, everybody's involved now, everybody's doing projects. Everybody, it's like a shotgun throwing everywhere. And it's very difficult to have one clearing house for information. So I thought, okay, I'm going to, this is just a mockup. We're going to print the first week in January. So these are just spreads that I'm putting together, throwing out, looking at different combinations. But you can see that we're going to be a practical magazine. We're going to talk about the industry and the news that's going on. And I had a bit of another epiphany, if you want to jump to the next slide, is how do I turn this industry into little bites? So I thought, what would happen if we took a section of the magazine? For example, this one is green hydrogen heavy industry. And I thought, well, if I built a corresponding website so that people could read the magazine, read the articles, but if they want to drill down on the backstory who did it, who financed it, who did the construction, all of those details, I thought, well, why not create a website under each section that you could go to online and drill into more information? So I started doing this and then I started registering for the websites and they kept going on and on and on. Well, I covered what I needed in these different sections. Now, heavy industry. You just never hear of green hydrogen and heavy industry in the same sentence. Well, the Europeans have an initiative. They're doing a green new deal and they call it a green hydrogen deal. They know what to do. And their initiatives, their renewable portfolio standards are double all of what Asia is now declaring. So Europe is definitely ahead in this. There's a great project in the Netherlands called Hermes where they are demonstrating every single aspect, a port facility, storage facilities, large electrolyzers, large fuel cells. And just to refer back to your first slide, that's what's so wonderful about this because when you have an electrolyzer, you basically have two inputs and two outputs. The two inputs are water and electricity, well, from newables, from solar wind. And then you have two outputs which is hydrogen and oxygen. Well, the fuel cell is just the opposite. You have two inputs and two opposites, but the inputs are hydrogen and oxygen and the output is electricity and water. So by putting those two together, you now have a total ecosystem. You have a water-based system and you can power anything. Jets will talk about Airbus and their new initiative. So when I was designing the idea for this magazine, I thought, okay, when I do a section, I'm gonna have the supporting website so we can really get to the information. And now when we have information, we know what bin to put it in. And my philosophy is the smaller the bin, the more comprehensive the content. So this would allow us a tool. In fact, I have a very ambitious notion of building out these websites through this trade journal and becoming the world's largest app. Ah, how about that? Now, that's me waving my arms. And none of these things are built yet, but they're under construction and we'll be going live one by one with the websites as they come up to speed. But the magazine will be coming out in January. I would encourage everyone, please get a copy of it and contribute. If you have articles, if you have projects. So I'm very excited about this and what's happening. So, well, let's go on to the next slide. Let's just keep going. Okay, then one of these is transition. We don't want to strand our assets. When we get to the capital part, one of the things I heard in these conferences is they're very much involved with the fossil fuel industry. And 90% of the world runs on fossil fuels, which really leads us to the big question in the world. Here's the fork in the road.  That's the choice. If you pick carbon, because we saw in our last episodes, the energy's in the hydrogen, not the carbon. Cold is mostly carbon, little hydrogen, low energy. Oil less carbon, but more hydrogen, higher energy. Natural gas even less carbon, but more hydrogen proportionally, higher energy. And then forget the carbon, you don't need it at all. Just look at hydrogen and it has the highest energy. So the whole world runs on hydrogen, but it's stuck to a carbon atom. Exactly, and then we pollute the atmosphere when we get rid of the carbon atom. The only way we can get rid of that is attaching it to oxygen. Exactly, and as you pointed out in the earlier show, you're in Hawaii. You're surrounded with the ocean of hydrogen stuck to oxygen, water. So why would you import hydrogen stuck to carbon from some other place? This is what your work demonstrates. And the fact that you're using large scale solar arrays, making clean energy, potent energy for public transit in your application, which is brilliant because now you're actually fulfilling your state's mandate of saying you're gonna be clean by 2045. Now you're doing it right now. And that's what's brilliant about the work you do and the role Hawaii has played and I believe will play pushing forward in the future. And really Hawaii has a lot of skin in the game because 10 years ago they put together the hydrogen fund and it was a $10 million fund. So that doesn't sound so big when you're looking at some of the projects today. But if you look at the pop on a population basis, as I think I've said before on a per person basis and you compare it to say the population of California, which is what about 35 million people and we're about like slightly over a million not counting visitors. That'd be like a $330 million investment if you kind of project it based on population, which is a huge deal and a huge investment. And even now we've got additional investment from the, for example, from the county of Hawaii, the big island, just invested in some hydrogen buses that we've got to upgrade them and a significant investment for them, especially given the economic times we're in right now. So yeah, Hawaii's got definite skin in the game and they're pretty, they're very serious about it. And particularly on the big island, they're very aware of hydrogen. I've got a really excellent support group out there that's working with me to kind of get the word out to particularly the school children. So that's going to be coming up in January where we're going to be running a contest, an art contest with all the students so that we can put a really interesting graphics on the buses. But the kids are going to be involved as a contest through the prizes. And if you get the kids involved, guess what? The parents are automatically involved. So I like to really leverage, you know, the opportunity here and get the kids being creative and thinking about it. So that's just one of the many tools in the toolbox. So anyway, this is about your show here. No, no, but your message is the greatest message for everybody. And particularly the younger generation. There is a solution.  They don't have to grow up in a world where they can't go out and play in the forest and the streams and the fields as we did when we were kids. This is really it. And I'm so impressed with the Asia Pacific platform and the activity here because they really, around the world, they actually really get it. And they are in a race now to put in as many projects in the ground as possible. Hawaii has been the heart and soul of all of this because you guys have declared a hydrogen economy as your goal nearly 20 years ago, I think it is. Correct, yes. Yeah, so you beat everybody because it's, well, in the Hawaiian language, I think the phrase is opono, do the right thing. And so, you know, pono is really in the heart of Hawaii and look at the world's benefit. You know, you did all the hard work to open the door and now the world is running through that door. But they're not gonna push us down because there's room for everybody. And the great opportunity is to put as many shovel-ready projects in front of these capitalists as possible. You don't have to come up with the money. The underwriters have all the money you need. In fact, this is new to me, I didn't realize this, but there's a program in the magazine, I'll put all the contacts. There's a program from the DOE, Department of Energy, where they have a $2 billion fund. It's already been funded, it's sitting there. I think most of it is still ready to be placed. And they're looking for shovel-ready projects that are not R&D, but in the next stage, demonstration phase. Now, this is perfect for what you're doing because as your buses come in and you demonstrate this, we can go now to Oahu, to the bus, the transit authority, and say, look, let's put a program in. Here's the funding for it. And let's do clean hydrogen for all your public transit. Let's look at what we need to do, and let's do it. And so you're in the perfect position because you did the hard lifting, the heavy lifting. You demonstrated in the environment of Hawaii that you can make this work and you've done it. That's amazing, really. Well, let's look at your next slide. I'm really excited for you there, Toby. Thank you. Okay, another section I'm gonna do is greenhydrogenclimate.com, of course, in the magazine. And then when you go to the.com, we'll drill into it. But the climate issues here, this is an actual way to demonstrate what your first slide has been saying. It's a water cycle, and that's not only good for humans, it's good for every species on the planet. In fact, if we don't do green hydrogen, our planet will slip away. The toxicity loading that we're doing is unfathomable. It's just out over the top, almost to the point of reckless insanity. We have to understand that, you know, it's like everyone, imagine everyone in the world in a swimming pool, and everyone's doing number one and number two, at what point do you go, ew? We've reached that point. I'm there now. I'm never going swimming in a pool again. Well, we're in this together. And so we have to find, here's the bottom line in my view. If we want to solve global problems, the only way it can be done is if we all employ the same local solution. And green hydrogen, what you do, making hydrogen from renewables and from water, stores safely when you use it, you get the energy out in the water back. You know, that is the future for the world. And the young people of the world have to understand that they've been fed all of this propaganda that hydrogen is dangerous. That's the safest fuel. I mean, look, NASA has been working with liquid hydrogen since the 60s. Have you ever heard of a hydrogen accident from NASA? No, they rail it down from Canada all the time. Have you ever heard of a derailment that caused a town to be that? No, hydrogen doesn't work that way. It's so lightweight that if it breaches, it goes straight up and out.    So the whole idea is that it's foolproof. You've reasoned it out and you understand in the work you do that water is the feedstock and water is the end result. So it's a cycle. And it's potent, safe, available to everyone in the world and a hundred percent non-toxic. You know, anything else, do you want coffee in the morning? Yeah, I bet we could make it make coffee in the morning, but it does what we need to do. So in this magazine, I want to use all the things that I grew up with Loving Magazines was the visuals of images, the compelling content. For example, let's go to the next slide. Let's just try and go through them. Now, this is on capital. We're gonna have greenhydrogencapital.com but in the magazine, we're gonna talk about what the capitalists are doing. They see an opportunity. They see $30 trillion of infrastructure that needs to be replaced. They make money placing money, placing money to work. Tronches of capital are available. I'm astounded at what is being mobilized and how sophisticated the venture capitalists are today. They understand risk assessment. They understand asset management. They understand projections. And what they're really drilling down on is this new emerging market called green hydrogen offtake. Now offtake agreements are, of course, when people buy the hydrogen that you're going to produce. Now, a little sidebar in Japan, Mitsubishi Heavy Industries, they're finishing a port at Kobe, the Kobe port, where they're putting a facility to then receive hydrogen from ships. Now, that's gonna open up. The Australians are salivating because they export more coal than anyone in the world. But as you remember, last year, they burned up. Just like we did a few months ago in Oregon and the West Coast, we burned up. They realized, okay, instead of coal, we've got to move to an export that we can do. We have lots of renewables. Let's make hydrogen and ship it to Singapore. And the Japanese said, wonderful, we'll build you a port facility to receive it. So the opportunity on the big island, particularly for you to manufacture, produce hydrogen from water and export it to the Asia Pacific region, would make you the Saudi Arabia of clean energy and wonderful. So I've been going through these sections from capital to infrastructure to even transition. The capitalists are talking about stranded assets. They don't want to strand assets, so they're trying to work it out. But I would remind everyone this, more than just hardware, there's human assets. So we have to have a transition for people to have new training, new education, new jobs. So I registered green hydrogen training, green hydrogen education, green hydrogen jobs. And the idea is now we have a place where people to go and they can figure out what they want. I even registered green hydrogen RFP, Request for Proposal, green hydrogen RFQ. These are where you could request a quote. This is where the industry can consolidate. And what I recognize is just this moment in time where the industry is just being formed that we really do need a trade journal, a trade magazine. It has to be professional, it has to be beautifully done. The slides you're seeing are kind of my mock-ups, my spreads, but I'll hire a real graphic artist to do the final master. But I think it'll be a wonderful opportunity to interview you. I'm gonna have a section called Hydrogen in Hawaii. It's gonna be wonderful. We're gonna talk about your coal plants and their conversions, the projects you're doing. The whole world would love to see what you do. And I intend to make sure they see it. Awesome. Well, I like your slides and your mock-ups are pretty good. Thank you. It'll just get better. You're doing well already. Well, we've got a good start. And let's go to the next slide. What do we have next? Okay, this is what my favorite is maritime. So I registered green hydrogen maritime and greenhydrogenmarine.com. I haven't decided which one yet. Doesn't matter. What you see here and what I was amazed to learn out of the Asia Pacific Conference is how much the shipping industry is going to be transformed. Port facilities, even airports for aviation fuel. And so one of the articles I'm preparing for this section is we're gonna take an example of a typical cargo ship from MERSC. We'll look at its range. We'll look at the fuel capacity and we'll examine if we use hydrogen, how would that impact the amount of space, the amount of range? And they have to go 14,000 nautical miles at 29 days. And then you have to have enough fuel for 29 more days because of contingency, weather. These are real things. And Kawasaki is now modifying liquid natural gas ships to transport hydrogen, very safe because hydrogen can only go up. They vent it if there's a problem. It's really the safest. I would feel very comfortable sleeping on a ship with the hydrogen in it because I know the physics of it. I would not be comfortable on a liquid natural gas ship. If you had a catastrophic explosion it'll blow up a square mile. So there's no surviving that but hydrogen goes straight up so I'd sleep well. So the point is these heavy industries are now in play where I must admit I didn't see this before. You sensed it, put me onto it and then when I looked at it I went, oh my goodness. This is not just a little bit of an interest or just a propaganda play. These guys are in to win. And it's a $6 trillion a year business and they want every bit of it. That's what I find very interesting indeed. What do we have next? Let's talk about aviation. Oh, this is wonderful. Okay. It looks like a shark with sunglasses on it. This is Airbus. Airbus announced they're doing a hydrogen transportation hydrogen jets, turbo fans, turbo props. They're doing a whole sequence of three. I'm only showing one of them. This is their kind of molded wing design. They are going big on hydrogen. And the reason is the whole world has really kind of matured and it's been a lot of hard work from people like you pushing projects pushing policy considerations pushing the renewable portfolio standards. And what we're seeing now is Airbus is responding as the Europeans are with great vigor. And they're going to take over the eight, in the next decade, things are gonna be Airbus. I predict unless Boeing decides to understand the moment in history, they better not wait. They're not waiting now. Just FYI there was a US Department of Energy put on H2 at airport's workshop I think it was last week of the week before last. And it was a three day online conference and they started out with the major aviation players like there was Airbus, there was Boeing, Sikorsky and some others, not too many more I guess because there aren't that many aircraft manufacturers but they all have like you said they all have very active hydrogen programs. So it was astounding. And then the next day was looking at what kind of technologies do we need? And then the third day was looking at the infrastructure. And for example, back of the envelope as I recall the number was for LAX to convert that. And everybody pretty well agreed that, well, it's had no question that the aircraft are gonna have to fly on liquid hydrogen. I mean, you got to ask us hydrogen for that. But they would have to have like, I think the number was 130 tons of liquid hydrogen per day. So I'm going to talk about infrastructure how do you get 130 tons of hydrogen per day into the airport? So there were a lot of ideas about how that might be done. So they'll come out with a report sometime in January, February, what happened. And a lot of the presentations will be presented in that right now they're just seeking permission from the, it was a bi-invitation only kind of a conference. So the US DOE is right on top of it, I'm happy to say. Pete Devlin and his team and Sunita are really, you know, being proactive and getting after it just as you said. So yeah, very encouraging. And I think Airbus said they were gonna have a test plane up and running in 2023, like in three years. So pretty interesting. Now they're moving. And I'm happy to hear you mentioned Sunita at DOE. She was one of the guest speakers at the Green Hydrogen Coalition out of Berkeley. And she's brilliant. What a resource. They were calling her the hydrogen rock star. And after hearing her speak. I understand. I'm a groupie. Oh yeah, yeah. She's been very effective at moving the program, you know through lots of barriers and getting it done. And even some of the times we're going through now. So that's right. And she has, she's sitting on $2 billion of funding and she'd love to place it. That's her job. And part of what she oversees. There are other groups in whether it's alone. Yeah, I don't think she has $2 billion. No, no, no, but her position in the DOE knows where it is. She knows where to go. Yeah, that own program you were talking about. Exactly, exactly. So the whole world is waking up with your prediction of a tremor, something happening. Something is near. It is a sea change. I've been around the hydrogen industry for 27 years. And I've never seen anything like this. It seems like every decade we get a surge. And then the powers that be kind of pushed back on it. Most recently with all the investment going to lithium ion, which at best might do 2% of transportation. Whereas fuel cells will do 98%. OK, there's a lot of. So we've got about a minute to go. I don't want to curb the enthusiasm, which is great. But let's have the last slide. We're going to talk about the different colors, very quickly, different colors of hydrogen. You've got that slide up there. Yeah, absolutely. So we talked about hydrogen being the real energy and fossil fuels. So in the world, in the Europeans and in the Asian world, we really talked about green hydrogen. And I thought I'd put a chart up of what we really So there's black hydrogen. That's fossil fuels. That's 90% of the world. Gray hydrogen is how most of the hydrogen in the world is made today. 98% of it, 70 million tons a year. And what that is is they use SMR, steam methane reforming. And this is making it from a fossil fuel, from methane. Now, the problem is you get one ton of hydrogen, but you're going to produce 10 tons of CO2. So that's not really a great solution, but that's where we are at the moment. So talk about hydrogen green. We've got to move fast. Oh, yeah, very good. OK, blue is where they say, OK, we're going to make it from fossil fuels, but we're going to capture all the CO2. That's CCS, carbon capture and storage. But it's green hydrogen. You're still fossil fuels. But green hydrogen, and that's the one because it's made from renewables on the front end, running an electrolyzer, turning water into hydrogen fuel. That's the answer. That's what the world understands. That's what you've been doing for decades. And bless your heart for all the work you do. And the leadership in Hawaii, it's going to really make a big difference in informing everyone in our country that we ought to follow what you do and get to it. Oh, we're all in that canoe behind me together. So last slide, how do we contact you? Oh, thanks. If anyone has any questions or they want to submit an article or comments or anything like that, please contact me, Toby Kincaid, at gmail.com. There's a number there. If you want to see some of the books on Amazon, you have to search Christopher Kincaid, which is my given. I publish under my given name. So not many people know me by Christopher because I've always been called Toby. But just in case, so if someone wanted to search Amazon, that's what you need to do. I'm going to move the back. Amazing enthusiasm. Thank you so much. You'll be back real time very soon. Toby, I love having you on the show.  So this is Mitch Ewan signing off and aloha for Hawaii, the state of clean energy.


Thank you for the invitation, Hamels, to speak here. So issues within vivo models, cell and tissue culture. Let's not make the same mistake we make in animal experiments. And the main driver for this talk, and we have heard it before, is the reproducibility crisis that Mona Baker already published in 2016. I'm quite happy that reproducibility was discussed already this morning, because it's still a great problem. Also, in vivo studies, in laboratory animal science, we knew already long before 2016 that when working with animals, we have reproducibility problems. And I will very briefly discuss two of those issues. The first one is feed composition. And the way we feed animals. So already in, oh, I have to go back. Yeah, already in 1985, Anton Bain has showed that what you feed the animals has an effect on their physiology and will affect the results of your study. And it's not only what you give the animals, but also how you feed them. Because at limited feeding, what is normal with rats and mice, gives obese mice, they live shorter. And they're not as healthy as animals that get intermittent feeding. So we are actually studying non-healthy mice, but the logic mice. So how can we relate the results to, for instance, in preclinical studies to human situations? So feed is very important, and the environment we keep the animals. The other one, and we have heard that before, is substrains. We also have substrains in animals. And the nicest example is the black-six mice. People refer always, well, I do my experiments on black-six mice. But some of them are not aware that there are several black-six strains. So there is a black-six strain from Jackson. And there is a black-six strain from the NIH. And they originally got the same mice, but because of genetic drifting, they showed a different phenotype over the time. And we still see people, I'm an assessor of animal protocols, that use different strains, so either the Jackson or the one of NIH. And they still want to compare those, and they're not really comparable. So in animals, substrains, you get very quickly a substrate. So as this article show, in as little as three generations, so there's really fast, you are already getting a genetic drifting and a new substrate. So you have to regularly back cross to a bona fide black-six line to maintain your original characteristics. Okay, now we go to the in vitro studies. Because when Mona Baker released her study, I started to realize that actually we have the same problems with in vitro studies. I mean, I'm not a vitro person, scientist, and I always claim that in vitro studies are much better than animal studies, also with regard to reproducibility. But actually we have a lot of issues ourselves. And we had a very nice example of that, I have to mention, it is also not only important this reproducing results, but also reproducing quality of products or production consistency. So when you use the cell lines, for instance, for the production of vaccines, etc., it's very important that you use always a nice cell of which you know what it actually is. So in 2021, there was an internal laboratory study published. And they studied the variability of A5 for nine cells. So there were two laboratories, one in Switzerland, one in the UK. They followed the same SOPs. They got the cells from the same supplier, ATCC, and they got fetal bovine serum from the same supplier, Sigma. So those are the two issues I'm going to focus on. And these are the results, just the basic cell properties. Cell viability was different, number of cells per square centimeter was different, and the trans-epidereal electrical resistance of the cells of the two cultures were different, significantly different. And then they started to investigate what's the problem behind that, what is causing this difference. And the first culprit they found was FBS. And they found out that they didn't get the same FBS, they got FBS from different lots. And so they say that FBS can strongly affect the outcome. And in the results they refer to our 2010 paper, where we discussed that batch to batch variability related to variations in the concentration of serum components because it's a biological product. So it can't always have the same composition. In addition to the unknown exact composition of FBS, we still don't know what's the exact composition. Can also ultimately lead to experimental variability and limit inter-laboratory reproducibility. And that led the authors to conclude that if you use FBS in different studies, it will be very hard to get the same results because it will be very hard to get FBS in all the different laboratories from the same lot. So it's very likely you get FBS from different lots, and it's very likely that you get different results. So they suggest that we should go to alternative approaches, and not involving field bovine serum. So animal free serum preferably. And that there is a batch to batch difference was already known at the time that the first paper was published showing that FBS was a nice growth medium for the cells. Because Puck who invented it in the same paper already observed that there was variability of performance when serum was collected in winter or in spring and compared to the other seasons. And we now know that there are a lot of difference not only to the different seasons, but also to the areas where we raised the hurts. And in addition, and that's important nowadays, we have when we produce pharmaceuticals, he also noted that there are toxic factors in serum. And that's for instance nowadays one of the reasons why EMA here in Europe and EPA don't allow serum in the medium when you are going to produce pharmaceuticals with in vitro methods. Okay, serum provides proteins, vitamins, hormones. Shea force protection, attachment factors and trace elements. And it's very important for its growth factors, so almost all cells grow. With serum, it has a limited number of antibodies and therefore it's regarded as a universal medium. So that's why it's so popular still at the moment. But there are scientific problems. So the composition of FBS is unknown, so you don't know what you add to your cells and what is influencing the compounds that you might be testing. We have seen that qualitative and quantitative variation between different serum batches may contain different amounts of endotoxins, hemoglobin and other adverse factors and may be contaminated with viruses, bacteria, fungi, microplasma and prions. And that lead to reproducibility problems in experiments and safety of the products. In addition, there are animal welfare issues. So there are problems with the transportation of the pregnant cows, which is a problem nowadays in Europe because they have restricted the transportation of pregnant cows. But also the withdrawal of blood from the fetus is a problem. But we cannot always get the guarantee from the slaughterhouse that the fetuses are already dead at the moment they do the heart puncture to collect the blood. So the conclusions when considering supplementing cell and tissue culture with animal serum actually do not, unless principle, should be applied. Preferentially the medium should not contain any animal derived component unless it was proved to be an absolute requirement. So how do you replace fetal bovine serum? Well, one of the solutions is go to the FCS-free database. We have established the FCS-free database together with the annual free research UK. And if you go into the FCS-free database, we have 286 different cell types there at the moment, so it's still rather small for which we have 651 different medium. The reason that it is small is that only until recently people haven't developed CM3 media since the last five to ten years people start to be developing those CM3 media. And it involves a lot of time before you have CM3 medium. So you go, the FCS-free database is composed of commercially available products, that was the original basis. And now also of literature based records. So modifications of commercially available products and formulations. The problem we have with commercially available products is that you still don't know what's in there. It could be still and will base products in the commercially in the media. And also they change the formulations without informing the customers. This is what the database looks like. You can here fill in the cell line that you're interested in. Then you get, if you have the cell line CHO, you get all the CHO cell lines here. And when you press on one of the records. Here's the CHO, all the CHO results. When you press on one of the records, you get more information on that particular CM3 media and a link to the source, either the journal or the commercial supplier of it. Also, if there is not a CM3 medium available for your cells, you may go to the reference and review paper part of the website. And there we have a lot of papers that helps you show different strategies to develop CM3 medium for your own cell. But also, and that's really important, some people tend to forget that, the adaptation procedure. I mean, if you have been able to obtain CM3 medium for your cells, don't put your cells directly in the CM3 medium, they will die. They need to get accustomed, adapted to the new medium. And that's a very careful procedure to do that. And that's described in, for instance, very well described in the bottom paper. So conclusions, preferentially, the medium should not contain any animal derived component, unless it was proved to be an absolute requirement. And that brings us also to another very popular supplement, a medium in which we grow, for instance, organoids, and that's matrigel, because matrigel is also animal derived. And because it's animal derived, we also have batch to batch differences. But again, a lot of people claim that they use organoids and the other things we, now let's first claim what is matrigel. It's a basement membrane matrix produced by Corning Life Sciences. And it's used for 3D culture of organized cultures, stem cell growth and differentiation, angiogenesis essays, tumor essays, and a lot more essays. But it's animal derived, and it may have different compositions. And may contain also xenobiotic contaminants and growth factors. So the origin is the Engelbreth-Holm-Swan-Sacoma, and that is maintained and grown in mice. In the base of the tail, they inject the sacoma and there it is grown. For every five milliliters of matrigel, one mouse is killed. So you see already when you claim that you are using organoids to replace animal experiments, and you do it in matrigel.  And there is a lot of suffering involved, because the tumor can be bloody or contains a lot of pus, and they inject the matrigel. At a low temperature and injecting a low temperature in a warm animal is causing suffering, so there's also suffering involved. And the tumor size is a problem, so they have to be very alert on the size of the tumor and also, again, as it says here, there could be pus involved. So an infection of the animal. So both for FBS and matrigel, it holds that also in vitro methods may involve animals and animal suffering. The production of the substance may involve animal suffering again. Many animals are killed for these substances. And there are batch to batch differences, and therefore, with respect to results reproducibility issues. And preferably in vitro methods are xenofree and animal component free. And chemically defined, that would be the best situation. Okay, so far, so much for the environment in which we grow the cells. Now about the cells themselves, and there will be some repetition of what you have heard this morning. Because another culprit that caused that difference in results between the two laboratories was, they obtained the different batch of cells. A different lot of cells, they were not informed by that. They had to go back to the ATCC to find out that they obtained a different lot. And again, different batches of cells potentially introduce experimental variability because the exact differences between the batches is not disclosed by the provider. And one of the causes, and we have heard that before is genetic drifting. And Cuevado found that that is prevalent in almost cell lines with a medium of 4.5% to 6.1% of the total genomes. So ATCC also recommends to passing the cell line for no more than two months of continual culturing. Because after the two months, the cells may have diverted so much away from the original cell that it is not really representative anymore of the original cell. So that's what was used also concluded. They no longer represent reliable models of the original source material. So you should not overpassage your cells. And then the one that has already a lot of attention this morning is the cell that you're working with, really, your cell. And then we refer to the cell of Sauer's database, where we have the problematic cell lines and we have seen this already. It's already in the menu. And when you go to the, you get a whole list of the problematic cell lines where you can check of your cell, whether your cell is problematic. And that is really flagged, clearly flagged in the cell of Sauer's database. And the cell contamination, and Amanda has already showed that, is not new, it is already known for a very long time. So in 1962, Brent already showed that several human, monkey, rabbit, swine, calf, and whatever. Tissues are actually either mouse or human species. And we heard the name Gardler this morning. Also already, and he showed in 1960, that four cell lines, three stated of human origin and one of rabbit origin, were actually mouse origin. A probably consisting of L cells, so the contamination. We have that already for a long time. And also, Gardler showed that different cell lines were not the cell lines that were supposed to be, they were actually the cervical cancer cell line, Hila, and that's, well, we know, that's a very problematic cell line that affects a lot of other cell lines. So go to the cell of Sauer's database. So what are the cost of unauthenticated cell lines? Between 18 and 36 of cell lines may be contaminated or misidentified at the moment, at the moment of 2018 when News published his results. So that's quite a lot, between 80% and 36%. So that really shows what others have already argued this morning, that you have to have your cells regularly checked, either by DSMZ, ATCC, and whatever organization that provide these services. Then one thing I would like to draw attention that has been mentioned this morning also, that there are gender differences between cells. You may recall that in 2014, NIH introduced a new policy where they required that there is a balance between the sexes when you do cell culture and animal studies. Because men and women are different, and that's well known. So here you see an example of 2015 where, as the air studies were done, of 855 annotated female cells, actually 331 were males. The other way around is a little bit less of 600 annotated males, only 10 were females. So also here in cell we misidentify the sex of the cells. And sex of the cells is different, two examples. There are gender-specific differences in expression in human lympho, blastoid cell lines, and we do have the gender of cells really matter when screening for novel anti-cancer drugs.  So in conclusion, over subculturing cells leads to genetic drifting. There's a problem with misidentification of cells and sex of gender of cells matter. And there are a lot more issues with in vitro methods, which I can't discuss now. Thomas Hartung already discussed all these issues in 2007, so there are a lot more issues related to in vitro methods that we should be aware of. And luckily enough, like with animal studies, nowadays we have guidelines that helps us identifying those issues and improving our studies. So those are the different good cell culture practices guidance and toward good in vitro reporting standards. So ECFOM produced good cell culture practice, two different reports for just ordinary cell culture, but later on they also produced good cell culture practices for stem cells and stem cell derived models. And good cell culture practice for human primaries, stem cell derived, and organoid models as micro physiological systems. And actually for me, the best and the most extensive guidance report was published in 2018 by the OECD, where they not only focus on the actual experiment, but also all the conditions around the in vitro studies, like the roles and responsibility of in vitro method developers, quality consideration, the facilities, strategies to avoid cross-contamination, requirements for all the materials and reagents, and so on and so on. Oh, staff training and development also very important. So it's really interesting to read this extensive paper and see whether you can learn something from that. Because most of the time we are so stuck with our routine practices that we forget that there are also other issues that we have to take care of. And then the last warning in vitro reporting standards. So now Barozova found out that the two different laboratories had different strains for their studies. They now reported not only what cell they used, they also report that they have different lots and they reported the different numbers. So that in case you want to repeat their studies, and you've got different results, you can tell that you might have a problem with the lot you obtained from the ATCC. So we are pushing that with animal experiments, but we have to push that again, and it was said this morning already. And Anita has said that also, the materials and methods have to be meticulously written down, because otherwise we have a reproducibility problem. Okay, so in overview, we have a problem with medium, particular FCS and matric gel, cell identification, genetic drift, the general sex of the cells. So also in vitro methods face comparable issues as animal experiments, so we can learn from them as well. And performance of good and well-informed research could avoid these problems. And very important there is experimental design and reporting on the experimental mental design and knowing the ins and outs of your model. A regularly updated good cell practice, culture practice could facilitate best in vitro practices and detailed reporting contributes to reusability. And I hope then that in a couple of years, we can answer this question with a no. And with that, I thank you for your attention. Thank you very much. And I think it's very, very important for all people working in labs that all this consideration. I was thinking about something because in the lab and I could notice that also a very important aspect is the time when you are doing your experiment, especially when you are working on metabolism, for instance, the cells are submitted to circadian clock. And do you think that this is also something that we should consider? And we should maybe also give this kind of information in the papers. Yeah, yeah. We have seen, well, I didn't have time to discuss that, but in a much longer presentation, I had that as an example and I showed you their papers where they showed different results dependent on the time of the day because indeed cells do have a circadian written. Yeah. Thank you for the talk. It was really interesting and informative. Do you think it would be a good step towards having FCS alternatives if providers like ECAC, ATCC, when you buy a cell line instead of giving the cell and the media standard one with the FPS, they give you a steady alternative immediately how to grow the cells. Because a pushback that we have all the time is, oh, I've gathered all my data with this condition and they won't be any more reproducible. Yeah. Which I understand, but if you start from scratch and you go to ATCC. Absolutely. When you start a new cell line, I think it's an absolute must to start it with CM3 medium. Yeah. It will be good to have when you buy them that the first option is actually the serum free. Yeah, yeah. And then not the serum plus. And you know the problem is that some people say, well, when I move away from fetal calf serum, my cells get different properties. And then my question is, well, maybe those are the properties of the original in vivo cell because I think fetal calf serum introduces a lot of artifacts that are not related to original in vivo cell. Absolutely. And also the other thing that I personally believe that should be an overall completely of the culturing condition in general, because for example, the amount of glucose in the media is a lot.  It's true because 3.5 grams liters is a lot. And maybe considering a hypoxic condition instead of a normoxia and maybe CO2 content. So I think it would be nice to have this starting, this topic starting on all to create a real better reproducible system as a baseline for animal studies. Yeah. Thank you. One question, I mean, in your list of programs you didn't have time to talk, so was zombies, can you in one or two sentences? Well, that has to do with the traditional way of 2D culture in which we change the medium one or two times a week. So the cells are not stimulated, they're just sitting there, the zombies. And the biggest problem then arises when you change the medium. They suddenly get pushed again and then again they turn into zombies because they have to wait for another couple of days before the medium is refreshed and before they get all the nudges then. And they're flowing around in their own waste material which also make them zombies. Thank you very much. Sorry, so where's the... I'm sorry, yeah. I have a question. So I wonder how people made serum-free medium. How they make it? So I mean, recently some research group create tissue culture medium based on the data of RNA sequencing data. So meaning that we can optimize tissue culture medium based on the proteome or transcriptome data. So do you think we need to improve the serum-free culture medium? Sure. And do you know somebody who... Well, go to the page I showed you. There are several papers that show different ways to get to serum-free medium for your particular cell type. It's a tedious process, I have to say. The other thing you can do, Stina Ortson, I think it's last year or the year before but I can give you the reference, produce a serum-free medium and according to her result, several different cell types already grew in that medium because for the ones who are not familiar with serum-free medium, up until now the new medium you develop was really particular for each cell type. Each cell type needs its own serum-free medium. So it was a challenge now to find a universal serum-free medium and it seems that Stina Ortson has found a serum-free medium. So that's worth trying. It's a gift to full formulation in a paper so you can easily use that as a basis for your cells. Thank you.


Clay, what happened on that first pitch to the young? Yeah, I think something that's made me successful in years, just limiting the type of contact of the varieties. And he was able to, you know, the first pitch there. Pretty, pretty good swing on it. And then got to double down the line. Obviously, it's, you know, that type of contact.  It's, you know, it can't happen. And probably just need to make a better picture in the count there. So how tough is it to digest just the way that rally formulated with a weak hit and then a walk? Yeah, that's kind of baseball some days. Maybe the potential senior, and then threw some good pitches there to a nil, and fouled a pretty good one off there, 3-2, and oh, you just got to be able to make a pitch there. And like I said, Dylan got me running the count, and it kind of made me pay for that for the walk. Aaron Goon felt like you were making better pitches than you were maybe on Sunday. Do you feel like you were making quality pitches? Yeah, I definitely felt a lot better out there tonight. But, you know, at the end of the day, you have two runs scored, and that's really the only thing that matters. And I don't know if nights like this are kind of tough, especially when you do feel pretty good.  But one able to do it tonight.